Flow cytometry enables rapid evaluation of novel, new and niche antimicrobial agents.

The global rise of multidrug-resistant bacteria necessitates the development of new antimicrobials and faster diagnostic tools. Conventional antimicrobial susceptibility testing is slow, relying on culture-based methods that delay effective treatment, often with fatal consequences in severe infections. In this study, we evaluate flow cytometry as a rapid, culture-minimal method to assess bacterial responses to six antimicrobials: ceftazidime-avibactam, meropenem-vaborbactam, cefiderocol, doxycycline, omadacycline, and lefamulin. Across 165 evaluable antibiotic-isolate combinations, essential agreement between flow cytometry and broth microdilution minimum inhibitory concentrations was 90.71%. Assessable categorical agreement, determined using the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute breakpoints, was 92.59% for doxycycline, 91.67% for omadacycline, and 100% for meropenem-vaborbactam. Cefiderocol exposure was associated with substantial cell elongation, demonstrating cellular-level antimicrobial effects observed using confocal microscopy and imaging flow cytometry. These findings demonstrate the potential of flow cytometry for novel antimicrobial evaluation, offering rapid insights into drug efficacy with potential to improve clinical outcomes in patients.