Journal: Communications Biology

Article Title: Single-cell phenotypic profiling and backtracing exposes and predicts clinically relevant subpopulations in isogenic Staphylococcus aureus communities

doi: 10.1038/s42003-024-06894-z

Figure Lengend Snippet: A Schematic overview of the CPPT setup for comparative analysis of VISA strain Mu50 and a VSSA control. Cells were labelled with Vancomycin-BFL and PI. PI (+) -cells are depicted in gray, PI (–) -cells with different degrees of Vanco-BFL-labeling are indicated in light and dark green. PI (–) -cells were sorted onto Mueller-Hinton Agar (MHA) supplemented with different concentrations of vancomycin and colony formation was observed at different time points and for individual plates by time-lapse imaging. For VISA cells growth on a 0.25× MIC plate was used for cell fate determination (regular colony, delayed, smaller colony or no colony) prior to phenotypic backtracing of the ancestral phenotypic signatures in the flow cytometry dot plot. The exemplary dot plot shows all recorded Vanco-BFL ( +) , PI (–) events in light green, Vanco-BFL (+) , PI (+) -events in grey. The individually colored dots correspond to backtraced, sorted cells that were determined to belong the ´regular colony´ cell fate category. B Flow cytometry histogram showing the fluorescence signal in the Vanco-BFL-A channel of unstained and Vanco-BFL-labelled VSSA (blue) and VISA cells (red)´. C Percentage of sorted events that gave rise to colonies on agar plates with different concentrations of vancomycin. D Violin plot showing the cellular Vanco-BFL signal intensity of VISA cells for the different cell fate groups determined after growth on 0.25xMIC vancomycin. The graph shows the results of N = 960 sorted events from a single pre-sorting culture and is representative for 4 biological replicates. Statistical significance testing was performed by Kruskal–Wallis test in combination with Dunn´s multiple comparisons test. E Colony growth dynamics of sorted VISA cells in the presence (0.25xMIC) and absence of vancomycin as analyzed by time-lapse imaging analysis (compare Supplementary Movies , ). Growth ( Y -axis) was measured as contour size on the edge of the colony and is plotted against cultivation time. Raw data and calculations are available in Supplementary Data . The figure was created with BioRender.

Article Snippet: Single colonies (3 biological replicates) of the vancomycin susceptible S. aureus strain ATCC 29213 and representative clinical MRSA-VISA strain Mu50 (ATCC 700699) were grown overnight in TSB at 37 °C.

Techniques: Control, Labeling, Imaging, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: Single-cell phenotypic profiling and backtracing exposes and predicts clinically relevant subpopulations in isogenic Staphylococcus aureus communities

doi: 10.1038/s42003-024-06894-z

Figure Lengend Snippet: Bacterial strains used in this study

Article Snippet: Single colonies (3 biological replicates) of the vancomycin susceptible S. aureus strain ATCC 29213 and representative clinical MRSA-VISA strain Mu50 (ATCC 700699) were grown overnight in TSB at 37 °C.

Techniques: Isolation, Plasmid Preparation

Journal: Scientific Reports

Article Title: Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein

doi: 10.1038/s41598-021-97844-w

Figure Lengend Snippet: Identification of PBP2a mecA protein from MRSA cell extract by LC–MS/MS. ( a ) Intact wild-type PBP2a mecA MS spectrum from a representative MRSA strain (ATCC 33,591) separated by LC. Intact protein precursor ion charge states are labelled from 80 to 110. Data are representative for multiple MRSA strains (n ≥ 3). ( b ) Recombinant His 6 -PBP2a mecA MS spectrum acquired using direct infusion. Identical precursor ion charges states are labelled as wild-type PBP2a mecA spectrum. ( c ) Intact wild-type PBP2a mecA MS spectrum produced during LC separation of cell extract and PTCR-mediated separation of superposed protein ion populations. Isolation window was centered at m / z 777 with a width of 5 m / z . Precursor ion charge states are labelled from 80 to 95. Data are representative of multiple technical replicates from the same MRSA cell extract (ATCC 33,591). ( d ) MS/MS spectrum of intact wild-type PBP2a mecA precursor ion at m / z 793 (charge state = 102), using 1.5 m / z isolation window and fragmented with an HCD collision energy of 10 eV. Abundant N-terminal ( b -ions) and C-terminal ( y -ions) fragment ions are labelled. Fragmentation data are representative for multiple charge states and different MRSA strains (n ≥ 100). ( e ) Associated b- and y-ion fragment location for MS/MS of intact wild-type PBP2a mecA protein indicated by purple vertical lines. Red colored amino acids indicate protein coverage (72.2%) for complementary bottom-up peptide analysis and protein characterization. Protein coverage associated with peptide data are from multiple analyses (n = 12).

