Antimicrobial resistance (AMR) represents a growing global health threat, driven in part by the overuse of broad-spectrum antibiotics and the limited effectiveness of conventional diagnostics and therapeutic approaches. This Thesis addresses a dual strategy to address these challenges: (i) rapid, enzyme-responsive diagnostics to enable early detection and mitigate antibiotic misuse, and (ii) targeted antimicrobial photodynamic therapy (aPDT) to enhance treatment efficacy. For bacterial detection, the strategy of autonomously reporting chitosan-based sensors was further developed, focusing on E. coli detection using β-glucuronidase as a biomarker. A paper-based carrier for chitosan hydrogels was integrated with smartphone-assisted RGB analysis, enabling visible, low-cost, and user-friendly point-of-care diagnostics. The system relies on color intensity changes of a released reporter dye, modulated by enzyme concentrations corresponding to bacterial load. To improve β-glucuronidase sensitivity and facilitate scalable fabrication, enzyme-responsive chitosan-based electrospun nanofibers were investigated. At a fixed enzyme concentration, these nanofibers exhibited a 3.4-fold increase in the initial enzymatic hydrolysis rate compared to the hydrogel system and achieved a limit of detection of 4.7 nM after 60 min, in contrast to 16 nM for the hydrogel system. The enhanced sensitivity of the nanofiber platform was further validated through in vitro experiments using pathogenic E. coli, demonstrating its potential utility in real biological samples. For bacterial eradication, a Ru(II)-based photosensitizer (Ru(phen)₃) encapsulated in polymeric nanocarriers was evaluated against P. aeruginosa in its planktonic form, in biofilms and in artificial sputum medium (ASM), which simulates the Cystic Fibrosis (CF) lung environment. Ru(phen)3-loaded vesicles exhibited a greater photodynamic inactivation of planktonic P. aeruginosa compared to Ru(phen)3-loaded micelles, attributed to the higher loading capacity of Ru(phen)3. The biological complexity of ASM significantly influenced aPDT efficacy, with Ru(phen)₃-loaded vesicles showing reduced activity compared to the neat Ru(phen)₃ at similar overall Ru(phen)3 concentrations. In biofilm eradication assay, a 4.8 log reduction was achieved with neat Ru(phen)3 while Ru(phen)3-loaded vesicles resulted in 2 log reduction after 120 min of irradiation. Notably, prolonged pre-incubation times appeared to facilitate deeper photosensitizer penetration into biofilms, thereby improving aPDT outcomes. This approach was further applied to multidrug-resistant (MDR) isolates of P. aeruginosa and S. aureus from CF patients, as well as pathogenic E. coli. The results underscore the therapeutic potential and versatility of Ru(phen)3-loaded vesicles in treating MDR infections, particularly those associated with CF pathology. Overall, this Thesis contributes to the development of effective, enzyme-responsive diagnostic tools and optimized aPDT treatment strategies for combating bacterial infections to fight AMR.
Kawaljit Kaur (Wed,) studied this question.
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