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Antibiofilm Function-Spacer-Lipid Coatings on Medical Surfaces

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Antibiofilm Function-Spacer-Lipid Coatings on Medical Surfaces ANTIBIOFILM FUNCTION- SPACER-LIPID COATINGS ON MEDICAL SURFACES Pavithra Raghuraman Thesis submitted to the fulfilment of requirement for the degree of Doctor of Philosophy MARCH, 2021 AUCKLAND UNIVERSITY OF TECHNOLOGY New Zealand Table of contents The need for novel antimicrobials to prevent implant infection ................................... 4 Antibiotic resistance by bacteria ............................................................................ 4 Biofilm bacteria are difficult to treat ..................................................................... 6 Challenges in sampling and treating implant infection .......................................... 7 The process involved in biofilm formation .................................................................... 9 Adhesion ................................................................................................................ 9 Colonisation ......................................................................................................... 11 Biofilm .................................................................................................................. 11 Dispersion and Infection ...................................................................................... 12 Microorganism in infection ......................................................................................... 14 Gram-positive bacteria ........................................................................................ 14 Gram-negative bacteria ....................................................................................... 16 Factors affecting bacterial attachment onto the surface ............................................ 17 Current surface coating strategies to combat bacterial biofilm formation ................. 18 Antibiofilm and antimicrobial coating .................................................................. 18 Antibiofilm and antibacterial combined coating .................................................. 32 Coating technologies used for rendering an antimicrobial medical surface................ 33 FSL technology an opportunity to deliver an antimicrobial surface ............................ 34 Potential advantages of using an FSL molecule to render a surface with antimicrobial features ................................................................................................................................... 38 Aims ............................................................................................................................. 39 i FSL constructs used ..................................................................................................... 40 Benign FSL ............................................................................................................ 40 FSL -Zero (FSL-Z) .................................................................................................. 41 Bioactive antimicrobial FSLs................................................................................. 41 Zeta potential of FSL constructs .................................................................................. 43 FSL constructs interaction with biological surfaces ..................................................... 44 Microorganism interaction with FSL .................................................................... 44 Single-celled fungi ................................................................................................ 47 Non-biological surfaces labelling with benign FSL ....................................................... 50 BAND-AID® ........................................................................................................... 50 Contact lens surfaces ........................................................................................... 54 Non-biological surface labelling with antimicrobial FSL .............................................. 55 Antimicrobial FSL-Se constructs on BAND-AID ® Surfaces ................................... 55 FSL-SPM detection on BAND-AID® ....................................................................... 60 Summary ..................................................................................................................... 66 Bacterial cultivation and inoculum preparation .......................................................... 68 Bacterial concentration and resazurin colour development ....................................... 69 Minimum inhibitory concentration with resazurin as an indicator ............................. 70 Antimicrobial activity of FSL-Se in solution .......................................................... 70 Antimicrobial activity of FSL-SPM in solution ....................................................... 72 Antimicrobial effect of FSL-RIP ............................................................................ 73 Summary ..................................................................................................................... 74 Ablative layer plus adhered layer ................................................................................ 75 The antimicrobial ability of FSL-Se on BAND-AID® ............................................... 75 Antimicrobial effect of monolayer ....................................................................... 83 Antimicrobial activity of FSL-SPM ........................................................................ 84 Antibiofilm efficacy of FSL-RIP labelled surfaces .................................................. 90 Summary ..................................................................................................................... 95 ii Positive charge surface association with [-]Agnp ........................................................ 96 Antimicrobial efficacy of FSL-SPM associated with negative charged silver nanoparticles ....................................................................................................... 96 Capture of positive charged compounds with negative charged FSLs ........................ 99 Negative charged FSL association with positive charged silver nanoparticles [+]Agnp .............................................................................................................. 100 Negative charge surfaces associate positive charge crystal violet ..................... 100 Negative charged FSLs association with positive Cv and then negative charge [-]Agnp ............................................................................................................... 105 Antimicrobial efficacy of FSL+Cv+AgNO3 ........................................................... 109 Antimicrobial efficacy of FSL-Z and FSL-Bn premixed with [Cv+AgNO3] before applying on to the surfaces ................................................................................ 111 Methodology used and its limitations ....................................................................... 130 Micro-organisms and methods for their detection ........................................... 131 Surfaces and baseline FSLs ................................................................................ 133 Covalent FSL-antimicrobials ...................................................................................... 134 FSL-Spermine (FSL-SPM) .................................................................................... 134 FSL-Selenium (FSL-Se) ........................................................................................ 137 FSL-antimicrobial peptide RIP (FSL-RIP) ............................................................. 141 FSL secondary capture of antimicrobials ................................................................... 142 Negatively charged FSL with captured Cv .......................................................... 142 Positively charged FSL-SPM with captured [-]Agnp ........................................... 143 Antimicrobial efficacy of negatively charged FSL captured Cv captured [-]Agnp143 Antimicrobial efficacy of negatively charged FSL captured Cv, captured AgNO3 144 Negatively charged FSL captured [Cv and AgNO3] ............................................. 145 Outcomes of this research ........................................................................................ 146 Future prospects ....................................................................................................... 149 iii Abstract The establishment of bacteria on surgical implants as a biofilm is a major problem and will significantly affect the morbidity and potentially the mortality of the patient. There are two basic approaches to prevent this from occurring being: (i) to prevent it from happening and/or (ii) to treat it afterwards. The first approach is the most desirable. Surgical implant infections number in the range up to 4% of surgeries and new methods to prevent this are needed. The main objective of this research was to examine the potential of Kode FSL constructs to prevent the establishment of a biofilm. To determine this, FSL-antimicrobials needed to be designed and constructed; the ability of the FSL constructs to modify surgical surfaces established and then their ability to prevent the establishment of biofilms evaluated. During the research it was discovered that the best results were obtained by using FSLs to secondarily capture antimicrobials and this new approach was extensively evaluated. It was found that a combined product of crystal violet with silver captured by a charged
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