Diversity and antifungal susceptibility yeast in the selected rivers in the North West Province M.E. Monapathi 23680490 Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in Environmental Sciences at the Potchefstroom Campus of the North-West University Supervisor: Prof. C.C. Bezuidenhout Co-supervisor: Mr. O.H.J. Rhode November 2014 TABLE OF CONTENTS TABLE OF CONTENTS ____________________________________________________ i ABSTRACT______________________________________________________________ iv ACKNOWLEDGEMENTS __________________________________________________ v DECLARATION __________________________________________________________ vi LIST OF FIGURES _______________________________________________________ vii LIST OF TABLES _______________________________________________________ viii CHAPTER 1 - INTRODUCTION _____________________________________________ 1 1.1 Introduction and problem statement ________________________________________ 1 1.2 Research aim _____________________________________________________________ 3 1.3 The objectives of the study were: ___________________________________________ 3 CHAPTER 2 – LITERATURE REVIEW _______________________________________ 4 2.1 State of the water resources in South Africa _________________________________ 4 2.2 State of water resources in the North West Province _________________________ 4 2.2.1 Pollution in the North West Rivers __________________________________________________ 5 2.3 Water quality ______________________________________________________________ 8 2.3.1 Temperature _____________________________________________________________________ 9 2.3.2 Nutrient enrichment ______________________________________________________________ 10 2.3.3 pH _____________________________________________________________________________ 11 2.3.4 Total Dissolved Solids (TDS) and Electrical conductivity (EC) _________________________ 12 2.3.5 Chemical oxygen demand (COD) _________________________________________________ 12 2.3.6 Microbiological quality ___________________________________________________________ 13 2.4 Selected rivers in the North West __________________________________________ 13 2.4.1 Mooi River catchment ____________________________________________________________ 13 2.4.2 The Harts River _________________________________________________________________ 14 2.5 Characteristics of yeasts __________________________________________________ 15 2.6 Yeast in water ____________________________________________________________ 15 2.7 Yeasts as water quality indicators _________________________________________ 16 2.8 Yeast infections in immune compromised people ___________________________ 17 2.9 Antifungal agents ________________________________________________________ 18 2.9.1 Mechanism of action of azoles ____________________________________________________ 20 2.9.2 Antifungal resistance ____________________________________________________________ 21 2.9.3 Mechanism of resistance to azoles ________________________________________________ 22 2.9.4 Mechanism of resistance in polyenes and allylamines _______________________________ 24 2.9.5 Mechanism of resistance to echonocandins ________________________________________ 24 2.10 Yeast pathogenicity _____________________________________________________ 24 2.10.1 Thermotolerance _______________________________________________________________ 24 i 2.10.2 Cell wall components and capsules ______________________________________________ 25 2.10.3 Adhesion ______________________________________________________________________ 25 2.10.4 Extracellular enzymes production ________________________________________________ 25 2.11 Isolation of yeasts from the water sources ________________________________ 26 2.11.1 Sampling of yeasts _____________________________________________________________ 26 2.11.2 Enumeration and isolation of yeasts ______________________________________________ 26 2.12 Determination of physico-chemical parameters ____________________________ 26 2.13 Biochemical tests used for yeast identification ____________________________ 28 2.13.1 Diazonum Blue B (DBB) test _____________________________________________________ 28 2.13.2 Disk diffusion method ___________________________________________________________ 28 2.14 Identification of yeasts __________________________________________________ 29 2.15 Summary of the literature ________________________________________________ 31 CHAPTER 3 - MATERIALS AND METHODS ________________________________ 32 3.1 Study site ________________________________________________________________ 32 3.2. Sampling and physico-chemical parameters _______________________________ 34 3.3. Isolation and enumeration of yeasts _______________________________________ 34 3.4. Characterisation and identification of yeasts _______________________________ 34 3.4.1. Diazonium blue B (DBB) _________________________________________________________ 34 3.5. Molecular identification __________________________________________________ 35 3.5.1 DNA isolation ___________________________________________________________________ 35 3.5.2 Agarose gel electrophoresis of isolated DNA _______________________________________ 35 3.5.3. DNA amplification _______________________________________________________________ 36 3.5.4 Sequencing _____________________________________________________________________ 36 3.5.5 Phylogenetic tree ________________________________________________________________ 36 3.6 Haemolysin production on blood agar _____________________________________ 36 3.7 Extracellular enzyme production __________________________________________ 37 3.7.1 Proteinase ______________________________________________________________________ 37 3.7.2 Lipase _________________________________________________________________________ 37 3.7.3 Dnase _________________________________________________________________________ 37 3.8 Antifungal susceptibility tests _____________________________________________ 38 3.9 Statistics ________________________________________________________________ 38 CHAPTER 4 - RESULTS __________________________________________________ 39 4.1 Introduction______________________________________________________________ 39 4.2 Water samples collected in Mooi River _____________________________________ 39 4.2.1 Physico-chemical parameters _____________________________________________________ 39 4.2.2 Colony counts __________________________________________________________________ 41 4.3 Water samples collected in Harts River 2014 _______________________________ 43 4.3.1 Physico-chemical parameters _____________________________________________________ 43 4.3.2 Colony counts __________________________________________________________________ 44 4.4 Diazonium Blue B (DBB) testing ___________________________________________ 45 4.5 Haemolysin production ___________________________________________________ 45 ii 4.6 Extracellular enzyme production __________________________________________ 45 4.7 Molecular identification of yeast isolates ___________________________________ 46 4.7.1 DNA Isolation of pure yeast cultures _______________________________________________ 46 4.7.2 DNA amplification _______________________________________________________________ 46 4.7.3 Sequencing _____________________________________________________________________ 47 4.7.4 Phylogenetic tree ________________________________________________________________ 50 4.8 Distribution of yeasts amongst the sampling site ___________________________ 52 4.9 Correlation of physico-chemical parameters and yeast in the Mooi and Harts Rivers ______________________________________________________________________ 55 4.10 Antifungal susceptibility testing __________________________________________ 57 4.11 Summary of the results __________________________________________________ 70 CHAPTER 5 -DISCUSSION ____________________________________________________ 72 5.1 Introduction______________________________________________________________ 72 5.2 Physico-chemical parameters _____________________________________________ 72 5.2.1 Temperature ____________________________________________________________________ 72 5.2.2 pH _____________________________________________________________________________ 73 5.2.3 Total Dissolved Solids ___________________________________________________________ 73 5.2.4 Nitrates ________________________________________________________________________ 74 5.2.5 Phosphates _____________________________________________________________________ 75 5.3 Yeast levels ______________________________________________________________ 76 5.4 DBB and extracellular enzyme test ________________________________________ 77 5.4 Molecular identification and distribution of yeasts __________________________ 78 5.5 Antifungal susceptibility tests _____________________________________________ 81 CHAPTER 6 - CONCLUSION AND RECOMMENDATIONS ____________________ 85 6.1 Conclusion ______________________________________________________________ 85 6.1.1 Physico-chemical parameters _____________________________________________________ 85 6.1.2 Yeast levels ____________________________________________________________________ 85 6.1.3 Yeasts isolation at 37°C __________________________________________________________ 86 6.1.4 Identification of yeast isolates using biochemical tests and 26S rRNA gene sequencing _ 86 6.1.5 Antifungal susceptibility tests _____________________________________________________ 87 6.2 Recommendations _______________________________________________________