The application of next generation sequencing to profile microbe related cheese quality defects

High throughput next generation sequencing, together with advanced molecular methods, has considerably enhanced the field of food microbiology. By overcoming biases associated with culture dependant approaches, it has become possible to achieve novel insights into the nature of food-borne microbial communities. In this thesis, several different sequencing-based approaches were applied with a view to better understanding microbe associated quality defects in cheese. Initially, a literature review provides an overview of microbe-associated cheese quality defects as well as molecular methods for profiling complex microbial communities. Following this, 16S rRNA sequencing revealed temporal and spatial differences in microbial composition due to the time during the production day that specific commercial cheeses were manufactured. A novel Ion PGM sequencing approach, focusing on decarboxylase genes rather than 16S rRNA genes, was then successfully employed to profile the biogenic amine producing cohort of a series of artisanal cheeses. Investigations into the phenomenon of cheese pinking formed the basis of a joint 16S rRNA and whole genome shotgun sequencing approach, leading to the identification of Thermus species and, more specifically, the pathway involved in production of lycopene, a red coloured carotenoid. Finally, using a more traditional approach, the effect of addition of a facultatively heterofermentative Lactobacillus (Lactobacillus casei) to a Swiss-type cheese, in which starter activity was compromised, was investigated from the perspective of its ability to promote gas defects and irregular eye formation. X-ray computed tomography was used to visualise, using a non-destructive method, the consequences of the undesirable gas formation that resulted. Ultimately this thesis has demonstrated that the application of molecular techniques, such as next generation sequencing, can provide a detailed insight into defect-causing microbial populations present and thereby may underpin approaches to optimise the quality and consistency of a wide variety of cheeses.

The microbiome and the pathophysiology of asthma

Asthma is a chronic respiratory disease whose prevalence is increasing in the western world. Recently research has begun to focus on the role the microbiome plays in asthma pathogenesis in the hope of further understanding this respiratory disorder. Considered sterile until recently, the lungs have revealed themselves to contain a unique microbiota. A shift towards molecular methods for the quantification and sequencing of microbial DNA has revealed that the airways harbour a unique microbiota with apparent, reproducible differences present between healthy and diseased lungs. There is a hope that in classifying the microbial load of the asthmatic airway an insight may be afforded as to the possible role pulmonary microbes may have in propagating an asthmatic airway response. This could potentially pave the way for new therapeutic strategies for the treatment of chronic lung conditions such as asthma.

Molecular analysis of evolutionary relationships of Phaseolus dumosus with the gene pool of common bean

Valuable genetic variation for bean breeding programs is held within the common bean secondary gene pool which consists of Phaseolus albescens, P. coccineus, P. costaricensis, and P. dumosus. However, the use of close relatives for bean improvement is limited due to the lack of knowledge about genetic variation and genetic plasticity of many of these species. Characterisation and analysis of the genetic diversity is necessary among beans' wild relatives; in addition, conflicting phylogenies and relationships need to be understood and a hypothesis of a hybrid origin of P. dumosus needs to be tested. This thesis research was orientated to generate information about the patterns of relationships among the common bean secondary gene pool, with particular focus on the species Phaseolus dumosus. This species displays a set of characteristics of agronomic interest, not only for the direct improvement of common bean but also as a source of valuable genes for adaptation to climate change. Here I undertake the first comprehensive study of the genetic diversity of P. dumosus as ascertained from both nuclear and chloroplast genome markers. A germplasm collection of the ancestral forms of P. dumosus together with wild, landrace and cultivar representatives of all other species of the common bean secondary gene pool, were used to analyse genetic diversity, phylogenetic relationships and structure of P. dumosus. Data on molecular variation was generated from sequences of cpDNA loci accD-psaI spacer, trnT-trnL spacer, trnL intron and rps14-psaB spacer and from the nrDNA the ITS region. A whole genome DArT array was developed and used for the genotyping of P. dumosus and its closes relatives. 4208 polymorphic markers were generated in the DArT array and from those, 742 markers presented a call rate >95% and zero discordance. DArT markers revealed a moderate genetic polymorphism among P. dumosus samples (13% of polymorphic loci), while P. coccineus presented the highest level of polymorphism (88% of polymorphic loci). At the cpDNA one ancestral haplotype was detected among all samples of all species in the secondary genepool. The ITS region of P. dumosus revealed high homogeneity and polymorphism bias to P. coccineus genome. Phylogenetic reconstructions made with Maximum likelihood and Bayesian methods confirmed previously reported discrepancies among the nuclear and chloroplast genomes of P. dumosus. The outline of relationships by hybridization networks displayed a considerable number of interactions within and between species. This research provides compelling evidence that P. dumosus arose from hybridisation between P. vulgaris and P. coccineus and confirms that P. costaricensis has likely been involved in the genesis or backcrossing events (or both) in the history of P. dumosus. The classification of the specie P. persistentus was analysed based on cpDNA and ITS sequences, the results found this species to be highly related to P. vulgaris but not too similar to P. leptostachyus as previously proposed. This research demonstrates that wild types of the secondary genepool carry a significant genetic variation which makes this a valuable genetic resource for common bean improvement. The DArT array generated in this research is a valuable resource for breeding programs since it has the potential to be used in several approaches including genotyping, discovery of novel traits, mapping and marker-trait associations. Efforts should be made to search for potential populations of P. persistentus and to increase the collection of new populations of P. dumosus, P. albescens and P. costaricensis that may provide valuable traits for introgression into common bean and other Phaseolus crops.

Molecular composition of organic aerosols in central Amazonia: an ultra-high-resolution mass spectrometry study

The Amazon Basin plays key role in atmospheric chemistry, biodiversity and climate change. In this study we applied nanoelectrospray (nanoESI) ultra-high-resolution mass spectrometry (UHRMS) for the analysis of the organic fraction of PM2.5 aerosol samples collected during dry and wet seasons at a site in central Amazonia receiving background air masses, biomass burning and urban pollution. Comprehensive mass spectral data evaluation methods (e.g. Kendrick mass defect, Van Krevelen diagrams, carbon oxidation state and aromaticity equivalent) were used to identify compound classes and mass distributions of the detected species. Nitrogen- and/or sulfur-containing organic species contributed up to 60 % of the total identified number of formulae. A large number of molecular formulae in organic aerosol (OA) were attributed to later-generation nitrogen- and sulfur-containing oxidation products, suggesting that OA composition is affected by biomass burning and other, potentially anthropogenic, sources. Isoprene-derived organosulfate (IEPOX-OS) was found to be the most dominant ion in most of the analysed samples and strongly followed the concentration trends of the gas-phase anthropogenic tracers confirming its mixed anthropogenic–biogenic origin. The presence of oxidised aromatic and nitro-aromatic compounds in the samples suggested a strong influence from biomass burning especially during the dry period. Aerosol samples from the dry period and under enhanced biomass burning conditions contained a large number of molecules with high carbon oxidation state and an increased number of aromatic compounds compared to that from the wet period. The results of this work demonstrate that the studied site is influenced not only by biogenic emissions from the forest but also by biomass burning and potentially other anthropogenic emissions from the neighbouring urban environments.