A Novel Antibiofilm Treatment for Surgical Site Infections

Abstract

Surgical site infections (SSIs) are the most common postoperative complication, causing frequent hospital readmissions, significant mortality and representing an economic burden for the healthcare system. The pathogens typically associated with SSIs are Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Prophylaxis and treatment of SSIs with antibiotics frequently fail due to increased antibiotic-resistance of bacteria and their ability to reside as clusters within a matrix. This form of life is called a biofilm and offers the bacteria protection against antibiotics and the immune system. To eradicate biofilms, high systemic concentrations of antibiotics are needed, often causing toxic side effects, emphasising the need for novel therapeutic strategies that can be administered locally on SSIs. Repurposed diethyldithiocarbamate (DDC-), a metabolite of the old anti-alcoholic drug Disulfiram, previously showed antibacterial activity against mycobacteria and streptococci in combination with copper ions (Cu2+) but had not been investigated against S. aureus and S. epidermidis biofilms. In this thesis, the antibacterial and antibiofilm activities of DDC- with Cu2+ were examined and an injectable gel containing the combination was developed for application on SSIs. The antibacterial and antibiofilm activity of DDC- was strictly Cu2+-dependent and linked to the formation of Cu(DDC)2 (2:1 molar ratio of DDC- and Cu2+) with excess Cu2+. This combination inhibited bacterial growth, multiple steps in the biofilm formation, including bacterial attachment and bacterial aggregation, and reduced biofilm viability. Furthermore, DDC- and Cu2+ demonstrated either synergistic or additive antibiofilm effects against the staphylococci tested and synergised with multiple antibiotics. In vitro cell culture studies, at relevant concentrations, showed no toxicity of the DDC- and Cu2+ combination in human dermal fibroblast cells. Antibacterial activity and non-toxicity of Cu(DDC)2 + Cu2+ was also confirmed in vivo in a Galleria mellonella infection model. To enable prolonged exposure of the combination at surgical sites, a depot system was developed for controlled drug release. As Cu(DDC)2 is water-insoluble, the complex was first encapsulated into PEGylated liposomes. Cu2+-liposomes were produced by thin-film hydration and extrusion, then Cu(DDC)2-liposomes were formed by diffusion of DDC- into the liposomal core and complexation with encapsulated Cu2+. Like free Cu(DDC)2 + Cu2+, the liposomal combination showed antibiofilm activity in vitro and antibacterial effects and non-toxicity in vivo, making it a water-soluble formulation for Cu(DDC)2. Following lyophilisation, the liposomes were stable below 6 °C for over six months and able to be incorporated within a gel. A biocompatible mixture, comprising chitosan and beta-glycerophosphate, was fluid at ambient temperature and formed a gel at body temperature. These thermosensitive properties were maintained following sterilisation, storage at -20 °C and liposomes incorporation. The liposomal gel prevented biofilm formation and reduced S. aureus and S. epidermidis biofilm viability. The combination of DDC- and Cu2+ has potential as antibacterial and antibiofilm treatment against S. aureus and S. epidermidis. An application at the surgical site is possible when liposomal Cu(DDC)2 + Cu2+ is incorporated into an injectable gel. This antibacterial gel represents an innovative therapeutic approach for the prophylaxis and treatment of SSIs.