A molecular-based analysis of the antibiotic biosynthetic potential of selected marine Micromonosporaceae Species

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2019Introduction: The discovery of the first antibiotic, penicillin, paved way for many other discoveries and developments much to the benefit of antibiotic treatment against bacterial infections. As years went by, the irresponsible use of antibiotic compounds in the medical, agricultural and veterinary fields, as well as lack of robust infection control protocols in clinical spaces, led to the emergence of antibiotic tolerant and resistant microorganisms. In an attempt to arrest the crisis, the discovery of new bioactive compounds that can be developed into potent novel antibiotics has been of importance. Amongst the many sources of bioactive compounds that have been researched for decades, microorganisms of the genus Micromonospora have been well documented as producers of potent bioactive metabolites, many of which have been successfully developed into novel commercial antibiotics. There is, however, a dearth of research information concerning the possibility of novel bioactive metabolites isolated from Micromonospora species from South Africa. Therefore, this study aimed to investigate the antibiotic biosynthetic potential of selected marine Micromonospora species isolated from the Algoa Bay region in Port Elizabeth, South Africa. Method: A total of 30 Micromonospora strains isolated from Algoa Bay region, Port Elizabeth in South Africa were provided as frozen stock cultures at the Cape Peninsula University of Technology’s Biocatalysis and Technical Biology (BTB) research unit. The strains were first cultured on SGG and 172 F solid and liquid media, with and without artificial sea water (ASW). The Gram stain was performed to ensure purity of strains and to evaluate microscopic morphology before extracting DNA. Multi-Locus Sequence Analysis (MLSA) of the rpoB and gyrB housekeeping genes was performed as well as 16S rRNA gene analysis. Phylogenetic analysis was performed using MEGA X and phylogenetic trees were constructed to this effect. Eight antibiotic biosynthetic gene clusters (BGCs) were screened for via PCR. Gaps in the current primer sets available for BGC screening were analysed. Genomic data for 44 Micromonospora strains was retrieved from EzBiocloud and antiSMASH and these assisted with primer designing after assessing primer-knowledge gaps. The designed primers were designed to target BGCs encoding for bacteriocins and lanthipeptides and were tested on five selected Micromonosporaceae strains. The antibacterial activity of the top five strains was also investigated using overlay studies on solid media cultures and bioautography studies in liquid media cultures. In addition, the efficacy of antibiotic extraction was tested through the use of five different antibiotic extraction techniques. Results: Our results demonstrated that all the strains under study were viable Micromonospora species. Phylogenetic analysis of the five strains chosen for further analysis identified their closest related validly published type strain as Micromonospora aurantiaca ATCC 27029. Furthermore, one of the eight BGCs that were screened for, the Type II PKS BGC, was positive in 28 out of the 30 strains. Genomic information of the genus Micromonospora was retrieved from antiSMASH which assisted in assessing gaps in current primer knowledge. Ultimately this led to the design of new primers to target bacteriocin and lanthipeptide BGCs. Four of the five strains tested gave a positive PCR result, albeit with multiple bands. The multiple bands on the agarose gel signified non-specificity in the binding capacity of the designed bacteriocin BGC primers hence there was no exclusive and convincing evidence of existence of this gene cluster. Negative PCR results were observed for the Lanthipeptide primer set. Antibacterial activity analysis on solid and in liquid culture media proved that the five selected strains produced bioactive compounds that were active against Gram-positive (Bacillus cereus ATCC 10876) and Gram-negative (Escherichia coli ATCC 25922) pathogens as well as yeast (Candida albicans ATCC 24433). Conclusion: The Micromonosporaceae species that were under investigation in this study show great potential as sources of bioactive metabolites with broad spectrum antibacterial activity as well as antifungal activity. These should be considered as suitable candidates for whole genome sequencing as well as comparative genome sequence analysis for greater insights into the M. aurantiaca group