Single-cell morphological responses as predictors of ciprofloxacin resistance in

The rise of antimicrobial resistance (AMR) coincides with a declining pipeline of new antibiotics, underscoring the urgent need for rapid and accurate antibiotic susceptibility testing (AST). Single-cell AST approaches offer the potential to shorten diagnostic turnaround times; however, reliably distinguishing antibiotic-sensitive from antibiotic-resistant strains at the single-cell level remains challenging. In this study, we employed bacterial cytological profiling (BCP), a single-cell imaging technique previously used for mechanism of action identification, to quantitatively analyze the morphological responses of ciprofloxacin (CIP)-sensitive and CIP-resistant IMPORTANCE: Antimicrobial resistance represents a growing challenge for clinical management, in part because current antibiotic susceptibility testing can take days to determine which antibiotics will work. This study demonstrates that early, single-cell morphological responses to antibiotic exposure can serve as informative phenotypic indicators of resistance. By quantitatively analyzing how individual bacterial cells respond to antibiotic treatment, we show that resistant and susceptible strains can be reliably distinguished within 1 h of antibiotic exposure. Importantly, this approach remains effective across multidrug-resistant and clinical isolates. Together, these findings highlight rapid, imaging-based, single-cell phenotypic responses as a promising complement to current approaches for the future development of faster and more informative antimicrobial susceptibility diagnostics.