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Isolation of Bacteriophages Obtained from Limestones Cave Soils And

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Isolation of Bacteriophages Obtained from Limestones Cave Soils And ISOLATION OF BACTERIOPHAGES FROM LIMESTONE CAVE SOILS AND EVALUATION OF THEIR POTENTIAL APPLICATION AS BIOCONTROL AGENTS OF PSEUDOMONAS AERUGINOSA By HASINA MOHAMMED MKWATA A thesis presented in fulfillment of the requirements for the degree of Master of Science (Research) School of Chemical Engineering and Science, Faculty of Engineering, Computing and Science SWINBURNE UNIVERSITY OF TECHNOLOGY 2018 ABSTRACT The emergence and frequent occurrence of multidrug-resistant and extremely drug- resistant bacteria have raised a major concern because infections caused by these bacteria are often associated with high mortality rates, prolonged hospitalization, and high treatment costs. This situation is predicted to worsen in the future due to a massive decline in the development of new antibiotics in recent years. Bacteriophages and their derivatives have long been exploited as powerful and promising alternative antibacterial agents in phage therapy and biocontrol applications. Limestone caves remain relatively unexplored as a source of novel lytic bacteriophages compared with other environments, despite being one of the most propitious sources for the discovery of novel antimicrobial compounds. This research presents, for the first time, the screening and isolation of lytic bacteriophages targeting different pathogenic bacteria from limestone caves of Sarawak, and evaluation of their potential application as biological disinfectants to control P. aeruginosa infections. A total of 33 lytic bacteriophages were isolated from samples obtained from FCNR and WCNR targeting bacterial strains Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Escherichia coli and Vibrio parahaemolyticus, using enrichment culture method. Phage amplification was performed, and lysates were obtained, and spot tested on lawns of various bacteria strains to assess their lysis spectrum. The result revealed that P. aeruginosa and V. parahaemolyticus infecting phage isolates showed the broadest host range among all the phage isolates. An interesting feature observed, was the ability of some phage isolates to exhibit trans-subdomain infectivity between gram positive and gram-negative bacterial hosts. Phage bacteriolytic activity was investigated in an in-vitro co-culture assay with P. aeruginosa PAO1 strain using five multiplicity of infection (MOI) ratios. Viable P. aeruginosa PAO1 cells that survived phage infection were enumerated at 6 hrs and 24 hrs post-infection and the counts were compared with those of untreated control. Bacteriophages FCPA3 (MOI 105), WCSS4PA (MOI 105) and Cocktail (MOI 104) showed the highest bacterial inactivation among all the tested phages at the end of 6 hrs of incubation. The highest bacterial log10 CFU/mL reduction was 11.82 equivalent to 100% reduction in bacteria observed in cultures treated with phage cocktail (Cocktail, MOI 104) at the end of 6 hrs of i incubation. Similarly, surviving bacterial counts assessed 24 hrs post-infection showed 4 2 that phages WCSS4PA (MOI 10 ) and Cocktail (MOI 10 ) had the highest bacterial log10 CFU/mL reduction of 10.5 and 10.86 respectively, equivalent to 100% reduction in bacterial cells. Some of the phages did not show any reduction in bacterial cells at 6 hrs and 24 hrs post-infection, instead the cells rebounded and surpassed those of the untreated control. Assessment of phage’s ability to be utilized as a biological disinfectant was performed on P. aeruginosa PAO1 contaminated sand samples. The sand samples served as a simulant of any environmental surface exposed to contamination with P. aeruginosa. Surviving bacterial cells following treatment with bacteriophages FCPA3, WCSS4PA and Cocktail were enumerated at 0 hr, 6 hrs, 24 hrs and 48 hrs post-treatment, and the counts were compared with those of untreated control. Over 99% reduction in bacterial cells were observed on all phage treated sand samples harvested at 6 hrs post- treatment. No reduction in bacterial cells was observed in sand samples harvested at 24 hrs and 48 hrs post-treatment despite phage recharge, instead, the cells rebounded and surpassed those of untreated controls. The ‘Bacterial rebound’ phenomenon mentioned in this study indicates that the bacteria evolved resistance against the infecting phage. This study suggests that P. aeruginosa bacteriophages obtained from Sarawak limestone caves (FCNR and WCNR) may present potentials to be developed into biological disinfectants to control P. aeruginosa infections, upon further exploration. ii ACKNOWLEDGEMENT Foremost, I am deeply grateful to my coordinating supervisor, Assoc. Prof Dr Peter Morin Nissom for his