Utilising metagenomics to investigate presumptive novel actinomycete spp isolated from the Great Salt Plains, Oklahoma

Actinomycetes have historically provided mankind with secondary metabolites that have led to the development of the majority of our antimicrobial agents, with Rifampicin and Streptomycin to name a few. The increased requirement for the discovery of new therapeutic and commercially significant antimicrobial agents has stimulated the need for the selective isolation of novel actinomycetes. In the present study, isolates obtained from 9 soil samples recovered from the Great Salt Plains via modified selective isolation were subjected to repetitive sequence-based polymerase chain reaction (BOX-PCR) to dereplicate and group isolates according to strain differentiation. Out of the 654 isolates that form 10 distinct clades 58 isolates were shown to be of interest. Salt and pH tolerance of the isolates were tested at pH 7, pH 8 and pH 9 with KCl and NaCl gradients at 1% increments (0 to 6 %(w/v)). Isolates grew better at pH 8 and 9 on NaCl, whereas all strains grew well at pH 7, 8 and 9 on KCl. Where diffusible pigments were seen, strains were screened for bioactive compounds against known pathogens using a modified agar plug diffusion assay. Zones of inhibition were observed, across all pH and salinity levels with antimicrobial activity recorded against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). All isolates produced halos of varying sizes depending on the pathogen, pH and salinity (NaCl or KCl). The 58 dereplicated isolates were subjected to 16S rRNA gene sequencing to ascertain their identity and novelty within the taxonomic family, Actinomycetales, with the majority residing within the genus Streptomyces with 18 of the 53 Streptomyces isolated were found to be presumptive novel isolates. Next generation sequencing showed the community profiles were different between the soils but did show come commonality.