# AgNPs-tailored antimicrobial surfaces against Pseudomonas aeruginosa biofilm formation under dynamic conditions

Maya Rima, Christina Villeneuve-Faure, Laurent Boudou, Fatima El Garah, Ludovic Pilloux, Christine Roques, Kremena Makasheva

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## To cite this version:

Maya Rima, Christina Villeneuve-Faure, Laurent Boudou, Fatima El Garah, Ludovic Pilloux, et al.. AgNPs-tailored antimicrobial surfaces against Pseudomonas aeruginosa biofilm formation under dynamic conditions. IEEE International Conference on Nano/Molecular Medicine & Engineering (IEEE NANOMED 2025), Dec 2025, Hong Kong, China. hal-05449803

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# AgNPs-tailored antimicrobial surfaces against *Pseudomonas aeruginosa* biofilm formation under dynamic conditions*

M. Rima, C. Villeneuve-Faure, Member, IEEE, L. Boudou, F. El Garah, L. Pilloux, C. Roques, and K. Makasheva, Senior Member, IEEE

***Abstract***—Although medical devices, whether implanted or not, are intended to ensure well-being, they may turn into a reservoir for the colonization and growth of microorganisms, thus supporting the onset of a resilient biofilm and ultimately contributing to serious infections. A promising strategy to address this challenge is to advance in the development of medical devices with anti-biofilm surface properties. Here, we explore nanocomposite materials based on silver nanoparticles (AgNPs) immobilized on the surface of a thin silica (SiO₂) layer for their potential impact on the adhesion, detachment, viability, biofilm formation and resilience of *Pseudomonas aeruginosa* PAO1-Tn7-gfp under dynamic conditions. It is found that the flat bacterial layer that forms on the surface of the AgNPs-based biomaterials after 72 h of dynamic culture is made up of dispersed cells and appears very sensitive towards application of increasing wall-shear stresses, thus demonstrating no or poor resilience under dynamic conditions.

## I. INTRODUCTION

The development of new biomaterials has considerably increased in the last decade due to the enlarged demand by many biomedical applications. A thorough description of the biomaterial properties requires consideration of their exposure and interaction with microorganisms, since the microbial adhesion and subsequent biofilm formation significantly contribute to hospital-acquired infections (HAIs) [1, 2]. These infections often lead to septic complications, and even lethal issues, and entail large economical losses for the health-care systems. In this context, new strategies oriented to the prevention of environmental contamination of medical devices, catheters, implants, etc., are under scrutiny.

Pursuing high efficiency, silver nanoparticles (AgNPs) have gained popularity in the biomedical domain due to their inherent antimicrobial properties against bacteria, viruses, and eukaryotic microorganisms [3, 4]. Development of AgNPs-based nanocomposite surface coatings that locally provide a controlled amount of biocide agent and unceasing inhibition of the microbial adhesion and biofilm formation over a long term is thus a promising strategy for preventive or curative treatments. The current contribution focuses on development of AgNPs-tailored antimicrobial surfaces and assessment of their efficacy against *Pseudomonas aeruginosa* PAO1-Tn7-gfp adhesion and subsequent biofilm formation under dynamic conditions.

## II. EXPERIMENTAL

### A. Elaboration of AgNPs-based biomaterials by a plasma process and their structural characterization

The AgNPs-based biomaterials contain a monolayer of AgNPs immobilized on the surface of 100 nm-thick SiO₂ layers, thermally grown on Si-substrates. The synthesis of AgNPs was performed in a hybrid plasma (electrical discharge) process successfully combining in the same reactor Ag-sputtering and plasma polymerization [4, 5]. This deposition method allows a strict control over the size and surface density of the AgNPs. The plasma elaborated AgNPs-based biomaterials have been structurally characterized by Atomic Force Microscopy (AFM) for the surface topography and by Scanning Electron Microscopy (SEM) for the size, interparticle distance and distribution on the surface of the AgNPs (Figure 1(a)).

### B. Bacterial strain and culture media

The bacterial strain used in this study was the opportunistic bacterium *Pseudomonas aeruginosa* (PAO1-Tn7-gfp strain) [6]. In all assays, the used final concentration of cell suspension was 10⁷ CFU/mL prepared in Water for Injectable Preparations (WIP). For biofilm formation assays, a low nutritive medium, named minimum biofilm broth (MBB) was used, in order to create stressful conditions and subsequently promote biofilm formation and growth of adherent bacteria rather than planktonic growth. Details on the followed procedure are given elsewhere [5]. The observations on microbial adhesion and biofilm formation were conducted using a Zeiss-Axiotech epifluorescence microscope with a 20X (Zeiss, EC Plan-Neofluar) objective, equipped with an HXP 120 C light source. The analysis is based on the recorded fluorescent intensity (FI).

