Universal-Bac3Gel: 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-Bac3Gel 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-Bac3Gel 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-h post-inoculation via spot plating, while viability, spatial distribution, and organization were assessed by confocal laser scanning microscopy. All ESKAPE strains successfully grew throughout the structure of Universal-Bac3Gel. Distinct 3D biofilm architectures were observed across species, ranging from diffuse colonization to compact microcolony formation, in agreement with species-specific biofilm patterns. Ciprofloxacin susceptibility assays revealed reduced susceptibility of bacteria cultured within Universal-Bac3Gel compared with their planktonic counterparts, supporting the development of biofilm-associated tolerance phenotypes. Consistent with these findings, crystal violet staining confirmed the accumulation of biofilm-associated biomass within the hydrogel. Notably, the platform's ready-to-use 96-well format allowed direct comparison of these high-priority pathogens under standardized conditions, highlighting species-specific biofilm traits that would be difficult to discern in conventional two-dimensional culture systems. This work highlights the versatility of Universal-Bac3Gel as a biofilm-relevant in vitro platform for studying pathogen colonization, biofilm development and antimicrobial susceptibility under controlled conditions.