Attenuation of Pseudomonas aeruginosa Virulence by Pomegranate Peel Extract

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen often responsible for biofilm-associated infections. The high adhesion of bacterial cells onto biotic/abiotic surfaces is followed by production of an extracellular polysaccharidic matrix and formation of a sessile community (the biofilm) by the release of specific quorum-sensing molecules, named autoinducers (AI). When the concentrations of AI reach a threshold level, they induce the expression of many virulence genes, including those involved in biofilm formation, motility, pyoverdine and pyocyanin release. P. aeruginosa embedded into biofilm becomes resistant to both conventional drugs and the host's immune response. Accordingly, biofilm-associated infections are a major clinical problem underlining the need for new antimicrobial therapies. In this study, we evaluated the effects of pomegranate peel extract (PomeGr) in vitro on P. aeruginosa growth and biofilm formation; moreover, the release of four AI was assessed. The phenolic profile of PomeGr, exposed or not to bacteria, was determined by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. We found that bacterial growth, biofilm production and AI release were impaired upon PomeGr treatment. In addition, the PomeGr phenolic content was also markedly hampered following incubation with bacterial cells. In particular, punicalagin, punicalin, pedunculagin, granatin, di-(HHDP-galloyl-hexoside) pentoside and their isomers were highly consumed. Overall, these results provide novel insights on the ability of PomeGr to attenuate P. aeruginosa virulence; moreover, the AI impairment and the observed consumption of specific phenolic compounds may offer new tools in designing innovative therapeutic approaches against bacterial infections.

Keywords: Pseudomonas aeruginosa; anti-biofilm; autoinducers; phenolic compounds; pomegranate; virulence.

Figure 1

The PomeGr effects on P. aeruginosa growth and biofilm formation. P. aeruginosa (10⁶/mL, 100 µL/well) was exposed for 24 h to PomeGr or neg-C (100 µL/well) at dilutions of 1:8, 1:16 and 1:32. By BLI assay, the total microbial load was kinetically measured every hour (Panel A: 1:8 dilution; Panel B: 1:16 dilution; Panel C: 1:32 dilution). The samples were then washed and the biofilm formation was quantified by an additional bioluminescence reading (Panel D). The results shown are the mean (±SEM) of the RLU from 9–12 replicates of three independent experiments. The asterisk indicates p ≤ 0.001 (PomeGr vs. neg-C).

Figure 2

Phenolic compounds decrease upon PomeGr exposure to P. aeruginosa. Overlapped peaks of 5 phenolic compounds in PomeGr extract, assessed alone (black line) or upon exposure to P. aeruginosa (grey line). The data were obtained by a pool of 4 replicates from a representative experiment. Each panel also shows the chemical structure of each compound.