title: Metabolomic profiling of Burkholderia pseudomallei using UHPLC-ESI-Q-TOF-MS reveals specific biomarkers including 4-methyl-5-thiazoleethanol and unique thiamine degradation pathway.
abstract: BACKGROUND
  Burkholderia pseudomallei is an emerging pathogen that causes melioidosis, a serious and potentially fatal disease which requires prolonged antibiotics to prevent relapse. However, diagnosis of melioidosis can be difficult, especially in culture-negative cases. While metabolomics represents an uprising tool for studying infectious diseases, there were no reports on its applications to B. pseudomallei. To search for potential specific biomarkers, we compared the metabolomics profiles of culture supernatants of B. pseudomallei (15 strains), B. thailandensis (3 strains), B. cepacia complex (14 strains), P. aeruginosa (4 strains) and E. coli (3 strains), using ultra-high performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). Multi- and univariate analyses were used to identify specific metabolites in B. pseudomallei.
  
  
  RESULTS
  Principal component and partial-least squares discrimination analysis readily distinguished the metabolomes between B. pseudomallei and other bacterial species. Using multi-variate and univariate analysis, eight metabolites with significantly higher levels in B. pseudomallei were identified. Three of the eight metabolites were identified by MS/MS, while five metabolites were unidentified against database matching, suggesting that they may be potentially novel compounds. One metabolite, m/z 144.048, was identified as 4-methyl-5-thiazoleethanol, a degradation product of thiamine (vitamin B1), with molecular formula C6H9NOS by database searches and confirmed by MS/MS using commercially available authentic chemical standard. Two metabolites, m/z 512.282 and m/z 542.2921, were identified as tetrapeptides, Ile-His-Lys-Asp with molecular formula C22H37N7O7 and Pro-Arg-Arg-Asn with molecular formula C21H39N11O6, respectively. To investigate the high levels of 4-methyl-5-thiazoleethanol in B. pseudomallei, we compared the thiamine degradation pathways encoded in genomes of B. pseudomallei and B. thailandensis. While both B. pseudomallei and B. thailandensis possess thiaminase I which catalyzes degradation of thiamine to 4-methyl-5-thiazoleethanol, thiM, which encodes hydroxyethylthiazole kinase responsible for degradation of 4-methyl-5-thiazoleethanol, is present and expressed in B. thailandensis as detected by PCR/RT-PCR, but absent or not expressed in all B. pseudomallei strains. This suggests that the high 4-methyl-5-thiazoleethanol level in B. pseudomallei is likely due to the absence of hydroxyethylthiazole kinase and hence reduced downstream degradation.
  
  
  CONCLUSIONS
  Eight novel biomarkers, including 4-methyl-5-thiazoleethanol and two tetrapeptides, were identified in the culture supernatant of B. pseudomallei.
authors.affiliation: State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Room 423, University Pathology Building, Queen Mary Hospital, Pok Fu Lam, Hong Kong ; Research Centre of Infection and Immunology, The University of Hong Kong, Pok Fu Lam, Hong Kong ; Carol Yu Centre for Infection, The University of Hong Kong, Pok Fu Lam, Hong Kong ; Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Susanna K P
authors.initials: SK
authors.lastname: Lau
authors.affiliation: Department of Pathology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Ching-Wan
authors.initials: CW
authors.lastname: Lam
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Shirly O T
authors.initials: SO
authors.lastname: Curreem
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Kim-Chung
authors.initials: KC
authors.lastname: Lee
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Wang-Ngai
authors.initials: WN
authors.lastname: Chow
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Candy C Y
authors.initials: CC
authors.lastname: Lau
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Siddharth
authors.initials: S
authors.lastname: Sridhar
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Sally C Y
authors.initials: SC
authors.lastname: Wong
authors.affiliation: Ocean Park Corporation, Aqua City, Hong Kong.
authors.forename: Paolo
authors.initials: P
authors.lastname: Martelli
authors.affiliation: Ocean Park Corporation, Aqua City, Hong Kong.
authors.forename: Suk-Wai
authors.initials: SW
authors.lastname: Hui
authors.affiliation: State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Room 423, University Pathology Building, Queen Mary Hospital, Pok Fu Lam, Hong Kong ; Research Centre of Infection and Immunology, The University of Hong Kong, Pok Fu Lam, Hong Kong ; Carol Yu Centre for Infection, The University of Hong Kong, Pok Fu Lam, Hong Kong ; Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
authors.affiliation: State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Room 423, University Pathology Building, Queen Mary Hospital, Pok Fu Lam, Hong Kong ; Research Centre of Infection and Immunology, The University of Hong Kong, Pok Fu Lam, Hong Kong ; Carol Yu Centre for Infection, The University of Hong Kong, Pok Fu Lam, Hong Kong ; Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
authors.forename: Patrick C Y
authors.initials: PC
authors.lastname: Woo
keywords: Biomarkers; Burkholderia pseudomallei; Metabolomics; Specific