| Summary: | Rapid antimicrobial susceptibility testing (AST) is an effective measure in the treatment of infections and the prevention of bacterial drug resistance. However, diverse antibiotic types and bacterial characteristics have formed complicated barriers to rapid diagnosis. To counteract these limitations, we investigated the interactions between antibiotic-treated bacteria and functionalized microbeads in optical diffusometry. The conjugation with bacteria increased the effective microbead complex size, thereby resulting in a temporal diffusivity change. The yielded data were sorted and analyzed to delineate a pattern for the prediction of antimicrobial susceptibility. The outcome showed that a completed rapid AST based on the trend of microbead diffusivity could provide results within 3 h (2 h measurement + 1 h computation). In this research, we studied four bacterial strains, including <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, <i>Klebsiella pneumoniae</i>, and <i>Staphylococcus aureus</i>, and six antibiotics. Despite the different inhibitory effects caused by various antibiotics, similar trends in diffusivity alteration for all susceptible and resistant cases in the last 40 min of the 2-h measurement period were deduced. In addition, the AST results obtained using optical diffusometry showed good agreement with those acquired from the commercial instrument and conventional culture methods. Finally, we conducted a single-blinded clinical test, and the sensitivity, specificity, and accuracy of the system reached 92.9%, 91.4%, and 91.8%, respectively. Overall, the developed optical diffusometry showcased rapid AST with a small sample volume (20 μL) and low initial bacterial count (10<sup>5</sup> CFU/mL). This technique provided a promising way to achieve early therapy against microbial diseases in the future.
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