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Biofilms are omnipresent in clinical and industrial settings and most of the times cause detrimental side effects. Finding efficient strategies to control surface-growing communities of micro-organisms remains a significant challenge. Rhamnolipids are extracellular secondary metabolites with surface-active properties mainly produced by Pseudomonas aeruginosa. There is growing evidence for the implication of this biosurfactant in different stages of biofilm development of this bacterium. Furthermore, rhamnolipids display a significant potential as anti-adhesive and disrupting agents against established biofilms formed by several bacterial and fungal species. Their low toxicity, biodegradability, efficiency and specificity, compared to synthetic surfactants typically used in biofilm control, might compensate for the economic hurdle still linked to their superior production costs and make them promising antifouling agents.