title: Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal.
abstract: BACKGROUND
  Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance.
  
  
  RESULTS
  From the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four vanA-containing E. faecium isolates in this study, and two of them harboured the purK1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of vanA-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.
  
  
  CONCLUSIONS
  The comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors.
authors.affiliation: Institute for Biotechnology and Bioengineering, Center of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. gigrejas@utad.pt.
authors.forename: Hajer
authors.initials: H
authors.lastname: Radhouani
authors.forename: Patrícia
authors.initials: P
authors.lastname: Poeta
authors.forename: Luís
authors.initials: L
authors.lastname: Pinto
authors.forename: Júlio
authors.initials: J
authors.lastname: Miranda
authors.forename: Céline
authors.initials: C
authors.lastname: Coelho
authors.forename: Carlos
authors.initials: C
authors.lastname: Carvalho
authors.forename: Jorge
authors.initials: J
authors.lastname: Rodrigues
authors.forename: María
authors.initials: M
authors.lastname: López
authors.forename: Carmen
authors.initials: C
authors.lastname: Torres
authors.forename: Rui
authors.initials: R
authors.lastname: Vitorino
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.forename: Gilberto
authors.initials: G
authors.lastname: Igrejas