Antimicrobial resistance classification using MALDI-TOF-MS is not that easy: lessons from vancomycin-resistant Enterococcus faecium
Authors: M. Brackmann, S.L. Leib, M. Tonolla, N. Schürch, M. Wittwer
Journal: Clinical Microbiology and Infection (2019)

Matrix-assisted Laser-Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a routinely applied technique to identify bacterial species. The acquired spectra show predominantly the molecular weights of ribosomal proteins. Once acquired, the spectra are compared with a database of either previously acquired spectra for which the organism is known or theoretical spectra based on predicted masses from sequence data, and identified based on identical peaks. Many efforts are undertaken to expand the information that can be extracted from these spectra to deduct further features regarding the antibiotic resistance phenotype or even the presence of virulence factors. To achieve this, large data sets of spectra of bacteria with different features are computationally analysed and their differences in masses are assumed to be distinctive for absence or presence of this feature. Enterococci are commensal bacteria of animal intestines but may also cause nosocomial infections in immunocompromised patients [[1]Vu J. Carvalho J. Enterococcus: review of its physiology, pathogenesis, diseases and the challenges it poses for clinical microbiology.Front Biol. 2011; 6: 357Crossref Scopus (39) Google Scholar]. Enterococcus faecium is naturally resistant against a number of antibiotics and easily acquires resistance to more antibiotics, e.g. vancomycin, by horizontal gene transfer. Even though alternative antibiotics for treatment of patients with vancomycin-resistant enterococcus (VRE) infections exist, VRE infections remain difficult to treat. Enterococcus faecium is a member of the ESKAPE group of pathogens that frequently display antibiotic resistance and are considered a major threat to modern medicine [[2]Pendleton J.N. Gorman S.P. Gilmore B.F. Clinical relevance of the ESKAPE pathogens.Exp Rev Anti-Infect Ther. 2013; 11: 297-308Crossref PubMed Scopus (613) Google Scholar]. Griffin et al. analysed the MS spectra of Enterococcus faecium from a hospital outbreak and correlated these with the presence or absence of vanA- or vanB-type resistance against vancomycin. Using a supervised learning approach, the study found a strong correlation between the presence of a set of peaks and the antibiotic resistance [[3]Griffin P.M. Price G.R. Schooneveldt J.M. Schlebusch S. Tilse M.H. Urbanski T. et al.Use of matrix-assisted laser desorption ionization–time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak.J Clin Microbiol. 2012; 50: 2918-2931Crossref PubMed Scopus (120) Google Scholar]. Here we investigate the suitability of this approach further by examining a peak at 5092 m/z in a spectrum of vancomycin-resistant Enterococcus faecium. Enterococcus faecium ST796 (vanB+) and a vancomycin-sensitive Enterococcus faecium (Orla-Jensen, ATCC 19434) were grown in liquid medium or on plates and lysed in a 1:1 mixture of 70% formic acid in water and acetonitrile. MALDI-TOF spectra were acquired from 3 kDa to 20 kDa. Swiss E. faecium ST796 showed a peak at the same mass as published by Griffin et al. [[3]Griffin P.M. Price G.R. Schooneveldt J.M. Schlebusch S. Tilse M.H. Urbanski T. et al.Use of matrix-assisted laser desorption ionization–time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak.J Clin Microbiol. 2012; 50: 2918-2931Crossref PubMed Scopus (120) Google Scholar]. For intact, exact mass measurements, proteins of the lysate were precipitated using 20% trichloroacetic acid and resuspended in a 1:1 mixture of water and acetonitrile. A fraction of the sample was reduced by addition of Tris(2-carboxyethly)phosphine. One microlitre of the reduced and untreated samples was spotted on the target plate and overlaid with α-cyano-4-hydroxycinnamic acid, (Bruker Daltonik GmbH, Bremen, Germany) (see Supplementary material, Appendix S1). The high signal intensity of the peak at 5092.12 m/z after protein precipitation allowed us to determine the protein sequence using MS/MS to a large extent and led to its identification as hiracin (Uniprot Q0Z8B6) or bacteriocin T8, a sec-dependent secretory protein [[4]Kwaadsteniet M.D. Fraser T. Reenen C.A.V. Dicks L.M.T. Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus.Appl Environ Microbiol. 2006; 72: 4761-4766Crossref PubMed Scopus (52) Google Scholar] (Fig. 1). The pre-protein has a theoretical molecular weight of 8156.65 Da; however, without the signal peptide, its theoretical average mass (MH+avg/red/theo) is 5094.73 Da, which is in agreement with what is published by Griffin et al. (5094.7 m/z) (Fig. 1c) [[3]Griffin P.M. Price G.R. Schooneveldt J.M. Schlebusch S. Tilse M.H. Urbanski T. et al.Use of matrix-assisted laser desorption ionization–time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak.J Clin Microbiol. 