The Anticancer Agent 3,3'-Diindolylmethane Inhibits Multispecies Biofilm Formation by Acne-Causing Bacteria and Candida albicans

Abstract

The Gram-positive anaerobic bacterium Cutibacterium acnes is a major inhabitant of human skin and has been implicated in acne vulgaris formation and in the formation of multispecies biofilms with other skin-inhabiting organisms like Staphylococcus aureus and Candida albicans. Indoles are widespread in nature (even in human skin) and function as important signaling molecules in diverse prokaryotes and eukaryotes. In the present study, we investigated the antibacterial and antibiofilm activities of 20 indoles against C. acnes. Of the indoles tested, indole-3-carbinol at 0.1 mM significantly inhibited biofilm formation by C. acnes without affecting planktonic cell growth, and the anticancer drug 3,3'-diindolylmethane (DIM) at 0.1 mM (32 μg/mL) also significantly inhibited planktonic cell growth and biofilm formation by C. acnes, whereas the other indoles and indole itself were less effective. Also, DIM at 0.1 mM successfully inhibited multispecies biofilm formation by C. acnes, S. aureus, and C. albicans. Transcriptional analyses showed that DIM inhibited the expressions of several biofilm-related genes in C. acnes, and at 0.05 mM, DIM inhibited hyphal formation and cell aggregation by C. albicans. These results suggest that DIM and other indoles inhibit biofilm formation by C. acnes and have potential use for treating C. acnes associated diseases. IMPORTANCE Since indoles are widespread in nature (even in human skin), we hypothesized that indole and its derivatives might control biofilm formation of acne-causing bacteria (Cutibacterium acnes and Staphylococcus aureus) and fungal Candida albicans. The present study reports for the first time the antibiofilm and antimicrobial activities of several indoles on C. acnes. Of the indoles tested, two anticancer agents, indole-3-carbinol and 3,3'-diindolylmethane found in cruciferous vegetables, significantly inhibited biofilm formation by C. acnes. Furthermore, the most active 3,3'-diindolylmethane successfully inhibited multispecies biofilm formation by C. acnes, S. aureus, and C. albicans. Transcriptional analyses showed that 3,3'-diindolylmethane inhibited the expressions of several biofilm-related genes including lipase, hyaluronate lyase, and virulence-related genes in C. acnes, and 3,3'-diindolylmethane inhibited hyphal formation and cell aggregation by C. albicans. Our findings show that 3,3'-diindolylmethane offers a potential means of controlling acne vulgaris and multispecies biofilm-associated infections due to its antibiofilm and antibiotic properties.

Keywords: 3,3'-diindolylmethane; Cutibacterium acnes; antibiofilm; indole-3-carbinol; multispecies biofilms.

FIG 1

Antibiofilm activities of indole derivatives against C. acnes. Screening of C. acnes biofilm formation in the presence of indole derivatives at 0.1 mM (a). Cell growths in the presence of indole (b), DIM (c), and indole-3-carbinol (d). Antibiofilm activities of indole (e), DIM (f), and indole-3-carbinol (g). Error bars indicate standard deviations. *, P < 0.05 versus nontreated controls.

FIG 2

C. acnes biofilm inhibition by antibiotics in the presence or absence of DIM. Biofilm inhibitions by benzoyl peroxide (a), gentamicin (b), and ciprofloxacin (c) were measured after incubation for 6 days in 96-well plates at 37°C under anaerobic conditions. Antibiofilm activities of benzoyl peroxide (d), gentamicin (e), and ciprofloxacin (f) in the presence of 3,3′-diindolylmethane (DIM) were measured after incubation under identical conditions. Error bars indicate standard deviations. *, P < 0.05 versus nontreated controls.

FIG 3

Antibiofilm activities of 3,3′-diindolylmethane (DIM) against C. albicans and S. aureus under anaerobic and aerobic conditions. Biofilm formation by C. albicans (a) and S. aureus (b) was measured after incubation for 24 h in 96-well plates at 37°C under aerobic conditions. Biofilm formations by C. albicans (c) and S. aureus (d) were measured after incubation for 6 days in 96-well plates at 37°C under anaerobic conditions. Error bars indicate standard deviations. *, P < 0.05 versus nontreated controls.

FIG 4

Antibiofilm effects of DIM on C. acnes. The antibiofilm activity of DIM against C. acnes was determined after culture for 6 days under anaerobic conditions. Re-created C. acnes biofilm color-coded 2-D and 3-D images after culture in the presence of DIM (0, 0.02, or 0.05 mM) (a), and CLSM images (b) and COMSTAT analysis results (c) of C. acnes biofilm inhibition by DIM. Scale bars in panel b represent 100 μm. *, P < 0.05 versus nontreated controls. SEM images of C. acnes biofilms formed in the presence or absence of DIM (0, 0.02, or 0.05 mM) (d). Yellow and white scale bars represent 3 μm and 750 nm, respectively in panel d. None: nontreated control.

FIG 5

Effects of DIM on polymicrobial biofilms. Antibiofilm activities of DIM against polymicrobial biofilms of C. acnes and S. aureus (a), C. acnes and C. albicans (b), and C. acnes, C. albicans, and S. aureus (c) biofilms after culture under anaerobic conditions. Color-coded 2-D and 3-D images of 3-component C. acnes, C. albicans, and S. aureus biofilms cultured in the presence of DIM (d), CLSM images (e), and COMSTAT analysis results (f) of three species biofilms cultured in the presence of DIM. Scale bars represent 100 μm. *, P < 0.05 versus nontreated controls. SEM images of three species biofilms formed in the presence or absence of DIM (g). White and yellow scale bars represent 10 and 3 μm, respectively in panel g. None: nontreated control.

FIG 6

Effects of indole, DIM, and indole-3-carbinol on EPS production by C. acnes. Extracellular polymeric substance production by C. acnes in the presence of indole (a), DIM (b), or indole-3-carbinol (c). *, P < 0.05 versus nontreated controls.

FIG 7

DIM inhibited hyphal filamentation and aggregation by C. albicans. Inhibition of filamentous hyphal growth in PDB medium (a) and cell aggregation in RPMI medium containing 10% fetal bovine serum (b). Hyphae were visualized after incubation for 24 h at 37°C without agitation. The scale bars represent 100 μm in panels a and b. None: nontreated control.

FIG 8

Relative transcriptional profiles of C. acnes cells treated with DIM. C. acnes cells grown for 3 days were treated with DIM at 0.1 mM for 24 h without shaking. Transcriptional profiles were analyzed by qRT-PCR. Fold changes represent transcriptional changes of treated versus untreated C. acnes. The experiment was performed in duplicate (six qRT-PCRs were performed per gene). *, P < 0.05 versus nontreated controls (None).

FIG 9

Diagram of the putative functions of indole-3-carbinol and 3,3′-diindolylmethane in single and polymicrobial biofilms.