Pomegranate Peel Extracts Suppress the lasI/lasR and rhlI/rhlR Quorum Sensing Systems, eta Gene Expression, Virulence, and Biofilm Formation in Multidrug-Resistant Pseudomonas aeruginosa

Abstract

The increasing prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa has reduced the effectiveness of conventional antibiotics and necessitated the development of alternative therapeutic approaches. Quorum sensing (QS), a bacterial cell-to-cell communication system that regulates virulence factor production and biofilm formation, represents a promising target for anti-virulence therapy. Pomegranate peel (Punica granatum) is rich in bioactive phytochemicals and has been reported to possess antimicrobial and quorum-quenching properties. Objective: To evaluate the anti-quorum sensing, anti-virulence, and antibiofilm activities of pomegranate peel extracts against MDR clinical isolates of P. aeruginosa. Methods: Pomegranate peels were sequentially extracted using hexane (PG-1), chloroform (PG-2), ethyl acetate (PG-3), n-butanol (PG-4), ethanol (PG-5), and water (PG-6). The extracts exhibited MIC values of 20, 25, 25, 15, 10, and 10 mg/mL, respectively, while corresponding SICs were 10, 20, 20, 10, 5, and 5 mg/mL. SICs were used to investigate their effects on bacterial growth, protease activity, exoprotease production, pyocyanin synthesis, motility, biofilm formation, and the expression of QS-related genes (lasI, lasR, rhll, and rhlR) and the exotoxin A (eta) gene using RT-qPCR. Results: The selected SICs did not significantly affect bacterial growth, indicating that the extracts acted through virulence attenuation rather than growth inhibition. All extracts significantly reduced QS-regulated virulence factors (P < 0.0001). Total protease activity decreased from 0.706 in untreated cultures to 0.18 and 0.32 following treatment with PG-4 and PG-5, respectively. Exoprotease production was completely inhibited by both fractions, while pyocyanin production decreased from 5.317 μg/μg protein to 0.17 and 2.6 pg/pg protein, respectively. Swarming motility decreased from 36.7 mm to 6.2 and 7.5 mm, while swimming motility decreased from 54.33 mm to 11.83 and 13.10 mm following treatment with PG-4 and PG-5, respectively. Biofilm formation was markedly reduced by all extracts, with the n-butanol fraction exhibiting the strongest antibiofilm activity. RT-qPCR analysis demonstrated significant downregulation of lasI, lasR, rhll, rhlR, and eta genes. Notably, the n-butanol and ethanol fractions completely suppressed the expression of all tested QS and exotoxin A genes. Conclusion: Pomegranate peel extracts effectively attenuated QS-controlled virulence, motility, biofilm formation, and exotoxin production in MDR P. aeruginosa without affecting bacterial growth. The n-butanol and ethanol fractions showed the greatest activity and completely inhibited the expression of key QS genes, highlighting their potential as natural anti-virulence agents for the management of antibiotic-resistant P. aeruginosa infections.