Antibiofilm properties of Clitoria ternatea flower anthocyanin-rich fraction towards Pseudomonas aeruginosa

Abstract

In Asia, Clitoria ternatea flowers are commonly used as a traditional medicinal herb and as a food colourant. Their bioactive compounds have anti-inflammatory, anti-microbial and anti-biofilm activities. Pseudomonas aeruginosa is one of the major pathogens that cause biofilm-associated infections resulting in an increase in antimicrobial resistance. Hence, the aim of this study was to investigate if the anti-biofilm properties of the anthocyanin-rich fraction of C. ternatea flowers were effective against P. aeruginosa . The effect of the anthocyanin-rich fraction of C. ternatea flowers on P. aeruginosa biofilms formed on a polystyrene surface was determined using the crystal violet assay and scanning electron microscopy (SEM). The anthocyanin-rich fraction reduced biofilm formation by four P. aeruginosa strains with a minimum biofilm inhibitory concentration value ranging between 0.625 and 5.0 mg ml^-1. We further show that the biofilm-inhibiting activity of C. ternatea flowers is not due to the flavonols but is instead attributed to the anthocyanins, which had significant biofilm inhibitory activity (64.0±1.1 %) at 24 h in a time-response study. The anthocyanin-rich fraction also significantly reduced bacterial attachment on the polystyrene by 1.1 log c.f.u. cm^-2 surface based on SEM analysis. Hence, anthocyanins from C. ternatea flowers have potential as an agent to decrease the risk of biofilm-associated infections.

Keywords: anthocyanin; antimicrobial; biofilm-inhibiting activity; blue pea; crystal violet; flavonol.

Fig. 1.

Effect of the C. ternatea anthocyanin-rich fraction on biofilm formation at 24 h of P. aeruginosa (a) ATCC 10145, (b) ATCC 9027, (c) ATCC 27853 and (d) ATCC BAA-47. Different letters indicate significant differences in biofilm formation at P<0.05, as analysed by one-way ANOVA. Bacterial suspension without treatment was the negative control while bacterial suspension treated with sodium hypochlorite was used as the positive control.

Fig. 2.

Effect of the C. ternatea anthocyanin-rich fraction on biofilm disruption at 24 h of P. aeruginosa (a) ATCC 10145, (b) ATCC 9027, (c) ATCC 27853 and (d) ATCC BAA-47. Different letters indicate significant differences in biofilm disruption at P<0.05, as analysed by one-way ANOVA. Bacterial suspension without treatment was the negative control while bacterial suspension treated with sodium hypochlorite was used as the positive control.

Fig. 3.

Impact of the C. ternatea anthocyanin-rich fraction (5 mg ml⁻¹) on bacterial growth of P. aeruginosa ATCC 9027 over 24 h. There was no significant difference in bacterial growth over the respective time points (P>0.05) as analysed by an independent t-test.

Fig. 4.

Biofilm formation by P. aeruginosa ATCC 9027 treated with the C. ternatea anthocyanin-rich fraction and flavonol fraction at 5 mg ml⁻¹ (24 h) and the corresponding biofilm biomass stained with crystal violet (0.1%). Significant differences relative to the negative control are marked (*P<0.05), as analysed by one-way ANOVA. The negative control was bacterial suspension with BHI broth (no treatment) while the positive control was bacterial suspension with the addition of sodium hypochlorite.

Fig. 5.

Micrographs (1000× magnification; bars, 100 µm) of crystal violet- (0.1%) stained P. aeruginosa (ATCC 9027) attached to (a) polystyrene and (b) glass surfaces after 1 h in the presence or absence of C. ternatea anthocyanin-rich fraction (5 mg ml⁻¹).

Fig. 6.

Biofilm formation of P. aeruginosa (ATCC 9027) treated with C. ternatea anthocyanin-rich fraction (5 mg ml⁻¹) at 1, 4, 8, 12 and 24 h. Significant differences from the negative control group at respective time intervals are marked (*P<0.05), as analysed by one-way ANOVA. The negative control was bacterial suspension with BHI broth (no treatment) while the positive control was bacterial suspension with the addition of sodium hypochlorite.

Fig. 7.

SEM images of biofilm formation. Biofilm formation by P. aeruginosa (ATCC 9027) treated with and without C. ternatea anthocyanin-rich fraction (5 mg ml⁻¹) at (a) 2000× (bar, 20 µm) and (b) 5000× (bar, 10 µm) magnification at 24 h.

Fig. 8.

RT-qPCR results of gene expression of P. aeruginosa (ATCC 9027) treated with C. ternatea anthocyanin-rich fraction for 24 h. Expression of genes in both the control and experimental groups was normalized to proC and rpoD as the reference genes. The control group expression level is designated as 1, so values >1 indicate up-regulation and values <1 indicate down-regulation. There was no significant difference between the control and treated group at P<0.05, as analysed by an independent t-test.