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Novel one-step approach to a universal anti-adhesion coating to prevent P. mirabilis induced catheter-associated… Alzahrani, Amal Ahmed 2020

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Novel one-step approach to a universal anti-adhesion coating to prevent P. mirabilis induced catheter-associated urinary tract infection  by  Amal Ahmed Alzahrani  B.Sc., Taif University, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   July 2020  © Amal Ahmed Alzahrani, 2020 ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled:   Novel one-step approach to a universal anti-adhesion coating to prevent P. mirabilis induced catheter-associated urinary tract infection  submitted by Amal  Ahmed Alzahrani in partial fulfillment of the requirements for the degree of Master of Science In Experimental Medicine  Examining Committee: Dr. Dirk Lange, Professor, Department of Urologic Sciences, UBC Supervisor  Dr. Jayachandran Kizhakkedathu, Professor, Department of Pathology and Lab Medicine, UBC   Supervisory Committee Member  Dr. Ben Chew, Professor, Department of Urologic Sciences, UBC Supervisory Committee Member Dr. Horacio Bach, Professor, Department of Medicine, UBC Additional Examiner  iii  Abstract Introduction: Catheter-associated urinary tract infections (CAUTIs) account for a vast number of hospital-acquired infections and are a significant burden to healthcare systems. In the USA and Canada, more than 33 million catheters are inserted each year. Urinary catheters provide ideal surfaces for bacterial attachment and biofilm formation. Several attempts to change catheter biomaterial design to prevent biofilm formation have met with poor success. We have developed a two-component coating that is highly biocompatible and effective in preventing bacterial biofilm formation. Here, we present data showing the efficacy of the coating against a difficult to treat uropathogen, Proteus mirabilis and show decreased biofilm formation and encrustation in vitro and in a murine model of CAUTI . Materials and methods: A novel binary coating composed of polydopamine (PDA) and poly (N,N-dimethylacrylamide)(PDMA) was developed and applied via a simple dip coating mechanism. The antifouling activity was determined in vitro following incubation of coated and uncoated catheter material to P. mirabilis at 4, 8, 12, and 24 hours post-exposure. Adherent bacteria were quantified via colony forming units (CFU) counts. The in vivo efficacy of our coating was determined using a murine model of CAUTI. Briefly, 4 mm (24G) catheters (coated and uncoated) were introduced into the mouse bladder percutaneously with ultrasound guidance followed by inoculation of 105 P. mirabilis. Adherent bacteria and struvite formation were quantified following 3-days post-infection.  Results: The in vitro study showed that our novel coating decreased P. mirabilis adhesion to polyurethane (PU) surfaces by 99% reduction compared to uncoated surfaces. Furthermore, in vivo studies showed an 87.9% reduction in P. mirabilis adhesion on coated iv  compared to uncoated catheters. In both in vitro and in vivo models, the accumulation of struvite and calcium oxalate encrustations on the coated surfaces were significantly reduced.  Conclusions: Using relevant in vitro and in vivo models, we have shown our novel binary coating to be highly efficacious at decreasing P. mirabilis attachment and subsequent biofilm formation and inorganic crystal accumulation on urinary materials. Further testing of this novel coating in validating the results in a porcine infection model will be important. v  Lay Summary  Urinary tract infection one of the serious health care issues that faced the patient who stay in the hospital for surgery and patient with a urological problem. These patients usually need catheterization for the short term or long term. Synthetic surfaces used in urinary catheters are mostly responsible for urinary tract infection. Simple explanation, bacteria itself attaches to catheter surfaces that lead to the urinary tract infection associated with catheterization. Our lab developed a special coating for catheters, which is safe and effective in preventing the infection. In addition, this coating can also prevent the formation of stone-like material on the catheter surface. Aim of this project is to evaluate the efficacy of the coating to prevent infection associated with bacteria known to cause urinary tract infection and is difficult to treat. Moreover, it plays a role in preventing encrustation formations on the catheter surface.   vi  Preface Amal Alzahrani was responsible for designing, testing, and analyzing this project data under the direct guidance of Dr. Dirk Lange with assistance from Dr. Jayachandran Kizhakkedathu at The University of British Columbia. Specially, Dr. Kai Yu from the Kizhakkedathu lab was responsible for the synthesizing PDA/PDMA coating that used throughout the project. For all animal work, Dr. Igor Moskalev from the Vancouver Prostate Centre provided assistance and guidance performed.  All animal work, which performed in this study, Ultrasound-guided intravesical implantation of urinary devices, protocol (A17-0297), was approved by The University of British Columbia Animal Care Committee.   vii  Table of Contents Abstract ..................................................................................................................................... iii Lay Summary .............................................................................................................................. v Preface ........................................................................................................................................ vi Table of Contents ..................................................................................................................... vii List of Tables ............................................................................................................................. xi List of Figures .......................................................................................................................... xii List of Abbreviations ............................................................................................................... xvi Acknowledgements ............................................................................................................... xviii Dedication ................................................................................................................................ xix Chapter 1: Introduction ............................................................................................................... 1 1.1   Catheter-associated urinary tract infections ..................................................................... 1 1.1.1   Urinary Catheters: What they are and why they are used .................................... 1 1.1.2   Contamination with catheterization ..................................................................... 4 1.1.3   Bacterial adhesion and biofilm formation ............................................................ 4 1.1.4   Mechanism of adhesion ....................................................................................... 6 1.2   Proteus mirabilis and how it infects the urinary tract ...................................................... 8 1.3   Strategies for the prevention of CAUTI ........................................................................... 9 1.3.1   Limiting use of urinary catheters ......................................................................... 9 1.3.2   Reducing the duration of catheterization ........................................................... 10 1.3.3   Using the right techniques for insertion and maintenance of urinary catheters . 10 1.3.4   Antimicrobial urinary catheter coating .............................................................. 10 1.4   Novel anti-adhesion coating to prevent CAUTIs ........................................................... 16 viii  1.4.1   Our Coating: What is it? .................................................................................... 16 1.4.2   The mechanism of action of PDA/PDMA coating ............................................ 18 1.4.3   Using PDA/PDMA coating to prevent CAUTI ................................................. 18 1.5   Thesis aims ..................................................................................................................... 20 Chapter 2: In vitro evaluation of the catheter coating ............................................................... 21 2.1   Bacterial adhesion ........................................................................................................... 21 2.1.1   Synthesis of PDMA ........................................................................................... 21 2.1.2   Development of an anti-fouling coating ............................................................ 21 2.1.3   Bacterial strain and culture condition ................................................................ 21 2.1.4   Anti-adhesion testing of PDA/PDMA coating .................................................. 24 2.1.4.1   Determining bacterial concentration by CFU counting .............................. 24 2.1.4.2   Use of scanning electron microscopy (SEM) to observe bacterial biofilm formation… ............................................................................................................... 25 2.1.5   Statistical analysis .............................................................................................. 25 2.2   Struvite encrustation formation ...................................................................................... 25 2.2.1   SEM and EDX spectroscopy-static model ......................................................... 26 2.2.2   SEM ................................................................................................................... 26 2.2.3   XPS analysis – static model and in vitro flow model ........................................ 27 2.3   Calcium oxalate encrustation .......................................................................................... 29 2.3.1   Sample fixation for SEM imaging ..................................................................... 32 2.3.2   AAS .................................................................................................................... 32 2.3.3   Statistical analysis .............................................................................................. 33 Chapter 3: Results ..................................................................................................................... 34 ix  3.1   Bacterial adhesion results ............................................................................................... 34 3.2   Struvite encrustations formation results ......................................................................... 38 3.2.1   SEM analysis ..................................................................................................... 38 3.2.1.1   Characterization of struvite encrustation by SEM-EDX analyses .............. 40 3.2.1.2   XPS analysis ............................................................................................... 47 3.3   Calcium oxalate encrustation .......................................................................................... 52 3.3.1   SEM images ....................................................................................................... 52 3.3.2   Calcium deposition ............................................................................................ 54 Chapter 4: In vivo analysis of catheter infection and encrustation ........................................... 55 4.1   Bacteria and animals ....................................................................................................... 55 4.2   Modification of catheters ................................................................................................ 55 4.3   Coating 24G PU catheters (PDA/PDMA) for in vivo study ........................................... 