A systematic approach to the interrogation and sharing of standardised biofilm signatures

Publicado em "6th International Conference on Practical Applications of Computational Biology & Bioinformatics", ISBN 978-3-642-28838-8The study of microorganism consortia, also known as biofilms, is associated to a number of applications in biotechnology, ecotechnology and clinical domains. A public repository on existing biofilm studies would aid in the design of new studies as well as promote collaborative and incremental work. However, bioinformatics approaches are hampered by the limited access to existing data. Scientific publications summarise the studies whilst results are kept in researchers’ private ad hoc files. Since the collection and ability to compare existing data is imperative to move forward in biofilm analysis, the present work has addressed the development of a systematic computer-amenable approach to biofilm data organisation and standardisation. A set of in-house studies involving pathogens and employing different state-of-the-art devices and methods of analysis was used to validate the approach. The approach is now supporting the activities of BiofOmics, a public repository on biofilm signatures (http://biofomics.org).The authors thank, among others, Rosario Oliveira, Maria Joao Vieira, Idalina Machado, Nuno Cerca, Mariana Henriques, Pilar Teixeira, Douglas Monteiro, Melissa Negri, Susana Lopes, Carina Almeida and Helder Lopes, for submitting their data. The financial support from IBB-CEB, Fundacao para a Ciencia e Tecnologia (FCT) and European Community fund FEDER (Program COMPETE), project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480, are also gratefully acknowledged

BiofOmics: A Web Platform for the Systematic and Standardized Collection of High-Throughput Biofilm Data

Background: Consortia of microorganisms, commonly known as biofilms, are attracting much attention from the scientific community due to their impact in human activity. As biofilm research grows to be a data-intensive discipline, the need for suitable bioinformatics approaches becomes compelling to manage and validate individual experiments, and also execute inter-laboratory large-scale comparisons. However, biofilm data is widespread across ad hoc, non-standardized individual files and, thus, data interchange among researchers, or any attempt of cross-laboratory experimentation or analysis, is hardly possible or even attempted. Methodology/Principal findings This paper presents BiofOmics, the first publicly accessible Web platform specialized in the management and analysis of data derived from biofilm high-throughput studies. The aim is to promote data interchange across laboratories, implementing collaborative experiments, and enable the development of bioinformatics tools in support of the processing and analysis of the increasing volumes of experimental biofilm data that are being generated. BiofOmics data deposition facility enforces data structuring and standardization, supported by controlled vocabulary. Researchers are responsible for the description of the experiments, their results and conclusions. BiofOmics curators interact with submitters only to enforce data structuring and the use of controlled vocabulary. Then, BiofOmics search facility makes publicly available the profile and data associated with a submitted study so that any researcher can profit from these standardization efforts to compare similar studies, generate new hypotheses to be tested or even extend the conditions experimented in the study. Significance BiofOmics novelty lays on its support to standardized data deposition, the availability of computerizable data files and the free-of-charge dissemination of biofilm studies across the community. Hopefully, this will open promising research possibilities, namely: the comparison of results between different laboratories, the reproducibility of methods within and between laboratories, and the development of guidelines and standardized protocols for biofilm formation devices and analytical methods.The financial support from the Institute of Biotechnology and Bioengineering - Center of Biological Engineering (IBB-CEB), Fundacao para a Ciencia e Tecnologia (FCT) and European Community fund FEDER (Program COMPETE), project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480 and PhD grant of Idalina Machado (SFRH/BD/31065/2006) are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Green synthesised Zinc Oxide Nanoparticles and their antifungal effect on Candida albicans Biofilms

Magister Scientiae Dentium - MSc(Dent)Candida albicans is a clinical fungal isolate that is most frequently isolated from different host niches, and is implicated in the pathogenesis of several fungal infections, including oral candidiasis. The pathogenesis and antifungal resistance mechanisms of Candida species are complex and involve several pathways and genes. Oral candidiasis incidence rates are rapidly increasing, and the increase in resistance to conventional antifungals has led to the need to develop innocuous and more efficacious treatment modalities. The purpose of this study was to explore a single pot process for phytosynthesis of zinc oxide nanoparticles (GZnO NPs) and to assess their antifungal potential

Fleas of fleas: The potential role of bacteriophages in Salmonella diversity and pathogenicity.