### C. Biofilm formation of P. aeruginosa PAO1-Tn7-gfp on AgNPs-based biomaterials under dynamic conditions

The impact of AgNPs on the adhesion, viability and biofilm formation of *P. aeruginosa* PAO1-Tn7-gfp was evaluated under dynamic conditions using a shear-stress flow chamber (Figure 1(b)) [7].

## III. RESULTS AND DISCUSSION

The results show that the AgNPs-based biomaterials contain a monolayer of randomly distributed on the SiO₂-

<footnote>*Research supported by l'Agence Nationale de la Recherche in France, project ANR BENDIS (ANR-21-CE09-0008).</footnote>

<footnote>C. Villeneuve-Faure, L. Boudou, and K. Makasheva are with LAPLACE (Laboratoire Plasma et Conversion d'Energie), University of</footnote>

<footnote>Toulouse, CNRS, INPT, Toulouse, France (e-mail: kremena.makasheva@laplace.univ-tlse.fr).</footnote>

<footnote>M. Rima, F. El Garah, L. Pilloux and C. Roques are with LGC (Laboratoire de Génie Chimique), University of Toulouse, CNRS, INPT, Toulouse, France (e-mail: fatima.el-garah@utoulouse.fr).</footnote>
<figure><img src="image_3.png" /><figcaption>Figure 1. (a) Structure of the AgNPs-based biomaterials, characterization of their surface topography by AFM and SEM-analysis of the AgNPs; (b) Experimental setup for study of biofilm formation under dynamic conditions and (c) Epifluorescence microscopy images of 72 h-mature biofilms of *P. aeruginosa* PAO1-Tn7-gfp on SiO₂-surfaces and AgNPs-based biomaterials, and AFM-analysis of the interaction of bacteria with the AgNPs.</figcaption></figure>

surface AgNPs with average size of 18.9 nm and interparticle distance of 5.6 nm (Figure 1(a)). One notices a rapid bactericidal effect of AgNPs, clear within 30 min of bacterial exposure. The presence of some residual viable cells leads to delayed surface colonization with no structured biofilm, even after 72 h (3 days) of dynamic culture. Only dispersed cells, without any sign of 3D-organization, are observed on the AgNPs-based biomaterials in contrast to the thick and well-structured biofilms settled on the SiO₂-surfaces (Figure 1(c)). Progressive increase of the wall shear-stress from 0.3 to 16.7 Pa does not modify the 2D-biofilm morphology for the SiO₂-surfaces. On the contrary a progressive detachment of the residual sessile bacteria is detected for the AgNPs-based biomaterials, with no visible bacteria resisting the last condition, thus demonstrating no or poor resilience under dynamic conditions.

## IV. CONCLUSION

The main finding of this study is that in direct contact with AgNPs-based biomaterials, *P. aeruginosa* PAO1-Tn7-gfp does not organize in a structured biofilm, even after 72 h of culture under dynamic conditions. It underlines the promising potential of AgNPs-based coatings in the conception of novel antimicrobial/antibiofilm surfaces.

## ACKNOWLEDGMENT

The authors thank Mr. Parsek from the Department of

Microbiology, University of Washington, USA for kindly providing the *P. aeruginosa* PAO1-Tn7-gfp strain. The authors acknowledge support from UAR Centre de MicroCaractérisation Raimond Castaing of the University of Toulouse and CNRS, and thank Mr. Stéphane Le Blond du Plouy for the SEM observations.

## REFERENCES

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[6] M. J. Kirisits, L. Prost, M. Starkey, and M. R. Parsek, "Characterization of Colony Morphology Variants Isolated from *Pseudomonas aeruginosa* Biofilms," *Appl. Environ. Microbiol.*, vol. 71, no. 8, pp. 4809-4821, Aug. 2005.

[7] M. Rima et al., "Towards a better understanding of the effect of protein conditioning layers on microbial adhesion: a focused investigation of fibronectin and bovine serum albumin layers on SiO₂ surfaces," *Biomater. Sci.*, vol. 12, no. 12, pp. 3086-3099, 2024.