Anti-Virulence Activity of 3,3′-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound Healing Benefits

Abstract

Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3′-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM.

3,3′-Diindolylmethane (DIM): A Potential Therapeutic Agent against Cariogenic Streptococcus mutans Biofilm

“…Recently, we explored the effects of a bis-indole derivative, DIM, on the biofilm formation of different Gram-negative pathogens. This compound was found to be a potential candidate for anti-biofilm properties and virulent attenuation [ 19 ]. In general, indole plays important roles as a signal molecule participating in biofilm formation and in multiple physiological pathways in many bacteria [ 13 ].…”

“…In order to attain a greater understanding of DIM efficacy to prevent biofilm formation under shear flow conditions, a continuous culture flow cell was employed. S. mutans biofilm was allowed to form in the presence of DIM (0.5 μM) or an equivalent concentration of DMSO as a control on the inner surface of channels of the Ibidi flow cell (µ-slide sizes of 17 × 3.8 × 0.4 mm) for 48 h at 37 °C [ 19 ]. To initiate biofilm growth, the cells were inoculated with an overnight culture of S. mutans diluted with 0.9% NaCl to an OD 600 of 0.05.…”

“…A number of indole derivatives, including naturally occurring and synthetic ones, have been reported to act as signaling molecules that affect the behavior of different bacteria [ 14 ]. In addition, several indole derivatives have been reported to have more potent antimicrobial and anti-biofilm activities than a basic indole such as 3,3′-Diindolylmethane (DIM) [ 19 , 20 , 21 , 22 ]. Furthermore, recent evaluations of a number of natural compounds that affect S. mutans biofilm and virulence factors have suggested that these indole compounds may have therapeutic properties [ 23 , 24 , 25 , 26 ].…”

Broccoli: A Multi-Faceted Vegetable for Health: An In-Depth Review of Its Nutritional Attributes, Antimicrobial Abilities, and Anti-inflammatory Properties

“…By inhibiting biofilm formation, bacterial load is reduced. In this context, 3,3'-diindolylmethane (DIM), a bioactive component of broccoli, may act as an inhibitor of biofilm formation and cause a reduction in bacterial load [74]. Bacterial contamination in food is a common cause of foodborne illnesses.…”

Artificial Neural Network Prediction of Antiadhesion and Antibiofilm-Forming Effects of Antimicrobial Active Mushroom Extracts on Food-Borne Pathogens

“…There are even fewer compounds that can inhibit biofilm formation without exhibiting antimicrobial activity at the same time. Among substances of natural origin, most are phytochemicals such as carvacrol, hordenin, ginkgolic acid, epigallocatechin-3-gallate, isosteviol, phytol, curcumin, tannic acid, and 3,3′-diindolylmethane, to name a few [ 10 , 11 , 12 ].…”