Non-typhoidal salmonellosis is an important foodborne and zoonotic infection, that causes significant global public health concern. Diverse serovars are multidrug-resistant and encode several virulence indicators, however, little is known on the role prophages play in driving these characteristics. Here, we extracted prophages from 75 Salmonella genomes, which represent the 15 most important serovars in the United Kingdom. We analysed the genomes of the intact prophages for the presence of virulence factors which were associated with; diversity, evolution and pathogenicity of Salmonella and to establish their genomic relationships. We identified 615 prophage elements from the Salmonella genomes, from which 195 prophages are intact, 332 being incomplete while 88 are questionable. The average prophage carriage was found to be more prevalent in S. Heidelberg, S. Inverness and S. Newport (10.2-11.6 prophages/strain), compared to S. Infantis, S. Stanley, S. Typhimurium and S. Virchow (8.2-9 prophages/strain) and S. Agona, S. Braenderup, S. Bovismorbificans, S. Choleraesuis, S. Dublin, and S. Java (6-7.8 prophages/strain), and S. Javiana and S. Enteritidis (5.8 prophages/strain). Cumulatively, 2760 virulence factors were detected from the intact prophages and associated with cellular functionality being linked to effector delivery/secretion system (73%), adherence (22%), magnesium uptake (2.7%), resistance to antimicrobial peptides (0.94%), stress/survival (0.4%), exotoxins (0.32%) and antivirulence (0.18%). Close and distant clusters were formed among the prophage genomes suggesting different lineages and associations with bacteriophages of other Enterobacteriaceae. We show that diverse repertoire of Salmonella prophages are associated with numerous virulence factors, and may contribute to diversity, pathogenicity and success of specific serovars

Raman spectroscopy for point of care urinary tract infection diagnosis

Urinary tract infections (UTIs) are one of the most common bacterial infections experience by humans, with 150 million people suffering one or more UTIs each year. The massive scale at which UTIs occurs translates to a tremendous health burden comprising of patient morbidity and mortality, massive societal costs and a recognised contribution to expanding antimicrobial resistance. The considerable disease burden caused by UTIs is severely exacerbated by an outdated diagnostic paradigm characterised by inaccuracy and delay. Poor accuracy of screening tests, such as urinalysis, lead to misdiagnosis which in turn result in delayed recognition or overtreatment. Additionally, these screening tests fail to identify the causative pathogen, causing an overreliance on broad-spectrum antimicrobials which exacerbate burgeoning antimicrobial resistance. While diagnosis may be accurately confirmed though culture and sensitivity testing, the prolonged delay incurred negates the value of the information provided doing so. A novel diagnostic paradigm is required that that targets rapid and accurate diagnosis of UTIs, while providing real-time identification of the causative pathogen. Achieving this precision management is contingent on the development of novel diagnostic technologies that bring accurate diagnosis and pathogen classification to the point of care. The purpose of this thesis is to develop a technology that may form the core of a point-of-care diagnostic capable of delivering rapid and accurate pathogen identification direct from urine sample. Raman spectroscopy is identified as a technology with the potential to fulfil this role, primarily mediated though its ability to provide rapid biochemical phenotyping without requiring prior biomass expansion. Raman spectroscopy has demonstrated an ability to achieve pathogen classification through the analysis of inelastically scattered light arising from pathogens. The central challenge to developing a Raman-based diagnostic for UTIs is enhancing the weak bacterial Raman signal while limiting the substantial background noise. Developing a technology using Raman spectroscopy able to provide UTI diagnosis with uropathogen classification is contingent on developing a robust experimental methodology that harnesses the multitude of experimental and analytical parameters. The refined methodology is applied in a series of experimental works that demonstrate the unique Raman spectra of pathogens has the potential for accurate classification. Achieving this at a clinically relevant pathogen load and in a clinically relevant timeframe is, however, dependent on overcoming weak bacterial signal to improve signal-to-noise ratio. Surface-enhanced Raman spectroscopy (SERS) provides massive Raman signal enhancement of pathogens held in close apposition to noble metal nanostructures. Additionally, vacuum filtration is identified as a means of rapidly capturing pathogens directly from urine. SERS-active filters are developed by applying a gold nanolayer to commercially available membrane filters through physical vapour deposition. These SERS-active membrane filter perform multiple roles of capturing pathogens, separating them from urine, while providing Raman signal enhancement through SERS. The diagnostic and classification performance of SERS-active filters for UTIs is demonstrated to achieve rapid and accurate diagnosis of infected samples, with real-time uropathogen classification, using phantom urine samples, before piloting the technology using clinical urine samples. The Raman technology developed in this thesis will be further developed toward a clinically implementable technology capable of ameliorating the substantial burden of disease caused by UTIs.Open Acces

