Investigating bacterial diversity employing a bTEFAP approach resulting in isolation and characterization of an actinomycete library from Atlantic Canada for antimicrobial and anticancer drug discovery

Members of the order actinomycetales a re known to produce over 10,000 bioactive compounds, the majority of which are produced by Streptomyces species (70%). Due to the commercial success of many of these metabolites as human and animal therapeutics Streptomyces continue to be a valuable resource when bioprospecting for novel bioactive metabolites with potential commercial applications. The isolation of novel Streptomyces species from hitherto unexplored habitats is essential to fuel the continued search for novel metabolites as it is generally accepted that biological diversity directly translates to chemical diversity when searching for novel natural products (NPs).;454-massively parallel pyrosequencing of small subunit rRNA (16S) amplicons was employed to assess overall bacterial diversity in 24 sediment samples. This is the first systematic drug discovery-orientated examination of bacterial diversity using this innovative culture independent technique in Canada. In total over 174, 000 sequences were obtained from these analyses. Interestingly, all sediments exhibited significant bacterial diversity; however, Atlantic Canadian sediments exhibited greater Actinobacterial diversity in comparison to sediments collected from San Salvador Island, thus highlighting the potential of Atlantic Canadian sediments as an exciting source of novel Actinobacteria. To exploit the metabolic potential of Actinobacteria present in the Atlantic Canadian sediments a selective isolation strategy was applied to 16 sediment samples from the Bay of Fundy, New Brunswick (n=8) and Bonne Bay, Newfoundland (n=8). A combination of three sediment pretreatments and five selective media led to the isolation of 826 actinomycetes. Small subunit rRNA gene sequencing revealed that the majority of cultured isolates belonged to the genus Streptomyces. As a first step to evaluate the potential of this library to produce novel natural products a small subset (10) were fermented in a range of media and the resulting fermentation extracts screened for anticancer activity against a primary breast cancer cell line (HMT390958; AvantiCell Science, Ayr, Scotland) and tested for antimicrobial activity against seven pathogens. Extracts exhibited exceptional cytotoxicity when compared to assay controls and a range of antimicrobial activity against all pathogens tested. Fermentation extracts of RKKD0868, an Actinoalloteichus sp. isolated from Newfoundland sediment exhibited the highest anticancer activity and extensive antimicrobial activity. Initial chemical investigation of these extracts suggests that the metabolites produced by RKKDO868 are potentially novel.;The library of actinomycetes generated from this research is a rich resource for future chemical investigations, and the discovery of bioactive NPs. The exceptional bioactivity of select actinomycetes has been exemplified by the extensive antimicrobial and high anticancer activity observed from preliminary investigations. The establishment of such a diverse collection of Streptomyces from a relat ively small number of sediments has demonstrated the potential of Atlantic Canada as a resource for filamentous actinomycetes for future drug discovery.