(613b) Antibiofouling Activity of Natural Phenolic Compounds against Gram Negative Bacteria

Biofouling is caused by adhesion of microorganisms to solid surfaces with the production of extracellular polymers (polysaccharides and proteins) that results in namely a biofilm. Biofouling causes serious problems in chemical, medical, pharmaceutical industries, and health care fields. In the medical industry, for example, very high concentrations of antibiotics are used to kill the cells growing in a biofilm, which may lead to antibiotic resistance problems. Because it is difficult to kill or remove biofilm, it is considered better approach to prevent microbial cells from forming. Recent findings indicate that some natural phenolic compounds found in plant extracts have an effect on biofilm formation of Gram-negative bacteria. Natural phenolic compounds are known to possess strong anti-oxidant and anti-microbial activities. In this study, we investigated the effect of anti-oxidant activities of natural phenolic compounds on biofilm formation of Pseudomonas Aeruginosa, Gram-negative bacteria. Pseudomonas Aeruginosa was grown to form biofilm in the presence of several natural phenolic compounds such as phenol, anacardic acid, cardanol, salicylic acid, ethyl linoleate, polyanacardic acid, polycardanol, polysalicylic acid, ascorbic acid, catechin, epigallocatechin, tannic acid and tocopherol. None of the above mentioned phenolic compounds were found affecting the growth of Pseudomonas Aeruginosa below the concentration of 15 ppm. The biofilm formation test was performed using a modified microtiter plate assay. Biofilms were fixed with methanol, stained with crystal violet dye, then the bound dye was released with 33% glacial acetic acid and optical density was measured at 570 nm. The optical density readings give an estimate of amount of biofilm formed on the surfaces of the microtiter plate. Bacteria coordinate their interaction with higher organisms and the surrounding environments by intercellular communication systems, namely quorum sensing. For Gram-negative bacteria, one type of communication system functions via N-acyl homoserine lactone (AHL) signal molecules. In order to investigate the effect of phenolic compounds on AHL, a mutant cell, E. coli JB525, was used. E. coli JB525 is deficient of AHL-producing gene, and harboring green fluorescent protein (GFP) producing gene. Therefore, under normal conditions, E. coli JB525 cannot produce GFP, because AHL is not produced. When AHL is provided from outside, GFP-producing gene sequence is triggered by AHL and GFP is produced. Experiments were performed to compare the different phenolic compounds; in one beaker, E. coli JB525 was cultured with AHL, N-3-oxohexanoyl-L-homoserine lactone at 100 nM (Control). In the other beakers, the cells were cultured with AHL and with various phenolic compounds. Green Fluorescence was measured at excitation wavelength of 475 nm and emission detection at 515 nm. Difference in GFP was observed in the result. This result indicates that different phenols inhibit quorum sensing of the cells. Figure 1 shows the GFP intensity measurement for the phenolic compounds in terms of time. Figure 1. GFP fluorescence measurement for the phenolic compounds in the E. coli JB525 cultures. The control, which had no phenolic compounds (square marks), showed the highest fluorescence while the eppigallocatechin polyphenol (triangle marks) showed the lowest fluorescence. It is thought that the phenolic compounds interfered with AHL molecules, which resulted in low GFP fluorescence. The results show that biofouling is influenced by quorum sensing, and inhibiting or interfering with quorum sensing molecules by natural phenolic compounds may help the system prevent or reduce biofouling.