Exploring ambient mass spectrometry capacities for rapid detection and phenotyping of pseudomonas aeruginosa infection in cystic fibrosis patients

Cystic fibrosis (CF) patients mostly succumb to respiratory failure, usually caused by Pseudomonas aeruginosa infection. Early, but not chronic, infection can be eradicated, mandating timely detection. However, current methods are insufficiently sensitive and unsuitable for children. This thesis explores the potential of novel ambient mass spectrometric (MS) tools for the phenotyping of P. aeruginosa and its detection in non-invasive samples. The hypotheses are that bacterial expression evolves as infection progresses and that the composition of host body fluids is impacted by P. aeruginosa acquisition. Rapid evaporative ionisation MS (REIMS) was applied to clinical isolate cultures including common CF pathogens. The detected metabolic profiles differentiated between species and led to the identification of virulence-associated metabolites in P. aeruginosa: quorum sensing molecules and rhamnolipids. Repeated analysis revealed differences between P. aeruginosa strains that allowed isolate classification. Although classification according to genetically-defined types was not achieved, REIMS potentially provides a finer resolution on quickly evolving virulence features. Exploration of intra-species disparity showed a higher metabolic diversity in chronic respiratory than in acute infections, attributed to lengthy and site-specific adaptation. The bacterial population was more disparate between than within CF patients. Variations in virulence factor levels between early and chronic isolates provided insight into P. aeruginosa‘s metabolism and adaptation, supporting the first hypothesis. These findings may support future clinical strategies. REIMS and desorption electrospray ionisation MS (DESI-MS) were evaluated as direct-from-sample diagnostic tools using sputum as a reference, and urine and skin secretions as easily accessible samples. REIMS could not detect P. aeruginosa in sputum and urine pellets, nor could DESI-MS on skin secretions. Nevertheless, the rich sputum metabolic profile and skin lipidome contained potentially highly relevant information about host physiology that may assist clinicians in the future. Although the second hypothesis was not verified, the data reported will generate future clinically relevant hypotheses.Open Acces

Trophic ecology of intertidal harpacticoid copepods, with emphasis on their interactions with bacteria

