Effect of co-culture on the activity profile of marine actinobacteria

Abstract

Over the past few years, there has been a dramatic increase in the number of antimicrobialresistant pathogens. However, this increase has not been matched with the discovery of new antibiotics for the treatment of diseases or infections caused by these antimicrobial-resistant pathogens. In order to address this, marine actinobacterial diversity was explored as a potential source of novel antibiotics. Metabarcoding analysis of sediment samples collected at three subsites, designated as ‘Dry’, ‘Ocean’ and ‘Rocky’, from a tidal pool located within the Table Mountain National Park (TMNP2), reflected a core actinobiome consisting of unculturable and unidentified actinobacteria. In order to access this untapped diversity, actinobacteria were isolated from sediment samples. Initial bioactivity screening of twenty actinobacterial isolates was conducted against a set of ESKAPE pathogens: Acinetobacter baumannii ATCC 19606 and ATCC BAA-1605, Enterococcus faecalis ATCC 29212 and ATCC 51299, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC BAA-700603, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213 and ATCC 33591. Out of the twenty actinobacterial isolates, eight isolates displayed strong activity against ESKAPE pathogens when cross-streaked on M19, M19 with Red Sea salts (M19+), International Streptomyces Project medium 2 (ISP2), and ISP2 with Red Sea salts (ISP2+) media. These isolates were then identified by 16S rRNA sequencing, and it was found that all eight belong to the genus Streptomyces. The eight actinobacterial isolates were also cultured as mono-cultures in liquid media to determine their capacity to produce bioactive compounds. A time-based study was performed to determine at which time point (day 1, day 3, day 5, or day 10) the best activity was observed. Extracts of the mono-cultures were prepared, and filter disc assays were performed to determine the activity of the extracts against the ten ESKAPE pathogens. Only two strains, R-30 and R-35, exhibited strong activities against S. aureus ATCC 29213 and A. baumannii ATCC 19606 when cultured in ISP2. These two strains also showed activity against all ten test strains on solid media. Strains R-30 and R-35 were therefore chosen for further study, where they were indirectly cocultured with each other (separated by a membrane filter to ensure non-contact) and directly co-cultured with ‘alive’ and ‘dead’ Mycobacterium aurum A+. The strains were first grown separately to ensure growth and thereafter added together in one flask to determine if there was a change in the bioactivity of R-30 and R-35. When R-30 and R-35 were co-cultured together, both isolates showed an increase in activity on day 1 (T1). When co-cultured with ‘alive’ and ‘dead’ M. aurum A+, only R-35 showed good activity against four test strains. When co-cultured with different concentrations of M. aurum A+, the extracts prepared from R-35 showed very good activity against eight of the test strains. Metabolomic analyses via tandem mass spectrometry (MS/MS) fingerprinting were performed on the extracts prepared from the mono-cultures and the co-cultures. It was observed that antimycin was one of the main secondary metabolites present in the extracts prepared from the indirect co-cultures of R-30 and R-35. MS/MS fingerprinting of extracts prepared from the direct co-cultures of R-35 and M. aurum A+, showed the presence of desferrioxamines and N-acetyltyramine. Due to the promising results obtained, whole genome sequencing of strain R-35 was performed to identify potential biosynthetic gene clusters involved in secondary metabolite production under different co-culture conditions. The ability of strain R-35 to produce antimycin and desferrioxamine was confirmed by the antiSMASH results, which indicated the presence of biosynthetic gene clusters that encode for antimycin, desferrioxamine E and desferrioxamine D. The antiSMASH report also indicated the presence of naphthyridinomycin, curamycin, aurantimycin, albaflavenone, himastatin, nigericin, and desferrioxamine B biosynthetic gene clusters, but these compounds were not identified in the MS/MS data analyses. The molecular networking analysis of the metabolomics data also confirmed the presence of multiple unknown secondary metabolites. The outcome of this study, therefore, confirms that secondary metabolite production is enhanced when two marine actinobacterial isolates are co-cultured together or when they are co-cultured with other bacteria, such as the mycolic acid-producing M. aurum A+. Future studies will be aimed at isolating and identifying the compounds responsible for the activity seen in the co-culture experiments and determining if the activity seen is caused by one individual compound class or if it is a group of compounds working together to produce the activity.