Universal-Bac 3 Gel ® : a 3D Biofilm-Relevant Matrix that Supports In Vitro Growth and Biofilm Formation of ESKAPE Pathogens

Human microbiota is increasingly considered to shape health and disease, drawing interest of pharma and biotech industries in advanced models of in vitro human microbiome to streamline drug development. In this context, Universal-Bac 3 Gel ® represents a new generation of 3D biomaterials designed to mimic the properties of human mucus and biofilm features, including micro-gradients that replicate the heterogeneous environments colonized by microorganisms in the human body. To evaluate the suitability of Universal-Bac 3 Gel ® for studying clinically relevant species in antimicrobial resistance, the so-called ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae ) were cultured within this 3D environment. Bacterial growth was monitored at 24- and 48-hours post-inoculation via spot plating, while viability, distribution and organization were assessed using confocal laser scanning microscopy. All ESKAPE strains successfully grew throughout the structure of Universal-Bac 3 Gel ® . Distinct 3D biofilm architectures were observed across species, ranging from diffuse colonization to compact microcolony formation, in agreement with species-specific biofilm patterns. Notably, the platform’s ready-to-use 96-well format allowed direct comparison of these high-priority pathogens under identical conditions, highlighting species-specific biofilm traits that would be difficult to discern in traditional 2D or animal models. This work highlights the versatility of Universal-Bac 3 Gel ® as a platform for studying pathogen colonization under a biofilm-relevant environment and underscores its potential for translational applications, including antimicrobial drug testing, personalized medicine, and reduction of animal model use in preclinical research.