In the era of rapidly changing natural environments, there is an urgent need for understanding biodiversity-ecosystem functioning. Trophic interactions play a key role in structuring the ecosystem. In marine systems, the feeding ecology of the larger-sized species i.e. (top) predators of food webs, is fairly well documented, yet at the basal level of food webs, the complexity of grazer interactions is much higher and trophic linkages are potentially still missing. In this PhD thesis, the focus is on trophic interactions between harpacticoid copepods and bacteria. The meiofauna (organism size between 38 µm and 1 mm), and in particular harpacticoid copepods (the second most abundant group of the meiofauna, after nematodes), transfer microphytobenthos to higher trophic levels, predominantly larval and juvenile fish. Harpacticoids have highly complex feeding habits. They are able to consume a wide range of food sources, e.g. microalgae, cyanobacteria, flagellates, ciliates, mucoid substances, fungi and heterotrophic bacteria. They are therefore sometimes called ‘indiscriminate feeders’. However, laboratory feeding experiments and recently obtained in situ data, demonstrate their highly selective behaviour and their species-specific food preferences. Under experimental conditions, harpacticoids discriminated among substrates with poor- versus well-developed bacterial films although copepods are considered to be morphologically incapable of consuming small-sized particles such as bacteria. Based on their potential interaction with a broad range of food sources, the general importance of their herbivorous feeding strategy is being questioned and the recent development of dietary tracers such as stable isotopes and fatty acid analysis, now allows to investigate direct assimilation of bacterial matter or that of other food sources. Interest into the pathways of transfer of benthic bacterial matter upwards the food web, for instance with harpacticoids as intermediates, is due to the lack of insight into the general fate of the extensive benthic bacterial biomass and potential pathways of transfer to higher trophic levels are still unknown. A proportion of energy flows back to the microbial-detrital pool as copepod fecal pellets which are efficiently degraded and remineralized. Beside the upward fluxes of biomass and energy, the degradation and reminerilization of waste products resulting from grazing activity is crucial for the recycling of biomass and thus the efficiency of ecosystem functioning. We gained insight into food utilization by harpacticoids in a heterogeneous estuarine intertidal ecosystem, into species-specific feeding patterns and into the trophic importance of bacteria for harpacticoids. Feeding ecology was studied based on both field data of the harpacticoid species assemblages and on experimental laboratory data using individual species. The process of fecal pellet degradation was investigated with focus on the contribution of internal and external fecal pellet bacteria during early degradation and remineralisation of fecal pellets. In an attempt to gain information on the presence of internal bacteria and their position in the fecal pellet, Atomic Force Microscopy and Laser Scanning Confocal Microscopy (AFM-LSCM), a high-resolution imaging tool, was applied. It offers new prospects for studying microbial degradation of copepod fecal pellets. Harpacticoid assemblages for chapters 2 and 3 were studied at the Paulina intertidal area (Schelde estuary, The Netherlands) encompassing a gradient from tidal flat to salt marsh, from seasonal periods at five stations differing in sediment characteristics, tidal exposure, presence of vegetation, etc. To address the structural role of environmental factors, including abiotic sediment characteristics and food-related factor, for harpacticoid assemblages and disclose resource partitioning within harpacticoid assemblages, chapter 2 presents a field study into spatial-temporal heterogeneity in resource availability and in intertidal copepod assemblage structure (density, diversity and composition). An in-depth analysis of the most influential factors for species distribution added relevant information to the autoecology of intertidal harpacticoid species. Spatio-temporal harpacticoid assemblage variation was assigned to variables relating to total organic matter, microphytobenthic biofilms (characterised by pigments and their degradation products), differences in detrital origin and NH4+, suggesting a primary influence of food availability and quality. Nevertheless, harpacticoid assemblages of tidal flats were seemingly more structured by abiotic factors (granulometry and tidal exposure) and especially the harpacticoid assemblage of the sand flat (station H2, species Paraleptastacus spinicauda, Asellopsis intermedia) which was highly station-specific and constant over time. Assemblages of salt marsh stations are considerably similar in copepod family composition despite of differences in salt marsh granulometry, suggesting a primary influence of food availability and food quality. Variability in Microarthridion littorale abundances is related to microphytobenthos biomass. For Ectinosomatidae and Tachidius discipes, the limited number and low correlations for all biotic and abiotic factors indicates a generalistic occurrence. For some species, linkages between habitat characteristics and species distributions were little decisive (e.g. Platychelipus littoralis, Paronychocamptus nanus, Amphiascus sp. 1). Overall, high intercorrelation between a broad range of environmental factors hinders us to draw strong conclusions about the main regulating factors for harpacticoids distribution. Analysis of spatio-temporal variability in resource utilization by harpacticoid copepods, by means of copepod carbon isotopic profiles and fatty acid profiles (chapter 3), proved that the majority of intertidal harpacticoid species relied strongly on microphytobenthos (MPB) as a food source, with potentially fine-scaled selectivity among diatom species or other MPB components. In addition, harpacticoids spanned two trophic levels suggesting also an indirect pathway of MPB transfer to harpacticoids. Furthermore, intertidal harpacticoids showed dietary differences and species-specific spatio-temporal variability in food utilization with contributions of suspended particulate organic matter (Paronychocamptus nanus, Amphiascus sp. 1, Microartridion littorale), flagellates (M. littorale), and bacteria (Delavalia palustris) but not of Spartina detritus. Resource partitioning by harpacticoid assemblages occurred in all stations