Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates

• Whole-cell MALDI-TOF MS analysis of 120 bacterial isolates (60 Enterococcus spp. and 60 Escherichia coli)
• Streamline protocol from the bacteria seed, up to the statistical analysis of 1200 mass spectra by MassUp program
• Specific mass peaks that discriminate between the bacterial genus studied and from 3 species of Enterococcus
• Differentiation of spectra from Gram-positive and -negative bacteria proven by principal component analysis and clustering
• Insufficient discriminatory power of MALDI/statistical protocol for the differentiation of enterococci species

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.Biological significanceThis 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.

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