SINGLE BASE EXTENSION REACTIONS AND THEIR APPLICATIONS IN ENVIRONMENTAL MICROBIOLOGICAL STUDIES HONG PEI YING NATIONAL UNIVERSITY OF SINGAPORE 2009 SINGLE BASE EXTENSION REACTIONS AND THEIR APPLICATIONS IN ENVIRONMENTAL MICROBIOLOGICAL STUDIES HONG PEI YING (B. Eng (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISION OF ENVIRONMENTAL SCIENCE AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I am grateful for the presence of many special people throughout these five years. They encourage, inspire and provide me with ample opportunities to discover my passion for research. A big thank you to the following persons: Professor Liu Wen-Tso, thank you for providing all the right formulations to groom me. Professor F. Michael Saunders. He is the one who encouraged me to ask questions and is always there to render ready assistance whenever needed. Dr Wu Jer-Horng. He is a great mentor who tolerated with my ignorance when I first started my candidature. Dr Zhang Rui for his sincere advice. Dr Zang Kaisai for the FISH images displayed at the last chapter of this dissertation. The great seniors: Ezrein Shah Bin Selamat is my Yoda in microarray fabrication; Dr Johnson Ng Kian Kok provides his excellent guidance in writing; Dr Pang Chee Meng gives blunt criticisms and thought-provoking scientific discussions to improve myself; Dr Chen Chia Lung shows me how patience is the key to eventual success. The ESE administrative and lab staff. They shoulder the administrative headaches for me. i All my lab friends and the cat. Thank you for your great company! My family. My attitude towards research has made me a stranger to home. Thank you for showering me with your never-ending understanding and concern. For every ending lies a new beginning. ii Table of Contents Acknowledgements… ………………………………….………………….…….…….…i Table of Contents…………….…….……………… ……………… …….….……… iii Summary……………………………………………………………………… …… . ix List of Tables………………………………………………………………… ….……. xii List of Figures………………………………………………………………… ………xiv Abbreviations………………………………………………………………….….… . xvii Publications……………………………………………………………………… … xix Chapter 1: Introduction 1.1 Background and problem statement 1.2 Objectives and aims . 1.3 Organization of thesis 11 Chapter 2: Literature Review 2.1 In the Land of the One-Eyed King, we see the whole picture? 13 2.2 Limitations in the SSU rRNA-based approach . 13 2.2.1 Sampling and storage bias 14 2.2.1.1 Sampling design . 14 2.2.1.2 Sample storage . 16 2.2.2 DNA extraction bias . 17 2.2.3 PCR bias . 19 2.2.3.1 False-negatives and false-positives 19 2.2.3.2 Microbial variability . 21 iii 2.2.3.3 Oligonucleotide primers . 22 2.2.4 Whole genome amplification (WGA) bias . 24 2.3 Molecular tools 26 2.3.1 Identifying and profiling the change in microbial community . 27 2.3.1.1 Microarray 27 2.3.1.2 16S rRNA gene libraries and metagenomics . 30 2.3.1.3 Terminal restriction fragment length polymorphism . 32 2.3.2 Quantifying the abundances of bacterial targets 33 2.3.2.1 Whole cell hybridization 33 2.3.2.2 Quantitative PCR (Q-PCR) 35 2.3.2.3 Hierarchical oligonucleotide primer extension (HOPE) 36 2.4 Environmental microbiological studies . 40 2.4.1 Human gut and fecal microbiota 40 2.4.2 Water quality and human health . 41 2.5 Concluding remarks 44 Chapter 3: Materials and Methods 3.1 Fecal samples 45 3.1.1 Human feces . 45 3.1.2 Pig feces . 46 3.1.3 Cow feces . 46 3.1.4 Dog feces 46 3.2 Contaminated aqueous samples . 47 3.2.1 Municipal wastewater . 47 iv 3.2.2 Swine wastewater . 47 3.2.3 Aqueous medium contaminated with cow and dog feces 47 3.2.4 Blind samples . 47 3.2.5 Environmental water samples 48 3.3 Bacterial reference strains . 49 3.3.1 Bacteroides spp. . 50 3.3.2 Bifidobacterium spp. 50 3.3.3 Animal-specific uncultivated Bacteroidales 51 3.3.4 Non-Bacteroides and Non-Bifidobacterium spp. . 