Assessment of Antibiotic Sensitivity in Biofilms Using GelMA Hydrogel Microspheres

Conventional antibiotic susceptibility testing (AST) primarily assesses planktonic bacteria. However, the three-dimensional architecture and barrier properties of biofilms mean that the minimum inhibitory concentration (MIC) for planktonic cells is typically far lower than the antibiotic exposure required for biofilm eradication. In this study, gelatin methacryloyl (GelMA) microspheres were used to create a three-dimensional biofilm microenvironment for the quantitative evaluation of biofilm tolerance. Escherichia coli K-12 MG1655 was immersed in GelMA microspheres and subjected to a series of antibiotic concentration gradients. Bacterial viability was inferred from time-dependent changes in microsphere diameter. The results demonstrated substantial tolerance of the resulting biofilms to ampicillin, ciprofloxacin, and ceftriaxone, with biofilm antibiotic tolerance values exceeding 200 μg/mL, 10-50 μg/mL, and 20-50 μg/mL, respectively. Relative to planktonic MICs, these tolerance levels are elevated by one to two orders of magnitude and surpass the standard clinical breakpoint thresholds. This methodology includes a high-throughput platform, involving only several hundred microspheres and achieving completion within 24 h, thereby offering a useful platform for investigating biofilm resistance mechanisms and guiding antibiotic treatment strategies.