Author: Gia Han Tran
Tran, Gia Han, 2024 Nanoengineered charge gradients as a high throughput tool to study antimicrobial drug tolerance, Flinders University, College of Medicine and Public Health
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Implantable medical devices are essential in healthcare systems to improve human health and life expectancy. However, the colonisation of pathogenic microorganisms on the surface of these devices is a significant concern as they should be removed or replaced frequently from the body of the patient to prevent complete colonisation. Currently, antibiotics have been the main method to prevent and control infections from pathogenic microorganisms; however, the overuse and misuse of antibiotics lead to the emergence of antibiotic tolerance, challenging in treatment with this conventional method. To combat this challenge, previous studies have modified the surface to change the surface properties by coating an antimicrobial compound on the material. Among these compounds, quaternary ammonium compounds (QACs) are demonstrated to show broad-spectrum antimicrobial activity and be less toxic to human cells. This compound is widely used to coat surfaces for the creation of contact-active killing bacterial material, killing harmful microorganisms upon direct contact. However, previous studies have not investigated deep into the potential of immobilised QACs in reducing antibiotic tolerance, especially in the context of electrostatic properties. The surfaces with a strong positive charge are demonstrated to enhance the interaction with the negative charge cell membrane of bacteria. These interactions are critical as they facilitate the disruption of bacterial membranes, enhancing direct antimicrobial effects. In addition, these electrostatic properties can enhance the efficacy of antibiotics by facilitating their interaction with bacterial surfaces. Therefore, in this project, we investigated a biomaterial to address the antibiotic tolerance of creating an antimicrobial coating on surfaces with immobilised QACs gradient density. This modified surface was developed by the deposition of allylamine using plasma polymerisation to generate an amine-rich surface. Glycidyltrimethylammonium chloride (GTAC), a type of quaternary ammonium compound, was coated onto the plasma polymerised allylamine (ppAA) surface via the reaction between epoxy groups of QAC and amine groups of allylamine. The primary samples in this study were the surfaces with a concentration gradient of GTAC created by dip coating. The surface characterisation of the modified surface confirmed the presence of QAC and a gradient in QAC density on the surface through ellipsometry, water contact angle measurement and fluorescence analysis. In general, the addition of GTAC on ppAA surface changed the surface properties including thickness, wettability, fluorescence intensity due to the presence of NR4+ of QAC. The antimicrobial surface tests demonstrated that increasing the concentration density of QAC significantly enhanced the antibacterial efficacy against Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15692 compared to the control surface (ppAA only). Especially, after 3 hours of incubation with pathogenic microorganisms, the modified surface exhibited a reduction in the viability of S. aureus (13.23% 1.55) and P. aeruginosa (23.78% 3.81) at the 8-10 mm position remained in the GTAC solution for the longest time (6 hours). In contrast, the lowest viability of fungi (Candida albicans ATCC 10231) was observed at 0-2 mm position, which corresponds to the region immersed in GTAC solution for the shortest duration. The data also revealed that QAC surface immobilisation density did not significantly impact the attachment of the microorganisms, indicating that enhanced antimicrobial effect was primarily due to reduced bacterial viability rather than decreased microorganism adhesion. This study focused on the antibiotic tolerance of S. aureus on QAC surface concentration density gradient under vancomycin, daptomycin levofloxacin, and kanamycin treatment for 24 hours. Overall results revealed that the presence of QAC improved the antibacterial activity of antibiotics in reducing bacteria viability, especially at higher gradient positions (8-10 mm). Particularly, kanamycin reduced S. aureus viability by 55.21% 3.03, while vancomycin showed a similar reduction of 47.47% 1.21. Levofloxacin and daptomycin were the most effective, reducing viability by 31.08% 6.78 and 34.03% 2.14, respectively. In overall, the results demonstrated that the activity of four antibiotics was improved, highlighting the effect of QAC surface immobilisation density on antibiotic treatments in combating bacterial tolerance.
Keywords: Antibiotic tolerance, antimicrobial activity, charge density, gradient surface, GTAC, QAC
Subject: Medical Biotechnology thesis
Thesis type: Masters
Completed: 2024
School: College of Medicine and Public Health
Supervisor: Andrew Hayles