51 3.4 DNA extraction . 52 3.5 Oligonucleotide primers 52 3.5.1 Oligonucleotide primers for PCR . 52 3.5.2 Oligonucleotide primers for HOPE 53 3.5.3 Oligonucleotide primers for Q-PCR . 57 3.5.4 Oligonucleotide probes for FISH-flow cytometry . 59 3.6 Polymerase chain reaction . 59 3.7 Hierarchical Oligonucleotide Primer Extension (HOPE) . 60 3.7.1 Applied Biosystems PRISM 3130 60 3.7.2 Beckman Coulter CEQ 8000 61 3.7.3 Calculation of relative abundances of bacterial targets 62 3.8 Quantitative PCR (Q-PCR) . 63 3.9 Fluorescent in-situ hybridization- flow cytometry (FISH-FC) . 63 3.9.1 Cell fixation 63 v 3.9.2 Cell permeabilization . 64 3.9.3 Hybridization 64 3.9.4 Flow cytometry . 64 3.10 Statistical and in silico analyses 65 3.10.1 Primer design 65 3.10.2 In silico analysis of probe specificity . 65 3.10.3 Multi Dimensional Scaling . 66 3.10.4 Correlation analysis 66 3.11 Nucleotide sequence ascension number 66 Chapter 4: Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension (HOPE) 4.1 Abstract 67 4.2 Introduction 68 4.3 Results 74 4.3.1 PCR amplification 74 4.3.2 Specificity of primer extension 76 4.3.3 Calculation of calibration factor (CF) values . 77 4.3.4 Electropherograms of tested samples . 78 4.3.5 Relative abundance of Bacteroides spp. as determined by HOPE . 80 4.3.6 Abundances of Bacteroides spp. as quantified by Q-PCR . 86 4.3.7 Relative abundance of Bacteroides spp. in wastewaters 86 4.4 Discussion……………………………………………………………………. 89 vi Chapter 5: A High-throughput and Quantitative Hierarchical Oligonucleotide Primer Extension (HOPE)-based Approach to Identify Sources of Fecal Contamination in Water Bodies 5.1 Abstract . 94 5.2 Introduction . 95 5.3 Results . 99 5.3.1 Primer specificities . 99 5.3.2 Detection limits of PCR assays and HOPE 102 5.3.3 HOPE-based approach on feces . 102 5.3.4 HOPE-based approach on aqueous samples 106 5.3.5 HOPE-based approach on blind samples . 107 5.3.6 HOPE-based approach on environmental waters . 110 5.3.7 Validation by Q-PCR . 111 5.3.8 Validation by fecal coliform test 113 5.4 Discussion 114 Chapter 6: Hierarchical Oligonucleotide Primer Extension (HOPE) as a Time- and Cost-effective Approach for the Quantitative Determination of Bifidobacterium spp. in Infant Feces 6.1 Abstract 118 6.2 Introduction 119 6.3 Results 120 6.3.1 Primer and probe specificities 120 6.3.2 Detection limits of newly designed HOPE primers . 122 vii 6.3.3 Relative abundance of Bifidobacterium spp . 123 6.3.4 Statistical correlation analysis 126 6.4 Discussion 127 Chapter 7: Conclusion and Recommendations 7.1 Conclusion 131 7.2 Recommendations 135 7.2.1 Method development 135 7.2.2 Environmental study . 136 7.2.2.1 Human gut and fecal microbiota 136 7.2.2.2 Water quality and fecal source tracking . 136 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Appl Environ Microbiol 62(12): 4504-13. 158 [...]... utilized to look into this hypothesis The abundances of host-specific Bacteroidales in feces and contaminated aqueous samples are first quantified, and used to derive a database that can aid in subsequent identification of the fecal contamination sources in unknown samples 8 The method is then applied to blind samples and environmental waters, and the findings are validated against Q-PCR and conventional... use of HOPE in feces and fecal-contaminated environment through the use of genus Bacteroides as a model group of bacterial targets The genus Bacteroides is highly predominant in human feces and can play a significant role in maintaining the host health (Guarner and Malagelada, 2003) For example, species like B vulgatus and B caccae can colonize the surface area of intestinal mucosa and inhibit adherence... primer is calculated and used to determine the relative