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Daniel O'sullivan B.Sc M.Sc The Application of Next Generation Sequencing to Profile Microbe Related Cheese Quality Defects A thesis presented to the National University of Ireland for the Degree of Doctor of Philosophy by Daniel O’Sullivan B.Sc M.Sc Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland School of Food and Nutritional Sciences, University College Cork, Co. Cork, Ireland Date: 16.10.2015 Head of School: Prof. Yrjo Roos Research Supervisors: Dr. Diarmuid Sheehan, Dr. Paul Cotter, Dr. Linda Giblin & Prof. Paul McSweeney There is no substitute for hard work - Thomas Edison I Table of Contents Declaration ............................................................................................................................ IX Abstract ................................................................................................................................. .X Glossary of Terms .................................................................................................................. XII List of Tables ....................................................................................................................... ..XV List of Figures .................................................................................................................... ….XIX Chapter 1: Literature Review Nucleic acid-based approaches to investigate microbial-related cheese quality defects . 1 1.0. Abstract ............................................................................................................... 2 1.1. Introduction ........................................................................................................ 3 1.2. Defects Associated with Cheese and the Bacteria Responsible ......................... 5 1.2.1. Aroma and Taste Defects ................................................................. 5 1.2.2. Gas Defects: Split Defects and Secondary Fermentations ............... 7 1.2.3. BA Formation ................................................................................... 10 1.2.4. Mineral Deposition Defects ............................................................. 12 1.2.5. Cheese Pinking ................................................................................. 13 1.3. Detection Methods ............................................................................................. 14 1.3.1. Molecular Approaches to Study Cheese Defects ............................. 17 1.3.1.1. Conventional and Quantitative Real Time PCR (qRT-PCR) .. 17 1.3.1.2. Denaturing Gradient Gel Electrophoresis (DGGE) and Temporal Temperature Gel Electrophoresis (TTGE) ........... 20 1.3.1.3. Single Strand Conformation Polymorphism (SSCP) ............. 23 1.3.1.4. Fluorescence In Situ Hybridisation (FISH) ............................ 24 II 1.3.1.5. Amplified Ribosomal DNA Restriction Analysis (ARDRA) and Terminal Restriction Fragment Length Polymorphism (T- RFLP) .................................................................................... 25 1.3.1.6. Ribosomal Intergenic Spacer Analysis (RISA) ...................... 26 1.3.1.7. Denaturing High Performance Liquid Chromatography…….26 1.3.1.8. DNA Microarrays ................................................................. 27 1.4. New Detection Methods: Next Generation Sequencing (NGS) .......................... 29 1.4.1. Roche 454 FLX Pyrosequencer ......................................................... 30 1.4.2. Illumina/Solexa Genome Analyzer ................................................... 32 1.4.3. ABI SOLiD ......................................................................................... 33 1.4.4. Ion Torrent Personal Genome Machine (PGM) ............................... 33 1.5. Conclusions and Future Perspectives ................................................................. 34 1.6. Acknowledgements ............................................................................................ 36 1.7. References .......................................................................................................... 37 Chapter 2: Temporal and Spatial Differences in Microbial Composition During the Manufacture of a Continental-type Cheese .............................................................................................. 63 2.0. Abstract .............................................................................................................. 64 2.1. Introduction ....................................................................................................... 65 2.2. Materials and Methods ..................................................................................... 68 2.2.1. Cheese Production, Sampling and Nucleic Acid Extraction ............... 68 2.2.2. PCR Amplification of the Microbial 16S rRNA Gene .......................... 69 2.2.3. High-throughput Sequencing and Bioinformatic Analysis ................. 70 2.3. Results ................................................................................................................ 71 III 2.3.1. α and ß Diversity of Microbial Populations in Early and Late Day Production Cheeses ..................................................................................... 71 2.3.2. Cheese Composition .......................................................................... 72 2.3.3. High-throughput Sequencing Reveals Differences in Microbial Taxa Between Cheeses Produced Early and Late in the Production Day ............. 72 2.3.4. Distribution of Microbial Communities Present in the Core and Rind of a Brine Salted Continental-type Cheese .................................................. 74 2.5. Discussion .......................................................................................................... 75 2.6. Acknowledgements ........................................................................................... 80 2.7. References ......................................................................................................... 81 Chapter 3: High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses ........................................................................... 104 3.0. Abstract ........................................................................................................... 105 3.1. Introduction .................................................................................................... 107 3.2. Materials and Methods .................................................................................. 111 3.2.1. Sample Collection ........................................................................... 111 3.2.2. Determination of BA Content of Cheese ........................................ 111 3.2.3. Determination of Cheese pH, Salt and Moisture Contents ............ 112 3.2.4. DNA Extraction from Selected Cheeses .......................................... 112 3.2.5. PCR Detection of hdc and tdc Gene Fragments using Selected Primer Sets ............................................................................................................ 113 3.2.6. Cloning of PCR Amplicons ............................................................... 114 3.2.7. High-throughput Sequencing .......................................................... 114 3.2.8. Bioinformatic Analysis .................................................................... 115 3.3. Results ............................................................................................................. 117 IV 3.3.1. Sanger Sequencing Reveals the Identity of Bacteria with Histaminogenic Potential .......................................................................... 117 3.3.2. Sanger Sequencing Reveals the Identity of Bacteria with Tyraminogenic Potential ........................................................................... 118 3.3.3. α-diversity of Artisanal Cheese Microbiota with BA-producing Potential as Revealed by Next Generation DNA Sequencing ................... 119 3.3.4. High-throughput Ion PGM Sequencing Reveals the Presence of Amine Forming Communities in Different Cheese Varieties ............................... 120 3.3.5. Cheese Characterisation ................................................................. 121 3.4. Discussion ....................................................................................................... 122 3.5. Acknowledgements ........................................................................................ 128 3.6. References ...................................................................................................... 129 Chapter 4: Thermus and the Pink Discolouration Defect in Cheese ............................................... 162 4.0. Abstract ........................................................................................................... 163 4.1. Introduction .................................................................................................... 164 4.2. Materials and Methods .................................................................................. 166 4.2.1. DNA Extraction from Cheeses ......................................................... 166 4.2.2. Generation of 16S rDNA Amplicons for High-throughput Sequencing .................................................................................................................. 166 4.2.3. 16S rDNA Sequencing and Bioinformatic Analysis .......................... 167 4.2.4. Shotgun Metagenomic Sequencing and Gene Function Analysis
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