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

Moura H, Woolfitt AR, Carvalho MG, Pavlopoulos A, Teixeira LM, Satten GA, Barr JR - FEMS Immunol. Med. Microbiol. (2008)

Bottom Line: Reference strains of other bacterial species were also analyzed and compared with the GAS isolates.Statistical analysis confirmed method reproducibility.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341, USA.

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.

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Related in: MedlinePlus

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.
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fig01: 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.

Mentions: Three batches of S. pyogenes genome strain grown in liquid media were analyzed by MALDI-TOF MS. Spectra obtained on 106–108 whole bacteria μL−1 revealed complex spectral patterns with 20–40 peaks in the m/z range corresponding to 1000–6000 Da or 2000–14 000 Da. Observed mass spectra are believed to consist primarily of protonated peptide and protein signals, although some signals below m/z 4000 may represent other materials. The mass range corresponding to 2000−14 000 Da was preferred for this study because the peaks in this region were more abundant and more consistent. A representative example of S. pyogenes mass spectra is shown in Fig. 1a, while Fig. 1b depicts the reproducible patterns from denoised summed spectra revealed using multispec viewer. A γ-irradiated subset from batch 2 is included in Fig. 1b and shows few changes in the summed spectra as compared with the MALDI-TOF spectra obtained on whole live organisms. Visual analysis of processed spectra using multispec viewer revealed only a minor variation from day to day and from well to well in the quadruplicate analysis of the suspensions containing 106 or 107 bacterial cells. Figure 2 shows the spectra and a gel view representation of the mass spectra obtained from a single colony and from pools of 2, 3, 4, 5, and 30 colonies of S. pyogenes grown on blood agar and loaded directly onto the MALDI target. These spectra showed altered peaks, along with a set of similar but noisier peaks when compared with those obtained from cells grown in liquid media. Although a higher level of background noise was present in cells grown on blood agar, a key set of peaks corresponding to m/z 4451, 5951, 6310, 7962, 8181, and 9030 and representing potential biomarkers were consistently seen in all wells, as is easily observed in the denoised spectra depicted in Fig. 2.


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

Moura H, Woolfitt AR, Carvalho MG, Pavlopoulos A, Teixeira LM, Satten GA, Barr JR - FEMS Immunol. Med. Microbiol. (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2658026&req=5

fig01: 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.
Mentions: Three batches of S. pyogenes genome strain grown in liquid media were analyzed by MALDI-TOF MS. Spectra obtained on 106–108 whole bacteria μL−1 revealed complex spectral patterns with 20–40 peaks in the m/z range corresponding to 1000–6000 Da or 2000–14 000 Da. Observed mass spectra are believed to consist primarily of protonated peptide and protein signals, although some signals below m/z 4000 may represent other materials. The mass range corresponding to 2000−14 000 Da was preferred for this study because the peaks in this region were more abundant and more consistent. A representative example of S. pyogenes mass spectra is shown in Fig. 1a, while Fig. 1b depicts the reproducible patterns from denoised summed spectra revealed using multispec viewer. A γ-irradiated subset from batch 2 is included in Fig. 1b and shows few changes in the summed spectra as compared with the MALDI-TOF spectra obtained on whole live organisms. Visual analysis of processed spectra using multispec viewer revealed only a minor variation from day to day and from well to well in the quadruplicate analysis of the suspensions containing 106 or 107 bacterial cells. Figure 2 shows the spectra and a gel view representation of the mass spectra obtained from a single colony and from pools of 2, 3, 4, 5, and 30 colonies of S. pyogenes grown on blood agar and loaded directly onto the MALDI target. These spectra showed altered peaks, along with a set of similar but noisier peaks when compared with those obtained from cells grown in liquid media. Although a higher level of background noise was present in cells grown on blood agar, a key set of peaks corresponding to m/z 4451, 5951, 6310, 7962, 8181, and 9030 and representing potential biomarkers were consistently seen in all wells, as is easily observed in the denoised spectra depicted in Fig. 2.

Bottom Line: Reference strains of other bacterial species were also analyzed and compared with the GAS isolates.Statistical analysis confirmed method reproducibility.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341, USA.

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.

Show MeSH
Related in: MedlinePlus