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Typhimurium biofilms were grown and treated as described in B and EC 50 values for biofilm disruption with JG-1 and M4 were calculated using GraphPad Prism 8 to plot normalized compound activity (percent biofilm remaining) as a function of log 10 drug concentration and fitting the dose response curve (log vs. Annotations above the data denote values that are significantly different from the vehicle control (gray bar), as determined by two-way ANOVA with Dunnett’s test for multiple comparisons ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Incubation was resumed for an additional 24 h, after which biofilm formation was assessed via crystal violet assay. Typhimurium biofilms were grown for 24h, after which media was removed and replaced with various concentrations (10–150 μM) JG-1 or M4 or a vehicle control (DMSO). Annotations above the data denote values that are significantly different from the vehicle control (gray bar), as determined by two-way ANOVA with Dunnett’s test for multiple comparisons ns p ≥ 0.05, *** p < 0.001, **** p < 0.0001. Biofilms were incubated for a total of 24 h, after which biofilm formation relative to a vehicle control (DMSO) was assessed via crystal violet assay. Typhimurium biofilms to achieve a final compound concentration of 10 μM. At either the time of inoculation (0h post-inoculation) or at various timepoints 1–12 h post-inoculation, JG-1 or M4 was added to the media of S. Typhimurium biofilms suggests anti-biofilm activity decreases as biofilm development progresses.
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All data are presented as the mean +/- SD.ĭisruption of preformed Salmonella biofilms by JG-1 and M4.Ī. Two-way ANOVA with the Tukey correction for multiple comparisons revealed no significant differences at any timepoint. At various timepoints, aliquots of cultures were serially diluted and plated onto LB for CFU enumeration. Typhimurium cultures were grown in the presence of JG-1, M4, or a vehicle control (DMSO) for a total of 24 h.
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JG-1 and M4 have no effect on the viability of planktonic S. Corresponding EC 50 values for JG-1 and M4 were calculated as described in B. Typhi biofilms were grown in cholesterol-coated microtiter plates for a total of 96h in the presence of a vehicle control (DMSO) or various concentrations (3.1–200 μM) of JG-1 or M4. Typhimurium biofilms were grown as described in A, and EC 50 values for JG-1 and M4 were calculated using GraphPad Prism 8 to plot normalized compound activity (percent biofilm formed) as a function of log 10 drug concentration and fitting a dose-response curve (log vs. Annotations above the data denote values that are significantly different from the vehicle control (gray bar), as determined by two-way ANOVA with Dunnett’s test for multiple comparisons ns p ≥ 0.05, **** p < 0.0001. Biofilm formation was measured using the crystal violet assay. Typhimurium biofilms were grown for 24 h in the presence of a vehicle control (DMSO) or various concentrations (2.5–100 μM) of JG-1 or M4 in order to assess the dose-dependency of biofilm inhibition. JG-1 and M4 inhibit Salmonella biofilm formation in a dose-dependent manner without affecting planktonic growth.Ī. Chemical structures of the small molecule biofilm inhibitors JG-1 (B) and M4 (C). Data are presented as mean +/- SD ** p < 0.01, * p < 0.05, as assessed by unpaired t-tests. Biofilm formation after 24 h was evaluated in a semi-quantitative manner using the crystal violet assay, and inhibition of biofilm formation was calculated by assessing the amount of biofilm formed relative to a vehicle control (DMSO). Typhimurium biofilms were grown in microtiter plates in the presence of various compounds at a concentration 5 μM.
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Typhimurium) biofilm formation via high-throughput screening of small molecule libraries. The compounds JG-1 and M4 were identified as inhibitors of Salmonella Typhimurium ( S. Identification of small molecule inhibitors of Salmonella Typhimurium biofilm formation.Ī.