title: A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied toCandida albicansandStaphylococcus aureusbiofilms.
abstract: BACKGROUND
  Combined infections fromCandida albicansandStaphylococcus aureusare a leading cause of death in the developed world. Evidence suggests thatCandidaenhances the virulence ofStaphylococcus-hyphae penetrate through tissue barriers, whileS. aureustightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state,C. albicansenhances the antimicrobial resistance characteristics ofS. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence.
  
  
  OBJECTIVE
  To explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards.
  
  
  METHODS
  OptimisedC. albicansandS. aureusmono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media.
  
  
  RESULTS
  The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation.
  
  
  CONCLUSIONS
  Evaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids.
authors.affiliation: Polyomics, University of Glasgow, 211 Wolfson Wohl Translational Cancer Research Centre, Garscube Campus, Glasgow, G61 1QH UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Weidt
authors.affiliation: Polyomics, University of Glasgow, 211 Wolfson Wohl Translational Cancer Research Centre, Garscube Campus, Glasgow, G61 1QH UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Haggarty
authors.affiliation: Oral Sciences Research Group, Glasgow Dental School, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Ryan
authors.initials: R
authors.lastname: Kean
authors.affiliation: ThermoFisher Scientific, Hemel Hempstead, UK.
authors.forename: Cristian I
authors.initials: CI
authors.lastname: Cojocariu
authors.affiliation: ThermoFisher Scientific, Hemel Hempstead, UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Silcock
authors.affiliation: Oral Sciences Research Group, Glasgow Dental School, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Ranjith
authors.initials: R
authors.lastname: Rajendran
authors.affiliation: Oral Sciences Research Group, Glasgow Dental School, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Gordon
authors.initials: G
authors.lastname: Ramage
authors.affiliation: Polyomics, University of Glasgow, 211 Wolfson Wohl Translational Cancer Research Centre, Garscube Campus, Glasgow, G61 1QH UK.
authors.forename: Karl E V
authors.identifier: 0000-0002-0881-715X
authors.initials: KE
authors.lastname: Burgess
keywords: Biofilm; Candida; Metabolomics; Staphylococcus