title: A novel metabolomic approach used for the comparison ofStaphylococcus aureusplanktonic cells and biofilm samples.
abstract: BACKGROUND
  Bacterial cell characteristics change significantly during differentiation between planktonic and biofilm states. While established methods exist to detect and identify transcriptional and proteomic changes, metabolic fluctuations that distinguish these developmental stages have been less amenable to investigation.
  
  
  OBJECTIVE
  The objectives of the study were to develop a robust reproducible sample preparation methodology for high throughput biofilm analysis and to determine differences between Staphylococcus aureus in planktonic and biofilm states.
  
  
  METHODS
  The method uses bead beating in a chloroform/methanol/water extraction solvent to both disrupt cells and quench metabolism. Verification of the method was performed using liquid-chromatography-mass spectrometry. Raw mass-spectrometry data was analysed using an in-house bioinformatics pipe-line incorporating XCMS, MzMatch and in-house R-scripts, with identifications matched to internal standards and metabolite data-base entries.
  
  
  RESULTS
  We have demonstrated a novel mechanical bead beating method that has been optimised for the extraction of the metabolome from cells of a clinical Staphylococcus aureus strain existing in a planktonic or biofilm state. This high-throughput method is fast and reproducible, allowing for direct comparison between different bacterial growth states. Significant changes in arginine biosynthesis were identified between the two cell populations.
  
  
  CONCLUSIONS
  The method described herein represents a valuable tool in studying microbial biochemistry at a molecular level. While the methodology is generally applicable to the lysis and extraction of metabolites from Gram positive bacteria, it is particularly applicable to biofilms. Bacteria that exist as a biofilm are shown to be highly distinct metabolically from their 'free living' counterparts, thus highlighting the need to study microbes in different growth states. Metabolomics can successfully distinguish between a planktonic and biofilm growth state. Importantly, this study design, incorporating metabolomics, could be optimised for studying the effects of antimicrobials and drug modes of action, potentially providing explanations and mechanisms of antibiotic resistance and to help devise new antimicrobials.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, The University of Glasgow, Glasgow, UK ; Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, The University of Glasgow, Garscube Estate, Bearsden, Scotland G61 1QH UK.
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Stipetic
authors.affiliation: Institute of Molecular Cell and Systems Biology, The University of Glasgow, Glasgow, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Dalby
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, The University of Glasgow, Glasgow, UK.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Davies
authors.affiliation: Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, The University of Glasgow, Garscube Estate, Bearsden, Scotland G61 1QH UK.
authors.forename: Fraser R
authors.initials: FR
authors.lastname: Morton
authors.affiliation: Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, The University of Glasgow, Glasgow, UK.
authors.forename: Gordon
authors.initials: G
authors.lastname: Ramage
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, The University of Glasgow, Glasgow, UK ; Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, The University of Glasgow, Garscube Estate, Bearsden, Scotland G61 1QH UK.
authors.forename: Karl E V
authors.initials: KE
authors.lastname: Burgess
keywords: Biofilms; Metabolite extraction; Metabolomics; S. aureus