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