UCC Library and UCC researchers have made this item openly available. Please let us know how this has helped you. Thanks! Title Dairybiota: analysing the microbiota of the dairy chain using next generation sequencing Author(s) Doyle, Conor J. Publication date 2017 Original citation Doyle, C. 2017. Dairybiota: analysing the microbiota of the dairy chain using next generation sequencing. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2017, Conor Doyle. http://creativecommons.org/licenses/by-nc-nd/3.0/ Embargo information No embargo required Item downloaded http://hdl.handle.net/10468/5524 from Downloaded on 2021-10-05T14:38:06Z Dairybiota: analysing the microbiota of the dairy chain using next generation sequencing A thesis presented to the National University of Ireland for the degree of Doctor of Philosophy By Conor Doyle B.Sc Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland School of Microbiology, University College Cork, Cork, Ireland 2017 Research supervisors: Dr. Paul Cotter and Professor Paul O’Toole i “And all this science, I don’t understand. It’s just my job five days a week” Rocket man “No number of sightings of white swans can prove the theory that all swans are white. The sighting of just one black one may disprove it.” Karl Popper ii Table of Contents Declaration .........................................................................................................................................x Abstract ..............................................................................................................................................xi Publications ........................................................................................................................................xiii Glossary of Terms ..............................................................................................................................xiv List of Figures .....................................................................................................................................xviii List of Tables ......................................................................................................................................xxii Chapter 1: Literature review Metagenome-based surveillance and diagnostic approaches to studying the microbial ecology of food production and processing environments 1.0 Abstract ........................................................................................................................................2 1.1 Introduction .....................................................................................................................3 1.2 The microbial ecology of crop production and livestock management ..........................4 1.3 Moving inside, biogeography of the food production and processing environment .....6 iii 1.4 Recent advances in understanding the compositional metagenomics of the food production and processing environment ..............................................................................8 1.5 “Terroir”–ising the microbiome .......................................................................................12 1.6 Food safety .......................................................................................................................13 1.7 Microbial Sourcetracking of communities through the food chain ................................14 1.8 Application of shotgun metagenomics and metatranscriptomics ..................................18 1.9 Conclusion ........................................................................................................................20 1.10 References .....................................................................................................................23 Chapter 2: Literature review Anaerobic sporeformers and their significance with respect to milk and dairy products 2.0 Abstract ........................................................................................................................................30 2.1 Introduction .....................................................................................................................31 2.2 Different groups of sporeforming bacteria ......................................................................33 2.2.1 Psychrotrophic thermophilic sporeformers ......................................33 2.2.1.1 Aerobic psychrotrophic thermophilic sporeformers ..........................................................................33 2.2.1.2 Anaerobic psychrotrophic thermophilic sporeformers .........................................................................35 iv 2.3 Taxonomy of Clostridium ....................................................................................................36 2.4 Sulphite reducing clostridia ................................................................................................38 2.4.1 Clostridium perfringens .....................................................................40 2.5 Clostridium botulinum .........................................................................................................46 2.5.1 Botulism and dairy powders .............................................................47 2.5.2 Botulism and other dairy products ...................................................51 2.5.3 Regulations ........................................................................................52 2.6 Butyric acid bacteria ...........................................................................................................53 2.6.1 Gas defects in cheeses ......................................................................54 2.7 Importance of Farming Practices ........................................................................................57 2.8 Conclusions .........................................................................................................................61 2.9 References ..........................................................................................................................62 Chapter 3: High-throughput metataxonomic characterization of the raw milk microbiota identifies changes reflecting lactation stage and storage conditions 3.0 Abstract ........................................................................................................................................82 3.1 Introduction .....................................................................................................................83 3.2 Materials and methods ....................................................................................................84 3.2.1 Experimental design.......................................................................................84 3.2.2 DNA extraction ...............................................................................................86 v 3.2.3 Quantitative PCR ............................................................................................86 3.2.4 16S rRNA amplicon preparation and high throughput sequencing ..............86 3.2.5 Bioinformatic and statistical analysis ...........................................................87 3.3 Results ..............................................................................................................................88 3.3.1 Sequences ......................................................................................................88 3.3.2 Lactation stage has the most considerable effect on the alpha and beta diversity of the raw milk microbiota .....................................................88 3.3.3 Taxonomic analysis highlights the influence of lactation stage and temperature on the raw milk microbiota ......................................................91 3.3.4 qPCR analysis to estimate total bacterial number in milk .............................93 3.4 Discussion .........................................................................................................................95 3.5 References .......................................................................................................................98 Chapter 4: Compositional metagenomic sequencing highlights the significant influence of seasonal housing and teat preparation on the raw milk microbiota 4.0 Abstract ........................................................................................................................................117 4.1 Introduction .....................................................................................................................118 4.2 Materials and Methods ....................................................................................................120 4.2.1 Treatment and Sample collection ..................................................................120 4.2.2 16S rRNA amplicon sequencing .....................................................................125 4.2.3 Bioinformatic and statistical analysis.............................................................126 4.2.4 Quantitative PCR ............................................................................................127 vi 4.3 Results ..............................................................................................................................128 4.3.1 Microbiota alpha and beta diversity of raw milk, teat surface swabs and environmental samples cluster according to habitat .............................128 4.3.2 Network analysis shows