Abstract
BackgroundAn accurate and timely identification of bacterial species is critical in clinical diagnostics. Species identification allows a potential first adaptation of empiric antibiotic treatments before the resistance profile is available. Matrix assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS) is a widely used method for bacterial species identification. However, important challenges in species identification remain. These arise from (i) incomplete databases, (ii) close relatedness of species of interest, and (iii) spectral quality, which is currently vaguely defined.MethodsWe selected 47 clinically relevant bacterial isolates from 39 species, which can be challenging to identify by MALDI-TOF MS. We measured these isolates under various analytical conditions on two MALDI-TOF MS systems. First, we identified spectral features, which were associated with correct species identification in three different databases. Considering these features, we then systematically compared spectra produced with three different sample preparation protocols. In addition, we varied quantities of bacterial colony material applied and bacterial colony age.ResultsWe identified (i) the number of ribosomal marker peaks detected, (ii) the median relative intensity of ribosomal marker peaks, (iii) the sum of the intensity of all detected peaks, (iv) a high measurement precision, and (v) reproducibility of peaks to act as good proxies of spectral quality. We found that using formic acid, measuring bacterial colonies at a young age, and frequently calibrating the MALDI-TOF MS device increase mass spectral quality. We further observed significant differences in spectral quality between different bacterial taxa and optimal measurement conditions vary per taxon.ConclusionWe identified and applied quality measures for MALDI-TOF MS and optimized spectral quality in routine settings. Phylogenetic marker peaks can be reproducibly detected and provide an increased resolution and the ability to distinguish between challenging species such as those within the Enterobacter cloacae complex, Burkholderia cepacia complex, or viridans streptococci.
Highlights
Matrix assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS) has revolutionised microbial diagnostics
In order to investigate spectral quality of the different datasets, we first assessed which spectral features are associated with a correct species identification with all databases and suitable as quantitative measures for spectral quality
The spectra analysed here include a range of mass spectral quality, and were acquired using all different sample preparation protocols examined in this study and for the species included in three databases (MALDI Biotyper, VitekMS, PAPMIDTM) (Supplementary Table S1)
Summary
Matrix assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS) has revolutionised microbial diagnostics. The mass of ribosomal subunits can be directly calculated from genomic sequences, as they are relatively conserved and rarely post-translationally modified Their potential to serve as MALDI-TOF MS biomarkers has been applied to clinically relevant phylogenetic groups (Hotta et al, 2010; Ziegler et al, 2015; Rothen et al, 2019), and multiple databases using marker masses predicted from genomic data are available (Kassim et al, 2017; Ojima-Kato et al, 2017; Tomachewski et al, 2018). These arise from (i) incomplete databases, (ii) close relatedness of species of interest, and (iii) spectral quality, which is currently vaguely defined
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