Development of Bioluminescent and Fluorescent Strains of Staphylococcus aureus

Abstract

Staphylococcus aureus (S. aureus) is an opportunistic Gram-positive pathogen that causes a wide range of diseases. The emergence and increase in antibiotic-resistant strains in nosocomial and community settings is an urgent problem. Furthermore, our ability to treat such dangerous strains is in jeopardy due to the lack of development of new antimicrobials. Hence there is an ongoing need for research on S. aureus to ensure that we will have the ability to combat any threat that may develop. An important approach for studying S. aureus is biophotonic imaging (BPI), which involves the use of bioluminescent and fluorescent reporters to visualise S. aureus in in vitro and in vivo settings. Current S. aureus reporter strains typically have reporter genes stably integrated into the genome or present on unstable plasmid-based systems. The aims of this thesis were to create stable plasmid-encoded bioluminescent and fluorescent strains of S. aureus that were as bright, if not brighter, than current strains and to test their utility in preliminary in vitro and in vivo studies. Growth, bioluminescence, fluorescence and plasmid stability in the absence of antibiotic selection was assessed. The streptococcal -- toxin-antitoxin system was introduced to our plasmids to confer post-segregational stability and different promoters were considered. The best candidate for further studies was firefly luciferase driven by the cap1A promoter and carried on the vector pRB473 (JffTA). The constitutive expression of bioluminescence was found to correlate with bacterial numbers in vitro. Preliminary studies involve an in vivo Galleria mellonella infection model and in vitro whole blood killing assays showed that bacterial numbers could be accurately interpolated from bioluminescence from JffTA without the need for traditional plating methodologies, therefore saving time and cost. This study shows that there is great utility of our reporter strain in infection models of S. aureus. The use of BPI in infection models could increase the efficiency of screening for potential therapeutic agents and hopefully lead to the discovery of new anti-staphylococcal agents.