Journal: bioRxiv

Article Title: Spatial pattern formation of bacterial colonies with social antibiotic resistance

doi: 10.1101/2024.08.21.608973

Figure Lengend Snippet: (A) Experimental setup to investigate homeland spatial organization. Left: A mixture of defined density and ratio of fluorescently labeled ampicillin-resistant E. faecalis bacteria expressing β-lactamase and ampicillin sensitive E. faecalis bacteria is added in a 2µl droplet to an agar plate containing growth media (Brain Heart Infusion), ampicillin, and spectinomycin to select for the fluorescent plasmids. Approximate scales of the plate diameter, droplet diameter and cell diameter are shown. Center left: The seeded colony grows overnight at optimal growth temperature, and is then imaged on a confocal fluorescence microscope, using a cover glass spacer to leave the colony at the agar-air interface unperturbed. The spatial arrangement inside the seeded colony area (the homeland) is analyzed. Center right: Overall density and ampicillin-resistant fraction of the colony, as well as ampicillin concentration were the input variables that were varied across different initial conditions to test their impact on spatial pattern formation. Right: Ampicillin-resistant and sensitive subpopulations are expected to have the following ecological interactions: Competition for space or nutrients within the colony area and ampicillin-induced death that is alleviated by degradation of antibiotic by the β-lactamase expressed by resistant bacteria. As these immotile bacteria maintain relatively fixed position in the spatially structured colony apart from cell-division and death processes, these ecological interactions are expected to be heterogeneous and neighborhood-dependent. Resistant cells are shown in green, sensitive cells in magenta and antibiotic concentration is encoded in blue. (B) Resistant and sensitive E. faecalis subpopulations form a diverse array of spatial patterns on agar. Representative images (maximum intensity z-projections) of spatial homeland organization of ampicillin-resistant (green, fluorophore mDasher) and ampicillin-sensitive (magenta, fluorophore mRudolph) bacteria after overnight growth. Scale bars = 500 µm; unless otherwise noted, all images in the same panel are at the same scale. “Rings” of varied intensity of the green signal at high ampicillin concentrations and low initial resistant fractions are imaging artifacts arising from the maximum intensity projection in combination with non-uniform illumination across angled surfaces.

Article Snippet: Single colonies of relevant strains were inoculated into ∼3ml Brain Heart Infusion (BHI) media (Remel) with 120 μg/ml spectinomycin sulfate (Thermo Fisher Scientific, 430.43 g/mol) to select for pBSU101 plasmids over night at 37C.

Techniques: Labeling, Bacteria, Expressing, Fluorescence, Microscopy, Concentration Assay, Imaging