CHARACTERIZATION OF MYCOBACTERIA SPP. AND ANTIMYCOBACTERIAL ACTIVITIES OF DERIVED COMPOUNDS FROM FAMILY

by

PRUDENCE NGALULA KAYOKA

Submitted in accordance with the requirements

for the degree of

DOCTOR OF PHILOSOPHY

in the subject

ENVIRONMENTAL SCIENCE

at the

UNIVERSITY OF SOUTH

SUPERVISOR: Prof L J MCGAW

SUPERVISORS: Prof J N ELOFF AND PROF C L OBI

NOVEMBER 2016

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DECLARATION

Name: Dr P N PRUDENCE KAYOKA

Student number: 44414021

Degree: PhD in Environmental Science

Exact wording of the title of the thesis as appearing on the copies submitted for examination:

CHARACTERIZATION OF MYCOBACTERIA SPP. AND ANTIMYCOBACTERIAL ACTIVITIES OF DERIVED COMPOUNDS FROM ANACARDIACEAE FAMILY

I declare that the above thesis is my own work and that all the sources that I have used or quoted have been indicated and acknowledged by means of complete references.

Signed 8/02/2017

______

SIGNATURE DATE

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DEDICATION

This research is dedicated to all sufferers. I also dedicate this work to my late father, Raphael Kabongo for teaching me resilience and work ethics, to my mother, Therese Kabongo for teaching me the values that are leading my life and to my son, Axel Kayoka, and daughter, Benissa Kayoka, my best friends; you have been part of my life’s goals motivation from the time you were born up to date. May this work inspire you to persevere in reaching your goals and live your dreams to the mutual benefit of communities.

Happy is the man who finds wisdom and the man who gains understanding: Proverbs 3:13.

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ACKNOWLEDGEMENTS

There is no stand-alone individual in the universe as we are the result of so many interactions at each stage of our lives. I will not be able to remember each factor that has contributed to this PhD. Nevertheless, I wish to express my appreciation to the following individuals and/organizations:

Promoter, Prof L.J. McGaw: for your unconditional availability, your guidance, technical inputs and overall support throughout the course of this study.

Co-promoter, Prof J.N. Eloff: for the advice, technical input, mentoring and guidance. Your open door policy and guidance step by step through some of the experiments were very helpful in the understanding of the assays.

Co-promoter, Prof C.L. Obi: for believing in me and my research proposal from the initial stage and encouraging me to pursue my degree despite all the difficulties encountered and for your support, guidance and technical inputs.

Dr Matt Ekron: for organizing sample collection from infected herds.

Drs Johann Kotze, Jacoba Wessels and Melinda Hansen: for providing samples.

Ms Emmerentia Mkhize and Mrs Tharien de Winnaar: for carrying out administrative work in the Phytomedicine Programme.

My fellow postgraduate students: for maintaining a team spirit and an environment conducive to learning in the Phytomedicine Programme.

Dr Ahmed Aroke Shahid: for assisting during the last round with the laborious separation of compounds.

Prof Vinesh Maharaj, Dr Mamoalosi Selepe from University of Pretoria and Dr Chris Van der Westhuyzen from CSIR: for assisting with the identification and structural elucidation of the isolated compounds.

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Mr Reckson Ramuageli and Mr William Mokgojane: for providing clean glassware and always ready to assist with relevant items when requested.

Ms Annette Venter: for making sure that work in the tissue culture laboratory ran smoothly in maintaining the tissue culture collection.

National Research Foundation (NRF) for financial support.

Department of Agriculture, Forestry and Fisheries (DAFF): for giving the authorization for my work to be conducted.

National Health Laboratory Services (NHLS): for access to their laboratory facilities and providing ATCC strains of tuberculosis H37Ra, ATCC strain of Mycobacterium avium and clinical isolates of Mycobacterium tuberculosis.

Prof Nontombi Mbelle, Ms Kathy Lindeque and Mrs Omowunmi Onwuegbuna: for assisting with all items needed to perform my work in the laboratory.

Onderstepoort Veterinary Institute: for access to laboratory facilities and providing BCG vaccine strain of .

Research Center for Zoonosis Control, Hokkaido University, Japan: for access to laboratory facilities. I am grateful to Professors Chie Nakajima and Suzuki who assisted with the gene sequencing and spoligotyping of the Mycobacterium isolates.

Tohoku University, Graduate School of Medicine, Department of Emerging Infectious Diseases, Japan: for access to the relevant laboratory facilities and financial support. I am grateful to Prof Toshio Hattori for organizing financial support during my stay in Japan and for being a reliable mentor in all activities while in Japan.

University of Pretoria: for access to laboratory facilities in the Department of Paraclinical Sciences, Phytomedicine Programme and access to my promotors.

University of : for allowing me time out to complete my studies and financial support.

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Lastly, my two best friends, Axel and Benissa Kayoka, my children, my motivators for your encouragement, support and love during this journey.

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ABSTRACT

The treatment of tuberculosis (TB) is currently a challenge due to multi- and extensively drug resistant strains of Mycobacterium tuberculosis. Mycobacterium bovis and M. tuberculosis cause clinically indistinguishable tuberculosis in humans. Both M. bovis and M. tuberculosis have been isolated from humans and animals. Plant species contain antimicrobial compounds that may lead to new anti-TB drugs. To conduct in vitro antimycobacterial assays, it is important to include current clinical isolates as new strains of bacteria might be circulating under the ongoing climate change environment. The overall goal and objectives of this study were to isolate and characterize mycobacteria species from South Africa, to test some selected plant species of the Anacardiaceae family for antimycobacterial activity using some of the newly isolated and reference strains of mycobacteria followed by cytotoxicity evaluation of the most active plant species, and finally the isolation and characterization of at least one compound from the most active and least toxic plant.

This study led to the discovery of a new isolate of Mycobacterium Avium Complex species from black wildebeest. Other non-tuberculous mycobacteria and M. bovis isolates were identified from other animal species. Five out of 15 plant species screened showed good activity against Mycobacterium species. Five antimycobacterial compounds were isolated from undulata, the most active plant species. Two out of the five compounds were identified, and one compound appears to be novel, but both compounds have been isolated for the first time from Searsia undulata. An incidental finding was the potential anticancer property of extracts of Searsia undulata.

Recommended future activities include isolation and identification of more active compounds from Searsia undulata which were visible in bioautography analysis, as well as synergy evaluation of antimycobacterial activities of the different compounds with current anti-tubercular drugs.

Key words: Characterization, Antimycobacterial, NTM mycobacteria, Black wildebeest, MDR-M. bovis, MDR-M. tuberculosis, Anacardiaceae, Searsia undulata, Betulonic acid.

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The project involved the following steps: <