Two-Sided Antibacterial Cellulose Combining Probiotics and Silver Nanoparticles

Abstract

The constant increase of antibiotic-resistant bacteria demands the design of novel antibiotic-free materials. The combination of antibacterials in a biocompatible biomaterial is a very promising strategy to treat infections caused by a broader spectrum of resistant pathogens. Here, we combined two antibacterials, silver nanoparticles (AgNPs) and living probiotics (Lactobacillus fermentum, Lf), using bacterial cellulose (BC) as scaffold. By controlling the loading of each antibacterial at opposite BC sides, we obtained a two-sided biomaterial (AgNP-BC-Lf) with a high density of alive and metabolically active probiotics on one surface and AgNPs on the opposite one, being probiotics well preserved from the killer effect of AgNPs. The resulting two-sided biomaterial was characterized by Field-Emission Scanning Electron Microscopy (FESEM) and Confocal Laser Scanning Microscopy (CLSM). The antibacterial capacity against Pseudomonas aeruginosa (PA), an opportunistic pathogen responsible for a broad range of skin infections, was also assessed by agar diffusion tests in pathogen-favorable media. Results showed an enhanced activity against PA when both antibacterials were combined into BC (AgNP-BC-Lf) with respect to BC containing only one of the antibacterials, BC-Lf or AgNP-BC. Therefore, AgNP-BC-Lf is an antibiotic-free biomaterial that can be useful for the therapy of topical bacterial infections.

Keywords: Ag nanoparticles; antibiotic-resistant bacteria; bacterial cellulose; probiotics.

Figure 1

(A) Graphical representation of the experimental protocol used to obtain the two-sided BC. The first step involves the adsorption of probiotics followed by the impregnation of the opposite BC side with AgNPs. The resulting material is referred to as AgNPs-BC-Lf. (B) The UV-vis spectrum showed the expected absorbance band centered at 420 nm, in agreement with AgNPs with a mean diameter of 50 nm. The inset in B corresponds to a picture of an AgNPs solution. (C) HAADF-STEM micrograph of AgNPs (scale bar is 100 nm). (D) Diameter distribution of AgNPs (n = 100).

Figure 2

CLSM of BC-Lf at different incubation times in AgNPs solution. Samples were stained with SYTO9/PI dyes. The images are maximum intensity projections of 20 μm in-depth of (A) BC after Lf adsorption (BC-Lf), (B) BC-Lf after 15 min of incubation in AgNPs (AgNP-BC-Lf), and (C) BC-Lf after 30 min of incubation in AgNPs. Scale bars = 10 μm. (D–F) images are three-dimensional reconstructions of (A–C), respectively.

Figure 3

FESEM images of the two-sided biomaterials. (A) The side containing Lf exhibited the typical rod-like morphology of this bacterium. (B) BC side containing AgNPs. (C) Lf-functionalized BC surface, containing a higher amount of AgNPs (30 min of incubation in the AgNPs solution). Arrows show the cell wall damage. Scale bars: 2 μm.

Figure 4

Inhibitory activity of the composite materials against PA. Inhibition zones of BC, AgNP-BC, BC-Lf and AgNP-BC-Lf. The corresponding BC side in contact with agar is marked in bold and underlined. The diameter of the Petri dish is 9 cm.