The effects of chromolaena odorata ethanolic extract on pseudomonas aeruginosa biofilm formation / Wan Mohamad Afifi Wan Mohd Zawawi

Chromolaena odorata is known to possess antimicrobial effects against wide range of microorganisms including Pseudomonas aeruginosa. However, the inhibitory effects of C. odorata extracts against the biofilm growth mode of Pseudomonas aeruginosa remain uncertain. Therefore, this study was carried out to determine the antibiofilm activity of C. odorata extracts against P. aeruginosa under aerobic and anaerobic conditions. Phytochemical screening using Gas Chromatography Mass Spectrometry (GCMS) revealed the major constituents in Chromolaena odorata ethanolic extracts (COEE) as Germacrene D, Caryophyllene and δ- Cadinene. Microbroth dilution assay showed that absence of oxygen did not affect MIC and MBC of COEE against P. aeruginosa. However, antibacterial susceptibility test showed that the size of inhibition zone of COEE against P. aeruginosa were slightly different between the aerobic and anaerobic conditions. Colony forming unit counting of biofilm cells showed inhibition greater than 50% at COEE test concentration of 50mg/ml and 200mg/ml under both aerobic and anaerobic conditions. Also, COEE treatment resulted in changes in the biochemical composition of P. aeruginosa biofilm extracellular matrixes under both experimental conditions as indicated by variation in the infrared spectra in the region between 1700 and 900cm-1. A combination of SDS polyacrylamide gel electrophoresis and densitometry was used to analyze the total protein profile of P. aeruginosa biofilm under aerobic and anaerobic conditions. Treatment with COEE was found to alter the total protein profile of P. aeruginosa biofilm under aerobic and anaerobic conditions. Densitometric analysis between 400 and 750 nm demonstrated higher expression of proteins of 16 kDa, 34 kDa and 44 kDa under aerobic condition whilst lower expression of 25 kDa, 41 kDa and 55 kDa proteins was observed under anaerobic condition. Meanwhile, two dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with MALDI TOF-TOF successfully identified 19 biofilm proteins under both experimental conditions. The three highest functional categories of identified P. aeruginosa biofilm proteins were found to be responsible for translation, ribosomal structure and biogenesis (56%), energy production and conversion (22%) and redox homeostasis (11%).