Metagenomic guided isolation of sporoactinomycetes from soil and screening for antimycobacterial activity

Hewat, Samuel

Metagenomic guided isolation of sporoactinomycetes from soil and screening for antimycobacterial activity

Thesis / Dissertation

2025

Publisher

University of Cape Town

Department

Department of Molecular and Cell Biology

Faculty

Faculty of Science

Abstract

Metagenomic analysis was used to analyse the actinobacterial diversity in the Kramer (K) and Tennis Court (TC) soil samples collected at the University of Cape Town (UCT). Fifty (50) and 52 16S-rRNA gene sequences were amplified for the K and TC samples, respectively, from environmental DNA (eDNA) using actinobacterium-specific primers. EzBioCloud analyses of the 16S-rRNA gene sequences showed a total of 10 orders present in each of the K and TC samples with the orders Acidimicrobiales, Pseudonocardiales and Propionibacteriales being the most abundant in both samples. Rarefaction curves showed that the full actinobacterial diversity of the samples was not captured. Using selective media chosen based on the results of the metagenomic analyses, a total of 63 presumptive actinobacterial strains was isolated from the two soil samples, but 21 of these strains were discarded. Of the 42 actinobacteria studied, partial 16S-rRNA gene sequences were analysed for 40 of the 42 presumptive actinobacterial strains: 23 strains from the K sample and 17 strains from the TC sample. The 40 strains were identified to the genus level revealing 38 Streptomyces strains and two non-Streptomyces strains: Actinomadura strain 1KK1 and Nonomuraea strain 7TK1. The phylogenetic position of each identified strain was investigated by generating phylogenetic trees. Thirty (30) of the 38 Streptomyces strains identified were potential-clones, which were initially divided into three potential-clone groups. Clone analysis carried out on 23 of the 30 Streptomyces potential-clone strains revealed that the three potential-clone groups divided into five clone groups and eight distinctive Streptomyces strains based on their partial gyrB gene sequences. Forty (40) of the 42 isolates studied were screened for antimycobacterial activity against Mycobacterium aurum strain A+ on four different media. A total of 35 strain-medium combinations (from 25 different strains) showed strong or very strong antimycobacterial activity. These 35 strain-medium combinations were tested for activity against Escherichia coli strain ATCC 25922 and Staphylococcus aureus strain ATCC 25923 to investigate the antibiotic spectrum of each active actinobacterial strain. Crude extracts of the most active actinobacterial strains were subjected to Global Natural Products Social (GNPS) molecular networking analyses to assess if the compounds produced are known compounds. None of the active actinobacterial strains showed activity against E. coli ATCC 25922, with just two strains (Actinomadura strain 1KK1 on R2A medium and Streptomyces strain 49TA3 on 7H9 and R2A media) showing strong or very strong activity against S. aureus ATCC 25923. Crude extracts of 26 of the 35 strain-medium combinations tested returned no matches on the GNPS database, with nine combinations having matches to know compounds. These no-match combinations could potentially be novel antibiotics with activity against Mycobacterium tuberculosis.

Keywords

Metagenomic, sporoactinomycetes

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