Nanoparticle Coatings on Glass Surfaces to Prevent Pseudomonas fluorescens AR 11 Biofilm Formation

Abstract

Microbial colonization of surfaces is a sanitary and industrial issue for many applications, leading to product contamination and human infections. When microorganisms closely interact with a surface, they start to produce an exo-polysaccaridic matrix to adhere to and protect themselves from adverse environmental conditions. This type of structure is called a biofilm. The aim of our work is to investigate novel technologies able to prevent biofilm formation by surface coatings. We coated glass surfaces with melanin-ZnO₂, melanin-TiO₂, and TiO₂ hybrid nanoparticles. The functionalization was performed using cold plasma to activate glass-substrate-coated surfaces, that were characterized by performing water and soybean oil wetting tests. A quantitative characterization of the antibiofilm properties was done using Pseudomonas fluorescens AR 11 as a model organism. Biofilm morphologies were observed using confocal laser scanning microscopy and image analysis techniques were used to obtain quantitative morphological parameters. The results highlight the efficacy of the proposed surface coating to prevent biofilm formation. Melanin-TiO₂ proved to be the most efficient among the particles investigated. Our results can be a valuable support for future implementation of the technique proposed here in an extended range of applications that may include further testing on other strains and other support materials.

Keywords: antibiofilm; antimicrobial; biofilm; cold plasma; confocal laser scanning microscopy; hybrid nanoparticles; wetting.

Figure 1

XRD spectra of Mel/TiO₂ and Mel/ZnO nanoparticles.

Figure 2

TGA curves of Mel/TiO₂ (A) and Mel/ZnO (B) nanoparticles.

Figure 3

Side views of 30 μL droplets of water and soybean oil. Experiments are conducted in triplicates for each type of sample (nanoparticles of melanin/ZnO, TiO₂, and melanin/TiO₂).

Figure 4

Confocal laser scanning microscopy scans performed with LIVE/DEAD staining of 48 h biofilm growth on bare glass (control) and functionalized samples (nanoparticles of melanin/ZnO, TiO₂, melanin/TiO₂). Live bacteria are reported in green, dead in red.

Figure 5

Confocal laser scanning microscopy Z-scan projections performed with live/dead staining of 48h biofilm growth on bare glass (control) and functionalized samples (nanoparticles of melanin-ZnO, TiO₂, melanin TiO₂). For each sample, top view (right) and side view (left) display overlaid images. Live bacteria are reported in green, dead in red.

Figure 6

Area occupied in each layer of the Z-scan acquisition by dead cells (in red) and live cells (in green). The grey line represents the total surface area. The height represents biofilm thickness starting from the bottom surface layer to the last biofilm layer detected for each sample. Area is evaluated by processing CSLM stack images of biofilms cultivated under different coatings: bare glass, melanin/ZnO, TiO₂, melanin/TiO₂. Average values ± standard deviation calculated from at least triplicate independent experiments are shown.