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

Ivana Aleksic,Branka Vasiljevic,Lidija Senerovic,Milos Jovanovic,Jasmina Nikodinovic-Runic,Milos Petkovic,Dusan Milivojevic

doi:10.3389/fmicb.2017.02454

Abstract

A new strain, namely Lysinibacillus sp. BV152.1 was isolated from the rhizosphere of ground ivy (Glechoma hederacea L.) producing metabolites with potent ability to inhibit biofilm formation of an important human pathogens Pseudomonas aeruginosa PAO1, Staphylococcus aureus, and Serratia marcescens. 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 Lysinibacillus sp. BV152.1 were more potent in biofilm inhibition, both cell adhesion and biofilm maturation, than commercial di-rhamnolipids inhibiting 50% of P. aeruginosa 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 S. aureus, 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.

Highlights

Microbial biofilms are prevalent in nature, especially in medical, industrial and environmental settings where they cause undesirable effects due to their pathogenicity and resistance toward antibiotics or biofouling technologies (Berlanga and Guerrero, 2016; Holscher and Kovacs, 2017)
Culture extracts from these isolates were screened for their ability to inhibit biofilm formation in P. aeruginosa PAO1 using crystal violet (CV) based assay in 96-well polystyrene plates
The crude cell extract of the isolate BV152.1 showed the most potent ability to inhibit 60% of biofilm formation when applied in the concentration of 500 μg mL−1 while no bactericidal activity of the extract was observed

Summary

Introduction

Microbial biofilms are prevalent in nature, especially in medical, industrial and environmental settings where they cause undesirable effects due to their pathogenicity and resistance toward antibiotics or biofouling technologies (Berlanga and Guerrero, 2016; Holscher and Kovacs, 2017). Biofilms play an important role in virulence of many pathogenic bacteria including Pseudomonas aeruginosa, Staphylococcus aureus, and Serratia marcescens (Costerton et al, 1999; Singh et al, 2000; Bryers, 2008). They can be formed in the patients’ tissues or on the surface of medical. Rhamnolipids as a group of anionic microbial glycolipids, consisting of L-(+)-rhamnose and β-hydroxyalkanoic acids, have been considered in a wide range of applications such as health care, cosmetics, pharmaceutical processes, food and beverage processing, detergents and polymer industry, cryo-protectants, biofuels, microbial fuel cells, and bioremediation, due to their unique characteristics such as low toxicity, surface activities, sustainability, and biodegradability (Li, 2017; Varjani and Upasani, 2017)

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