Fig 1.
Data from the high-through screening (HTS) of the AnalytiCon MEGx library.
The natural products were screened against C. difficile at 3 μM. The natural product chemotypes that exhibited greater than or equal to 95% bacterial growth inhibition were deemed as hits. The HTS assay identified 9 hits.
Table 1.
Minimum Inhibitory Concentration (MIC) values of natural products against a panel of clinical and hypervirulent strains of C. difficile.
Table 2.
Minimum Bactericidal Concentration (MBC) values of natural products against a panel of clinical and hypervirulent strains of C. difficile.
Fig 2.
Killing kinetics of hit natural products, vancomycin, and fidaxomicin (5X MIC) against C. difficile.
The standard deviation values for the triplicate samples of each natural product/control antibiotic have been represented by error bars.
Fig 3.
C. difficile toxin inhibition by natural products and control antibiotics (vancomycin and fidaxomicin).
C. difficile was incubated with subinhibitory concentrations of drugs for 12 hours. Viable cells (log10 CFU/ml, bars) in each sample were determined by plating and the amount of toxin in each supernatant (OD450-OD620, lines) was assessed using ELISA. The data represents the mean and standard deviation for triplicate samples of each treatment. Asterisk (*) indicates a statically significant difference in the toxin content of the supernatant between the fidaxomicin- or natural product-treated samples and the untreated control.
Fig 4.
Cytotoxicity assay of natural products against Caco-2 cell line.
The percentage of viable Caco-2 cells was measured as the ratio of average absorbance of each sample relative to the negative control (DMSO). The absorbance values represent the mean of three samples of each natural product compound with error bars representing standard deviation values for the absorbance.
Fig 5.
(A) Schematic of mice model. 6-week-old female mice were randomly divided into groups (n = 5 per group). Mice were given antibiotic water consisting of kanamycin (0.4 mg/ml), colistin (850 U/ml), gentamicin (0.035 mg/ml), vancomycin (0.045 mg/ml), and metronidazole (0.215 mg/ml) for 5 days. Clindamycin (10 mg/kg) was injected intraperitoneally a day before infection. C. difficile spores were administered via oral gavage on Day 0. Treatment was initiated on Day 0 and continued for 5 days. A single dose of each drug was administered via oral gavage daily. (B) The Kaplan-Meier survival curve was analyzed using a log-rank (Mantel-Cox) test. Significant difference was noted between the survival curves of the mice treated with NP-003875 vs. untreated control (100% vs. 20%; p = 0.0144).