A rapid bacterial labeling method for phenotypic antimicrobial susceptibility testing

Bacterial infections pose significant diagnostic and treatment challenges due to the increasing prevalence of multidrug-resistant pathogens. Current Antimicrobial Susceptibility Test (AST) workflows, including automated systems, require bacterial isolation which is a time-consuming process. Providing timely AST results can lead to better patient outcomes, effective antibiotic stewardship, and significant cost savings. This study evaluates a novel diagnostic method, the Bacteria Labeling Antibiotic Susceptibility Test (BLAST), using a selection of strains that typically cause urinary tract infections. The BLAST test provides AST results within 6 hours, demonstrated here using reference pathogens grown in broth, agar plates, or as bacteria-contrived urine samples. BLAST is a metabolic labeling method that uses click-chemistry to tag labeled, newly synthesized proteins in bacteria and measure signal inhibition by frequently prescribed antibiotics. The method was conducted over a range of starting inocula (10⁵ to >10⁸ CFU/mL) to determine the labeling response using two strains. Minimum Inhibitory Concentration (MIC) values for multiple Gram-positive and Gram-negative pathogens against eleven antibiotics were then determined using a fluorescent reader in a 96-well filtration plate format. BLAST MIC values were compared to the reference broth microdilution (rBMD) MICs. It is significant that the BLAST method can detect the inhibition of new protein biosynthesis for disparate classes of antibiotics, with MIC values in the range expected for CLSI quality control strains. Testing of bacteria-contrived urine cultures using BLAST also demonstrated agreement with rBMD results. Our results indicate that the BLAST method offers a novel pathway to a rapid and scalable solution for AST.