Combating Antimicrobial Resistance via Single-cell Diagnostic Technologies Powered by Droplet Microfluidics

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Antimicrobial resistance is a global threat that if left unchecked could lead to 10 million annual mortalities by 2050. One factor contributing to the rise of multi-drug-resistant (MDR) pathogens is the reliance on traditional culture-based pathogen identification (ID) and antimicrobial susceptibility testing (AST) that typically takes several days. This delay of objective pathogen ID and AST information to inform clinical decision making results in clinicians treating patients empirically often using first-line, broad-spectrum antibiotics, contributing to the misuse/overuse of antibiotics. To combat the rise in MDR pathogens, there is a critical demand for rapid ID and AST technologies. Among the advances in ID and AST technologies in the past decade, single-cell diagnostic technologies powered by droplet microfluidics offer great promise due to their potential for high-sensitivity detection and rapid turnaround time. Our laboratory has been at the forefront of developing such technologies and applying them to diagnosing urinary tract infections (UTIs), one of the most common infections and a frequent reason for the prescription of antimicrobials. For pathogen ID, we first demonstrated the highly sensitive, amplification-free detection of single bacterial cells by confining them in picoliter-scale droplets and detection with fluorogenic peptide nucleic acid (PNA) probes that target their 16S rRNA (rRNA), a well-characterized marker for phylogenic classification. We subsequently improved the PNA probe design and enhanced detection sensitivity. For single-cell AST, we first employed a growth indicator dye and engineered an integrated device that allows us to detect growth from single bacterial cells under antibiotic exposure within 1 h, equivalent to two to three bacterial replications. To expand beyond testing a single antibiotic condition per device, a common limitation for droplet microfluidics, we developed an integrated programmable droplet microfluidic device for scalable single-cell AST. Using the scalable single-cell AST platform, we demonstrated the generation of up to 32 droplet groups in a single device with custom antibiotic titers and the capacity to scale up single-cell AST, and providing reliable pathogen categories beyond a binary call embodies a critical advance. Finally, we developed an integrated ID and AST platform. To this end, we developed a PNA probe panel that can identify nearly 90% of uropathogens and showed the quantitative detection of 16S rRNA from single bacterial cells in droplet-enabled AST after as little as 10 min of antibiotic exposure. This platform achieved both ID and AST from minimally processed urine samples in 30 min, representing one of the fastest turnaround times to date. In addition to tracing the development of our technologies, we compare them with contemporary research advances and offer our perspectives for future development, with the vision that single-cell ID and AST technologies powered by droplet microfluidics can indeed become a useful diagnostic tool for combating antimicrobial resistance.

References

Hsieh, Kuangwen, et al. "Combating Antimicrobial Resistance via Single-Cell Diagnostic Technologies Powered by Droplet Microfluidics." Accounts of chemical research 55.2 (2021): 123-133.

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