Molecular Diversity of Bacteria from a Municipal Dumpsite: Implications to Public Health
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The Nelson Mandela AFrican Institution of Science and Technology NM-AIST Repository https://dspace.mm-aist.ac.tz Computational and Communication Science Engineering PhD Theses and Dissertations [CoCSE] 2016 Molecular Diversity of Bacteria from a Municipal Dumpsite: Implications to Public Health Mwaikono, Kilaza Samson The Nelson Mandela African Institution of Science and Technology http://dspace.nm-aist.ac.tz/handle/123456789/56 Provided with love from The Nelson Mandela African Institution of Science and Technology MOLECULAR DIVERSITY OF BACTERIA FROM A MUNICIPAL DUMPSITE: IMPLICATIONS TO PUBLIC HEALTH Kilaza Samson Mwaikono A Dissertation Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Life Sciences and Bioengineering of the Nelson Mandela African Institution of Science and Technology Arusha, Tanzania April, 2016 MOLECULAR DIVERSITY OF BACTERIA FROM A MUNICIPAL DUMPSITE: IMPLICATIONS TO PUBLIC HEALTH Kilaza Samson Mwaikono A Dissertation Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Life Sciences and Bioengineering of the Nelson Mandela African Institution of Science and Technology Arusha, Tanzania April, 2016 ABSTRACT Despite known risks of inappropriate disposal of solid wastes; most cities in developing countries dispose waste in open dumpsite where humans, animals and microbes interact. This study was done in Arusha municipal dumpsite, Tanzania to investigate the abundance and diversity of bacteria, compare the faecal microbiota of pigs scavenging on dumpsite and indoor reared pigs, and also determine the resistance profile of enteric bacteria from the dumpsite. Domestic wastes, solid biomedical wastes, sludge from the river near the dumpsite and faecal materials of pigs were sampled. Total DNA was extracted and the variable region four (v4) of the 16S rRNA gene was sequenced using high throughput Illumina MiSeq platform. The quality control of sequences and the statistical analyses was performed using Mothur platform. A total of 8,469,294 quality sequences were generated. The mean of bacterial species per sample was 8,243. Diversity was high with an average InvSimpson index of 44.2. Thirty-five bacterial phyla dominated by Firmicutes (38%), Proteobacteria (35%), Bacteroidetes (13%) and Actinobacteria (3%) were found. Overall, 76,862 operational taxonomic units (OTUs) dominated by Acinetobacter (12.1%), Clostridium sensu stricto (4.8%), Proteinclasticum and Lactobacillus (each 3.4%), Enterococcus (2.9%) and Escherichia/Shigella (1.7%) were revealed. There was a significant difference in faecal microbiota between scavenging and indoor reared pigs. Pathogenic genera like Brucella, Rickettsia and Listeria were exclusive to scavenging pigs. In solid biomedical waste, 36.2% of OTUs were related to dehalogenation, 11.6% degraders of aromatic hydrocarbons, 8.5% chlorophenol degradation and Atrazine metabolism 8.3%, and bacteria related to pathogens were 34%. Escherichia/ Shigella, Bacilli and Proteiniclasticum were predominant enteric bacteria. Some bacteria in scavenging pigs had 99% sequence similarity to pathogenic Escherichia furgosonii, Shigella sonnei, Enterococcus faecium and Escherichia coli O154:H4. Over 50% of the isolates were multidrug resistant. The study provides a comprehensive report on diversity of bacteria in Arusha municipal dumpsite. The high species richness shows the complexity of this man-made ecosystem, and calls for further research to assess for a link between human diseases and the dumpsite. This would provide insight into proper disposal of the waste, as well as, limit the risks to human health associated with the dumpsites. i DECLARATION I, Kilaza Samson Mwaikono do hereby declare to the Senate of the Nelson Mandela African Institution of Science and Technology that this dissertation is my own original work and that it has neither been submitted nor being concurrently submitted for degree award in any other institution. Kilaza Samson Mwaikono Name and signature of candidate Date The above declaration is confirmed Prof. Paul S Gwakisa Name and signature of supervisor Date ii COPYRIGHT This dissertation is copyright material protected under the Berne Convention, the Copyright Act of 1999 and other international and national enactments, in that behalf, on intellectual property. It must not be reproduced by any means, in full or in part, except for short extracts in fair dealing; for researcher private study, critical scholarly review or discourse with an acknowledgement, without a written permission of the Deputy Vice Chancellor for Academic, Research and Innovation, on behalf of both the author and the Nelson Mandela African Institution of Science and Technology. iii ACKNOWLEDGEMENT First and foremost, I would sincerely like to thank my supervisor, Professor Paul Gwakisa for his patience and wealth of knowledge for the entire period of this study. Countless hours have been spent by him critically reviewing this dissertation in its many stages, for which I am grateful. I would like to express my gratitude to Professor Vivek Kapur from Penn State University for his input and advice into the data analyses and linking me to bioinformatics experts at Penn State University. Both of them paved my way into the world of molecular biology. I humbly acknowledge the government of the United Republic Tanzania through COSTECH and The Nelson Mandela African Institution of Sciences and Technology for sponsoring my PhD studies including this research work, without which this would not have happened. I am very thankful to the African Biosciences Challenge Fund (ABCF) for offering me a six month research fellowship to access state of the art facilities at the BecA-ILRI hub Nairobi, Kenya, The fellowship made possible to execute part of this work. I also appreciate Dr. Robert Skilton from the same institute, for his logistic guidance, Prof. Morris Agaba for his advice into molecular techniques used in this work, Mr. Solomon Maina as my assistant researcher. It was a great time to work with them and I thank them for their advice and their friendship. I also acknowledge Mr Edson Rugaimukamu from the Department of Veterinary Microbiology and Parasitology of Sokoine University Agriculture (SUA) for his assistance in biochemical identification of bacterial isolates. Thanks to the Genome Science Centre at SUA for offering me a spacious room right from the designing stage of this research to the final write-up. I acknowledge my employer, Dar es Salaam Institute of Technology for giving me time to pursue PhD studies. I am indebted to my wife, Edith and our children and my parents for their unwavering support and patience. You may not have “understood” what I have been going through, but your encouragement has been priceless. Lastly but not the least, I acknowledge my almighty GOD for taking me through. iv DEDICATION To my family Edith R. Silayo, Sam, Mengo, Sabhogo and Blessing For patience endured when I was away for this business v TABLE OF CONTENTS ABSTRACT ..................................................................................................................................... i DECLARATION ............................................................................................................................ ii DEDICATION ................................................................................................................................ v TABLE OF CONTENTS ............................................................................................................... vi CHAPTER ONE ............................................................................................................................. 1 1.0. Background and literature review .................................................................................... 1 1.1 Global overview of municipal solid waste generation and management ......................... 1 1.2. Study of bacteria of public health importance from the environment.............................. 4 1.2.1. Conventional approaches to study bacteria............................................................... 4 1.2.2. Application of metagenomics to study bacteria ........................................................ 4 1.3. Study justification ............................................................................................................ 5 1.4. Research question ............................................................................................................. 5 1.5. Research Objectives ......................................................................................................... 5 1.6. Significance of the study .................................................................................................. 5 CHAPTER TWO ............................................................................................................................ 6 High-throughput sequencing of 16S rRNA gene reveals substantial bacterial diversity in Arusha municipal dumpsite ........................................................................................................ 6 Abstract ....................................................................................................................................... 6 2.1 Introduction ...................................................................................................................... 7 2.2. Materials and methods ....................................................................................................