Effects of Piper betle Leaf Extract on Biofilm and Rhamnolipid Formation of Pseudomonas aeruginosa

Abstract

High mortality rate and antimicrobial resistance are still becoming world-wide problems, due to Pseudomonas aeruginosa’s (P. aeruginosa) virulence and its ability to form biofilm. Biofilm’s formation is affected by the presence of rhamnolipid, whose production is regulated by quorum sensing systems. Piper betle (P. betle) possesses antimicrobial, antioxidant, anti-inflammatory and immunomodulatory properties. The aim of our study is to investigate the effects of P. betle leaf’s extract against biofilm formation and rhamnolipid production of P. aeruginosa. Active compounds of P. betle were identified using plate chromatography. Agar dilution method was used to determine the minimum biofilm inhibitory concentration (MBIC) of methanolic leaf extract of P. betle. A biofilm-producing P. aeruginosa isolate in the polystyrene plate adherence test was selected for confirmation of biofilm production by Scanning Electron Microscopy (SEM), after P. betle administration. Rhamnolipid detection and evaluation were performed by interpreting halo formed around the well. After administration of various concentrations of P. betle leaf extract on the microplate well, it was concluded that the MBIC of P. betle leaf extract on P. aeruginosa was 0.4%. Methanolic extract of P. betle leaf extract at concentration of 0.4% showed that P. aeruginosa could not form biofilm at all, although the bacteria could still aggregate and form a matrix. After linear regression analysis, beta-coefficient was obtained at -0.931 for P. betle leaf extract. It can be concluded that P. betle leaf extract was effective in inhibiting the growth of biofilm and formation of rhamnolipid by P. aeruginosa. The increase in concentration of P. betle leaf extract was inversely proportional to the diameter of the halo rhamnolipid formed. The higher the level of P. betel leaf extract, the smaller the diameter of the halo rhamnolipid formed.