The dynamic response of Cupriavidus metallidurans CH34 cells and biofilms to silver nanoparticles and the importance of biofilm maturation stage

Biofilms are in many sectors a persistent technical challenge that can lead to reduced efficiency in industrial processes, increased maintenance costs, and potential product contamination. Furthermore, biofilms can contribute to the development of antimicrobial resistance, posing health risks in medical and food production environments, as well as reducing the effectiveness of cleaning and sterilization efforts. Therefore, we investigated the impact of antimicrobial silver nanoparticles (AgNPs) on macroscopically visible biofilms of Cupriavidus metallidurans CH34, a metal-resistant and oligotrophic bacterium. We accelerated the time-consuming process of naturally forming macroscopic biofilms via a laboratory setup using porous synthetic surfaces. This setup simulates growth dynamics on air-exposed and nutrient-leaking substrates such as rocks, wood, polymeric coatings, joints and dripping pipes. Remarkably, 24-hour matured biofilms were unaffected by AgNPs at concentrations as high as 750 mg/L. Biofilm resilience to AgNPs was age-dependent and although densely populated, young biofilms (6–9 hours) were inactivated by AgNPs while older ones (14–24 hours) exhibited resistance in the same conditions. Additional experiments suggested that the resistance in older biofilms was not attributed to cell density, but potentially to other factors like extracellular polymeric substances (EPS) or their metabolic state. AgNPs induced oxidative stress in exposed biofilms but did not result in microscopically observable morphological changes in the biofilm-resident cells, indicating that inhibition mechanisms did not involve outer membrane disruption. The proteomic analysis of macroscopic biofilms exposed to AgNPs revealed an age-independent protein set that was consistently upregulated/down regulated across all analyzed biofilm stages as well as age-specific pathways. These findings suggest complex adaptive molecular mechanisms in C. metallidurans biofilms that confer resistance to AgNPs, involving oxidative stress management, iron metabolism, and electron transport chain adjustments.