Rapid direct disk diffusion testing for antibiotic resistance in urinary tract infections: a bacterial concentration-adjusted approach.

Urinary tract infections are among the most prevalent bacterial infections worldwide, typically diagnosed using clinical symptoms, dipstick tests, and laboratory methods requiring standardized bacterial suspensions for antibiotic susceptibility testing (AST). This study evaluated two rapid disk diffusion methods for urine samples that incorporate bacterial concentration into the analysis, eliminating the need for standardized suspensions. The threshold-adapted approach compares inhibition zones to concentration-specific breakpoints derived from reference strains, while the regression-based method transforms inhibition zones at various concentrations into predicted standard (0.5 McFarland) values using a linear model. In both methods, urine and antibiotic disks are applied to one agar plate for disk diffusion, while a separate plate determines bacterial concentration, enables isolation, and supports matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis. This simultaneous processing reduces diagnostic time by 18-24 hours. The approaches were compared with traditional disk diffusion testing using defined bacterial suspensions, reference strains, and clinical urine samples. In trials using defined suspensions from 0.5 McFarland to 10³ CFU/mL, the threshold-adapted method achieved 94.7% and 94.1% categorical agreement for susceptible and resistant bacteria, respectively; the regression-based method achieved 100% for susceptible and 88.2% for resistant bacteria. When urine was applied directly, both methods showed 93.7% true susceptible and 94.1% true resistant agreement with standard testing. By integrating inhibition zone size, antibiotic, species, and bacterial concentration, these rapid AST methods streamline urine diagnostics and show high agreement with standard testing-highlighting their potential as practical alternatives to conventional AST, particularly in resource-limited settings where reduced diagnostic time and simplified laboratory procedures can significantly improve patient care.IMPORTANCEAntibiotic susceptibility testing (AST) for urinary tract infections typically requires time-consuming standardization of bacterial suspensions, delaying targeted treatment. Prior direct susceptibility testing (DST) approaches have largely overlooked the inoculum effect, testing at a single, arbitrary concentration and risking misclassification of both resistant and susceptible isolates. This study presents two rapid disk diffusion methods that systemically incorporate bacterial concentration into the analysis, enabling direct testing from urine samples without prior inoculum adjustment. Both approaches demonstrated high agreement with standard AST and reduced diagnostic time by up to 24 hours. These concentration-aware methods may streamline susceptibility testing workflows, particularly in resource-limited settings, and represent a practical advancement toward faster, clinically reliable DST.