56 4.4   Day 0: Ultrasound-guided bladder catheter implantation ............................................... 57 4.5   Day 1: Bacterial injection into mouse bladder (infection induced) ................................ 59 4.6   Daily monitoring............................................................................................................. 59 4.7   Day 3: Experimental endpoint ........................................................................................ 59 4.7.1.1   Statistical analysis ....................................................................................... 60 4.8   Results ............................................................................................................................ 60 4.8.1   Bacterial adhesion .............................................................................................. 60 4.8.1.1   Coated and uncoated (4 mm) catheters successfully implanted into mice bladder ....................................................................................................................... 60 4.8.1.2   Urine pH levels ........................................................................................... 60 4.8.1.3   Anti-adhesive efficiency of PDA/PDMA coating in the animal model...... 61 x  4.8.2   Catheter encrustation ......................................................................................... 63 4.8.2.1   In vivo imaging ........................................................................................... 63 4.8.2.2   SEM images ................................................................................................ 65 4.8.2.3   XRD analysis .............................................................................................. 65 Chapter 5: Discussion ........................................................................................................... 67 Chapter 6: Conclusion and future direction .......................................................................... 71 Bibliography ......................................................................................................................... 73        xi  List of Tables Table 1.1: The main three types of urinary catheters .................................................................. 2 Table 1.2: Types of antimicrobial urinary catheter coatings .................................................... 13 Table 1.3: Thickness and composition of binary PDA/PDMA coating .................................... 17 Table 1.4: Chemical structure of PDA and PDMA .................................................................. 17 Table2.1: Artificial urine recipe ................................................................................................ 23 Table 2.2: Composition and concentration of artificial urine that used in CaOx encrustation formation ................................................................................................................................... 30 Table 3.1: Bacterial adhesion reduction by the time in AU media and LB media ................... 34 Table 3.2: Elements percentage at 2 days incubation, samples 1 and 2 were in flow model condition, and sample 3 was in static model ............................................................................. 49 Table 3.3: Elemental composition on the surface at 5 days incubation by XPS analysis. Sample 1 was in flow model condition, and sample 2 was in static model .............................. 51           xii  List of Figures Figure 1.1: A Foley catheter; (A) An overview parts of an indwelling urinary catheter (IUC) and (B) a diagram depicting an inserted Foley catheter. Note: Fig. A was a diagram from Urotoday website and Fig. B was from the HealthLink BC website (https://www.healthlinkbc.ca/healthtopics/zm6285)  ................................................................. 3 Figure 1.2: Biofilm formation steps (i) Reversible attachment of bacteria to surfaces. (ii) Irreversible attachment to surfaces. (iii) Formation of the external matrix. (iv) Biofilm maturation structure. (v) biofilm detachment. This figure was from Hindawi website(https://www.hindawi.com/journals/ab/2014/543974/) .................................................. 5 Figure 1.3: Antimicrobial coating for urinary catheters to prevent CAUTI. This diagram was from ‘A Review of the Recent Advacnes in Antimicrobial Coatings for Urinary Catheters’ [33]  ........................................................................................................................................... 12 Figure 1.4: Non-fouling coating to prevent CAUTI ................................................................. 19 Figure 2.1: Static experimental set up ....................................................................................... 27 Figure 2.2: (A) PU samples half-coated and half-uncoated. (B) Insert the samples inside the tube. C) AU with (∼5 × 103  CFU/mL) wild type Proteus mirabilis  is circulated through the peristaltic roller pump (Masterflex L/S variable-speed economy drive, 10-600 rpm, 115V) for 2 days and 5 days at 37 °C. Experimentation was performed using a flow rate of 3 mL/minute ................................................................................................................................................... 29 Figure 2.3: CDC bioreactor setup ............................................................................................. 31 Figure 3.1: : Proteus mirabilis adhesion on uncoated and coated PU surfaces in AU media A) within 4 hours incubation, B) within 12 hours incubation, C) within 24 hours incubation, and xiii  D) graph showed the percentage reduction of P. mirabilis. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001 .......................................................................................... 35 Figure 3.2: Proteus mirabilis adhesion on uncoated and coated PU surfaces in LB media A) within 4 hours incubation, B) within 12 hours incubation, C) within 24 hours incubation, and D) graph showed the percentage reduction of P. mirabilis. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001 .......................................................................................... 36 Figure 3.3: (A) SEM images of bacterial adhesion on uncoated PU surface (right image) and coated PU surfaces (left image) after 4 hours incubation in AU (B) SEM images of bacterial adhesion on uncoated PU surface (right image) and coated PU surfaces (left image) after 12 hours incubation in AU. (C) SEM images of biofilm on uncoated PU surface compared to PDA/PDMA coated PU surface (left image) after 24 hours incubation in AU ........................ 37 Figure 3.4: (A) SEM images of bacterial attachment on uncoated PU surface (right image) and coated PU surfaces (left image) after 4 hours incubation in AU (B) SEM images of bacterial attachment on uncoated PU surface (right image) and coated PU surfaces (left image) after 12 hours incubation in AU. (C) SEM images of biofilm on uncoated PU surface compared to PDA/PDMA coated PU surface (left image) after 24 hours incubation in AU ........................ 38 Figure 3.5: SEM images upper uncoated PU images and bottom are coated PU images (200 X magnification) ........................................................................................................................... 39 Figure 3.6: SEM images upper uncoated PU images and bottom are coated PU images ......... 39 Figure 3.7: SEM images of struvite crystals on uncoated surfaces after two days of incubation in AU with Proteus mirabilis. (A) SEM image (mag .8000X). (B) SEM image (mag.150X)  . 40 Figure 3.8: A) the percentages of elements for uncoated sample (2 days incubation), B) EDX uncoated samples (2 days)  ....................................................................................................... 41 xiv  Figure 3.9: A) the percentages of elements for coated sample (2 days incubation), B) EDX coated samples analysis (2 days)  ............................................................................................. 42 Figure 3.10: A) the percentages of elements for uncoated sample (5 days incubation)  .......... 44 Figure 3.10: B) EDX analysis – uncoated samples (5 Days)  ................................................... 45 .Figure 3.11: A) the percentages of elements for coated sample (5 days incubation), B) EDX coated samples analysis (5 days)  ............................................................................................. 46 Figure 3.12: XPS spectra: A) and B) flow model condition, and C) static model condition ... 48 Figure 3.13: XPS spectra: A) flow model condition and B) static model condition ................ 50 Figure 3.14: Scanning electron micrographs of encrusted PU samples from CDC bioreactor CaOx encrustation experiment A) within 3 weeks of incubation, B) within 6 weeks of incubation .................................................................................................................................. 53 Figure 3.15: Quantitative analysis of calcium oxalate encrustation formations PU surfaces by Atomic Absorption Spectroscopy (AAS). Comparison of uncoated and coated samples. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001 .................................... 54 Figure 4.1: Modification of 24G I.V. PU catheter for animal experiment  .............................. 56 Figure 4.2: Ultrasound-guided percutaneous catheter implantation into mouse bladder.(a) Anaesthetised mouse then put the mouse on this position while receiving isoflurane via nose cone we start the procedure (b). Inserting needle carefully (c) pushing the pusher into the mouse bladder and pulling gently the needle out at the same time. (d) Pulling the pusher out the bladder. (e) Catheter piece placed into bladders (as visualized by ultrasound)  ................. 58 Figure 4.3: Increasing PH level in mice urine during the experiment period, on day 0 the PH was 6.5 and on day 1 Proteus mirabilis was injected into mice bladders, which leads to increase the pH in urine to 9.2 at day 3  .................................................................................... 61 xv  Figure 4.4: Anti adhesive activity of PDA/PDMA coated catheters in vivo on (A) bacterial adhesion, and (B) bacterial planktonic in urine determined by CFU count. Each dot represent data from one mouse  ................................................................................................................ 63 Figure 4.5: Encrustation was visible on ultrasound in images of 4mm implanted catheter ..... 65 Figure 4.6: A) Appearance of catheter encrustation and stone formation after 3 days on uncoated catheter B) no encrustation formation on surface of the coated catheter .................. 65 Figure 4.7: catheters SEM images of in vivo study. A) SEM image of crystals formation on uncoated catheter, and B) PDA/PDMA coated catheter, after instillation of bacteria for 3days (scale bar = 300 µm)  ................................................................................................................ 66 Figure 4.8: XRD graph for uncoated and coated catheters, and B) With peaks numbers. C) a representative comparison graph of pure struvite which compared to our struvite on catheters (this graph found at Unique surface and internal structure of struvite crystals formed by Proteus mirabilis [52]  ............................................................................................................... 68    xvi  List of Abbreviations AMPs: Antimicrobial Peptides ATR-FTIR: Attenuated Total Reflectance Fourier Infrared AU: Artificial Urine CAUTIs: Catheter-associated urinary tract infections CFU: colony-forming units DNA: Deoxyribonucleic acid E. coli: Escherichia coli EDX: Energy-dispersive X-ray EPS: Extracellular Polymeric Substance FDA: Food and Drug Administration O/N: Over Night P. aeruginosa: Pseudomonas aeruginosa P. mirabilis: Proteus mirabilis  PBS: Phosphate-buffered saline PDA: Polydopamine PDMA: Poly (2,5-dimethoxyaniline) PU: Polyurethane RNA: Ribonucleic acid SCI: Spinal Cord Injury SEM: Scanning Electron Microscopy THP: Tamm-Horsfall Protein UBC: University of British Columbia xvii  uHMW: Ultra-High Molecular Weight USD: United States, Dollar UTI: Urinary tract infection XPS: X-ray Photoelectron Spectroscopy XRD: X-ray diffraction xviii  Acknowledgements First and foremost, I would like to thank my supervisor, Professor Dirk Lange, for his unflinching support, encouragement, and guidance has been invaluable throughout this project. Many thanks for my committee members Dr. Jayachandran Kizhakkedathu and Dr. Ben Chew, for their advices. Special thanks to Dr. Kai Yu and Dr. Igor Moskalev for their supports with animal works. I would like to express my deepest gratitude to all lab members, past and present, and all JBRC colleagues for a friendly and positive environment. Big thanks to Dr. Kymora Scotland; I am very thankful for all your valuable inputs and advice.    