3D bacterial culture and minimum inhibitory concentration measurement using limited alginate microsphere space

Bacterial resistance is a serious threat to global public health, creating a heavy burden on healthcare systems and patients. Traditional antimicrobial susceptibility testing (AST) is time-consuming, and two-dimensional (2D) culture systems fail to accurately replicate the three-dimensional (3D) microenvironment in which bacteria naturally grow. Moreover, conventional AST method only provide endpoint susceptibility results and cannot continuously monitor drug-bacteria interactions. In this study, we used a microfluidic chip to produce monodisperse alginate hydrogel microspheres, which were then used to isolate and encapsulate single Escherichia coli ( E. coli ) strain for 3D culture and AST. Our results demonstrate that the structure and limited space inside the microspheres provide a microenvironment similar to the natural 3D conditions for bacterial growth. Using this hydrogel microsphere-based 3D culture system, we performed rapid minimum inhibitory concentration (MIC) testing and continuous monitoring of E. coli strain to 3 antibiotics with different action mechanisms. This system enables to offer MIC results within 6 h, much faster than conventional methods (>16 h). We believe that combining microfluidic technology with 3D microsphere confined culture can not only effectively meet the needs of rapid clinical diagnosis and treatment by shortening the time needed for MIC testing, but also capture dynamic bacterial growth and antibiotic interactions, providing more valuable information for studying resistance mechanisms and developing treatment strategies.