MALDI-TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates

Abstract

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.

Fig. 1

MALDI-TOF mass spectra (m/z 2000–14 000 Da) for Streptococcus pyogenes ATCC 700294 whole cells grown in liquid media (THB). (a) Represents typical raw mass spectra of 10⁷ cells. (b) Depicts the reproducible patterns in denoised spectra revealed using multispec viewer after analyzing several runs of S. pyogenes (batches 1, 2, and 3), including a subset from batch 2 that was γ irradiated. Analyses were in quadruplicate in each case.

Fig. 2

multispec viewer depicting the influence of different colony numbers and culture media (blood agar and THB) in spectra variability of Streptococcus pyogenes ATCC 700294. (a) Mass spectra. Annotated denoised peaks are consistently seen in all wells. (b) Gel view feature to the mass spectra. From bottom to top, numbers 1, 2, 3, 4, 5, and 6 denote groups of four spectra obtained, respectively, from 1, 2, 3, 4, 5, and 30 colonies of S. pyogenes grown on blood agar and directly plated onto the MALDI target; numbers 7 and 8 are spectra obtained from washed cells from 30 colonies grown on blood agar (7) and 10⁸ cells grown in THB (8).

Fig. 3

multispec viewer of MALDI-TOF denoised mass spectra (m/z 2000–14 000 Da) for whole cells of (1) Streptococcus pyogenes ATCC 700294, (2) Streptococcus agalactiae, (3) Enterococcus faecalis, (4) Staphylococcus aureus, and (5) Escherichia coli, revealing differences among genus and species. Quadruplicate analyses are shown for each organism.

Fig. 4

Dendrogram obtained with past (http://folk.uio.no/ohammer/past/index.html) and corresponding gel view of mass spectra using multispec viewer, where differences between Streptococcus agalactiae and Streptococcus pyogenes could be detected visually. GAS isolates were clustered together and subclusters of invasive and noninvasive isolates of S. pyogenes could be detected. For this analysis, all spectra for each strain were summed to give one representative spectrum per organism.