Seagrass Halodule pinifolia active constituent 4-methoxybenzioic acid (4-MBA) inhibits quorum sensing mediated virulence production of Pseudomonas aeruginosa

Abstract

Biofilm associated, multidrug resistant Pseudomonas aeruginosa infection remain a challenging problem in the clinical field since the conventional antibiotic therapy are largely inefficient and new approaches are needed. Inactivating the QS virulence mechanism with anti-infective agent is an attractive approach to prevent bacterial infections without resistance development. Seagrass Halodule pinifolia (Miki) Hartog has been shown to exhibit potential antimicrobial activities against harmful pathogens. Our study investigated the effects of seagrass H. pinifolia leaf extract and its bioactive constituents on QS-mediated virulence factors and biofilm formation in P. aerugonasa PAO1. Preliminary screening on antibiofilm activity showed that the methanolic extract of H. pinifolia exhibited potential inhibition of biofilm formation (96%) as compared to the control respectively. Further, the potential extract was column fractionated and the active fraction was characterized by GC-MS. In total eight active compounds (protocatacheuic acid (69.3%), rosmarinic acid (63.5%), caffeic acid (59.18%), p-coumaric acid (59.08%), 4-methoxybenzoic acid (53.19%), naringenin (52.9%), vanillic acid (49.19%), 4-hydroxybenzoic acid (41.73%)) were profiled from fraction 2 and were purified by HPLC, structurally confirmed by NMR. Among the eight compounds studied, 4-methoxybenzoic acid (4-MBA) showed an effective inhibition of bacterial growth and was considered as a lead molecule with minimum inhibitory concentration (MIC) of 62.5 μg/mL. Further the effect of 4-MBA on QS mediated virulence factors demonstrated that the compound at MIC concentration reduced the virulence factor production such as elastase (87.5%), protease (79.38%), pyocyanin (91.46%), rhamnolipid (86%), alginate (86%), chitinase (55%), exopolysaccharide production (83.72%) and CSH (78.39%) over the control respectively. Moreover, 4-MBA down regulated the QS-mediated virulence transcript levels upon treatment with 4-MBA. The present findings suggests that seagrasses may act as a newer source for the marine based drug discovery and the lead compound 4-MBA derived from H. pinifolia may act as anti-infective agent against P. aeruginosa as it controls the QS-mediated virulence production.