Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens.

Jana Havlikova,Robin C May,Helen J Cooper,Iain B Styles

doi:10.1021/jasms.0c00466

Abstract

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species (E. faecium E745, K. pneumoniae KP257, A. baumannii AYE, and E. cloacae S11) as well as E. faecalis V583 (a close relative of E. faecium) and a clinical isolate of A. baumannii AC02 using an optimized solvent sampling system. In each case, top-down LESA MS/MS was employed for protein identification. In total, 24 proteins were identified from 37 MS/MS spectra by searching against protein databases for the individual species. The MS/MS spectra for the identified proteins were subsequently searched against multiple databases from multiple species in an automated data analysis workflow with a view to determining the accuracy of identification of unknowns. Out of 24 proteins, 19 were correctly assigned at the protein and species level, corresponding to an identification success rate of 79%.

Highlights

The ESKAPE pathogens are six clinically relevant bacterial species Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., of which two are Gram-positive (E. faecium, S. aureus) and the remainder are Gram-negative.[1]
Bacterial strains E. faecium E745, E. faecalis V583, and K. pneumoniae KP257 were obtained from Willem van Schaik (Institute of Microbiology and Infection (IMI), University of Birmingham, UK), S. aureus MSSA476 and P. aeruginosa PS1054 were obtained from Mark Webber (Quadram Institute, Norwich, UK), A. baumannii AYE and AC02 were obtained from Jessica Blair (IMI, University of Birmingham, UK), and E. cloacae S11 was obtained from Allan McNally (IMI, University of Birmingham, UK)
Initial liquid extraction surface analysis mass spectrometry (LESA mass spectrometry (MS)) experiments with the 50:45:5 extraction solvent system of Gram-positive Enterococci species resulted in the observation of no protein peaks for E. faecalis V583 and only a few, low abundance protein peaks for E. faecium E745 (Figure 1a,b)

Summary

Introduction

The ESKAPE pathogens are six clinically relevant bacterial species Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., of which two are Gram-positive (E. faecium, S. aureus) and the remainder are Gram-negative.[1]. The use of mass spectrometry (MS) for the analysis and identification of bacteria is well-established. The gold standard, FDA-approved mass spectrometry (MS) approach for identification of microbes is matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) MS together with dedicated software for spectral fingerprinting.[7] MALDI TOF MS has some drawbacks for bacterial analysis including sample preparation requirements and the fact that analysis takes place under vacuum conditions, precluding analysis of live colonies. Ambient ionization MS techniques overcome these limitations.[8−16] One such ambient technique is liquid extraction surface analysis (LESA) MS, a liquid microjunction sampling tool, based on diffusion of analytes into a droplet of solvent.[17] The droplet is subsequently introduced into the mass spectrometer via chip-based nanoelectrospray (nanoESI)

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