Combinatorial antibiofilm and anti-virulence effects of silibinin-BSA nanoparticles and gentamicin against Pseudomonas aeruginosa: Insights into quorum sensing-mediated molecular mechanisms.

The management of biofilm-associated infections caused by various strains of Pseudomonas aeruginosa using conventional antibiotics has become a challenge due to the emergence of multidrug resistance. Currently, phytochemicals, particularly their nano-formulations, have emerged as promising alternatives for the ability to target diverse bacterial signalling pathways involved pathogenesis. Here, silibinin, a flavonolignan of plant source was used, and the combinatorial antibiofilm effect of silibinin-loaded bovine serum albumin nanoparticles (SBN-BSA NPs) in combination with gentamicin on P. aeruginosa biofilms was investigated. The effect on biofilm architecture of these treatment strategies was evaluated by biochemical assays and microscopy. Results demonstrated a significant antibiofilm effect of combinatorial treatment of SBN-BSA NPs (70 μg/mL) and gentamicin (1 μg/mL). The combined treatment substantially inhibited bacterial swimming and swarming motilities, the key traits associated with bacterial virulence and biofilm development. Cytotoxicity assessment in HepG2 cells confirmed bio-compatibility and non-toxicity of SBN-BSA NPs. The combinatorial treatment also inhibited the production of major virulence factors of P. aeruginosa. RT-qPCR study revealed that this combinatorial treatment significantly affects the expression of genes involved in quorum sensing (QS) system of P. aeruginosa, including lasI, lasR, rhlI, rhlR, pqsA, and mvfR. Molecular docking studies indicated that silibinin effectively interacts with the active sites of the autoinducer synthases LasI, RhlI, PqsA, and respective transcriptional regulators, LasR, RhlR, and MvfR. In brief, the findings suggest that the SBN-BSA NPs and gentamicin combination could reduce biofilm development and virulence through QS-network, and projecting an alternative strategy to combat MDR P. aeruginosa infections.