abundance of the bacterial targets of interest (Wu and Liu, 2007) Wu and Liu further developed a 10-plexing reaction targeting Bacteroides spp at domain, group and species levels and applied the reaction to detect the relative abundances of Bacteroides spp in influent and effluent of domestic wastewater treatment plant (Wu and Liu, 2007) They successfully... origins of contamination in feces, contaminated aqueous samples, blind samples and environmental waters 11 • Chapter 6: Hierarchical oligonucleotide primer extension as a time- and costeffective approach for the quantitative determination of Bifidobacterium spp in infant stools This chapter demonstrates the use of HOPE to examine Bifidobacterium spp in the fecal microbiota of infants with and without eczema... be markedly different in the fecal microbiota of infants with and without eczema However, inconsistent findings in the abundances of Bifidobacterium spp have prevented x precise conclusions to their role in modulating the immune response of infants towards eczema HOPE is demonstrated as a time- and cost-effective method to determine the abundances of nine Bifidobacterium spp in 30 fecal samples It... spectrum (Levsky and Singer, 2003), Q-PCR can currently obtain a maximum of five-plexing in each reaction (Vet et al., 1999; http://www.biorad.com) This drives the need to develop alternative method that can complement Q-PCR, and to meet the needs for high-throughput quantitative measurement of bacterial targets Single base extension (SBE) reaction, which is primarily used in molecular biology studies for... abundances in feces and contaminated wastewaters The findings show that the 14 Bacteroides spp are human-associated and are present in human feces at abundances that are five and two-fold higher than in non-human feces and wastewaters, respectively The relative abundances of Bacteroides spp as quantified by HOPE are comparable to those reported in published literature and those obtained using conventional... type of single mismatch on single base extension Journal of Microbiological Methods 77: 267-275 Conference presentations 1 Liu, W.-T., and P.-Y Hong Hierarchical oligonucleotide primer extension (HOPE) for quantitative and qualitative analyses of biotic contaminants The 235th American Chemical Society National Meeting, April 6 to 10, 2008, New Orleans, USA (accepted for oral) xix 2 Hong, P.-Y., and W.-T... eczema (Mah et al., 2006; Mah et al., 2007) In particular, certain species in the genus may correlate closely to occurrence of eczema in infants at different stages of infancy For example, Bifidobacterium pseudocatenulatum is more commonly found in infants with eczema than in healthy infants (Gore et al., 2008) To draw conclusive findings to support this hypothesis, studies have to be performed on a statistically... J.-H Wu, and W.-T Liu (2008) Relative abundance of Bacteroides spp in stools and wastewaters as determined by hierarchical oligonucleotide primer extension Applied and Environmental Microbiology 74: 2882-2893 2 Hong, P.-Y., J.-H Wu, and W.-T Liu (2009) A high-throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)-based approach to identify sources of fecal contamination in water . SINGLE BASE EXTENSION REACTIONS AND THEIR APPLICATIONS IN ENVIRONMENTAL MICROBIOLOGICAL STUDIES HONG PEI YING NATIONAL UNIVERSITY OF SINGAPORE 2009 SINGLE BASE EXTENSION. targets in the human, pig and cow feces, together with those present in the influent of a municipal and swine wastewater treatment plant, were determined individually using multiplexing HOPE………………………………………………………………………. which in turn are important steps necessary to achieve homeostasis in both natural and engineered ecosystems. A small subunit (SSU) rRNA-based approach can bring new insights to our understanding