Novel approaches to preventing bacterial attachment and biofilm growth on biomedical devices.

Abstract

Biomaterials have greatly improved the quality of life for patients living with a wide range of medical conditions. Despite this, they are often susceptible to bacterial infections, which are a financial burden to healthcare systems alongside causing significant patient morbidity and mortality. The central aim of this thesis is to assess materials with the potential to prevent bacterial adhesion to surfaces to determine if they could be used to inhibit the development of device infections. The first study was to investigate the most appropriate means for the quantification of bacterial adherence to surfaces in vitro as the current gold standard of colony counting is time and resource consuming. The adherence of Staphylococcus aureus to 2-Hydroxyethyl methacrylate (HEMA) polymer films was first assessed with the colony counting method. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), resazurin and adenosine triphosphate (ATP) assays were chosen as an alternate means to enumerate viable bacterial cells, as they are more time-efficient than the colony counting method. These assays were performed on a range of concentrations of S. aureus to compare the signals measured with the cfu ml-1 to determine if they give the same result for bacterial quantification. It was found that, despite displaying varying sensitivity, neither of these assay methods could produce a signal in the concentration range corresponding to that of adherent bacteria on HEMA films. For this reason, the colony counting method was chosen as the most appropriate method to quantify bacterial adhesion. A large number of nosocomial infections are as a result of catheter-associated urinary tract infections (CAUTI) and these are associated with several complications, which arise from bacterial colonisation on device surfaces. The aim of the second study was therefore to modulate a low friction catheter coating by loading a commonly used antiseptic, chlorhexidine, to elute and prevent bacterial attachment. Bacterial adherence to coated sample surfaces was assessed in an infected artificial urine medium to simulate the in vivo environment. Model catheter surface samples consisting of polyvinyl chloride (PVC) substrate coated with either low friction coating without chlorhexidine or 0.1 % w/v chlorhexidine-loaded low friction coating were tested against common Gram-negative and Gram-positive urinary pathogens, Proteus mirabilis and S. aureus. Low friction coated samples without chlorhexidine showed a 27.0 % and 38.4 % reduction in adherence by 24 h against P. mirabilis and S. aureus respectively compared to PVC control. Chlorhexidine-loaded low friction coatings showed a significant reduction in adherence of 99.8 % and 99.4 % by 24 h against P. mirabilis and S. aureus respectively compared to PVC control. This corresponded to a significant reduction in bacterial adherence of over 2-log values against both bacterial species by 24 h. Further microbiological testing using time kill and agar diffusion assays indicated this coating had a significant effect against S. aureus but a lack of antimicrobial effect was demonstrated against P. mirabilis. This could be a result of the outer membrane, present on Gram-negative bacteria and not Gram-positive bacteria, reducing the effect of chlorhexidine. Further investigation into this effect was however recommended due to the promising reduction in adherence displayed against P. mirabilis. An additional aim of this study was to investigate the lubricity of the coating, as there is a significant level of urethral trauma and discomfort associated with the catheterisation process using many commercially available catheters. It was found that the coating has an extremely low friction profile of 0.0054. Furthermore, a long dry-out time of 140 minutes before the thickness of the coating is significantly reduced was determined and this has the potential to open non-surgical catheterisation to a larger patient market. Mechanically engineered nanomaterials have displayed great versatility in their applications and have demonstrated uses in the field of biomaterials to discourage microbial growth. The final study of this thesis therefore involved screening several materials produced by mechanical engineering and loaded with silver nanoparticles (AgNPs) including carbon nanotubes (CNTs), chitosan (CS) films and graphene oxide (GO)/CS scaffolds for antimicrobial activity. These were challenged with Escherichia coli and S. aureus, common pathogens implicated in a wide variety of device infections. Time kill and agar diffusion assays were used to assess the suitability of these materials for application as antimicrobial biomaterials. It was found that AgNP-loaded CNTs in solution did not exert an antimicrobial effect against E. coli over a 24 h period. AgNPs loaded into CS films and CS scaffolds indicated a wide spectrum of activity, producing zones of inhibition against E. coli and S. aureus and it was found that adding GO to the scaffolds did not result in an increase in antimicrobial activity. Due to the use of CS in wound healing applications, incorporation of these nanomaterials into anti-infective wound dressings was considered as an appropriate potential application.