All-In-One Escherichia coli Viability Assay for Multi-dimensional Detection of Uncomplicated Urinary Tract Infections

Area of Science:

• Biotechnology
• Microbiology
• Diagnostic Assays

Background:

• Antibiotic-resistant bacteria in urinary tract infections pose a significant public health challenge.
• Current culture-based diagnostics and antimicrobial susceptibility testing (AST) are slow and labor-intensive, contributing to antimicrobial resistance.
• Rapid and accurate diagnostics are crucial for effective treatment and infection control.

Purpose of the Study:

• To develop an all-in-one, rapid assay for detecting and determining antibiotic susceptibility of *Escherichia coli* in urine.
• To establish a digital β-d-glucuronidase (GUS)-AST assay utilizing a 3D microwell array chip.
• To enable timely and targeted antimicrobial therapies, thereby improving patient outcomes and reducing resistance.

Main Methods:

• An innovative digital β-d-glucuronidase (GUS)-AST assay was developed using a 3D microwell array chip.
• The assay leverages the GUS enzyme to convert bacterial presence into fluorescence, enabling bacterial quantification via digital counting of positive microwells.
• A streamlined workflow integrated urine processing, bacterial separation, and digital GUS-AST for direct urine analysis.

Main Results:

• The digital GUS-AST assay achieved a limit of detection of 39 CFU/mL within a 4.5-hour sample-to-result workflow.
• The assay demonstrated the ability to quantify *E. coli* concentrations across a 7-log dynamic range.
• The method successfully indicated antibiotic potency and determined minimum inhibitory concentrations.

Conclusions:

• The digital GUS-AST assay offers a rapid, sensitive, and efficient method for diagnosing *E. coli* UTIs and guiding antimicrobial treatment.
• This technology has the potential to significantly reduce the reliance on empirical therapies and mitigate the spread of antimicrobial resistance.
• Further development for broader pathogen detection and multiple antibiotic testing could enhance its clinical utility.