The effects of NX-AS-401 on methicillin resistant Staphylococcus aureus

Antimicrobial resistance is a global health concern, with once treatable infections becoming resistant to current standard of care antimicrobials. The search for new antimicrobials has led Neem Biotech Ltd. to manufacture NX-AS-401 an ajoene containing compound derived from Allium sativuum, commonly known as garlic. The research contained within this thesis aimed to identify the effects of NX-AS-401 on Methicillin Resistant Staphylococcus aureus (MRSA), one of the most well documented and commonly isolated antimicrobial resistant bacterial pathogens. A multi-stage approach was utilised, identifying how NX-AS-401 affects planktonic growth, biofilm development and virulence factor production. In Chapters 3 and 4 initial comparison between different NX-AS-401 formulations was performed in determined that ajoene content did not alter the antimicrobial effect of NX-AS-401. EUCAST broth microdilution compared NX-AS-401 to current standard of care antibiotic and determined effective inhibitory and bactericidal concentrations as 128 µg/ml and 2048 µg/ml respectively. When NX-AS-401 was used in combination with various antibiotic classes a synergistic effect was identified and the inhibitory concentrations of both agents were reduced. The primary focus on Chapter 5 was how NX-AS-401 affected S. aureus biofilm formation. NX-AS-401 concentrations of 32 µg/ml inhibited biofilm formation and a concentration of 512 µg/ml caused disruption of pre-established biofilms. These effects were confirmed using scanning electron microscopy and confocal microscopy with live/dead staining. In gene expression studies it was determined that the effects of NX-AS-401 on S. aureus biofilms were strain dependent and a target gene was not identified. Chapter 6 demonstrated that NX-AS-401 did not alter the production of Staphylococcus aureus exo-enzyme production in vitro during phenotypic studies. In Galleria mellonella low NX-AS-401 concentrations assisted in the recovery from S. aureus in a strain dependent manner, however, high concentrations caused increased Galleria mellonella fatality. NX-AS-401 altered the ability of S. aureus cells to invade human epithelial cells but did not prevent adhesion of S. aureus to the cells. NX-AS-401 has multiple effects on S. aureus with the ability to affect both planktonic cells and biofilm structure showing promise as an antimicrobial. Its main effects are growth inhibition and biofilm disruption rather than causing bacterial cell death. These attributes and the synergistic effects between NX-AS-401 and multiple antibiotic classes, indicate NX-AS-401 has potential as a strong antimicrobial adjuvant.