Fig 1.
High throughput screening and compound prioritization process.
Steps are shown as arrows with the name of the step to the left and the selection method to the right.
Fig 2.
Overview of the primary screen results.
The average residual growth of each compound is plotted against the Average B-score. The inset shows the application of an average residual growth cutoff of 0.70 and a B-Score cutoff of –17.5, (dashed line) to the whole screening campaign, which identified 206 Bce bioactives. The large scatter plot shows the 206 primary actives. For comparison reasons, 6 known antibiotics, are denoted with crossed rectangles, ceftriaxone sodium, (-121, 0.06); minocycline, (-109, 0.23); metampicilin, (-80.7, 0.08); chloramphenicol, (-68.8, 0.26); ciprofloxacin, (-35, 0.36); trimethoprim, (-29.6, 0.62). The two Bce bioactives with the most promising properties are indicated with diamonds and labeled.
Table 1.
Minimum inhibitory concentration (MIC) of the top 15 synthetic compounds against B. cenocepacia K56-2, the Survival100 (Surv100) concentration in C. elegans and the Surv100/MIC ratio.
Table 2.
MICs, MBCs and MBICs for the top 15 compounds tested against ten Bcc strainsa.
Fig 3.
Survival 100 (Surv100) assay, and the Surv100/MIC ratio determination.
(A) The Surv100 assay was conducted in a 96-well format, where each compound was serially diluted along the rows from its highest soluble concentration or the MIC. The last well for each compound was used as a DMSO control. Approximately, 5 to 10 OP50-fed worms were added to the wells containing LKM for a total assay volume of 100 μl. The number of worms was counted and recorded for each concentration on day 0 and again 24 hours later. Percent survival was determined for each concentration. The Surv100/MIC ratio was calculated. The compounds, which demonstrated a ratio of 1 and greater, were used in the in vivo antibiotic activity. (B) Photograph of the worms from the assay illustrating the difference between 0% survival and 100% survival.
Table 3.
Hemolytic activity (% hemolysis*) of the bioactive compounds that inhibit the growth of B. cenocepacia K56-2.
Fig 4.
C. elegans rescue assays. C. elegans was allowed to feed on B. cenocepacia K56-2 and OP50 for 16 hours.
The B. cenocepacia infected and OP50-fed worms were subsequently treated with (A) Trimethoprim (TP), Tetracycline (Tet), Meropenem (Mero), Chloramphenicol (Chl) and observed for 6 days every 24 hours and were compared to the non-treated (No Antibiotic) worms for survival. p < 0.0001 for all compounds. (B) The worms were treated with MAC-0013209, (p = 0.2503) with MAC-0151023 and MAC-0036650 at their respective MIC (128 μg/mL, 32 μg/mL and 16 μg/mL); p< 0.0001 for both MAC-0151023 and MAC-0036650 compounds. Trimethoprim was used as a control. p < 0.0001 for all concentrations.