To my family, I thank you all, especially my dear parents, Ahmed and Norah. From my bottom of my heat, I am thankful that I have my sisters Hanan, Bashayer, and Jawharah, and their providing constant inspiration, motivation, and unconditional love and support all the time. Jawharah, Big thanks for you, you're always supporting me to face all the challenges & keep moving forward.    Lastly, thanks to all my supportive friends and everyone who believes in me.    xix  Dedication To those who believe, science and dedication are a powerful combination that is making the world a better place.  1  Chapter 1: Introduction 1.1 Catheter-associated urinary tract infections Nosocomial (hospital-acquired) urinary tract infections (UTIs) are one of the most common healthcare-acquired infections [1, 2]; approximately 70-80% of these infections are associated with catheterization [1]. About 25% of hospitalized patients are receiving urinary catheters during their hospital stay [3]. In the United States, the cost to treat CAUTI amounts to about $350 million per year [4]. Between $1200 and $4700 USD is the cost of each CAUTI incidence [5]. Duration of catheterization increases the probability of catheter-associated urinary tract infection risk [5]. Urinary catheters are used to drain the patient bladder, however due to most patients with indwelling catheters developing bacteriuria the infection risk is high [6]. Prevention of the infection, which is attributable to catheters, is an important goal [6]. Several attempts to prevent CAUTI have been met with poor success. 1.1.1 Urinary Catheters: What they are and why they are used The urinary catheter is a hollow tube, which is inserted into the bladder by the physician or nurse, to drain the bladder and collect the urine in a drainage bag. Catheters are necessary for a patient who cannot empty their bladder in the usual way.  Urinary catheters are used in the following situations:   Urinary retention (inability to empty bladder)  Urinary incontinence (inability to control urination)   Measuring urine output during hospitalization  Patients who are under general anesthesia or in a coma   Surgery on prostate or genitals 2   Other medical conditions such as spinal cord injury or dementia [7].  There are three main types of urinary catheters. They include indwelling catheters, external catheters, and short-term catheters. Different types of catheters come in different materials, with a different design based on what they are used for. Table 1.1 provides a general description of these catheters.  Table 1.1: the main three types of urinary catheters Type of catheter: Uses Indwelling (urethral or suprapubic catheter) This catheter is used for long-term or short-term, urethral catheter inserts into the bladder through the urethra, and suprapubic inserts into the bladder through the abdomen.  External (condom catheter) This catheter usually used in male patients with neurogenic bladder, this type is placed outside the body [8]. Intermittent catheter Used to empty bladder after spinal cord injury (SCI), this type of catheterization reduces urothelial cancer, stone formation, and renal failure [8].   Indwelling catheters are one of the most common and significant risk factors for developing urinary tract infections [9]. Duration of catheterization is the major determinant for the development of bacteriuria, which is the presence of bacteria in urine [1].      3    Figure 1.1: A Foley catheter; (A) An overview parts of an indwelling urinary catheter (IUC) and (B) a diagram depicting an inserted,Foley catheter. Note: Fig. A was a diagram from Urotoday website and Fig. B was from the HealthLink BC website (https://www.healthlinkbc.ca/health-topics/zm6285). A. B. 4  1.1.2 Contamination with catheterization Catheterization leads to 3-7 % the risk of acquisition of bacteriuria per day [1]. Insertion of an indwelling catheter provides bacteria from the outside environment direct access to the urinary tract system [10]. The contamination happens by bacterial ascension from a contaminated catheter, drainage tube, or urine drainage bag. When the indwelling catheter is inserted, bacteria around the urethral can be carried into the bladder that causes bacteriuria (bacteria in the normal sterile urine), leading to develop biofilm formations on the catheter surface. Once the uropathogens are interceding into patient body started with bladder, the bacteria will buildup on the catheter surface and colonize developing biofilm formations which lead to catheter-associated urinary tract infection [10].  1.1.3 Bacterial adhesion and biofilm formation  The development of an effective coating for biomedical device surfaces to prevent CAUTI requires an understanding of the bacterial adhesion and biofilm formation mechanisms.  Bacterial biofilm plays a vital role in infections. Over 80% of all microbial infections are the result of biofilm formation [13], which is a layer of microorganisms on the biomedical surface [14]. The deposition of urinary components on the catheters leading to conditioning film is believed to facilitate the initial formation of biofilm [14]. Specifically, different urinary components such as Tamm-Horsfall glycoprotein, polysaccharides, and ions act as receptors for bacteria to attach [14]. Bacterial adhesion occurs via multiple mechanisms, including bacterial adhesins and electrostatic interactions [15]. Biofilms formed by urease-positive bacterial species such as Proteus mirabilis result in struvite encrustation further complicating CAUTIs [16]. 5  Biofilm is a complex multicellular structure formed by bacteria. Bacterial biofilm formations occur from significant steps that are represented in (Figure 1.2), which include the following steaps: bacterial attachment to surfaces (reversible then irreversible attachment), microcolony formation (complex layers of biomolecules and external matrix), biofilm maturation (a three-dimensional structure), and biofilm detachment (allowing new bacteria to attach to new areas) [13].    Figure 1.2: Biofilm formation steps (i) Reversible attachment of bacteria to surfaces. (ii) Irreversible attachment to surfaces. (iii) Formation of the external matrix. (iv) Biofilm maturation structure. (v) biofilm detachment. This figure is from the Hindawi website (https://www.hindawi.com/journals/ab/2014/543974/).    6  1.1.4 Mechanism of adhesion Bacterial adhesion is the first step in colonization and biofilm formation. Planktonic bacteria in urine initially adhere to the catheter surface via flagella, and via hydrophobic and electrostatic interactions [17].  The deposition of conditioning film on the biomedical devices such as proteins, electrolytes, and other organic molecules facilitates the initial stage of biofilm formation [17]. Conditioning film on the catheter surface is very important for allowing microorganisms to attach, colonize, and cause urinary tract infection. Bacteria bind to the device surface by specific receptors known as adhesins that bind to collagen, oligosaccharide residues of glycoprotein, glycolipid receptors, and fibronectin [18].  The colonization process is mediated by microorganism outer membrane structures which are known as adhesins, which play a role in recognizing and binding to specific moieties on cells or the device surface [18]. Adhesion is mediated via proteins or other surface structures, including fimbriae, pili, lipopolysaccharide, and capsular polysaccharide. Negative charges of both the pathogen and host cell or biomaterial surface lead to bacteria facing repulsive forces. Therefore both Gram-positive and -negative bacteria have developed cell surface structures located at the tip of hair-like filamentous surface appendages known as fimbriae or pili [18]. Once the bacteria attach to the surface, cell division occurs followed by the secretion of an extracellular polymeric substance (EPS) matrix composed of one or more extracellular polysaccharide, DNA and proteins, all of which form the three-dimensional structure of biofilms [17].  Escherichia coli is the most common bacterial species causing urological infection, and it has different factors that it uses to infect. Most E. coli strains have Type I pili, which is a virulence factor that leads significantly to bladder infection [20]. Another example is 7  fimbrial protein FimH, which is found on various bacteria (including E. coli and Klebsiella pneumonia), and binds to mannose containing molecules such as Tamm-Horsfall protein (THP), one of the most abundant proteins in the urine. THP is a protein that has been found to adhere to indwelling device surfaces [21].  Similarity, THP may bind to Proteus mirabilis, and Pseudomonas aeruginosa, despite THP regularly playing a role to inhibit bacterial adhesion and colonization.  Proteins such as collagen types I and IV, laminin, and fibronectin are recognized by FimH [22]. Various adhesins known as Dr adhesion family proteins have a role in E. coli adhesion to indwelling catheter surfaces, binding to integrins as well as collagen type IV [23]. Similarly, the protein Ace binds to collagen type IV and other extracellular matrix components that play role in the bacterial attachment to host and biomaterial devices via the interactions with collagen type I and IV [24, 25].  Interestingly, uropathogens have ability to alter the expression of surface structures includes lipopolysaccharide, capsular polysaccharide, or exopolysaccharide, to facilitate binding to biomedical device surfaces. This is important for medical devices such as indwelling ureteral stents, which are coated with a thin layer of the hydrophilic polymer to make the insertion easy and improve comfort. Pathogens initially attach to the polymer and bare surface of the stent by weak molecular interactions as mentioned above, initiating biofilm formation [26]. An example of bacteria using different chemical/physical characteristics of surface structures is P. aeruginosa. Differential expression of A-band lipopolysaccharide and B-band lipopolysaccharide give the bacteria both hydrophobic and hydrophilic surface characteristics that allow for interactions with hydrophilic polymers, or urinary proteins of varying physical characteristics such as urinary alpha 1-microglobulin and serum albumin [27, 28].  Likewise, the adhesion of E. faecalis, is facilitated by 8  subpopulations of this bacterial species expressing variable structures that yield different surface charges facilitating bacterial attachment and biofilm formation to surfaces with varying surface characteristics [29].  Researchers have found heterogeneous strains attach better to hydrophilic surfaces compare to homogenous strains [29]. Once bacteria adhere to indwelling device surfaces followed by biofilm formation, biofilm plays a very important role in CAUTI.  Past and current studies attempt to develop indwelling biomedical devices that prevent biofilm formation as well as prevent CAUTI.  1.2 Proteus mirabilis and how it infects the urinary tract  Proteus mirabilis HI4320 is a wild-type strain, one of the most common Gram-negative pathogens that infect the urinary tract system, especially patients with a long-term indwelling catheter. P. mirabilis has characteristic swarming motility to swarm across the surfaces [29]. Wild type P. mirabilis expresses the enzyme urease that catalyzes the hydrolysis of  urea to ammonia (NH3) and carbon dioxide (CO2), which leads to struvite crystal encrustation is (NH4MgPO4•6H2O) magnesium ammonium phosphate. A past study showed that crystalline biofilm formation due to increasing the risk of bladder stones formations. Moreover, 62% of the patient who had this kind of encrusted catheter that caused by P. mirabilis infection, were had bladder stones [30].  