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2016 ; 12
(ä): 75
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A novel metabolomic approach used for the comparison of Staphylococcus aureus
planktonic cells and biofilm samples
#MMPMID27013931
Stipetic LH
; Dalby MJ
; Davies RL
; Morton FR
; Ramage G
; Burgess KE
Metabolomics
2016[]; 12
(ä): 75
PMID27013931
show ga
INTRODUCTION: 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. OBJECTIVES: 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.