Synthetic Flavonoid BrCl-Flav-An Alternative Solution to Combat ESKAPE Pathogens

Abstract

ESKAPE pathogens are considered as global threats to human health. The discovery of new molecules for which these pathogens have not yet developed resistance is a high medical priority. Synthetic flavonoids are good candidates for developing new antimicrobials. Therefore, we report here the potent in vitro antibacterial activity of BrCl-flav, a representative of a new class of synthetic tricyclic flavonoids. Minimum inhibitory/bactericidal concentration, time kill and biofilm formation assays were employed to evaluate the antibacterial potential of BrCl-flav. The mechanism of action was investigated using fluorescence and scanning electron microscopy. A checkerboard assay was used to study the effect of the tested compound in combination with antibiotics. Our results showed that BrCl-flav displayed important inhibitory activity against all tested clinical isolates, with MICs ranging between 0.24 and 125 µg/mL. A total kill effect was recorded after only 1 h of exposing Enterococcus faecium cells to BrCl-flav. Additionally, BrCl-flav displayed important biofilm disruption potential against Acinetobacter baumannii. Those effects were induced by membrane integrity damage. BrCl-flav expressed synergistic activity in combination with penicillin against a MRSA strain. Based on the potent antibacterial activity, low cytotoxicity and pro-inflammatory effect, BrCl-flav has good potential for developing new effective drugs against ESKAPE pathogens.

Keywords: ESKAPE pathogens; anti-biofilm activity; antibacterial; low cytotoxicity; pro-inflammatory effect; synergistic effect; synthetic flavonoid.

Figure 1

Structure of flavonoid BrCl-flav.

Figure 2

The effects of BrCl-flav at different concentrations on the growth of S. aureus medbio1-2012 (a), S. aureus prxbio1 (b), E. faecium medbio2-2012 (c) and A. baumannii medbio3-2013 (d). Cells incubated in MHB medium supplemented with DMSO served as control. The data were presented as mean of three independent experiments. Bars indicate SEM.

Figure 3

The time-killing kinetics of BrCl-flav against S. aureus medbio1-2012 (a), S. aureus prxbio1 (b), E. faecium medbio2-2012 (c) and A. baumannii medbio3-2013 (d). The bacterial cells were exposed to BrCl-flav at concentrations equivalent to MBC. Untreated cells served as control. The data were presented as the means of three independent experiments. Bars indicate SEM.

Figure 4

The effects of the BrCl-flav on A. baumannii medbio3-2013 biofilms: (a) inhibition of biofilm formation (bacterial cells were incubated for 24 h in the presence of BrCl-flav); (b) disruption of mature biofilms (before exposure to BrCl-flav, the biofilms were pre-formed for 24 h). Values are the means of at least three replicates. Bars indicate SEM. Asterisks represent a significant difference (p < 0.05) vs. Control (** = p < 0.01, *** = p < 0.001, **** = p < 0.0001; ns—non-significant).

Figure 5

Effect of BrCl-flav exposure on S. aureus medbio1-2012 (a) and E. coli medbio4-2013 (b) cell membrane integrity. Exponential-phase cells were treated with the antibacterial agent concentration equivalent to MBC and stained with SYTO 9 and propidium iodide. Low levels of fluorescent cells were detected in the untreated control. Red fluorescent cells were detected in samples starting with 25 min of incubation with BrCl-flav, indicating membrane damages. Values are the means of three replicates. Bars indicate SEM. Asterisks denote a significant difference (p < 0.05) vs. Control (**** = p < 0.0001).

Figure 6

Fluorescent images of a time course experiment illustrating the S. aureus medbio1-2012 and E. coli medbio4-2013 cell membrane disruption effects due to BrCl-flav exposure, visualized by the influx of the fluorescent nuclear stain propidium iodide. The cells were stained using a Live/Dead BacLight Bacterial Viability Kit (Invitrogen, Waltham, MA, USA). The captured images are representative of a typical result.

Figure 7

The effects of BrCl-flav on the morphology of S. aureus medbio1-2012 and E. coli medbio4-2013. Control cells with normal morphology. Cells exposed for 6 h to concentrations equivalent to MBC. White arrows indicate morphological damages and cellular debris.

Figure 8

The time-kill curve of BrCl-flav and penicillin synergistic combination against an MRSA isolate. Values are the means of three replicates. Bars indicate SEM.

Figure 9

Effect of BrCl-flav on the viability of four human cell lines. Cells were incubated for 24 h with the BrCl-flav at increasing concentrations (0.1 to 100 µg/mL). The viability of the cells was evaluated by the measurement of mitochondrial hydrogenase activity assayed with CCK8 reagent. Means are presented ± SD (N = 2, n = 6). The molecule concentration required to cause 50% inhibition of the cell viability (IC₅₀) was determined using the nonlinear regression analysis function of GraphPad Prism.

Figure 10

Effect of BrCl-flav on LPS-induced secretion of tumor necrosis factor-alpha (TNF-α) and interleukin 10 (IL10) in U937-macrophages. U937 cells were differentiated into macrophages by incubation in the presence of 25 nM-PMA for 48 h, and then after 3 days of PMA weaning, cells were seeded in 12-well plates at the density of 2 × 10⁶ viable cells/well. Cytokine production was measured after 4 h of incubation with culture medium (black), LPS at 50 μg/mL (dark grey), or with a positive inflammation inhibition control (LPS at 50 μg/mL + dexamethasone, 20 µM; light grey) or with LPS (50 μg/mL) + different concentrations of BrCl-flav (n = 4). Statistical analysis of the data was performed using Mann–Whitney tests to compare the LPS control to the dexamethasone control and samples with GraphPad Prism software (* = p < 0.05). Results are presented as bar graphs (means and standard deviations).