Planktonic P. mirabilis is present in the contaminated urine after placed the biomaterial device, and these bacteria will begin to adhere to the surface.  These bacteria produce urease, which hydrolyze urea and increase pH urine. Urease, a urea-hydrolyzing enzyme expressed by P. mirabilis, plays a critical role in crystalline biofilm formation. Urease hydrolyses urea to carbonic acid and two molecules of ammonia, increasing urine pH [29].  The ammonia, which is a byproduct of the urease 9  reaction with urea, forms complexes with magnesium and phosphate to form crystals [30]. These processes are happening along with biofilm formation with layers of bacteria covered by crystals then the bacterial adhesion and the crystals growth increase on the surface of the biomaterial device which leads to catheter blockage. For this thesis, we focus on wild type P. mirabilis infections that generate crystalline biofilms and form urinary stones [29].    For the purposes of CAUTI, it is important to understand how P. mirabilis induces device-associated infections. The initial step involves P. mirabilis adherence on the catheter surface via fimbriae and other adhesins when a Foley catheter inserted into the patient [29]. Based on a past study, colonization on the catheter surface occurs within 6 hours of P. mirabilis entering the urine. The crystalline biofilm formations often lead to blockage of the catheter [32]. The fact that it forms crystalline biofilms in which bacteria are encased and protected by struvite makes it a complicated organism to treat and to prevent bacterial adhesion.   1.3 Strategies for the prevention of CAUTI 1.3.1 Limiting use of urinary catheters The first strategy of CAUTI prevention is reducing the use of unnecessary indwelling catheters. Other studies have shown that 21%-50% of catheterized patients received a catheter for inappropriate indications [29]. The most effective strategy is avoiding and limiting the insertion of indwelling urinary catheters and placing catheters only when medically necessary. On the other hand, there is an essential strategy for preventing catheter-associated urinary tract infections that should be followed by the nurse-directed, including institutional protocols for insertion, and in the perioperative setting [28] 10  1.3.2 Reducing the duration of catheterization  One of the critical interventions for prevention of CAUTIs is minimizing the duration of catheterization. Once the catheter is placed, it is essential that the indwelling catheter is removed as soon as possible. The awareness of physicians about the duration of urinary catheterization and whether their hospitalized patients had an indwelling catheter has been shown to result in increased UTI rates [32]. A past study showed that 28% of the physicians were unaware of their patients being catheterized [31]. Therefore, early removal of urinary catheters, checklists or daily plans, nurse-based interventions, and using electronic reminders are helpful to reduce the duration of catheterization [31].  1.3.3 Using the right techniques for insertion and maintenance of urinary catheters If the indwelling urinary catheter is necessary, it should be inserted by a trained professional using an aseptic technique. The aseptic technique for insertion and maintenance of urinary catheters are a sterile insertion, closed drainage system, and avoidance of routing of administration for the bladder irrigation [29]. Cleaning the meatus with soap and water is recommended. Using sterile lubricant for inserting the catheter is also recommended [33].  1.3.4 Antimicrobial urinary catheter coating While the needless insertion of catheters and duration of catheterization have decreased, and insertion techniques have improved, CAUTI still remains a significant problem. Therefore, researchers work in developing antimicrobial urinary catheter coatings to prevent infection. These coatings are designed to inhibit adhesion and bacterial growth.  Figure 1.3 shows the different antimicrobial materials that have been attempted over the past 10 years [33].  11  There are lots of studies on catheter coating to prevent CAUTI. Antimicrobial coating of a urinary catheter is one of these strategies and previous studies have used silver, nitrofurazone, or other antimicrobial materials to develop a coating that prevents CAUTI. However, these studies showed significant limitations such as short-term antimicrobial activity, the efficacy against only some of the bacterial species, and instability of the coating [34]. On other hand, the antibiotic catheter coating was showed significant rise in antibiotic resistance [35].    Moreover, to date several approaches have been attempted using antimicrobial peptides (AMPs) to develop antimicrobial indwelling device coatings; these were showed success activity against multiple bacterial species. Antimicrobial peptides (AMPs) were used to coat a variety of substrates such as nanoparticles, contact lenses, titanium-coated silicon, and catheters [36]. However, comparing to the antimicrobial soluble version the antimicrobial peptides coating very often showed lower activity and the possible reason is non-specific chemistry of the immobilization techniques utilized, leading to the changes of the peptide orientations on the surfaces, host cell toxicities, and also play role in inadequate surface concentration and high pH sensitivity which could be the possible reasons too [36, 37]. In addition, the AMPs coating still induced pathogens resistance [37].  Several studies of antifouling efficacies of hydrogel-coated catheters have been done. According to clinical criteria, the silver alloy hydrogel coating showed reducing in CAUTI symptoms [37]. Nevertheless, the hydrogel layer can increase the buildup of the planktonic cells and the encrustations formations on the surfaces which lead to catheter blockage [37].  Many types of antimicrobial urinary tract catheter coatings to prevent CAUTI have been tested (Table 1.2).  12              Figure 1.3: Antimicrobial coating for urinary catheters to prevent CAUTI [33].  13  Table 1.2: Types of antimicrobial urinary catheter coatings Type of coating Description Advantages Disadvantages Silver An antimicrobial agent for urinary catheter coating, Ag ions kill microbes [33].   Silver approved by the FDA [33].  This coating does not cause discomfort for patients [34].  It is useful for short-term catheterization [34].  At low concentration, silver coating is an effective antimicrobial agent [35].   It has less affectivity in long-term catheterization [33].  It may cause argyria with prolonged usage [36].   Antibiotics  They are low molecular weight compounds that kill or inhibit the growth of other organisms [33]. Examples of antibiotics coating are nitrofural and minocycline-rifampin (MR) [33].   Antibiotics are very well-studied agents.   Antibiotics coatings are good alternative to the cytotoxicity issues caused by silver-alloy catheter coatings [37].   Bacterial resistance. Chlorhexidine  A positively charged compound that binds and destabilize the cell wall of bacteria [33].  A very promising coating, it is more effective compare to antibiotics coating because does not develop bacterial resistance [33].   No enough studies to understand chlorhexidine release behavior [33]. Triclosan An enzymatic inhibitor of a reductase involved in bacterial fatty acid synthesis [38].  Active against Gram-negative and –positive bacteria [39, 40].    Not approved by the FDA [42].   Might play a role in increasing the antimicrobial resistance against antibiotics [33].   14  Type of coating Description Advantages Disadvantages Antimicrobial peptides Short strands of amino acids with antibacterial activities [33].  Damaging in bacterial membranes and kill the bacteria.  pH sensitivity, potential toxicity, and pricy [33].  Bacteriophages Releasing phages from catheter's coating and direct incorporation lead to prevent biofilm formation on catheter surfaces [43]. Phages are viruses that infect and kill bacteria [44].  Studies show that phages can prevent bacteria from developing resistance [33].  The biofilm matrix blocks the specific phage receptors, on the bacteria [45].   Activities can be affected by pH level and ions [45].  Enzymes   Hydrolytic enzymes play role in disrupting the biofilm formation via quorum sensing enzymes such as dispersin B, chitinases, beta-glucanases, lysozyme, alginate lyase, and alpha amylase [44].  Enzymes can kill specific bacteria without killing other necessary bacteria that means enzymes are specific for particular pathogesns [33].   Bacteria are rarely resistance to enzymes [33].    It is safer than other antimicrobial agents [33].   Enzyme coating is expensive.  Enzyme denaturation in extreme conditions such as transport, storage, etc [33].   Nitric Oxide Antimicrobial mechanisms of nitric oxide are tyrosine nitration and DNA cleavage, nitrosation of amines, and lipid peroxidation [46, 47].  Researchers found the effect of nitric oxide release polymer as an antimicrobial coating on catheter for the long term [33].    Need more studies.   Researchers concern about storage stability of NO because nitric oxide decomposed very fast. 0Needs to have a generation system [33].  Polyzwitterions  Zwitterions contain both positive and negative charges, creating hydration layers via electrostatic interactions. These hydration layers act against bacteria attachment. Zwitterions such as phosphorylcholines, sulfobetaines and carboxybetaines [33].  Polyzwitterions resist bacterial adhesion by repulsion [44].   The hydration layers on device surfaces are not stable over time.   Need more studies to improve the coating stability of catheters surfaces. 15  Type of coating Description Advantages Disadvantages Polymeric Coating Modifications Polymer coating  such as Polyvinyl chlorine (PVC), Modifications of polyurethane (PU), and Polytetrafluoroethylene (PTFE) that disrupts biofilm formation. Besides, polymers play a role in immobilizing many types of antimicrobials includes biocide releasing silver or antibiofilm agent releasing nitric oxide [33].  Polymeric coating decreases biofilm formation.  Bacteria are less likely to develop resistance with a polymeric coating [33].   Clinical trials are necessary [33]. Disadvantages Liposomes  Liposomes coatings effect on bacteria by inserting drugs within liposomes such as hydrophilic, hydrophobic, and amphipathic and delivered to target cells [33].   Results confirmed using an in vivo rabbit model [33].  Biocompatible comparing to other catheter coatings [33].     Need more studies to deliver a variety of antimicrobial drugs [33].      16  1.4 Novel anti-adhesion coating to prevent CAUTIs  1.4.1 Our Coating: What is it? Bacterial attachment and biofilm formation on indwelling catheters leads to CAUTIs. While most studies to develop catheter coatings to prevent CAUTI have met with poor success. To address the shortcomings of previous coatings, our collaborative research group developed a PDA/PDMA coating using two main components: polydopamine (PDA) which is a surface anchoring agent, and poly (N,N-dimethylamide) (PDMA), an antifouling hydrophilic polymer [48],. This novel coating is designed to prevent the first step of the infection, which is the formation of conditioning film on the implanted catheter as well as bacterial attachment.   The use of an ultra-high molecular weight (uHMW) PDMA resulted in significant stability of the coating on biomaterial surfaces and an ultrathin coating (∼19 nm) (ref). Thicknesses and other characteristics of this coating were measured by Kizhakkedathu's lab, University of British Columbia, via ellipsometry, X-ray photoelectron spectroscopy (XPS) (chemical composition), attenuated total reflectance Fourier infrared (ATR-FTIR) spectroscopy as well as wettability by water contact angle measurements (Table 1.3) [49]. The chemical structures of polydopamine (PDA) and poly (N,N-dimethylamide) (PDMA) are given in Table 1.4 [48].      