ATP quantification was performed on cells extracted from homogenized colony biofilms grown on YESCA agar for 11 days. One aliquot of biofilm cells was removed, and ATP concentration was determined using the Cell-Glo Titer kit (Promega) according to manufacturer’s protocols. Briefly, 50 μL of bacterial suspension was mixed with an equal volume of Cell-Glo Titer reagent and incubated with shaking at room temperature for 15 min. Luminescence was measured on a SpectraMax i3 plate reader (Molecular Devices) and converted to ATP concentration using a standard curve. A separate aliquot of the same sample was serially diluted for CFU enumeration. To account for differences in the number of cells between samples, ATP concentration was normalized to CFU per biofilm.

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