Elucidating constraints for differentiation of major human Klebsiella pneumoniae clones using MALDI-TOF MS

Abstract

The establishment of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) in routine microbial identification boosted many developments towards high-throughput applications, including bacterial typing. However, results are still controversial for different bacterial species. We aim to evaluate the suitability of MALDI-TOF MS for typing clinically relevant multidrug resistant (MDR) Klebsiella pneumoniae subsp. pneumoniae clones using routine conditions and a previously validated chemometric analysis workflow. Mass spectra of 83 K. pneumoniae clinical isolates representing major human MDR clones [11 sequence types (STs), 22 PFGE-types] recovered in Portugal and Spain during outbreaks and non-outbreak situations (2003-2012) were obtained from cell extracts (CE) and intact cells (IC), and analysed with different chemometric tools. We observed a highly consistent peak pattern among isolates from different clones either with CE or IC, suggesting a high degree of conservation of biomolecules analysed (a large part corresponding to ribosomal proteins). Moreover, the low degree of agreement between MALDI-TOF MS and other methods (from 34.9 % to 43.4 % of correct assignments for CE and from 40.8 % to 70.1 % for IC) corroborates the low discriminatory potential of the technique at infraspecies level. Our results suggest a low discriminatory power of MALDI-TOF MS for clinically relevant MDR K. pneumoniae clones and highlight the need of developing tools for high-resolution typing in this species.

Typing and Species Identification of Clinical Klebsiella Isolates by Fourier Transform Infrared Spectroscopy and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…The highly distinct capacities of FTIR and MALDI-TOF MS to delineate Klebsiella isolates support the notion that the "target structures" of the two methods are fundamentally different. Our findings are in line with a recent study that analyzed 83 K. pneumoniae isolates representing major multidrug-resistant clones from southern Europe and found low agreement between MALDI-TOF MS and other methods (40).…”

Overestimated discriminatory power of MALDI-TOF mass spectrometry for typing of carbapenem-resistantKlebsiella pneumoniaeclones

“…Two groups [15 16] reported that MALDI-TOF MS was a promising tool for this purpose but the low number of isolates (10 of 6 PFGE types) in one study [15] and the lack of calculation of the congruence between MALDI-TOF MS and other reference methods (WGS, PFGE or MLST) [16] limits firm conclusions to be made. The other three studies [17][18][19] found MALDI-TOF-based typing to be less discriminatory than whole genome sequencing, PFGE or MLST for typing K. pneumoniae. The limitations of MALDI-TOF-based typing were further pointed out by Spinali et al, [20] and Sauget et al, [21] who attributed its shortcomings as a typing tool to technological and biological factors such as strain sets, culture conditions, definition of specific MS peaks, statistical analysis, etc.…”

“…Several attempts have been made to apply MALDI-TOF MS to resolve between strains of K. pneumoniae, but with different conclusions [15][16][17][18][19]. Two groups [15 16] reported that MALDI-TOF MS was a promising tool for this purpose but the low number of isolates (10 of 6 PFGE types) in one study [15] and the lack of calculation of the congruence between MALDI-TOF MS and other reference methods (WGS, PFGE or MLST) [16] limits firm conclusions to be made.…”

Identification of Microorganisms by Liquid Chromatography-Mass Spectrometry (LC-MS1) and in silico Peptide Mass Data

“…While a large number of studies convincingly demonstrate successful discrimination and identification of pathogenic bacteria by MALDI-TOF MS at the species level, there is also ample evidence for limitations of the taxonomic resolution, particularly at the infraspecies level and when dealing with differentiation of genetically closely related species (Lasch et al, 2014;Sousa et al, 2015;Rodrigues et al, 2016;Grenga et al, 2019). For example, differentiation between Escherichia coli and Shigella (He et al, 2010;Paauw et al, 2015) or of Bacillus cereus and Bacillus anthracis (Dybwad et al, 2013;Lasch et al, 2015) required additional measures beyond the standard microbial identification workflow, such as custom reference libraries, higher levels of standardization, and/or sophisticated data analysis concepts (machine learning, etc.).…”

“…For example, differentiation between Escherichia coli and Shigella (He et al, 2010;Paauw et al, 2015) or of Bacillus cereus and Bacillus anthracis (Dybwad et al, 2013;Lasch et al, 2015) required additional measures beyond the standard microbial identification workflow, such as custom reference libraries, higher levels of standardization, and/or sophisticated data analysis concepts (machine learning, etc.). In these studies the reduced discriminatory power of MALDI-TOF MS has been attributed to the restricted m/z range (m/z 2000-20,000) and the dependence from mass patterns produced by a sub-proteome of small, abundant and basic proteins, mainly ribosomal subunit proteins (Rodrigues et al, 2016). Since the molecular evolution of these proteins is rather slow, ribosomal proteins are supposed to offer only limited taxonomic specificity.…”