17  Table 1.3: Thickness and composition of binary PDA/PDMA coating Coating Thickness (nm) Si (At%) C (At%) N (At%) O (At%) N/C PDA 32.2 ± 0.1 3.38 59.28 6.38 30.96 0.108 PDMA-795K 18.9 ± 1.3 0.45 71.67 10.67 17.21 0.149   Table 1.4: Chemical structure  of PDA and PDMA  Polydopamine (PDA)   Poly (N,N-dimethylacrylamide) (PDMA)    18  1.4.2 The mechanism of action of PDA/PDMA coating The novel PDA/PDMA coating is a non-fouling coating. A non-fouling surface reduces the ability of planktonic microbes to adhere and hence prevents the initial stage of biofilm formation.  The mechanism of action for this coating is not well understood. It is believed that the UHMW PDMA is a neutral hydrophilic polymer, which prevents the bacterial adhesion due to the steric repulsion [49]. In addition, the use of this polymer helps prevents biomolecule attachment associated with conditioning film deposition (Figure 1.4).  1.4.3 Using PDA/PDMA coating to prevent CAUTI To date, several attempts of changing catheter design via the development of coatings that prevent infections resulted in a limited success, due to reduced in vivo efficacy and limited application to different types of materials (see 1.3.4). Our coating overcomes these limitations, as the use of PDA as the binder allows for interactions with a variety of materials. This coating is safe and easily applicable to biomaterial devices via a simple one-step dip-coating approach based on two components PDA and UHMW PDMA.           19                                             Figure 1.4: Non-fouling coating to prevent CAUTI    20  1.5  Thesis aims The overall objective of this thesis was to test and validate the efficacy of our novel coating in preventing CAUTI and catheter failure associated with P. mirabilis infection.  Specifically, this involved testing the efficacy of the coating to: i. Prevent the conditioning film formation on the coated surfaces. ii. Prevent the adhesion of P. mirabilis to coated surfaces.  iii. Prevent struvite encrustation formation. iv. Prevent calcium oxalate encrustation formation. v. Test efficacy of the coating in an in vivo model of P. mirabilis CAUTI.  21  Chapter 2: In vitro evaluation of the catheter coating The first step was to perform in vitro assays to assess efficacy using “realistic” in vitro conditions. Here, there is a description of all of the experiments performed at different labs.   2.1 Bacterial adhesion  2.1.1 Synthesis of PDMA    PDMA was synthesized by atom transfer radical polymerization and kindly provided by Dr. Kai Yu from Kizhakkedathu lab at the University of British Columbia.  2.1.2 Development of an anti-fouling coating  Polyurethane sheets were used to prepare samples (1 cm x 0.5 cm). The samples were cleaned by exposure to 1.5% Alconox® detergent and sonicated for 10 minutes, followed by rinsing using Milli-Q water under sonication. After samples were dried, the cleaned surfaces were dipped into (1 mL) of the mixed PDA/PDMA solution (1:5 ratio in 10 mM Tris-HCl buffer, pH 8.5) in a 48 well-plate for 24 hours. The next day, the coated samples were rinsed with Milli-Q water and then dried with a N2 gun.     2.1.3 Bacterial strain and culture condition The bacterial species used for in vitro testing of coated and uncoated samples was P. mirabilis H14320 (source?). This strain produces urease which leads to the hydrolysis of urea to ammonia (NH3) with consequent increase in urine pH [50].  22  To prepare fresh bacteria for in vitro testing, two different cultures of P. mirabilis from freezer stock were cultured in 3 mL of Luria-Bertani broth (LB, 10 g tryptone, 5 g yeast extract, and 10 g NaCl/L) or sterilized artificial urine (Table 2.1) at 37C overnight (O/N). The next day, the cultures were sub-cultured in 3 mL of the same fresh broths at 37C O/N  The next day, approximately 5x103 colony-forming units (CFU)/mL was used after measuring the OD600 using the approximate equation of 0.1 OD600 = 108 CFU/mL.                  23  Table 2.1:  Artificial urine recipe Component Quantity (g) Peptone L37 1 Yeast extract 0.005 Lactic acid 0.1 (84 μL) Citric acid 0.4 Sodium bicarbonate 2.1 Urea 10 Uric acid 0.07 Creatinine 0.37 Calcium chloride 5.2 Sodium chloride 0.0012 (1 mL) Iron sulphate.2H2O 0.49 Magnesium sulphate.7H2O 3.2 Potassium dihydrogen phosphate 0.95 Di-potassium hydrogen phosphate 1.2 Ammonium chloride 1.3 Distilled water to 1 Liter   24  2.1.4 Anti-adhesion testing of PDA/PDMA coating Bacterial attachment was measured by the colony formation assay. Coated and uncoated polyurethane (PU) samples were disinfected by submersion in 1.5 mL of  70% ethanol in Eppendorf tubes for 5 minute followed by rinsing (×3) with 1 mL of sterile phosphate-buffered saline (PBS, add the components). The cleaned PU samples were transferred into 1 mL of prepared P. mirabilis culture (∼5 × 103 CFU/mL) in artificial urine or LB broths. All samples were incubated at 37°C on a 360° rotator at 16 rpm for 4, 12, and 24 hours.  2.1.4.1 Determining bacterial concentration by CFU counting At 4, 12, and 24 hours post-inoculation, samples (N = 4 per condition) were rinsed with sterile PBS buffer (× 3) to remove planktonic bacteria and transferred to a fresh 1 mL sterile PBS in new tube followed by sonication in a water bath for 10 mintues to remove adherent bacteria. After sonication, the samples were vortexed at high speed of 2800rpm for 10 seconds. Using a 96-well plate, bacterial suspensions were serially diluted in sterile PBS up to 10-7 dilution and plated on low salt LB agar plates (Tryptone 10 g, NaCl 5 g, Yeast Extract 5 g, agar 15 g, 1 L distilled water, then autocalve it and pure it in petri dishes) for CFU counting. All low salt LB agar plates were incubated at 37C O/N or until visible colonies formed. The averages of CFU counts between 3 to 30 colonies were recorded. The experiments using AU and LB media were repeated 5 times.   Note: Low salt LB agar was used because P. mirabilis has swarming motility. The use of low salt medium prevents swarming [54].   25  2.1.4.2 Use of scanning electron microscopy (SEM) to observe bacterial biofilm formation For each experiment, two (N=2) samples of each condition were taken at each time point for SEM analysis to visualize biofilm formation on the coated and uncoated PU surfaces. Samples were prepared by fixing the samples in 2.5% glutaraldehyde (1 mL) for 1 hour, then dehydrated using different concentration of ethanol (50%, 70%, 90%, and 100%) for 10 minutes each and 100% O/N. Samples were then dried. with a N2 gun. The imaging of the samples was performed with a scanning electron microscope (Phenom Pure Desktop Scanning Electron Microscope).    2.1.5 Statistical analysis  Data are shown as the mean ± SE. Statistical analysis was done using unpaired t-test. Statistical significance was set at P < 0.05. Statistical significance is P < 0.0001 with AU media.    2.2 Struvite encrustation formation Struvite encrustation is (NH4MgPO4•6H2O) magnesium ammonium phosphate, which forms in urine containing bacteria that produce the urease enzyme. Patients who have urinary tract infections by urease-positive bacteria are more likely to have struvite stones (ref). In this study, struvite composition of encrusting material was tested via Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), and X-ray photoelectron spectroscopy (XPS).     26  2.2.1 SEM and EDX spectroscopy- Static model   Static model was designed to test struvite encrustations on PU surfaces. In the static model, PU samples were cut (1 cm × 0.5 cm) and coated with PDA/PDMA (see 2.1.2 Development anti-fouling coating). Cleaned coated and uncoated samples were transferred to 70% ethanol (1 mL)  in Eppendorf tubes for 5 minutes then washed (× 3) in 1 mL sterile PBS. The samples were then transferred to 1 ml of P. mirabilis culture (∼5 ×103 CFU/mL) in AU broth. The samples were incubated at 37°C for up to 5 days using a 360° rotator at 16 rpm. Every 24 hours, the AU was removed from each tube and replenished with fresh 1 mL AU (no additional bacteria). At 4, 24, 48 hours, and five days, samples were removed, and  left to dry in a biosafety cabinet O/N. The next day, sputter coated with Carbon (20 nm) using (Leica EM ACE600 sputter coater) to enhance contrast of the images. In this study, the scanning electron microscope (JEOL JSM 5200) and EDX were used to examine the coating efficiency in the prevention of struvite encrustation formation at 150 X magnification.  2.2.2 SEM A static model was used (see 2.2.1) and samples were taken at time points 2, 5, 7, 10, 12, 14 days (figure 2.1). Samples were fixed in 2.5% glutaraldehyde (1 mL) for 1 h at 4C, and then dehydrated using an ethanol/water gradient (50%, 70%, 90%, 100%) for 10 minutes each and in 100% ethanol O/N. The samples were dried and imaged using the same instrument described above, to visualize biofilm formation. The experiment was repeated 2 times.   27                                                                          Figure 2.1: Static experimental set up 2.2.3 XPS analysis – static model and in vitro flow model XPS was used to determine the elemental composition of the surface. Polyurethane samples were cut (0.5 cm X 2 cm) and half of the surface was coated while the other half remained uncoated (see figure 2.2A). Samples were cleaned using 1.5% Alconox® detergent and sonication for 10 minutes, and then rinsed with Milli-Q water. Samples were dried, sterilized by submersion in 70% ethanol for 5 minutes and then rinsed (× 3) with 1.5 mL of sterile PBS.   Two different experimental models were set up for these experiments, a static model and a closed flow model. The static model uses a 360° rotator at 16 rpm. Each sample was 28  incubated in AU with P. mirabilis (∼5 × 103 CFU/mL) and incubated under rotation at 37 °C for 2 and 5 days.  AU was changed every 24 hours.     The closed flow model consisted of a silicon tube of approximately 300 cm in length with an inner diameter of 14 mm. The tube was connected to a one-liter flask containing the medium. Following autoclaving of all components, PU samples were placed inside the lumen of the tube which was connected to the side-arm flask. The tube was placed in a peristaltic roller pump (Masterflex L/S variable-speed economy drive, 10-600 rpm, 115V), and 400 mL of AU with (∼5 × 103 CFU/mL) wild type P. mirabilis was poured into 1 L flask (Figure 2.2 B). A flow rate of 3 mL/minute was chosen based on the pump's RPM settings. The AU in the flask was replaced with fresh AU (no additional bacteria) every 24 hours? under aseptic conditions. The flow model was run for 5 days, with samples removed at days 2 and 5 for analysis.  The samples were washed gently with PBS and dried for XPS analysis (PHI VersaProbe III).   29   Figure 2.2: (A) PU samples half-coated and half-uncoated. (B) Insert the samples inside the tube. C) AU with (∼5 × 103 CFU/mL) wild type P. mirabilis is circulated through the peristaltic roller pump for 2 and 5 days at 37°C. Experimentation was performed using a flow rate of 3 mL/minute.   2.3 Calcium oxalate encrustation  Indwelling device encrustation most commonly involves crystals made of calcium oxalate. Calcium oxalate stone is the most common type of kidney stone [51]. This type of stone usually forms when calcium combines with oxalate in urine. Calcium oxalate stones are not typically associated with bacterial infection; however, some struvite stones do contain a calcium oxalate component. As a result, we wanted to assess the ability for our novel coating to decrease calcium oxalate-based encrustation.  30  The experiment was designed using the Center for Disease Control (CDC) biofilm reactor to stimulate a urinary tract system. Calcium oxalate encrustation in AU was used. Two AU stock solutions were prepared separately (A & B), and the reason behind this was to prevent the pre-formation of calcium oxalate crystals that result from the AU components (Table 2.2) [51].   Table 2.2: Composition and concentration of AU that used in calcium oxalate encrustation formation  Substance Chemical formula Concentrations     (mmol/L) Quantity (g) Solution A  Sodium chloride NaCl 105.5 12.3308 Sodium dihydrogen phosphate NaH2PO4·H2O 3.2 0.7678 Sodium citrate Na3C6H5O7·2H2O 3.2 1.882 Magnesium sulphate MgSO4 3.9 1.922 Calcium chloride CaCl2 4 1.176 Solution B  Sodium sulphate  Na2SO4 17 4.828 Potassium chloride KC1 64 9.542 Sodium oxalate Na2C2O4 0.3 0.0804 Sodium nitrate NaNO3 1 0.170  PU samples  were cut (0.5 cm x 1cm) and  two groups, one coated and one uncoated group, were prepared. Each group consisted of 16 samples.  