Elsevier

Colloids and Surfaces B: Biointerfaces

Volume 157, 1 September 2017, Pages 325-334
Colloids and Surfaces B: Biointerfaces

Protocols
Antibacterial properties of sophorolipid-modified gold surfaces against Gram positive and Gram negative pathogens

https://doi.org/10.1016/j.colsurfb.2017.05.072Get rights and content

Highlights

  • Successful Adsorption of Sophorolipids on gold surfaces at various concentrations.

  • The initial concentration of Sophorolipids solutions affects the amount of grafted molecules monitored by XPS and QCM-D.

  • The biocidal activity was monitored by contact of bacterial strains on the coated substrates compared to bare surfaces.

  • Sophorolipid-modified gold surfaces are biologically active against Gram positive and Gram negative pathogens.

Abstract

Sophorolipids are bioderived glycolipids displaying interesting antimicrobial properties. We show that they can be used to develop biocidal monolayers against Listeria ivanovii, a Gram-positive bacterium. The present work points out the dependence between the surface density and the antibacterial activity of grafted sophorolipids. It also emphasizes the broad spectrum of activity of these coatings, demonstrating their potential against both Gram-positive strains (Enteroccocus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes) and Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhymurium). After exposure to sophorolipids grafted onto gold, all these bacterial strains show a significant reduction in viability resulting from membrane damage as evidenced by fluorescent labelling and SEM-FEG analysis.

Introduction

Carbohydrates are an interesting group of functional compounds with a variety of properties and applications in pharmacology (vaccines, drugs) [1], [2], [3] and diagnosis (glyco-microarrays for antibody recognition and cell adhesion) [4]. Their use as antimicrobials is of particular interest, with glycolipids or polysaccharides having broad activities for prevention of microbial contamination either via antiadhesive or biocidal properties [5]. For instance, amphotericin B [6] a complex glycolipid, is a commonly employed antifungal, while exopolysaccharides [7] are used for their antiadhesive properties. Other bioderived glycolipids like rhamnolipids, cellobioselipids or sophorolipids [8] are well-known for their antimicrobial properties. Their biocidal effect seems to depend on the synergistic effect of the sugar and lipid moieties in the so-called “surfactant-effect” [8], [9], [10], which is characterized by plasma membrane disruption, causing lysis and possible leakage of cytoplasm material [11], [12]. Antiadhesive properties are also commonly observed for polysaccharides and glycolipids which are either immobilized on a substrate (glycoarray) or free in solution [5], [13], [14].

The antimicrobial effects of carbohydrate-based compounds are worth studying because of their mild/low cytotoxicity and low ecotoxicological effects and their lower cost (compared to other antimicrobial agents like peptides) [15] which make them very appealing for large scale application, such as protection of outdoor infrastructure for instance. Moreover the diversity in this carbohydrate family promotes a broad spectrum of activity [16]. Despite these advantages and the extensive use of these compounds as antimicrobial agents, the role of the carbohydrate moiety, and in particular its interaction with the cell envelope is still not very clear. The interactions between carbohydrates and bacterial membranes have been studied for decades [17]. However, only recently, modeling studies have tried to put in evidence the impact of simple mono- and disaccharides on the liquid crystalline order of phospholipid bilayers [18], although experimental data are still lacking. In our recent work, we have shown for the first time that a self-assembled monolayer (SAM) composed of glucose β1,2 glycolipid stabilized on gold is able to pierce the cell envelope, and in particular the plasma membrane, of the non-pathogenic bacterial strain Listeria ivanovii [19]. This work has shown the important membrane-disrupting role of the disaccharide, which cannot be related to the so-called “surfactant-effect” since the glycolipid is covalently bond to the surface. However, we showed the impact of the disaccharide orientation on the efficiency of bacterial membrane disruption.

In the present work, we studied the effect of glycolipid grafting density on its biocidal properties. Indeed, sugar concentration appears as a key factor in sugar-membrane interactions [20] and grafting may enable to reach high concentrations in the vicinity of bacteria, thus enhancing its antimicrobial effects [3]; we also want to explore the spectrum of activity of such grafting towards several Gram positive and Gram negative bacterial strains.

Section snippets

Materials and methods

Cysteamine (cys), N-hydroxysuccinimide (NHS), 1-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), glutaraldehyde and sodium chloride (NaCl) were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Sophorolipids (SL) were derived from a commercial acidic and lactone mixture of sophorolipids purchased from Soliance (France) (Sopholiance S; batch number, 11103A; dry content, 60 ± 6%). To obtain a high purity form of the nonacetylated acidic sophorolipids only (>90 mol%,

Variation of SL grafting density

In order to explore the effect of glycolipid grafting density on biocidal properties, gold surfaces were functionalized with solutions at different sophorolipid concentrations. The first step was to assess the possible influence of the initial concentration on the modification of gold surfaces. The lowest concentration used was 5 mg L−1, ca. 0.8 μmol L−1; at this concentration, the solution contains theoretically enough molecules to fully saturate the surfaces with SL molecules (ca. 5 × 1015

Conclusion

In a recent publication we have shown that a monolayer of sophorolipids, microbial-derived glycolipids, can be chemically grafted on top of a gold substrate using a cysteamine primer and that such a material exhibits antibacterial properties against Listeria ivanovii, a non-pathogenic Gram+ bacterium. In this work, we go further and show that gold substrates engineered to tether modified-sophorolipids also damage the membrane of a series of pathogenic Gram+ (Enterococcus faecalis,

Acknowledgments

The authors would like to acknowledge the French state funds managed by the ANR within the Investissements d’Avenir program under Reference ANR-11-IDEX-0004-02 and more specifically within the framework of the Cluster of Excellence MATISSE for a Ph.D. Grant for CV. The authors acknowledge IMPC (Institut des Matériaux deParis Centre, FR2482) and the C’ Nano projects of the Region Ile-de-France, for SEM-FEG and Omicron XPS apparatus funding. The authors would also like to thank Dr. Souhir Boujday

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      Association of glycoconjugates to nanoscience contributed to develop the field of multidimensional glyconanomaterials like vesicles,[2] nanoparticles[3] or functional surfaces.[4] Interesting for their hydration,[5] interactions against lectins[6–8] or between carbohydrates themselves,[9] two-dimensional, supported, glyconanomaterials have found a specific interest for their tunable adhesive,[4] anti-adhesive[1,10] and antimicrobial properties[11,12] but also to better understand virus binding mechanisms.[13] Glycosylated surfaces are generally prepared by a classical self-assembled monolayer (SAM) process, involving chemisorption of thiolated glycoconjugates onto gold.[3,4,10,11]

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