Anti-biofilm Effect Of A Novel Exopolysaccharide From Marine Vibrio Sp QY101

In the past, bacterial cells were regarded as individual organisms growing in planktonic populations. However, it is now recognized that many or perhaps most bacteria have a strong propensity to form multicellular, matrix-enclosed assemblies, or biofilms, which are found on surfaces throughout the biological world. Cells within a biofilm have a number of advantages over their planktonic counterparts, including protection against the immune system and predation by protozoa, enhanced ability to transfer genetic information, and enhanced resistance to antimicrobial agents and other stresses. Thus, biofilm formation complicates a variety of chronic infections, including the devastating pulmonary infections that are caused by Pseudomonas aeruginosa in cystic fibrosis patients and other opportunistic infections by this organism. According to a public announcement by US National Institute of Health,"biofilms are medically important, accounting for over 80% of microbial infections in the body". These problems of sessile organisms predominate in most of the environmental, industrial, and medical problems and cause the interest of microbiologists. Yet bacterial biofilms remain poorly understood and the strategies for their control remain underdeveloped. Therefore, anti-biofilm substances have become the hotspots in medicine, material and other research fields.The aim of this study was to screen active substance from marine bacteria, which can inhibit biofilm formation and/or disrupt established biofilm; and to explore its structural character and anti-biofilm mechanism.Using micro tube bacterial biofilm model, we found that the supernatant of marine Vibrio sp QY101 significantly inhibited the biofilm formation of P. aeruginosa FRD1, and the inhibiting effect was dose-dependent in a certain concentration range. Further study showed that the active ingredient in supernatant did not relate to nucleic acid and protein, but to carbohydrate.A polysaccharide, namely A101, was purified from QY101 supernatant by acetone precipitation, dialysis and ion exchange chromatography. In order to characterize the polysaccharide structurally, many modern techniques were used which include elemental analysis, gas chromatography, infrared spectroscopy, light scattering and so on. As a result, exopolysaccharide A101 contains at lest 4 monosaccharide residues and sulfuric acid, amino and acetyl group. The average molecular weight of A101 is determined to be 1000 kD. A101 solution has colloidal character and strong absorption at 220nm. It significantly inhibited the biofilm formation of P. aeruginosa, Staphylococcus aureus and S. epidermidis.In order to monitor biofilm more directly and in real-time manner, the flow cell model was established. The development of flow cell-grown biofilm of P. aeruginosa FRD1 was investigated by confocal laser scanning microscopy and the structural development of the biofilms was quantified by the computer program COMSTAT. The exopolysaccharide A101 inhibited P. aeruginosa FRD1 biofilm formation by more than 90% and disrupted its established biofilm by 85%. In contrast to amikacin alone, the combination of amikacin and A101 increased the susceptibility of FRD1 biofilm by more than 16 times in minimum biofilm elimination concentration (MBEC) test. Our result suggested that A101 could significantly enhance the activities of antibiotics against biofilms.A101 did not inhibit planktonic FRD1 cell growth. On the contrary, it promoted cell growth without being used as carbon source. In addition, A101 prevent bacterial cells from aggregating, made them grow dispersedly, and bettered utilizing the environmental nutrition. This function of A101 may explain its anti-biofilm effect. Based on our results, we hypothesized that the mechanism of A101 anti-biofilm is mediated by blocking interaction between the cells.We demonstrated here an exopolysaccharide extract from marine Vibrio sp QY101 which can inhibit the biofilm formation and disrupt the established biofilm. This exopolysaccharide A101 prevented the formation of biofilms by a wide range of bacteria, particularly clinical infective P. aeruginosa, S. aureus, and S. epidermidis. Combined with exopolysaccharide A101, antibiotic was strengthened in killing bacterial cells in biofilm. This is the first report about a polysaccharide having these functions. A101 is expected to develop as an effective, broad-spectrum and safe anti-biofilm agent. It not only has wide application in the treatment of clinical biofilm-related infections and development of novel anti-biofilm materials, but also has important value in biofilm research fields, such as the formation and detachment of biofilm, the recognition and interaction among bacterial cells.