Samples were suspended in the AU solution within the CDC bioreactor by stringing them along a suture (Ethicon Prolene, polypropylene) that was attached to the top and to the bottom of the reactor vessel. For each group, two sutures containing 8 samples each were prepared. The reactor was then assembled and all samples were sterilized via plasma gas sterilization before use.   AU solutions (250 mL A & 250 mL B) were mixed with 500 mL double Milli-Q water to make 1 L of  AU and a pH of 6 was adjusted with 0.1 NaOH. The CDC bioreactor 31  was run under the following conditions: approximately 850 mL sterilized AU in the bioreactor (Figure 2.3), the rotator was stirred at 3 rpm, and both the CDC bioreactor and rotator were placed in an incubator at 37oC for two time-points (3 and 6 weeks) with AU being exchanged every 48 hours in a biosafety cabinet to maintain sterile conditions. At each time point, one suture for each group was removed and samples were gently dipped into sterile distilled water to remove any non-deposited crystaline material. Three PU samples from each group were used for SEM images; while five PU samples each group were placed in five separate 15 mL tubes and prepared for Atomic Absorption Spectroscopy (AAS) analysis.                                                                       Figure 2.3: CDC bioreactor setup  32  2.3.1 Sample fixation for SEM imaging  Within 3 weeks, three samples from each suture were removed from the reactor, placed in 2.5% glutaraldehyde for 4 h at 4C, and then dehydrated using different concentrations of ethanol (50%- 90%) with 10 minutes exchanges of each and then left at 100%. Samples were dried in a desiccator overnight. Samples analyzed by SEM using different magnifications. The same process was repeated for samples at the 6-week time point.     Note* that crystal formation will be significantly affected in areas where the PU sample was cut. These areas are not representative and were not considered   2.3.2 AAS The analysis was performed on five samples from each group at both time points using a Perkin Elmer PINAACLE 900 SERIES AAS. Calcium oxalate deposition was determined as the amount of calcium present on the surface of each sample. A standard curve using calcium standards was prepared using 1, 5, and 10 mg/L of CaCl2 stock solution (available as a companion product of the instrument) in 2% HNO3 (v/v) and 0.2% lanthanum chloride (La salt). HNO3 solutions with two different concentrations (5% HNO3 -2% HNO3) were used to dissolve the crystal deposits for AAS analysis. The materials needed for the preparation of the AAS solutions were lanthanum chloride (La salt), HNO3, and ultrapure water. A decalcifying solution (solution A) (1 L of 5% HNO3 (v/v) and 0.2% La salt solution was prepared. A beaker was filled with 929 ml ultrapure water and 71 mL of 70% HNO3, and 2 g La salt were added and mixed with a stir bar until the salt was dissolved.  33  In addition, a standards/diluent solution (solution B) (1L of 2% HNO3 (v/v), 0.2% lantLasalt solution) was prepared. A beaker was filled with 971 ml ultrapure water then 29 mL of 70% HNO3 and 2 g La salt were added to the beaker and mixed with a stir bar until the salt was dissolved.  For standards, the correct amount of calcium stock solution was added into the diluent solution to create 1, 5, and 10 mg/L Ca2+ solution standards.   Each sample was placed in a 15 mL tube to which 1 mL of decalcifying solution (solution A) was added followed by incubation at room temperature for 1 hour, and sonicated for 10 minutes. Then, 9 mL of diluent solution (solution B) was added to the samples. This process dissolved the crystal deposits. The AAS analysis using a Ca2+ lamp was calibrated with three standard Ca2+ solutions. Dilution of standard solutions of Ca2+ performed the linear calibration of the AAS. In addition, samples were diluted in solution B to aid measurement. 2.3.3 Statistical analysis  Data are shown as the mean ± SE. Statistical analysis was done using unpaired t-test. Statistical significance was set at P < 0.05. The significance * indicates P < 0.001, and ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001.         34  Chapter 3: Results 3.1 Bacterial adhesion results  The anti-biofilm efficiency of our coating (PDA/PDMA) was tested in vitro. In this study, we applied the coating on PU followed by exposure to wild type P. mirabilis in both media AU and LB for different time points (4, 12, and 24 hours). Overall, the decrease in adhesion was shown to be greater over time, with the greatest decrease being evident at 24 hours (Table 3.1) (Figure 3.1, 3.2). At this time point, the coating was shown to reduce bacterial adhesion by an average 99.1% compared to uncoated PU in AU media. In contrast, we only saw a 97.4% reduction in LB.    The fold reduction of P. mirabilis were 2.2-fold decrease at 4 hours, 18.4-fold decrease at 12 hours, and 116.22-fold decrease at 24 hours post-incubation in AU media (Figure 3.1 D). In contrast, the fold reduction of P. mirabilis were 2-fold decrease at 4 hours, 37.8-fold decrease at 12 hours, and 33-fold decrease at 24 hours post-incubation in LB media (Figure 3.2 D).   Table 3.1:  Bacterial adhesion reduction by the time in AU media and LB media Time point (hour) 4  12  24  Reduction (AU media) 53.8% 94.6% 99.1% Reduction (LB media) 50.1% 97.0% 97.4%   35                                                           Figure 3.1: P. mirabilis adhesion on uncoated and coated PU surfaces in AU media A) within 4 hours incubation, B) within 12 hours incubation, C) within 24 hours incubation,  and D) graph showed the fold  reduction of P. mirabilis within 4, 12, and 24 hours (at the top of each bar  the percentage reduction). * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001.    uncoated 4 hcoated 4 h110011011102110311041105CFU/mLuncoated 12 hcoated 12 h1100110111021103110411051106CFU/mL**uncoated 24 hcoated 24 h11001101110211031104110511061107CFU/mL***99.1% 0204060801001201404 hours 12 hours 24 hoursFold redease from uncoated samples  94.5% 53.7% A. B C. D36                                        Figure 3.2: P. mirabilis adhesion on uncoated and coated PU surfaces in LB media A) within 4 hours incubation, B) within 12 hours incubation, C) within 24 hours incubation,  and D) graph showed the fold  reduction of P. mirabilis within 4, 12, and 24 hours (at the top of each bar  the percentage reduction). * indicates P ≤ 0.05, ** indicates P ≤ 0.01.  uncoated 24 hcoated 24 h110011011102110311041105110611071108CFU/mL**97.0% 0510152025303540454 hours 12 hours 24 hoursFold decrease from uncoated samples  97.3% 50.1% A. B. C. D. 37  SEM was further used to determine bacterial attachment and subsequent biofilm formation on coated and uncoated PU samples. SEM imaging showed a thick biofilm on the uncoated PU surface in AU media (Figure 3.3), while only isolated bacterial colonies were found on the coated PU surface in LB media (Figure 3.4), respectively. In AU media, we can observe areduction in the P. mirabilis attachment on coated sample compared to uncoated samples. Images clearly show that the biofilm and struvite formed on the uncoated surfaces (control), while the coating reduced the bacterial attachment and struvite encrustation formation (Figure 3.3). In LB media, SEM images showed less bacterial attachment on coated PU surfaces than uncoated PU surfaces (Figure 3.4).    Figure 3.3: (A) SEM images of bacterial adhesion on uncoated PU surface (right image) and coated PU surfaces (left image) after 4 hours incubation in AU (B) SEM images of bacterial adhesion on uncoated PU surface (right image) and coated PU surfaces (left image) after 12 hours incubation in AU. (C) SEM images of biofilm on uncoated PU surface compared to PDA/PDMA coated PU surface (left image) after 24 hours incubation in AU (scale bar = 30µm).   30µm  30µm  30µm  30µm  30µm  30µm  38   Figure 3.4: (A) SEM images of bacterial attachment on uncoated PU surface (right image) and coated PU surfaces (left image) after 4 hours incubation in AU (B) SEM images of bacterial attachment on uncoated PU surface (right image) and coated PU surfaces (left image) after 12 hours incubation in AU. (C) SEM images of biofilm on uncoated PU surface compared to PDA/PDMA coated PU surface (left image) after 24 hours incubation in AU (scale bar = 30µm).  3.2 Struvite encrustations formation results  3.2.1 SEM analysis Struvite encrustations on the surfaces were detected via SEM imaging. Overall, struvite encrustation was evident on both uncoated and coated surfaces. Images shown are representative of 12 images taken per surface. At the 2, 5, and 7 day time points, struvite encrustation was found to be greater on uncoated compared to coated surfaces (Figure 3.5). This difference became less obvious with increasing incubation times. By days 10, 12, and 14 (Figure 3.6) no visual differences in the amount of encrustation on each surface was observed.    30µm 30µm  30µm  30µm  30µm  30µm  39                    2 Days                                         5 Days                                      7 Days          Figure 3.5: SEM images of uncoated PU (upper panel) and coated PU images (bottom panel) (200 X magnification) (scale bar = 300µm).                      10 Days                                      12 Days                                         14 Days          Figure 3.6: SEM images of uncoated PU (upper panel) and coated PU images (bottom panel) (200 X magnification) (scale bar = 300µm). 300µm 300µm  300µm  300µm  300µm  300µm  300µm 300µm 300µm 300µm 300µm 300µm 40  3.2.1.1     Characterization of struvite encrustation by SEM-EDX analyses Visual results of struvite formation and encrustation via the SEM analysis are given in Figures 3.7 A and B, after two days of incubation in AU with P. mirabilis. The EDX analysis (Figure 3.8 A and 3.9 A) showed the percentages of elements for uncoated and coated samples (2 days- time point).  Elements such as N and Na had small or undetectable peaks (1-10 wt %). The elements C and O had significant peaks on the spectrum indicating that they were present at amounts greater than 10% of the weight. Results for the uncoated samples are illustrated in Figure 3.8 B, while those for the coated surface are illustrated in Figure 3.9 B.  Overall, we observed high levels of C, which is attributed to the fact that samples were coated with carbon (20 nm) to get better images. Peaks for C and O were strong in all samples. Uncoated surfaces showed weak peaks for element Mg, N, and Na, indicating that they were present but at relatively low amounts and no P was detected that because these elements was below the EDX detection limit at this time point. In contrast, on coated surfaces, elements such as Mg, N, Na, and P were undetectable except for C and O, which showed strong peaks.  Figure 3.7: SEM images of struvite crystals on uncoated surfaces after two days of incubation in AU with P. mirabilis. (A) SEM image (magnification 8000X). (B) SEM image (magnification.150X). A B41   Element  (uncoated sample # 1) Weight % Atomic %  Intensity Mg  0.69   0.43 19.15 N  1.03   1.11 2.71 Na 1.92  1.26 50.49 P N/A N/A N/A  Element  (uncoated sample # 2) Weight % Atomic % Intensity Mg  0.15 0.08 4.78 N  3.20 3.14 10.04 Na 0.47 0.28 14.07 P N/A N/A N/A                       Figure 3.8: A) the percentages of elements for uncoated sample (2 days incubation), B) EDX uncoated samples (2 days).  A B42          Element  (coated sample #3)  Weight % Atomic % Intensity  C 77.58 81.77 1155.90 N 4.40 3.98 12.09 O 18.02 14.25 165.35  Element  (coated sample #4)  Weight % Atomic % Intensity  C 80.81 84.87 1113.73 O 19.19 15.13 168.45    Figure 3.9: A) the percentages of elements for coated sample (2 days incubation), B) EDX coated samples analysis (2 days).  A B 43  The EDX analysis results for the 5-day samples are shown in Figures 3.10B (uncoated) and Figure 3.11 B (coated). Results for uncoated surfaces showed peaks for P, C, O, N, and Fe. In contrast, on coated surfaces, only peaks for C, N, and O were detected, while peaks for Mg and P were absent and the reason behind this EDX may not be sensitive enough (Figure 3.10 B). These results are also compiled in (Figure 3-11 A), listing the percentage of each element that was present.  