Ruthenium metallotherapeutics: a targeted approach to combatting multidrug resistant pathogens

Britten, Nicole Sophie (2024) Ruthenium metallotherapeutics: a targeted approach to combatting multidrug resistant pathogens. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The discovery of antibiotics revolutionised healthcare practice. However due to overuse, inappropriate use, widespread prophylaxis therapy and the lack of new developments, the threat of antimicrobial resistance is now a major global threat to health. By 2050, it is estimated that mortality due to antimicrobial resistant infections will exceed 10 million people per annum, superseding cancer as the leading cause of global mortality. The use of drug repurposing to identify potential therapies which combat antimicrobial resistance is one potential solution. Metals have been used as antimicrobial agents throughout the history of medicine for a broad range of applications, including the use of Silver as an antimicrobial agent which dates back to antiquity. More recently, Ruthenium metallotherapeutic complexes have been shown to exhibit highly active antimicrobial properties by targeting a range of bacterial species, and in contrast to traditional antibiotics, these compounds are thought to elicit antibacterial activity at multiple sites within the bacterial cell, which may reduce the possibility of resistance evolution. This study aimed to evaluate the antimicrobial activity of a series of Ruthenium metallotherapeutic complexes against multidrug-resistant bacterial pathogens, with a focus on use within wound care applications. Antimicrobial susceptibility assays identified two lead candidates, Hexaammineruthenium (III) chloride and [Chlorido(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2-carbothioamide) ruthenium (II)] chloride which demonstrated activity against Pseudomonas aeruginosa and Staphylococcus aureus respectively with MIC values ranging between 4 μg mL-1 and 16 μg mL-1. Furthermore, Hexaammineruthenium (III) chloride demonstrated antibiofilm activity in both a time and concentration-dependent manner. Synergy studies combining lead complexes with antibiotics demonstrated the potential for use as resistance breakers. Subsequent in vitro infection modelling using scratch assays with skin cell lines, coupled with a 3D full thickness skin wound infection model was used to determine potential applied applications of Hexaammineruthenium (III) chloride for use as topical antimicrobial agent against P. aeruginosa infections. Antimicrobial mechanistic studies demonstrated that Hexaammineruthenium (III) chloride targeted the bacterial cell ultrastructure of P. aeruginosa strain PAO1 as cell perturbations were observed when treated cells were analysed by scanning electron microscopy. Furthermore, exposure of P. aeruginosa PAO1 to Hexaammineruthenium (III) chloride also resulted in a concentration dependent membrane depolarisation, which further supported the antimicrobial mechanistic role. Finally, global changes in gene expression following exposure of P. aeruginosa strain PAO1 to Hexaammineruthenium (III) chloride were explored by RNA sequencing. Genes involved in ribosome function, cofactor biosynthesis and membrane fusion were downregulated, which provided a further insight into the wider mechanisms of antibacterial activity. The research conducted in the present study indicated the potential use of Hexaammineruthenium (III) chloride (and derivatives) as a potential treatment option for chronic wounds infected with P. aeruginosa, which could be applied as either a direct treatment or used within antimicrobial wound care applications.