but was especially clear in the sand flat, comprising a diatom feeder (Asellopsis intermedia), a diatom feeder with temporal reliance on dinoflagellates (Tachidius discipes) and a bacterial feeder (Paraleptastacus spinicauda). In the muddy salt marsh, a trophic role of bacteria was found for Delavalia palustris and Cletodidae, the latter being unique by the utilization of a chemoautotrophic food source. In chapter 4, results from a microcosm feeding experiment using 13C-labelled bacteria, showed that bacterial feeding is linked to diatom grazing and that overall assimilation of bacterial carbon is low for all tested harpacticoid species. In contrast to the bacterivorous copepod Delavalia palustris, non-bacterivorous harpacticoid species (Nannopus palustris, Microarthridion littorale, Platychelipus littoralis) responded negatively on bacterial feeding, as deduced from their mortality and PUFA (polyunsaturated fatty acid) impoverishments. These findings suggest that bacterial biomass may complement feeding on MPB and that an exclusive bacterial diet does not meet copepod nutritional requirements. Delavalia palustris was able to biosynthesized PUFA from a bacterial diet but generally, bacteria represent a minor and low-quality food for these intertidal harpacticoid copepods. In a food-patch choice experiment with 13C-labelled bacterial biofilms (chapter 5), the ability of the harpacticoids Platychelipus littoralis and Delavalia palustris to select between bacterial species with potential different nutritional value (Gramella sp., Jannaschia sp. and Photobacterium sp.) was investigated. In line with chapter 4, bacteria carbon assimilation was low and significant bacterial fatty acid transfer was lacking. A low degree of selectivity was found (preference for Photobacterium sp.), and extracellular metabolites rather than biochemical content and bacterial densities are suggested to be the driver of selective feeding behaviour towards bacteria. Furthermore, the energetic cost of differential bacterivory resulted in a negative fatty acid balance for P. littoralis while D. palustris showed an improved fatty acid profile and thus a positive response to the low-quality bacterial food (similar as in chapter 4). As the ingested bacterial biomass is of limited use for the majority of harpacticoid species, the largest fraction of ingested bacteria returns to the microbial-detrital food web in the form of fecal pellets. Chapter 6 and 7 demonstrate that these ‘internal’ fecal pellet bacteria are viable cells with high densities and represent a diverse active community able to significantly participate in fecal pellet degradation and overall recycling of carbon for the grazer food web. In chapter 6, molecular (RNA-based PCR-DGGE) and metabolic profiling (Biolog Ecoplate assay) of freshly egested copepod fecal pellets proved the general presence of internal active bacteria with a broad metabolic potential in fecal pellets of different copepod species and with different fecal pellet content. The strong impact of the food source on the bacterial diversity of the fecal pellet, indicates the direct transfer of ingested food bacteria to the fecal pellets. Furthermore, the colonization of fecal pellets by bacteria from the surrounding water was relatively low. Consequently, internal bacteria diversity was not replaced by a new external bacterial assemblage. About half of the internal fecal pellet bacteria persisted after 60 h of fecal pellet degradation and, hence, internal fecal pellet bacteria deliver a significant contribution to fecal pellet recycling with Vibrio sp. as a potential important participant. These findings refute the hypothesis of high bacterial fp colonization, as observed for planktonic fecal pellets. AFM-LSCM (Atomic Force Microscopy - Laser Scanning Confocal Microscopy, chapter 7) imaging confirmed the presence of high densities of viable bacteria packed inside the fecal pellet. Furthermore, AFM—LSCM revealed the fibrillar network structure of the peritrophic membrane from a Paramphiascella fulvofasciata fecal pellet, similar to marine polysaccharides and α-chitin and allowed precise measurement of the membrane thickness (0.7-5.9 nm) and bacterial cell volumes (range 0.006-0.117 µm3, in liquid). This protocol enables high-resolution interrogation of biochemical structural changes and bacterial dynamics within the copepod fecal pellets and other heterogeneous particles such as marine snow under environmental conditions. AFM-LSCM generally allows studying bacterial cell size, cell shape and cell-cell interactions. Here it was applied (1) to visualize the ultra-structure of the peritrophic membrane and (2) to locate and quantify bacterial presence (cell size measurements) both inside and outside the fecal pellet. In conclusion, it is clear that the majority of intertidal harpacticoid species primarily relies on MPB, in particular diatoms, and their diet can include small contributions of other food sources such as suspended particulate organic matter, protozoa and bacteria. Despite indications that food availability and MPB shape harpacticoid assemblages on the spatio-temporal scale, the ’real’ importance of MPB, other food-related factors or physical habitat factors for structuring harpacticoid assemblages remains unclear. Furthermore, bacteria are a nutritional food source for some harpacticoid taxa but overall, transfer of bacterial biomass to harpacticoids seems rather limited and in the intertidal microbial food web, bacteria remain a sink. Although harpacticoids consume predominantly substrate-attached bacteria and often co-incidentally during grazing on a primary food source, there are indications that bacterivorous harpacticoids have special adaptations for consuming a poor-quality food source. Selective feeders Paraleptastacus spinicauda and Cletodidae proved, the latter consuming chemoautotrophic bacteria, proves harpacticoids ability to discriminate for bacteria and for bacterial groups but more fine-scale selectivity for bacterial species or for bacterial nutritional content, remains unclear. Although bacterial biomass is (passively) ingested by harpacticoids, the majority will be channeled back to the microbial-detrital food web in the form of fecal pellets. As a consequence of the relative higher contribution of ‘internal’ fecal pellet bacteria compared to external bacteria, the process of microbial degradation of benthic fecal pellets deviates from degradation of planktonic fecal pellets. This may imply that the functioning of the benthic microbial-detrital loop is not necessarily similar to the plantonic microbial-detrital loop