The results for the 5-day samples are compiled in Figure 3.10A (uncoated) and Figure 3.11 A (coated). For uncoated samples, P was present in minor amounts (1.02 wt%), while no Mg was detected. In the case of the coated samples, only C, O, and N were detected.               44   Element (uncoated sample # 1) Weight % Atomic % Intensity C  56.28 72.14 1069.45 N  3.05 3.35 14.57 O  19.04 18.32 301.27 Fe 20.61 5.68 24.89 P  1.02 0.51 18.93  Element (uncoated sample # 1) Weight % Atomic % Intensity C  60.36 74.36 926.48 N  3.21 3.39 11.67 O  18.94 17.52 228.94 Fe 16.82 4.46 16.25 Cl 0.68 0.28 8.85                                    Figure 3.10: A) the percentages of elements for uncoated sample (5 days incubation            A45                                              Figure 3.10:  B) EDX  analysis – uncoated samples (5 days).       B46             Element  (coated sample #3)  Weight % Atomic % Intensity  C 77.50 81.57 938.95 N 5.74 5.18 12.84 O 16.76 13.25 122.40  Element  (coated sample #3)  Weight % Atomic % Intensity C 71.70 76.59 1262.85 N 6.30 5.77 22.32 O 21.99 17.64 249.44  Figure 3.11: A) the percentages of elements for coated sample (5 days incubation), B) EDX coated samples analysis (5 days). BA47  3.2.1.2     XPS analysis  The chemical composition from the XPS survey scan showed strong C and O peaks, weak P and N peaks, while the C varied with the samples. No Mg was found on the survey scans (Figure 3.12) for samples incubated for 2 or 5 days (Figure 3.13). Despite this, the XPS data suggest that there are some differences between the coated and uncoated samples. Due to this, a ratio of % of each element with respect to C was calculated to tease out any differences (Table 3.2, Table 3.3). This was done for XPS analysis of samples incubated under flow and static conditions for each time point. Interestingly for all samples tested under flow, a decrease in the percentage of Ca was observed between uncoated and coated samples from 0.82% (uncoated) to 0.40% (coated) and 3.90% (uncoated) to 2.11% (coated) at the 2-day time point.  Under static conditions, however, the Ca percentage increased from 1.14% (Uncoated) to 1.76% (coated), while the P percentage on the coated surface was decreased from 1.20% (uncoated) to 1.04% (coated).  At the 5-day time point, results from the flow model showed the coating decreased Ca and P deposition from 5.98% (uncoated) to 2.35% (coated) for sample 1, and 3.89% (uncoated) to 1.70% (coated) for sample 2. In contrast, results from the static model, the results showed a decrease in the presence of Ca from 2.69% (uncoated) to 1.86% (coated) and increase in P percentage from 1.91% (uncoated) to 2.12% (coated). Overall, no Mg was detected by the XPS method. Based on XPS analysis, our PDA/PDMA coating played a promising role in preventing salt deposition.     48         Figure 3.12: XPS spectra: A) and B) flow model condition, and C) static model condition. A. B. C. 49    Table 3.2: elements percentage at 2 days incubation, samples 1 and 2 were in flow model condition, and sample 3 was in static model.   50                      Figure 3.13:  XPS spectra: A) flow model condition and B) static model condition. A. B. 51    Table 3.3:  Elemental composition on the surface at 5 days incubation by XPS analysis. Sample 1 was in flow model condition, and sample 2 was in static model.   52  3.3 Calcium oxalate encrustation  3.3.1  SEM images  PU sample exhibited significant levels of calcium oxalate encrustations that were clearly visible to the naked eye. The morphological characteristics of calcium oxalate encrustations were observed when representative samples were examined under the SEM at 250X, 1000X, and 2500X magnification, Results for time-point 3 weeks are shown in Figure 3.14 A, and for time-point 6 weeks in Figure 3.14 B.                  53                       250 X                               1000X                               2500X                              250 X                       1000X                       2500X           Figure 3.14: Scanning electron micrographs of encrusted PU samples from CDC bioreactor calcium oxalate encrustation experiment A) within 3 weeks of incubation, B) within 6 weeks of incubation. A.  Uncoated PU samples  Coated PU samples  B.  Coated PU samples  Uncoated PU samples  300µm 300µm  80µm  80µm  30µm  30µm 300µm  300µm  80µm 80µm  30µm  30µm  54  3.3.2 Calcium deposition  Quantification of calcium oxalate encrustation formation at the time point (3 weeks, 6 weeks) was performed using AAS (Figure 3.15). The presence of low levels of calcium concentration on coated PU surfaces was confirmed using AAS. However, the calcium concentration levels that deposited on uncoated PU surfaces were much higher compared to PDA/PDMA coated surfaces, based on AAS data analysis. These results came as confirmation to what was visible in the visual SEM results. Within 3 weeks, the average of calcium concentration deposition on uncoated and coated PU surfaces were 1.2 mg/L, 0.4 mg/L respectively. In addition, at the 6-week time point the average of the concentration of calcium deposition decreased on coated surfaces compared to the uncoated surfaces from 3.4 mg/L to 1.7mg/L.                              Figure 3.15: Quantitative analysis of calcium oxalate encrustation formations PU surfaces by Atomic Absorption Spectroscopy (AAS). Comparison of uncoated and coated samples. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001. uncoated 3 weeks coated 3 weeks uncoated 6 weeks coated 6 weeks01234Ca concentrationmg/L*****55  Chapter 4: In vivo analysis of catheter infection and encrustation  The ability of the PDA/PDMA coating to prevent biofilm formation and encrustation formation was tested.  The experiments were done using mouse urinary infection model using P. mirabilis. The murine percutaneous catheterization model was used in this project.   4.1 Bacteria and animals   P. mirabilis freezer stock bacteria were cultured in AU at 37C O/N, followed by a second O/N sub-culture the next day. Bacteria were then re-suspended in PBS at approximately 5 x 105 CFU/mL to be used for intravesical injection. All procedures were approved by The University of British Columbia animal care committee (A17-0297). A total of 20 male C57Bl/6 mice (Harlan®) at age 10 weeks were included in experiments.  4.2 Modification of catheters  Prior to animal procedures, 25 gauge angiocatheters (Terumo Surflash® Polyurethane I.V. Catheter 24G x 3/4", Cat. No. SRFF2419) were modified under strict aseptic conditions. Briefly, the needle portion of the catheter was temporarily removed, and cut off 4 mm from the tip of the PU catheter using sterile blades. For uncoated samples, the tip catheter piece (4 mm) and the remaining PU catheter were re-assembled onto the original needle (Figure 4.1) [52]. For coated samples, the tip 4 mm pieces were coated with PDA/PDMA coating (see section 2.1.4), then rinsed in 70% ethanol for 5 minutes, and washed in sterile PBS (x 3) times to re-assembled them back onto the needles.   56                             Figure 4.1: Modification of 24G I.V. PU catheter for animal experiments.    4.3 Coating 24G PU catheters (PDA/PDMA) for in vivo study Coated (PDA/PDMA) catheters were prepared by Dr. Kai Yu at Kizhakkedathu lab. For coating, the catheters were cleaned with N2 dipped into the coating solution consisting of (0.7 mL) of PDA and PDMA (1:5 ratio in 1mM Tris-HCl buffer, pH 8.5) for 24 hours. Catheters were rinsed by Milli-Q water and dried. To increase the thickness and coverage of the coating, the process was repeated with the same coating solution composition.  All coated catheters were transported to Jack Bell Research Centre (JBRC).  57   The uncoated catheters were prepared at JBRC. All coated and uncoated catheters were sterilized using plasma gas sterilization in the JBRC Animal Facility.   4.4 Day 0: Ultrasound-guided bladder catheter implantation  All mice were anesthetized via inhalational anesthesia with initial induction of 3% isoflurane. Once the mice were anaesthetized, the isoflurane was decreased to 2.5%, while the animals were positioned on a heating pad to keep their temperature at 38C. The hair of the abdominal area was removed, and a plastic belt was used to secure the mouse bladder in place. The Vevo 770® high-resolution imaging system was used after application of sterile ultrasound gel on the abdominal area, using the ultrasound probe to locate and visualize the mouse bladder. The modified 24G catheter, mounted on the original needle, was positioned above the pubic bone at a 30o angle with bevel directed to the anterior (Figure 3-2 A). Once the needle was visualized and properly aligned under ultrasound guidance, the needle was inserted carefully into the bladder (Figure 3-2 B). Ultrasound imaging was used to confirm the 4 mm catheter piece was entirely inside the bladder. Once this was confirmed, the needle was removed and the pusher was used to push the catheter piece slightly inward (Figure 3-2 C). This released the catheter piece into the lumen of the bladder (Figure 3-2 D). When the pusher was removed, the implanted 4 mm catheter piece is the only material left inside the mouse bladder (Figure 3-2 E).    58    Figure 4.2: Ultrasound-guided percutaneous catheter implantation into mouse bladder.(a) Anesthetized mouse then put the mouse on this position while receiving isoflurane via nose cone we start the procedure (b). Inserting needle carefully (c) pushing the pusher into the mouse bladder and pulling gently the needle out at the same time. (d) Pulling the pusher out the bladder. (e) Catheter piece placed into bladder (as visualized by ultrasound).    59  4.5 Day 1: Bacterial injection into mouse bladder (infection induced)   One day after catheterization, all mice were anesthetized as above. Under ultrasound guidance, P. mirabilis (5 x 105 CFU/mL in 50 L PBS) was percutaneously injected into the bladder lumen using a 30-gauge needle, changing needles between each mouse. Once successfully injected inside the bladder, mice were kept asleep at 1% isoflurane for 1 h to allow bacteria to attach onto the catheter piece. After an hour, all mice were recovered from anesthesia.  4.6 Daily monitoring  According to protocol (A17-0297), all mice were monitored daily for physical health assessments as well as appearance, behavior, and body weight. Moreover, urine samples were collected on day 1, 2 and 3 to measure pH levels.  4.7 Day 3: Experimental endpoint  At three days post-installation, all mice were euthanized as per the approved protocol of CO2 asphyxiation. Catheters were surgically collected on day 3 from all mice. For urine samples, the amount of P. mirabilis was quantified using serial dilutions and CFU counting. Catheter samples were collected in Eppendorf tubes, containing 100 L of sterile PBS and then transferred to new tubes containing 100 L of fresh PBS. Samples were sonicated at 50/60 Hz for 10 minutes in an ultrasonic water bath (No. 21811-820, VWR®) to aid biofilm dispersal. Next, the catheters were vortexed at high speed for 10 sec, and P. mirabilis numbers in the solution were determined by serial dilutions and CFU counting. XRD was used to analyze catheters uncoated and coated (2 pieces each) to determine the presence of struvite encrustation formations. XRD analysis was done at 4D labs at Simon Fraser University. 60  4.7.1.1 Statistical analysis  Data are shown as the mean ± SE. Statistical analysis was done using unpaired t-test. Statistical significance was set at P < 0.05.     4.8 Results  4.8.1 Bacterial adhesion 4.8.1.1 Coated and uncoated (4 mm) catheters successfully implanted into mice bladder The 24G (4mm) polyurethane coated and uncoated catheters were successfully introduced into the bladders followed by the induction of infection via instillation of P. mirabilis into mouse bladders. Successful insertion and instillation were confirmed using ultrasound guidance.   4.8.1.2 Urine pH levels  The urine pH increased up to the value of 9.2 during the experiment period (Figure 4.3). P. mirabilis has the ability to increase the urine pH (alkaline pH) because of the activity of its urease enzyme, which leads to hydrolyzing urea into ammonia and CO2 [50].   