Dairybiota: analysing the microbiota of the dairy chain using next generation sequencing
Doyle, Conor J.
University College Cork
Advances in DNA sequencing technologies, accompanied with developments in data analysis and interpretation, have provided novel insights into to the microbial ecology of foods and food production environments. By utilising these advances in technology, it is possible to overcome the biases associated with culture-based analysis. This has been achieved by targeting the metagenomic DNA of these environments using high-throughput sequencing (HTS). In this thesis, HTS was utilised to provide insights into factors which influence the microbial composition of raw milk, and reveal potential environmental sources of bacteria in the dairy chain. Firstly, a literature review explores the recent insights gained from applying HTS to study the microbial ecology of food production chains. Additionally, a second literature review focuses on sulphite reducing Clostridia (SRC), their taxonomy, toxigenicity and the prevalence in which they are detected in dairy products. The first research study focused on applying HTS to characterise the microbiota of blended raw bulk tank milk (BTM) stored at different temperatures at both mid and late-lactation. This highlighted that lactation stage had more of a significant impact on the raw milk microbiota compared to storage temperature. After this, in a second study, on-farm environmental niches were explored as possible reservoirs for bacteria to contaminate raw milk. Raw milk samples were collected from individual cows and from BTM, both when cows were housed indoors and when cows were grazing on pasture. Additionally, faecal and teat swab samples were collected from these cows over both periods, as well as environmental niches from both the indoor and outdoor habitats. Results from this study highlight that herd habitat drives the microbial composition of raw milk. In a subsequent investigation, shotgun metagenomic sequencing was employed to explore the cheese production microbiome for the presence of bacteria and phage, and this approach also facilitated strain level characterisation of starter bacteria. Production plant surfaces were found to harbour resident lactic acid bacteria and brine was identified as a potential reservoir for lactococcal phage. In a final study, whole genome sequencing and in-silico genome characterisation were used to determine the genes responsible for the SRC phenotype in dairy associated SRCs. Genome annotation facilitated the identification of two distinct pathways involved in the reduction of sulphite to sulphide in dairy associated isolates, asrABC mediated reduction in SRCs and cysJI mediated reduction in other sulphite reducing bacteria (SRBs). Ultimately this thesis will show that HTS can be a valuable tool for characterising the microbiota of food products and food production environments.
Microbiome , DNA sequencing , Source tracking , Food chain , Clostridium , Genomics , Metagenomics , Metagenostics
Doyle, C. 2017. Dairybiota: analysing the microbiota of the dairy chain using next generation sequencing. PhD Thesis, University College Cork.