Analysis of Actinobacterial Biodiversity in Marine Sediment from Gericke's Point (South Africa) and Screening of Isolates for Novel Antimycobacterial Compounds

Master Thesis


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Thirty-three (33) presumptive actinobacterial strains were isolated using traditional culturebased techniques from sediment taken from marine habitats (a subtidal zone, a rock pool and a beach area) at Gericke's Point (Garden Route National Park, Sedgefield, South Africa). Twenty-seven (27) of the 33 presumptive actinobacterial isolates were identified to the genus level: 26 Streptomyces strains and one Nocardia strain. The partial 16S-rRNA gene sequences obtained for each confirmed actinobacterial isolate were used to determine their phylogenetic positions within their respective genera. Further investigation of specific isolates was done utilising the gyrB gene to determine whether these isolates are clones. Metagenomic data generated from next-generation sequencing of 16S-rRNA amplicons were used to reveal the actinobacterial biodiversity of the Gericke's Point sediment that was not seen in the culture-dependent part of this study. A total of 1 541 544 actinobacterial partial 16S-rRNA gene sequences were identified using the SILVA 16S-rRNA gene database. Actinobacteria that could not be assigned to a class or order made up ~41% of the total actinobacterial strains found in the Gericke's Point sediment samples. The rest of the identified actinobacterial strains belonged to the orders Candidatus Microtrichales (~45%), Candidatus Actinomarinales (~9%), Propionibacteriales (~3%) and other actinobacterial orders that each made up less than one percent (<1%) of the actinobacterial strains found in Gericke's Point. The other actinobacterial orders include Bifidobacteriales, Euzebyales, Frankiales, Geodermatophilales, Micrococcales, Micromonosporales, Mycobacteriales, Pseudonocardiales, Streptomycetales and Streptosporangiales. This is one of the first detections of Frankiales strains in a marine environment. The majority (99%) of actinobacterial strains identified at Gericke's Point could not be assigned to a known genus. This represents an abundance of novel actinobacterial diversity that has yet to be revealed. Multidrug-resistance in Mycobacterium tuberculosis is a global threat to public health which has increased the need for new antibiotics to treat tuberculosis. In this study, all confirmed actinobacterial isolates and two presumptive actinobacterial isolates (29 strains in total) were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard agar overlay method. To investigate their spectrum of antibiotic activity, all isolates were also screened for activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Twenty-one (21) isolates (20 Streptomyces strains and one unidentified strain) displayed strong to very strong antimycobacterial activity (defined as a zone of growth inhibition of over 2000 mm2 ). In addition, two Streptomyces strains displayed strong to very strong activity against S. aureus ATCC 25923. Compounds that displayed strong antimycobacterial activity were analysed using High Performance Liquid Chromatography-Mass Spectrometry and resulting mass spectra were compared to those of known compounds within the Global Natural Products Social Molecular Networking (GNPS) database. Eighteen (18) strains produced compounds with no matches in the GNPS database indicating these compounds could be novel. One strain produced a potential analogue of abyssomicin L (a rare antibiotic). Overall, the results obtained in this study emphasize the potential of marine environments as a source of novel actinobacteria and novel bioactive compounds.