# A Culture-Free Lipidomics-Based Screening Test for Uropathogens

## TL;DR

This study introduces a rapid, culture-free test using lipidomics to detect both gram-negative and gram-positive uropathogens in urine, significantly reducing diagnostic time.

## Contribution

The novel use of lysozyme pretreatment enhances cardiolipin detection, enabling efficient identification of gram-positive bacteria in a lipidomics-based assay.

## Key findings

- Lysozyme pretreatment improved the detection of gram-positive bacteria to 95% sensitivity.
- The assay reduced analytical turnaround time by at least 90%.
- Polymicrobial infections were identified in two patients using the optimized method.

## Abstract

A rapid culture-free method is needed to improve diagnostic efficiency and guide timely antimicrobial therapy for urinary tract infections (UTIs). Previously, we utilized the lipidomics-based fast lipid analysis technique (FLAT) to screen for uropathogens by identifying distinctive microbial membrane lipid profiles, specifically lipid A in gram-negative and cardiolipin in gram-positive bacteria. This culture-free assay demonstrated high sensitivity (94%) in detecting gram-negative bacteria but poor sensitivity (51%) for gram-positive bacteria.

In this study, we pretreated urine pellets with lysozyme prior to FLAT analysis to break down the peptidoglycan layer bacteria, thereby promoting the efficient release of cardiolipin. The limit of detection (LOD) for 4 gram-positive bacteria and Escherichia coli was evaluated using contrived samples with known CFU/mL values and varying concentrations of lysozyme. Subsequently, we validated the optimized method in a clinical cohort of 76 urine samples known to contain gram-positive bacteria as confirmed by urine culture.

Optimal sensitivity was achieved by treating 1 mL of urine pellets with 100 µg lysozyme and incubating for 60 minutes, resulting in a 100-fold increase in cardiolipin LOD and a 95% detection rate for gram-positive bacteria. Signal-to-noise ratio for lipid A was also improved. Polymicrobial urine cultures with gram-negative and gram-positive species were identified in 2 patients.

The lysozyme-enhanced FLAT assay enables rapid and culture-free detection of both gram-negative and gram-positive uropathogens directly from urine. The unified workflow decreases the analytical turnaround time by at least 90% making it well-suited for high-throughput clinical laboratories.