title: Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates.
abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify intact bacteria than conventional microbiology and/or molecular biology methods. The MALDI-TOF MS method is potentially applicable in diagnostic laboratories to characterize commensal bacterial species, some of which are major pathogens, from human or animal gastrointestinal tracts. The aim of this study was to analyze at the cluster and statistical level the capacity of MALDI-TOF MS to distinguish between previously characterized enterococci and Escherichia coli isolated from wild birds of the Azores archipelago. Soluble proteins were extracted from intact cell cultures of 60 isolates of Enterococcus spp. and 60 isolates of E. coli by an expedient method. MALDI-TOF MS was used to obtain 1200 mass spectra that were statistically analyzed and compared. A total of 215 distinct mass-to-charge (m/z) peaks were obtained, including a peak at m/z 4428±3, which is exclusively found in spectra from Enterococcus isolates, and peaks at m/z 5379±3 and m/z 6253±3, which are only detected in spectra from E. coli isolates. By processing mass spectra and analyzing them statistically with MassUp software, including principal component analysis (PCA) and clustering, it was possible to correctly distinguish between isolates of Enterococcus and Escherichia genera. The results of the proteomic analysis confirm that these tools could be used to characterize whole bacterial cells. In the future, with an optimized protocol for obtaining plasmid-associated proteins and the further development of bioinformatics methods, it is likely that mass peak characteristic of antimicrobial resistance will be detected, increasing the potential usefulness of MALDI-TOF in routine clinical assays.
  
  
  CONCLUSIONS
  This study highlights the importance of MALDI-TOF MS in the rapid and reliable identification of bacteria by whole-cell analysis. The mass spectrometry approach performed in this study further contributes for the microbial biomarker discovery culminating in a preferable bacteria identification and antimicrobial resistance tool for the future of clinical microbiology. This article is part of a Special Issue entitled: HUPO 2014.
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Tiago
authors.initials: T
authors.lastname: Santos
authors.affiliation: Bioscope Group, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal; REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Capelo
authors.affiliation: Bioscope Group, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal; REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Santos
authors.affiliation: Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Irene
authors.initials: I
authors.lastname: Oliveira
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Marinho
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Gonçalves
authors.affiliation: Bioscope Group, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal; REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: José Eduardo
authors.initials: JE
authors.lastname: Araújo
authors.affiliation: Centre of Studies of Animal and Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Poeta
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. Electronic address: gigrejas@utad.pt.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Igrejas
mesh_terms: D000818:Animals; D001717:Birds / Q000382:microbiology; D016983:Enterococcus / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins
keywords: Bacterial identification; Enterococcus spp.; Escherichia coli; MALDI-TOF MS; MassUp; Statistical analysis