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

## TL;DR

This paper introduces two rapid methods for antibiotic susceptibility testing in urine samples that eliminate the need for bacterial standardization, reducing diagnostic time and improving accuracy.

## Contribution

The novel contribution is integrating bacterial concentration into direct disk diffusion methods, enabling faster and more accurate antibiotic resistance testing from urine.

## Key findings

- The threshold-adapted method achieved 94.7% agreement for susceptible and 94.1% for resistant bacteria.
- The regression-based method reached 100% agreement for susceptible and 88.2% for resistant bacteria.
- Both methods reduced diagnostic time by 18–24 hours and showed high agreement with standard testing.

## Abstract

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.

Antibiotic 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.