valuable advice, critics, challenges, encouragement, and directions throughout my research study. Special thanks goes to my co-supervisor, Dr Lee Tung Tan for accepting to co-supervise my MSc research project. I extend my appreciation to Sarawak Biodiversity Centre (SBC) and Sarawak Forestry Department (SFD) for issuing the permits (SBC-RA-0110-PMN) which enabled me to have access to soil samples from Fairy Cave and Wind Cave Nature Reserves, located in Bau, Kuching Division, Sarawak, Malaysia. I also wish to thank the science laboratory officers, Nurul Arina Salleh, Cinderella Sio and Marciana Jane Richard, as well as Chua JiaNi, biosafety officer, for their assistance with regards to the provision of the materials and apparatus throughout the course of my research. My heartfelt thanks extend to my boyfriend, Armstrong Ighodalo Omoregie for his unwavering support and encouragement throughout my research journey. I am sincerely grateful to my parents, Mohammed Mkwata and Fatma Mwenda for their unconditional love, care, advice and encouragement throughout my studies. I am profoundly thankful to my Dad, for his financial support used to partially fund my research. I am grateful to Almighty God, whose blessings have enabled me to successfully accomplish my research study. iii DECLARATION I hereby declare that this research entitled “Isolation of bacteriophages obtained from limestones cave soils and evaluation of their potential application as biocontrol agents of Pseudomonas aeruginosa” is original and contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of the examinable outcome; to the best of my knowledge contains no material previously published or written by another person except where due reference is made in the text of the examinable outcome; and where work is based on joint research or publications, discloses the relative contributions of the respective workers or authors. (HASINA MOHAMMED MKWATA) DATE: August 15, 2018 In my capacity as the Principal Coordinating Supervisor of the candidate’s thesis, I hereby certify that the above statements are true to the best of my knowledge. (ASSOCIATE PROFESSOR DR. PETER MORIN NISSOM) DATE: August 15, 2018 iv PUBLICATIONS Mkwata, HM, Tan, LT, & Nissom, PM, 2016, ‘Assessing the Diversity of Viruses in Soils Obtained from Limestone Caves,’ 15th International Peat Congress (IPC 2016), International Peatland Society, pp. 156-160. Mkwata, H.M., Omoregie, A. I., Musa, I. B., Suyuh, J., Yee, P.H., Sing, L.W., Tan, L. T. & Nissom, P. M. (2018), “A Laboratory Practicum on Screening for Lytic Bacteriophages from Soil Samples”, Transactions on Science and Technology, (6 pages), ISSN: 2289-8786, published by e-VIBS, Faculty of Science and Natural Resources, Universiti Malaysia Sabah. (Accepted). Omoregie, A. I., Siah, J., Pei, B. C. S., Yie, S. P. J., Weissmann, L. S., Enn, T. G., Rafi, R., Zoe, T. H. Y., Mkwata, H. M., Sio, C. A. & Nissom, P. M. (2018), “Integrating Biotechnology into Geotechnical Engineering: A Laboratory Exercise”, Transactions on Science and Technology, 13 pages), Volume 5, No. 2, ISSN: 2289-8786, published by e-VIBS, Faculty of Science and Natural Resources, Universiti Malaysia Sabah. CONFERENCE AND PRESENTATIONS Poster presenter, Assessing the Diversity of Viruses in Soils Obtained from Limestone Caves, 15th International Peat Congress (IPC 2016), International Peatland Society, 15- 19 August 2016, Kuching, Sarawak, Malaysia. Oral presenter, Phage Therapy: The forgotten cure, Three Minute Thesis (3MT) Competition, 17 June 2015, Swinburne University of Technology, Sarawak campus, Kuching, Sarawak, Malaysia. Participant, International Congress of the Malaysian Society for Microbiology, 7-10 December 2015, Batu Ferringhi, Penang, Malaysia. Participant, Asian Congress on Biotechnology, 15-19 November 2015, Kuala Lumpur, Selangor, Malaysia. v TABLE OF CONTENTS CONTENTS PAGE ABSTRACT ................................................................................................................. i ACKNOWLEDGEMENT ............................................................................................. iii DECLARATION ......................................................................................................... iv PUBLICATIONS ......................................................................................................... v CONFERENCE AND PRESENTATIONS ......................................................................... v TABLE OF CONTENTS ............................................................................................... vi LIST OF FIGURES...................................................................................................... ix LIST OF TABLES ......................................................................................................
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