Phylogenetic screening for possible novel antibiotic producing actinomycetes from rhizospheric soil samples collected from Ngaka Modiri Molema District in North West Povince, South Africa

PhD, North-West University, Mafikeng Campus, 2014

Infectious disease is the number one cause of death in developing countries, accounting for approximately half of all fatalities. Emerging and re-emerging infections are thought to be driven largely by socio-economic, environmental and ecological factors. These negative health trends call for a renewed interest in infectious disease as well as effective strategies for treatment and prevention. This work is designed to search for antibiotic(s) from actinomycetes to address the treatment of infectious diseases. In this study, 341 strains of actinomycetes were isolated from the rhizospheric soil samples collected from Ngaka Modiri Molema district in North West Province of South Africa. A combination of morphological, biochemical and physiological characteristics, sequencing of the 16S rDNA gene, and phylogenetic analysis of the nucleotide sequences determined from the I 6S rDNA gene were carried out and showed that 253 (73.4%) of the isolates belong to the genus Streptomyces and 88 (26.6%) were rare actinomycetes. According to molecular taxonomical analysis, these actinomycete isolates was dominated by Streptomyces spp., followed by Nocardia, Micromonospora, Rhodococcus, Streptosporangium, Nocardiopsis, Actinomadura, Pseudonocardia, Nonomuraea, Promicromonospora, Arthrobacter, Micrococcus, Rhodococcus, and Saccharothrix. The isolates were screened for antibacterial activity against pathogenic bacteria. This revealed that 92 (27%) out of the 341 strains showed antagonistic activity against at least five of the eleven test organisms; 21.4% of the isolates were assigned as potent Streptomyces spp, and 5.5% as potent rare actinomycetes. Polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) are biosynthetic systems involved in the synthesis of a large number of important biologically active compounds produced by microorganisms, especially actinomycetes. In order to assess the occurrence of these biosynthetic systems in these actinomycete isolates, degenerative PCR primers targeted to specifically amplify PKS-I, PKS-II and NRPS genes from actinomycetes were used. Sixteen isolates ( 4.6%) were identified to have PKS-1 gene positive strains, while the figures were 15.2% for PKS-II and 26% for NRPS gene. The amplification of the genes from some of the actinomycete isolates is an indication of their potential as antibiotic producers. Through the screening, it was found that Streptomyces have higher prevalence of PKS-I, PKS-II and NRPS genes compared to others genera. Phylogenetic analysis of the nucleotide sequences from the amplified biosynthetic genes confirmed that the isolates formed close phylogenetic relationship with known antibiotic producers. Three isolates (NWU 14, NWU49 and NWU9 I) were selected for fermentation optimization and partial characterization according to their phylogenetic diversity, antimicrobial activities and secondary-metabolite biosynthesis genes. For optimizing the bioactive secondary metabolite production from the 3 isolates, experiments with the supplementation of nutrients were conducted based on the method of one-factor-at-a-time. The chemical and physical parameters affecting the production of bioactive secondary metabolite were optimized. The result showed that the most effective carbon source was glucose and that the best sources of nitrogen were oatmeal and yeast extract. The results of this study showed that the temperature, pH, inoculum size, fermentation period and the culture medium directly influenced the production of bioactive secondary metabolites (antibiotics). Each isolate behaved differently, requiring specific conditions for the production of secondary metabolites. The results obtained allow an efficient production of bioactive secondary metabolites by the isolates of actinomycete used in this study. To investigate the bioactive secondary metabolites of actinomycete isolates in order to find novel compounds or effective components, the resultant crude extracts after fermentation fractions collected were determined. The ethyl acetate, petroleum ether, benzene and nhexane fractions of the crude extract from NWU91 , ethyl acetate fractions of NWU49 and NWU14 were found to be active and were subjected to Gas Chromatography-Mass Spectrometry analysis (GC-MS) to establish the chemical components of these active fractions. The bioactive compounds identified include furosemide, phellopterin, 4,5- dihydroxyanthraquinone-2-carboxylic acid and milbemycin B. The compound 4,5- dihydroxyanthraquinone-2-carboxylic acid produced by NWU91 was not reported earlier from actinomycete. This study demonstrates the significance of actinomycetes in the rhizosphere and their potential for producing biologically active compounds and novel material for genetic manipulation or combinatorial biosynthesis. These bioactive secondary metabolites have application in human medicine and agriculture.

Doctoral