Glycerophospholipid oxidation and production of aldehydes in oesophageal adenocarcinoma

Oesophageal adenocarcinoma (OAC) has an unmet clinical need, with five year survival in the UK remaining at 15%. There has been little improvement with advances in surgical practice or systemic chemotherapeutic regimens. Early diagnosis holds the key to radical treatment, the clinical utility of breath testing has highlighted aldehydes as a potential early marker of cancer. The emerging field of lipidomics has identified variations in lipid composition between cancer and benign tissue. These observed changes have highlighted phospholipids as particularly important class responsible for structural membrane stability, cell signalling and replication. In this research, multiple mass spectrometry techniques were implemented to identify and correlate lipid abundance with increased aldehyde quantitation. Desorption Electrospray Ionisation- Mass Spectrometry (DESI-MS) was utilised for lipid profiling in oesophageal adenocarcinoma tissue to reveal a prevalence of Phosphatidic acids (PA) and Phosphatidylglycerol (PG) species. Comprehensive bioinformatics analysis highlighted the PG pathway with significantly dysregulation and positive phenotype to PG production. The investigation of aldehydes was performed in vivo by lipid oxidation and corroborated in OAC tissue by a targeted Liquid Chromatography mass spectrometry (LC-MS) method. This identified medium and long chain aldehydes (Pentanal, Nonanal, Un-decanal) at particularly increased concentration. To investigate the lipid product correlation, the chemistry of lipid oxidation was defined and characterised. To explored the origin of the increased PA and PG a targeted LC-MS method was created and patient tissue and surface mucus samples were collected at paired sites. The analysis confirmed a relative increase of PAs and PGs in OAC tissue and mucus of representative intensities suggesting a correlation between mucus sampling and cell phospholipid concentration. These data highlight the Phospholipid products of a genetically dysregulated pathway in OAC, which may contribute to the production of unstable polyunsaturated lipids which are prone to oxidation and formation of aldehydes.Open Acces