Article Snippet: Figure 1 Identification of PBP2a mecA protein from MRSA cell extract by LC–MS/MS. ( a ) Intact wild-type PBP2a mecA MS spectrum from a representative MRSA strain (ATCC 33,591) separated by LC.

Techniques: Liquid Chromatography with Mass Spectroscopy, Recombinant, Produced, Isolation, Tandem Mass Spectroscopy

Journal: Scientific Reports

Article Title: Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein

doi: 10.1038/s41598-021-97844-w

Figure Lengend Snippet: PBP2a mecA protein detection using in-source generated peptide-like fragments and MS/MS fragmentation. (a , b ) Source-induced dissociated N-terminal peptide-like fragments from PBP2a mecA (ATCC MRSA isolate BAA-44) at precursor m / z 596.8961 (a) and m / z 653.4382 ( b ) for selected ion monitoring (insets) and associated representative targeted MS/MS spectra (n ≥ 100). ( c ) Synthetic peptide confirmation of source-induced dissociated peptide-like precursor m / z 596.8961 and associated MS/MS spectrum (inset spectrum series displays peptide spectrum without in-source energy, with in-source dissociation, and magnified spectrum of target precursor following in-source dissociation). ( d ) Synthetic peptide confirmation of source-induced dissociated peptide-like precursor m / z 653.4382 and associated MS/MS spectrum (inset spectrum series displays peptide spectrum without in-source energy, with in-source dissociation, and magnified spectrum of target precursor following in-source dissociation). Data collection for synthetic peptides was repeated multiple times on different MS instruments (n = 3).

Article Snippet: Figure 1 Identification of PBP2a mecA protein from MRSA cell extract by LC–MS/MS. ( a ) Intact wild-type PBP2a mecA MS spectrum from a representative MRSA strain (ATCC 33,591) separated by LC.

Techniques: Generated, Tandem Mass Spectroscopy

Journal: Scientific Reports

Article Title: Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein

doi: 10.1038/s41598-021-97844-w

Figure Lengend Snippet: PBP2a mecC protein detection using in-source generated peptide-like fragments and MS/MS fragmentation. ( a , b ) Source-induced dissociated N-terminal peptide-like fragments from PBP2a mecC (ATCC MRSA isolate BAA-2312) at precursor m / z 658.9042 ( a ) and m / z 715.4462 ( b ) for selected ion monitoring (insets) and associated representative targeted MS/MS spectra (n ≥ 100). ( c ) Synthetic peptide confirmation of source-induced dissociated peptide-like precursor m / z 658.9042 and associated MS/MS spectrum (inset spectrum series displays peptide spectrum without in-source energy, with in-source dissociation, and magnified spectrum of target precursor following in-source dissociation). ( d ) Synthetic peptide confirmation of source-induced dissociated peptide-like precursor m / z 715.4462 and associated MS/MS spectrum (inset spectrum series displays peptide spectrum without in-source energy, and magnified spectrum of target precursor following in-source dissociation). Data collection for synthetic peptides was repeated multiple times on different MS instruments (n = 3).

Article Snippet: Figure 1 Identification of PBP2a mecA protein from MRSA cell extract by LC–MS/MS. ( a ) Intact wild-type PBP2a mecA MS spectrum from a representative MRSA strain (ATCC 33,591) separated by LC.

Techniques: Generated, Tandem Mass Spectroscopy

Journal: Scientific Reports

Article Title: Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein

doi: 10.1038/s41598-021-97844-w

Figure Lengend Snippet: Evaluation of source-induced peptide detection of PBP2a across representative MRSA panel and clinical isolates. ( a ) Performance of source-induced detection method for PBP2a mecA (top panel) and PBP2a mecC (bottom panel) N-terminal peptide-like fragments for MRSA strains exhibiting different SCC mec and PFGE genetic backgrounds, as well as negative MSSA isolates (listed in Supplemental Table ). Strains were cultured on TSA plates and harvest after overnight growth. Colored bars represent the mean maximum intensity for each product ion over a 60-min LC protein separation. Results are from distinct biological replicates (n = 3). ( b ) Performance of MSSA and MRSA quality control strains along with clinical isolates over five-minute PBP2a mecA detection method using custom SPE trap and source-induced dissociation method. Strains were cultured on Blood Agar plates and harvested after overnight growth. Data are the mean maximum ion intensity for each product ion and calculated from distinct biological replicates (n = 2 or 3).

Article Snippet: Figure 1 Identification of PBP2a mecA protein from MRSA cell extract by LC–MS/MS. ( a ) Intact wild-type PBP2a mecA MS spectrum from a representative MRSA strain (ATCC 33,591) separated by LC.

Techniques: Cell Culture, Control