Figure 2. Continued metabolic activity of tetracycline-treated cells results in cell volume expansion.

(a) Fold-change of cell volume of surface-attached isolated cells grown in a microfluidic device, and planktonic cells grown in liquid culture with or without tetracycline (Tet) treatment, normalized to the untreated cell volume at 0 h. Values are displayed as means ± SEM, n = 3 independent biological replicates. (b) Tetracycline-treated cells are metabolically active, as shown by unlabelled ratios of hexose-6-phosphate (first intermediate of glycolysis), (c) phosphoenolpyruvate (late glycolysis intermediate) and (d) D-alanyl-D-alanine (cell wall precursor). Cells treated for 2 h with tetracycline (blue) or untreated control cells (red) were washed with C¹³-labeled medium for different times, ranging from 0-150 s (means ± SD, n = 3 independent biological replicates). (e) Fold-change in hexose-6-phosphate, phosphoenolpyruvate, and D-alanyl-alanine concentration between Tet-treated and untreated cells (means ± SE, n = 3 independent biological replicates). (f) Energy state for Tet treated and control cells (mean ± SEM, n = 3 independent biological replicates). (g) Fold-changes in cell volume and cell density (measured as volume fraction) for biofilms treated with Tet, trimethoprim (Tmp), Tet and Tmp, glucose removal (-glc), glucose removal and Tet treatment (-glc+Tet). Data shown as mean ± SEM, sample size (n) are 17, 5, 6, 3, 4, for +Tet, +Tmp, +Tet+Tmp, -glc, -glc+Tet, respectively; each sample corresponds to a different biofilm. For each treatment, the fold-change is calculated in relation to the 0 h treatment. For panels a and g statistical significances were calculated using a one-way ANOVA with Bonferroni’s correction for multiple comparisons. For panels e and f statistical significances were calculated using a two-sided unpaired t-test. Statistically non-significant differences (NS) in panel a correspond to p = 0.067, 0.99, 0.24, 0.78 (left to right), in panel e to p = 0.076, 0.071, 0.99 (left to right), in panel f to p = 0.70, in panel g to p = 0.99. *, ***, and **** indicate p <0.05, <0.001, and <0.0001 respectively.