Development of Methods for the Detection of Antibiotic Adjuvants

Abstract

The problem of antibiotic resistance is far-reaching and continuing to increase, necessitating the development of solutions to combat this threat. The lifespans of existing antibiotics have been extended through their combination with adjuvants to resensitize resistant bacteria, however, the emergence of newer resistance mechanisms is rendering these combinations ineffective. With few antibiotics in development and resistance towards existing antibiotics increasing, new antibiotics are desperately needed. The majority of antibiotics currently in use originate from microbial sources which are often seen as a used-up source of biodiscovery. Through a collaboration with the Crown Research Institute Manaaki Whenua – Landcare Research we have access to approximately 2000 endemic NZ fungi which have largely not been investigated for their antimicrobial properties. These fungi may potentially hold a wealth of undiscovered novel antibiotic compounds. Reviving the Waksman platform, I developed and validated a method for screening for antibiotics and antibiotic adjuvants which enhance the activity of beta-lactam antibiotics. Using the bacterium Streptomyces clavuligerus from which clavulanic acid, arguably the most successful antibiotic adjuvant in use, was discovered, I demonstrated that these methods can be used for the detection of similar compounds. With these methods, I screened fungal isolates for antibiotic and antibiotic-potentiating activity against two major groups of pathogenic bacteria: extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA). While I did not observe any major antibiotic-potentiating activity, of the 66 fungal isolates I screened, 52 were able to inhibit the growth of at least one of these pathogenic bacterial strains. Extracts were made from 19 of these fungi and tested to obtain minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) against my bacterial pathogens. Extracts from several fungal species, including Neofusicoccum australe, Ilyonectria torresensis A and Penicillium expansum demonstrated potent inhibition of the growth of E. coli and S. aureus strains at low concentrations. Analysis of 1H nuclear magnetic resonance (NMR) spectra from active extracts revealed several well-known antibiotic compounds, including curvularin, brefeldin A and taiwapyrone. My findings demonstrate the potential of this assay for the discovery of novel antibiotics against problematic bacterial pathogens.