Anti-biofilm Properties of Bacterial Di-Rhamnolipids and Their Semi-Synthetic Amide Derivatives

Abstract

A new strain, namely <i>Lysinibacillus</i> sp. BV152.1 was isolated from the rhizosphere of ground ivy (<i>Glechoma hederacea</i> L.) producing metabolites with potent ability to inhibit biofilm formation of an important human pathogens <i>Pseudomonas aeruginosa</i> PAO1, <i>Staphylococcus aureus</i>, and <i>Serratia marcescens</i>. Structural characterization revealed di-rhamnolipids mixture containing rhamnose (Rha)-Rha-C10-C10, Rha-Rha-C8-C10, and Rha-Rha-C10-C12 in the ratio 7:2:1 as the active principle. Purified di-rhamnolipids, as well as commercially available di-rhamnolipids (Rha-Rha-C10-C10, 93%) were used as the substrate for the chemical derivatization for the first time, yielding three semi-synthetic amide derivatives, benzyl-, piperidine-, and morpholine. A comparative study of the anti-biofilm, antibacterial and cytotoxic properties revealed that di-Rha from <i>Lysinibacillus</i> sp. BV152.1 were more potent in biofilm inhibition, both cell adhesion and biofilm maturation, than commercial di-rhamnolipids inhibiting 50% of <i>P. aeruginosa</i> PAO1 biofilm formation at 50 μg mL^-1 and 75 μg mL^-1, respectively. None of the di-rhamnolipids exhibited antimicrobial properties at concentrations of up to 500 μg mL^-1. Amide derivatization improved inhibition of biofilm formation and dispersion activities of di-rhamnolipids from both sources, with morpholine derivative being the most active causing more than 80% biofilm inhibition at concentrations 100 μg mL^-1. Semi-synthetic amide derivatives showed increased antibacterial activity against <i>S. aureus</i>, and also showed higher cytotoxicity. Therefore, described di-rhamnolipids are potent anti-biofilm agents and the described approach can be seen as viable approach in reaching new rhamnolipid based derivatives with tailored biological properties.