Novel Antibiofilm Inhibitor Ginkgetin as an Antibacterial Synergist against Escherichia coli

Abstract

As an opportunistic pathogen, Escherichia coli (E. coli) forms biofilm that increases the virulence of bacteria and antibiotic resistance, posing a serious threat to human and animal health. Recently, ginkgetin (Gin) has been discovered to have antiinflammatory, antioxidant, and antitumor properties. In the present study, we evaluated the antibiofilm and antibacterial synergist of Gin against E. coli. Additionally, Alamar Blue assay combined with confocal laser scanning microscope (CLSM) and crystal violet (CV) staining was used to evaluate the effect of antibiofilm and antibacterial synergist against E. coli. Results showed that Gin reduces biofilm formation, exopolysaccharide (EPS) production, and motility against E. coli without limiting its growth and metabolic activity. Furthermore, we identified the inhibitory effect of Gin on AI-2 signaling molecule production, which showed apparent anti-quorum sensing (QS) properties. The qRT-PCR also indicated that Gin reduced the transcription of curli-related genes (csgA, csgD), flagella-formation genes (flhC, flhD, fliC, fliM), and QS-related genes (luxS, lsrB, lsrK, lsrR). Moreover, Gin showed obvious antibacterial synergism to overcome antibiotic resistance in E. coli with marketed antibiotics, including gentamicin, colistin B, and colistin E. These results suggested the potent antibiofilm and novel antibacterial synergist effect of Gin for treating E. coli infections.

Keywords: EPS; Escherichia coli; antibacterial synergist; antibiofilm; ginkgetin; motility; quorum sensing.

Figure 1

Growth and metabolic activity of E. coli in the presence of Gin. The line graph shows the growth of E. coli with Gin using a microbroth dilution assay. The bar graph shows the metabolic activity of E. coli based on the AB assay. n = 3. Results from all experiments are presented as the mean ± SD of three replicates.

Figure 2

Gin’s effects on the growth of biofilms in E. coli. (A) Biofilm formation inhibition at various concentrations of Gin (6.25, 12.5, 25, 50, 100 200, and 400 μM) for 24 h. **** = p < 0.0001. (B) CLSM image of E. coli biofilm at different concentrations of Gin (12.5, 5, and 50 μM) for 24 h. n = 3. Results from all experiments are presented as the mean ± SD of three replicates.

Figure 3

The cytotoxicity of Gin in (A) Caco-2 cell and (B) IPEC-J2 cell. Gin was applied to cells at various concentrations (0, 6.25, 12.5, 25, 50, 100, and 200 µM) for 24 h. n = 3. Results from all experiments are presented as the mean ± SD of three replicates. ns: no significant, * = p < 0.05, **** = p < 0.0001.

Figure 4

Results of EPS inhibition (%) at various concentrations of Gin (0, 6.25, 12.5, 25, 50, 100, 200, and 400 µM) for 24 h. n = 3. Results from all experiments are presented as the mean ± SD of three replicates. ** = p < 0.01, **** = p < 0.0001.

Figure 5

Motility inhibition of E. coli with Gin. (A) Images of motility following incubation with E. coli at various concentrations of Gin (0, 6.25, 12.5, 25, and 50 µM). (B) Quantitative estimation of motility based on the diameter of the halo zone. n = 3. Results from all experiments are presented as the mean ± SD of three replicates. **** = p < 0.0001.

Figure 6

Inhibition of AI-2 activity treated with different concentrations of Gin (1.56, 3.13, 6.25, 12.5, 25, and 50 µM). n = 3. Results from all experiments are presented as the mean ± SD of three replicates.

Figure 7

Effects of Gin on transcription of biofilm-regulated genes. The qRT-PCR results revealed significant difference in ten genes (csgA, csgD, flhC, flhD, fliC, fliM, luxS, lsrB, lsrK, and lsrR) as compared with the control. Results from all experiments are presented as the mean ± SD of three replicates. **** = p < 0.0001.

Figure 8

Effects of Gin (50 μM) and Gentamicin on bacterial viability in six different E. coli strains. (A) ATCC 25922, (B) C83654, (C) XJ24, (D) O149, (E) O101, (F) KD-13-1. n = 3. Results from all experiments are presented as the mean ± SD of three replicates. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 9

Effects of Gin (50 μM) and Colistin B on bacterial viability in six different E. coli strains. (A) ATCC 25922, (B) C83654, (C) XJ24, (D) O149, (E) O101, (F) KD-13-1. n = 3. Results from all experiments are presented as the mean ± SD of three replicates. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 10

Effects of Gin (50 μM) and Colistin E on bacterial viability in six different E. coli strains. (A) ATCC 25922, (B) C83654, (C) XJ24, (D) O149, (E) O101, (F) KD-13-1. n = 3. Results from all experiments are presented as the mean ± SD of three replicates. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.