Multi-omics approaches to understanding Candida biofilms

Candida biofilms are a substantial clinical and human health burden which are still underappreciated. Benefits afforded by morphogenic switching from planktonic to biofilm communities include resistance to antimicrobials in the host’s immune system, and resilience to mechanical disruption, all of which complicate the treatment and management of infections. Biofilm formation in Candida spp. is influenced by numerous factors, including response to the host, pH, bacteria, and many other environmental factors. This is further complicated by inherent heterogeneity within candidal populations in respect to biofilm forming capabilities and its response to external stimuli. Biofilms are heterogeneous by their nature, formed of populations of yeast and hyphal cells in a consortium of morphological states. Many variables exist to influence biofilm heterogeneity in Candida spp. and can influence the clinical outcomes and observations. The overarching aim of this thesis was to explore factors that influences C. albicans biofilm formation using omics-based approaches. We performed analysis on the microbiome derived from three distinct oral niches in denture stomatitis patients. Alpha and beta diversity measures were extrapolated and compared to identify perturbations in the microbiome that were related to either the oral hygiene or the Candida burdenof the individuals. Correlation analysis between phyla, oral hygiene and fungal load were performed to identify significant relationships. Secondly, we selected clinical isolates, from our Scottish candidaemia study cohort, which were deemed high and low biofilm isolates as determined by biomass assays. We assessed the biofilm forming capabilities in media supplemented with and without serum. These assays consisted of crystal violet biomass assay and measurements ofhyphal. We also utilised SEM to visualise the phenotypes of the high and low isolates with and without serum. HBF and LBF isolates were then grown at 90min, 4h and 24h in the presence and absence of serum before being submitted to RNASequencing by Illumina. Differential expression analysis was performed using DESeq2 before over representation and gene set enrichment analysis. Cell wall proteomics on high and low isolates was also performed to identify computational changes in the cell wall. Thirdly, we performed metabolomic analysis both targeted and untargeted on the supernatants of HBF and LBF grown in the presence or absence of serum for 4 and 24h. Features were identified from the LC-MS peaks by PiMP and analysis of differentially abundant analytes between our isolates and growth conditions were performed. Functional analysis of the annotated analytes was then performed by pathway activity profiling. Finally, utilising our identified differentially expressed features from metabolomic and transcriptomic analysis we submitted both datasets to integrative analysis. Using a combination of conceptual, joint pathway analysis by MetaboAnalyst and multivariate Analysis using the MixOmics data integration package. From our microbiome analysis we observed that the oral hygiene measures had no significant effect on the diversity or composition of the oral microbiome. Candida similarly had no impact on the alpha diversity of the oral microbiome. However, we did observe some relationship in the beta diversity which correlated with Candida load. Further investigation identified correlations of genus including Lactobacillus with Candida load. From our high and low Candida candidaemia isolates we observed phenotypic switching of LBF in the presence of serum. We also found functional differences related to this phenotypic switching. The low biofilm response to serum included enrichment in fatty acid and aycl-coA metabolic pathways. Metabolomic analysis revealed changes in arachidonic acid metabolism in serum grown isolates and changes in the amino acid metabolism between LBF and HBF isolates. Integrating these data, we were able to observe overlaps in the metabolic reprogramming of C. albicans isolates in serum with joint pathway analysis confirming changes in the fatty acid metabolic response in both transcriptomic and metabolomic data. Multivariate analysis by sPLS-DA identified several highly covariate discriminatory features with and without the presence of serum. These included many genes of currently unknown function and a downregulation of specific genes in serum including zinc transport. Through the application of transcriptomics and metabolomics we have demonstrated that these holistic methodologies are invaluable to biofilm research. We identified Molecular processes and metabolomic reprogramming of C. albicans in response to the biofilm inducing stimulus of serum. We also highlight the current and potential benefits that integration of multiple omics data sets provides. Integration is not without its challenges, however, and we identify some key methodologies that could improve interpretability of omics datasets derived from microbial communities. As Candida spp. do not exist within a vacuum, and infectious disease aetiology is dependent on the interactions between fungal and bacterial species, understanding the mechanisms that govern these biofilm models will help us to identify important factors and potential therapeutic strategies