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

Area of Science:

• Microbiology
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Urinary tract infections (UTIs) require rapid diagnostics for effective antimicrobial therapy.
• Current culture-free methods like Fast Lipid Analysis Technique (FLAT) show high sensitivity for gram-negative bacteria but low sensitivity for gram-positive bacteria.
• Distinct lipid profiles (lipid A for gram-negative, cardiolipin for gram-positive) are key targets for FLAT.

Purpose of the Study:

• To enhance the sensitivity of the FLAT assay for detecting gram-positive bacteria in urine samples.
• To develop a unified, rapid, culture-free diagnostic workflow for both gram-negative and gram-positive uropathogens.

Main Methods:

• Urine samples were pretreated with lysozyme to degrade the bacterial peptidoglycan layer, facilitating cardiolipin release.
• The limit of detection (LOD) for gram-positive bacteria and Escherichia coli was assessed using varying lysozyme concentrations.
• The optimized method was validated on a clinical cohort of 76 urine samples.

Main Results:

• Optimal sensitivity for gram-positive bacteria was achieved using 100 µg lysozyme per 1 mL urine, with a 60-minute incubation.
• Lysozyme pretreatment increased cardiolipin LOD by 100-fold, achieving a 95% detection rate for gram-positive bacteria.
• The enhanced FLAT assay also improved the signal-to-noise ratio for lipid A and detected polymicrobial infections.

Conclusions:

• Lysozyme-enhanced FLAT provides a rapid, culture-free method for detecting both gram-negative and gram-positive uropathogens directly from urine.
• This unified workflow significantly reduces analytical turnaround time by over 90%.
• The optimized assay is suitable for high-throughput clinical laboratories, improving UTI diagnostic efficiency.