Selected flavonoids exhibit antibiofilm and antibacterial effects against Vibrio by disrupting membrane integrity, virulence and metabolic activities

Abstract

Vibrio parahaemolyticus is a high-risk foodborne pathogen associated with raw or undercooked seafoods and its biofilm forming potential has become a threat to food safety and economic values. Hence, this study aims to examine the antibacterial and antibiofilm activities as well as virulence inhibitory effects of selected flavonoids against V. parahaemolyticus. Out of the sixteen flavonoid derivatives, 6-aminoflavone (6-AF), 3,2-dihydroxyflavone (3,2-DHF) and 2,2-dihydroxy-4-methoxybenzophenone (DHMB) were found as active biofilm inhibitors. 3,2-DHF and DHMB had minimum inhibitory concentrations of 20 and 50 μg/mL respectively against Vibrio planktonic cells and displayed superior antibacterial activities to standard controls. Also, they disrupted preformed biofilms and suppressed virulence properties including motilities, cell hydrophobicity and aggregation. They impaired iron acquisition mechanism and hemolysin production at sub-MICs as supported by transcriptomic studies. Interestingly, the flavonoids interfered with the metabolic activity, cell division and membrane permeability to exert antibiofilm and antibacterial activities. 6-AF and 3,2-DHF were non-toxic in the C. elegans model and showed excellent capacity to protect shrimps from biodeterioration. Furthermore, the flavonoids inhibited biofilm formation by V. harveyi, Staphylococcus aureus and Salmonella typhimurium and the mixed-species biofilm with Vibrio. This study discovered flavonoid derivatives, especially 3,2-DHF as potential bioactive compounds capable of offering protection from risks associated with biofilm formation by V. parahaemolyticus and other food pathogens.

Keywords: Antibiofilm; Antimicrobial; Antivirulence; Flavonoid derivatives; Foodborne pathogens; Vibrio parahaemolyticus.

Fig. 1

Antibiofilm effects of flavonoid derivatives. The antibiofilm activities of the three active derivatives and the parent flavone at sub-MIC concentrations against V. parahaemolyticus(A), comparison with three standard controls (B) and the biofilm disrupting effects of the flavonoids (C). Bars represent biofilm formation and lines, the planktonic cell growth. * Denotes a significant difference at p < 0.05.

Fig. 2

Visualization of the antibiofilm activities of the flavonoids. Phase contrast microscope 3D images (A) and SEM images of flavonoid treated cells (B), effects of the analogues on biofilm metabolic activities (C). * Represents a significant difference (p < 0.05). Red and green arrows represent elongated and disrupted cells, respectively. Red and blue bars represent 10 and 5 μm, respectively.

Fig. 3

Effects of active flavonoids on V. parahaemolyticus survival and motility. Time killing effects of flavonoids against Vibrio exponential and stationary growth phases in mLB (A), PBS (B), and their effects on Vibrio swimming (C) and swarming motilities (D). * Represents a significant difference (p < 0.05). Exponential and stationary phase cells were obtained by culturing for 3 h and 14 h at 30 °C and 250 rpm, respectively before use.

Fig. 4

The effects of flavonoid derivatives on adhesion and virulence factors. Cell surface hydrophobicity (A), autoaggregation (B), extracellular indole (C), protease production (D), hemolysin (E) and siderophore production (F) in V. parahaemolyticus. *Represents a significant difference (p < 0.05).

Fig. 5

Effects of flavonoid derivatives on gene expression and seafood preservation. Relative gene expression analysis of Vibrio cells treated with 20 μg/mL of 6-AF, 3,2-DHF, and DHMB (A-B), antibacterial effects of the three derivatives on shrimps inoculated with V. parahaemolyticus(C), and biofilm eradication effects on squid (D). * Denotes a significant difference (p < 0.05). Sodium tripolyphosphate (STPP) served as the control.

Fig. 6

The inhibitory effects of the three flavonoid derivatives against biofilm formation and planktonic cells of important food pathogens. *Represents a significant difference at p < 0.05.

Fig. 7

The effects of flavonoid derivatives on mixed biofilm formation. The antibiofilm effects of 6-AF, 3,2-DHF, and DHMB against mixed V. parahaemolyticus - (Vp) and S. aureus – (Sa) biofilm formation (A), SEM observation of the activities against mixed biofilm formation (B). *Represents a significant difference (p < 0.05). The red and green arrows represent Vp and Sa while the yellow arrows indicate disrupted cells. Red and yellow bars represent 10 and 5 μm, respectively.

Fig. 8

Nucleotide leakages upon treatment with the derivatives (A), and in-vivo cytotoxic studies of the active flavonoids in C. elegans(B).

Fig. 9

A summary of various activities displayed by the flavonoid derivatives and their possible future applications.