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

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.

Keywords: amide derivative; biofilms; cell adhesion; di-rhamnolipids; rhamnolipids.

FIGURE 1

Structural characterization of di-rhamnolipids (F3) isolated from Lysinibacillus sp. BV152.1. (A) ¹H NMR spectrum of F3, (B) HPLC chromatogram of F3, and (C) thin layer chromatography of Pseudomonas aeruginosa rhamnolipids (R90), purified di-rhamnolipids from R90 (di-Rha) and di-rhamnolipids fraction isolated from Lysinibacillus sp. BV152.1 culture (F3).

FIGURE 2

Inhibition of P. aeruginosa PAO1 biofilm formation with (A) di-rhamnolipids produced by Lysinibacillus sp. BV152.1 (F3) and (B) P. aeruginosa (rhamnolipids mixture and purified di-Rha). ^∗P < 0.05.

FIGURE 3

Inhibition of cell attachment and biofilm formation with di-rhamnolipids from Lysinibacillus sp. BV152.1. Biofilms P. aeruginosa PAO1 were formed for 24 h on silicone catheter (A,B) or glass (C,D) in the presence of DMSO (0.1%) or F3 (50 μg mL^-1). Biofilms were analyzed by scanning electron microscopy (SEM; A,B) or fluorescent microscopy (C,D). In (C,D) bacteria labeled with Syto9 appeared green and bacteria stained with propidium iodide (PI) are red, scale bars represent 10 μm.

FIGURE 4

Chemical structures of di-Rha (C10-C10) and amide derivatives synthesized in this study with calculated parameters hydrophilic-lipophilic balance (HLB), acid dissociation constant (pKa), partition coefficient (logP).

FIGURE 5

Cytotoxic effects of rhamnolipids mixtures, di-rhamnolipids and their derivatives from Lysinibacillus sp. BV152.1 (A) and P. aeruginosa (B) on human fibroblasts (MRC5) measured by MTT method, following 48 h exposure. Values are representative of two independent experiments ± SD.

FIGURE 6

Pseudomonas aeruginosa PAO1 biofilm formation (A,B), cell adhesion (C,D), or biofilm disruption (E,F; %) in the presence of di-rhamnolipids isolated from Lysinibacillus sp. BV152.1 (A,C,E) or P. aeruginosa (B,D,F) and their derivatives. Values are presented as mean ± SD. ^∗P < 0.05.

FIGURE 7

Pseudomonas aeruginosa PAO1 biofilm formation on plastic surfaces in the presence of 0.1% DMSO (A), F3 (B), di-Rha-Mor derivative from Lysinibacillus sp. BV152.1 (C), or di-Rha (D), and di-Rha-Mor derivative from P. aeruginosa (E) at 50 μg mL^-1. Biofilms were stained with Syto9 (green) and PI (red), scale bars represent 10 μm.

FIGURE 8

Dispersion of P. aeruginosa PAO1 biofilms pre-formed on plastic surfaces (A) with 50 μg mL^-1 F3 (B), di-Rha-Mor derivative from Lysinibacillus sp. BV152.1 (C), di-Rha (D), or di-Rha-Mor derivative from P. aeruginosa (E). Biofilms were stained with Syto9 (green) and PI (red), scale bars represent 10 μm.