Using a polyphasic approach to investigate novel Streptomyces Spp. isolated from soil from the Great Salt Plains, Oklahoma, USA

Actinomycetes have historically provided mankind with secondary metabolites that have led to the development of the majority of our antimicrobial agents. 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. This present study isolated bacteria from 9 different soil sites of the Great Salt Plains in Oklahoma using a modified dispersion and differential centrifugation (DDC) method and plated onto media containing 3% and 10% NaCl at pH 7. DNA was extracted from all the isolates, dereplicated using BOX PCR, and the resulting fingerprints analysed using GelJ (Heras et al 2015), showing 10 distinct clades; 63 isolates were shown to be of interest. Selected isolates from these clades were subjected to bioactive screening against 6 pathogens, Bacillus cereus, Staphylococcus aureus, Methicillin resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli at varying pH and NaCl and KCl concentrations. All isolates produced halos of varying sizes depending on the pathogen, pH and salinity (NaCl or KCl). When subjected to varying percentages of NaCl and KCl at pH 7, pH 8 and pH 9 to determine tolerance levels, isolates grew better at pH 8 and 9 on NaCl, whereas all strains grew well at pH 7, 8 and 9 on KCl. This is interesting as both sodium and potassium concentrations in the soil where most of the isolates were recovered have extremely low levels (<0.2%). Presumptive identification of the isolates of interest was determined by 16S rRNA gene sequencing and phylogenetic analysis using MEGA 11 (Tamura et al 2021). Out of the twenty-four isolates tested, 16S rRNA gene sequencing found that they all resided in the genus Streptomyces, with sixteen strains forming 10 presumptively novel species within the genus Streptomyces.