2012; 50: 2918-2931Crossref PubMed Scopus (120) Google Scholar]. The high resolution of TOF mass spectrometers operating in reflectron-mode permitted us to measure the monoisotopic mass of the protein (MH+mono/ox) at 5089.64 Da (see Supplementary material, Fig. S1). This is below the theoretical monoisotopic mass (MH+mono/red/theo) of the reduced protein at 5091.51 Da, but fairly close to the monoisotopic mass of the oxidized protein at 5089.49 Da (MH+mono/ox/theo) (Fig. 1c and see Supplementary material, Fig. S1). Hiracin harbours two cysteines, which are expected to be oxidized when the protein is secreted. Reduction of the protein sample before analysis resulted in an increase of molecular weight by two hydrogen atoms (MH+mono/red 5091.65 Da) (see Supplementary material, Fig. S1). Additionally, after reduction, the sequence coverage by MS/MS was increased mainly in the region between the two cysteines (Fig. 1b). To further analyse the usability of this protein as a marker for the identification of vanB-type vancomycin-resistant E. faecium, we computationally analysed all available vanB- or hirJM79-encoding genomes on refseq (tBlastn on refseq, complete genomes, Enterococcus faecium, Uniprot accession numbers Q0Z8B6 (HirJM79) and P97205 (VanB), >90% query cover, >90% identity) and checked if these two genes co-occur by comparison of the strains' BioSample accession numbers (Fig. 1d). We found 17 vanB-harbouring genomes and 11 that contained the hirJM79 gene. Interestingly, they co-occurred in four strains, yielding a predictive power of 36% (4/11 vanB/hirJM79 to hirJM79, Fig. 1d) [[5]Arredondo-Alonso S. Top J. Schürch A.C. McNally A. Puranen S. Pesonen M. et al.Genomes of a major nosocomial pathogen Enterococcus faecium are shaped by adaptive evolution of the chromosome and plasmidome.BioRxiv. 2019; : 530725Google Scholar]. We have shown here the identity of a presumed indicative protein peak for vancomycin resistance using state-of-the-art MALDI-TOF-MS/MS. The corresponding hiracin gene is not directly linked to the presence of vanB-type vancomycin resistance, so cannot be routinely used to classify resistance phenotypes. However, we expect that it can be used to investigate and diagnose VRE in an outbreak setting, where the strain is sequenced and presence or absence of the hirJM79 gene has been established, for both the VRE-outbreak-causing strain and a reference background. We strongly advocate to investigate the functional relationship between MALDI-MS peaks and resistance phenotype other than by pure correlation, so that these results can potentially be transferred to other diagnostic centres. Our study is limited by the measurement of only two strains and MS/MS sequencing of one of their proteins and so lacks statistical implications. We also did not use the strain of the original publication [[3]Griffin P.M. Price G.R. Schooneveldt J.M. Schlebusch S. Tilse M.H. Urbanski T. et al.Use of matrix-assisted laser desorption ionization–time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak.J Clin Microbiol. 2012; 50: 2918-2931Crossref PubMed Scopus (120) Google Scholar]. However, this study underscores the need for characterization of MALDI protein peaks to deduce meaningful information about the strain's phenotype. Another caveat of our bioinformatic analysis is a regional bias, as many of the strains with complete sequence stem from the Netherlands (49 of 126) [[5]Arredondo-Alonso S. Top J. Schürch A.C. McNally A. Puranen S. Pesonen M. et al.Genomes of a major nosocomial pathogen Enterococcus faecium are shaped by adaptive evolution of the chromosome and plasmidome.BioRxiv. 2019; : 530725Google Scholar]. Analysis of more MS spectra of several clinical microbiology laboratories together with either genome sequencing or antimicrobial susceptibility testing will further enhance our understanding of the distribution of these genes as well as their suitability as markers for resistance. The authors declare no conflict of interest. No external funding was received for this study. The authors acknowledge Dr Peter Keller (Institute for Infectious Diseases, University of Bern) for kindly providing the strains as well as Prof. Dr Andrea Endimiani (Institute for Infectious Diseases, University of Bern) and Dr Samuel Lüdin (Federal Office for Civil Protection, Spiez Laboratory) for fruitful discussions. Dr René Brunisholz (Functional Genomics Centre, Zürich) is acknowledged for technical advice and fruitful discussions.