61                                                      Figure 4.3: Increasing pH level in mice urine during the experiment period.  4.8.1.3 Anti-adhesive efficiency of PDA/PDMA coating in the animal model At three days post-instillation, the number of bacteria attached to the 4 mm coated and uncoated catheters was measured by CFU counting. The average amount of P. mirabilis adhered onto uncoated (control) implanted catheters were 2.09 x 106 CFU/mL. In contrast, on coated catheters, the average of bacterial adhesion was 2.52 x 105 CFU/mL, the decrease is 8.29-fold. Hence, the bacterial CFU count was reduced by 88.0% for mice with coated vs uncoated catheters (Figure 4.4 A).   Similarly, the average planktonic CFU counts of P. mirabilis in the urine was decreased by 93.33% (15.01-fold) in animals containing coated catheters compared to those with uncoated catheters (Figure 4.4 B).    66.577.588.599.510Day 0 Day 1 Day 2 Day 3Urine pH 62                                                                  Figure 4.4: Anti-adhesive activity of PDA/PDMA coated catheters in vivo on (A) bacterial adhesion, and (B)bacterial planktonic in urine determined by CFU count. Each dot represents data from one mouse, and C) uncoatedcoated 110311041105110611071108CFU/catheter*2.09E+062.52E+0588.0% 0246810121416catheter urineFold redease from uncoated samples A BAC63  graph showed the fold reduction of P. mirabilis on catheter surfaces and in mice urine (at the top of each bar  the percentage reduction).       4.8.2 Catheter encrustation 4.8.2.1 In vivo imaging  Ultrasound images were taken at day 0 to confirm the success of catheters tip implantation. On days 1 and 3, ultrasound images were taken for all animals to visualize possible buildup of encrustation. Interestingly, the images showed that the encrustation rate was increased on the uncoated catheter on day three post-infection. However, no crystals were evident on the coated catheters after the same period of infection (Figure 4.5). These results were verified visually following retrieval of the catheter pieces on day 3, where white-yellowish encrustations were present on the ends of the uncoated catheter pieces but not on the coated catheter pieces (Figure 4.6).     64    Figure 4.5: Encrustation was visible on ultrasound in images of 4 mm implanted catheter.                                                                                               Figure 4.6: A) Appearance of catheter encrustation and stone formation after 3 days on uncoated catheter. B) No encrustation formation on surface of the coated catheter.     A B65  4.8.2.2 SEM images SEM analysis also confirmed reduction in crystals formation on the coated catheters (Figure 4.7).  Biofilm and crystals formation by P. mirabilis was evident on the uncoated catheter surface, compared to the coated catheter surface (Fig. 4.7).         Figure 4.7: catheters SEM images of in vivo study. A) SEM image of crystals formation on uncoated catheter, and B) PDA/PDMA coated catheter, after instillation of bacteria for 3days (scale bar = 300µm). 4.8.2.3 XRD analysis The XRD analysis of the powdered struvite crystals from uncoated and coated catheters shows peaks in the diffraction pattern. Struvite encrustation was evident on the uncoated catheter on the SEM images. X-ray diffraction patterns were obtained from uncoated and coated catheters (see Figure 4.8 A). The diameters of the rings in the diffraction patterns and their relative intensities in the uncoated surface were compared with the patterns in the coated surface (Figure 4.8 B). This comparison with struvite XRD graph as standard [52], allowed the element peaks to be identified as struvite (NH4MgP04.6H20). XRD data showed that the uncoated catheter diffraction peaks are related to MG, NH4, PO4, which were absent on the coated catheter pieces.  A B300µm  300µm 66      Figure 4.8: A) XRD graph for uncoated and coated catheters, and B) with peaks numbers. C) A representative comparison graph of pure struvite compared to our struvite on catheters [52].   A.  B.  C.  67  Chapter 5: Discussion  Catheter-associated urinary tract infection [53] is a serious problem that researchers have worked towards solving. Past studies have made several attempts to develop catheters that prevent CAUTI [55, 56, 57]. Various catheter coatings on catheter surfaces prone to bacterial adhesion and infection were tested such as silver, nitric oxide, antibiotics, quaternary ammonium compounds, and antimicrobial peptides [56, 57, 58].     Despite all of these attempts, no coating has been effective at preventing catheter-associated urinary tract infection and face challenges with long-term efficacy and research translation to clinical applications [59, 60]. For instance, bacterial resistance significantly complicates the translation of antibiotic-based coatings into clinical practice. This is where our novel coating has a significant advantage, as it does not contain any antimicrobial agents and acts by repelling bacteria. The focus of this thesis was to test the efficacy of our novel antifouling PDA/PDMA coating against P. mirabilis infection, both in vitro and in vivo. Using various relevant models, we have shown our thin coating to have significant potential in preventing P. mirabilis-induced indwelling device associated infections. In support of this, our coating was tested in different conditions and different media.  In vitro bacterial adhesion experiments showed a significant reduction in adhered bacteria within 24 hours on the coated samples. The greater bacterial adhesion reduction within 24 h was 99.1% in AU media, and was 97.4% in LB media; the difference between testing in AU and LB is because P. mirabilis produces struvite encrustations by the urease enzyme activity in AU media, which leads to provide more layers for bacterial adhesion on bare surfaces. However, LB media does not have urease and it is not expected to observe struvite 68  encrustation formations on surface; therefore, we tested the bacterial adhesion using different media. Moreover, our novel PDA/PDMA coating was designed to prevent bacterial adhesion and struvite encrustation formation. In addition, SEM images showed less bacteria attached to the coated samples compared to the uncoated ones, which means our coating has significant efficacy to prevent bacterial adhesion.   In terms of struvite encrustation, SEM images showed a difference between the coated and uncoated samples at early time points in artificial urine medium. On 10 and 14 days post-infection induction, similar struvite deposition was observed on uncoated and coated samples. This may be explained by the fact that with increasing incubation periods, crystal deposition on the coated surface reaches a level that is similar to that on the uncoated surface resulting in bacterial and crystal buildup on already deposited crystals eliminating the difference in deposition observed at earlier time points. EDX elemental analysis has been done to detect the elements deposition on PU surfaces that were incubated in AU media with the presence of P. mirabilis, the NH4 ions, calcium, and Mg produce crystallization such as struvite and carbonate apatite (Ca10(PO4)6CO3; CA). The results were similar to SEM results, with lower levels of magnesium and calcium being detected on samples at the 2-day time point on the coated compared to the uncoated surface. In contrast, at the 5-day time point, we found no magnesium or calcium on either uncoated or coated samples. While unexpected, it is possible that the amount of these elements was below the EDX detection limit at this time point.  Similarly, XPS analysis for struvite encrustation formations by P. mirabilis over a 5 day period also did not detect Mg on either coated or uncoated samples when incubated in static and flow condition, which may indicate that the amount of struvite deposited in this 69  simple in vitro model may be small and may be a limitation of this model. That said, 5 day results for the flow model did show less Ca and P deposition on the coated samples compared to the uncoated samples. The static data showed a similar trend to the flow model (less Ca on coated samples compared to uncoated). In addition, we found no Mg (or below the detection limit) but did find Ca based on XPS data. This is consistent with the EDX results, the reason could be in these experimental conditions the crystal formation with Ca and Mg may be different.    For the no infection encrustation, the calcium oxalate encrustation formation, distinct differences in Ca-based crystal deposition were observed at 3 and 6 weeks using SEM, with less deposit on coated versus uncoated samples. This is supported by the results from the AAS analysis, a quantification method, which showed a significant reduction in calcium deposition on the coated surfaces. Collectively these results suggest that the ability of this coating in preventing the encrustation formations in non-infections conditions (for instance, patients with calcium oxalate stones).   While the in vitro results suggested that the PDA/PDMA coating was efficient at reducing P. mirabilis adhesion and encrustation, in vivo testing was performed to test the efficacy of the coating in a realistic environment (in a mouse CAUTI model). For this, the coating was applied to 24G catheters and tested in a mouse model of catheter-associated urinary tract infection. Multiple experiments were performed, each consisting of 10 mice for the coated group and another 10 for uncoated group. At 3 days post-instillation of P. mirabilis, the CFU counting showed an 87.9% reduction in P. mirabilis adhered to coated catheters compared to the uncoated catheters (control) (see Figure 3-4 A).  Interestingly, we also observed a reduction in bacteria of 93% in the urine of animals with coated catheters 70  compared to the uncoated catheter group (Figure 3-4 B). This may be explained by the fact that the coating prevented bacterial attachment, leaving them in a planktonic form and resulting in their elimination via urination. Moreover, SEM images of catheters from the in vivo experiments showed obvious crystals deposited on the tips of uncoated catheter pieces, while none were evident on coated catheter pieces (see Figure 3-7). XRD results from crystals formed on uncoated samples were compared to those from a previous study analyzing struvite [52], and verified that the material on the uncoated samples were struvite (NH4MgPO4.6H2O).    71  Chapter 6:  Conclusion and future direction In this study, we have established the efficacy of thin antifouling PDA/PDMA coating, both in vitro and in vivo. By designing our effective anti-adhesion coating we found that the biofilm and encrustation formations are significantly decreased. Based on the results of this study, PDA/PDMA coating effectively prevents bacterial and crystals attachment, which is vital to prevent UTIs. Moreover, PDA/PDMA does not contain any antibacterial reagent which potentially eliminates the development of antibiotic resistance and eventual failure of the coating.  Another advantage of the PDA/PDMA is that it can be can easily applied to different types of biomaterials using a one-step dip-coating process. Importantly, our anti-adhesive coating is safe and stable on the medical devices based on the in vitro and in vivo experiments. Also, the results showed that P. mirabilis reduction in vitro was up to 99.1% within 24 hours in AU and 87.9% in vivo in the mouse CAUTI model.  The goals of this study are to prevent the infection by preventing bacterial attachment (P. mirabilis) and encrustation formations, which both were achieved. In this study, we tested our coating against P. mirabilis, which is a complicated bacterial species because it infects and causes crystal formation to which bacteria attach as well, blocks devices, causing them to fail. Indeed, for future studies, more in vitro and in vivo experiments must be performed to test the coating against different types of bacteria. In addition, further validation of the performance of our coating in preventing CAUTI in a pig model. 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