Figure 1.

Whole-cell phenotypic effects of carprofen treatment on M. smegmatis. (a) Inhibition of efflux pumps in the presence of subMIC concentration of carprofen (CRP) (0.25× MIC, 62.5 mg/L) as seen through the accumulation of EtBr and resulting increase in the fluorescence readout. Verapamil (VP) (0.25× MIC, 50 mg/L) was used as positive efflux pump inhibitor control. Untreated cells served as the negative control. The experiments were performed in triplicate (n = 3) and the graph was plotted using the averages. (b) Efflux of EtBr from pre-saturated cells in the presence of carprofen and verapamil, showing retention of the efflux pump substrate. (c) Complete inhibition of biofilm formation is observed in the presence of 250 mg/L (1× MIC) of carprofen in the PPE tubes and 96-well plates within a timescale of 5 days. Confocal images of stained extracellular DNA and lipids show distinct differences between carprofen-treated and untreated biofilms, especially their wrinkled surface, as captured in the z-axis images. (d) Concentration-dependent effect of carprofen on biofilm formation, as quantitated by crystal violet staining of the biofilms. Panels e, f and g show the amount of EPS, namely lipids, proteins and carbohydrates that make up the biofilms of the untreated control and carprofen-treated biofilms (0.25× MIC).