Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

Abstract

Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility.

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

“…Antimicrobial sensitivity and metabolic activity of individual bacterial cells can also be deduced using NGS. DropDx is a microfluidic-based technique in which single bacteria are encapsulated in droplets and briefly exposed to antibiotics before thermal lysis [153]. Then, these droplets are incubated with fluorescent probes designed to hybridize to genes encoding 16S rRNA of bacterial pathogens.…”

A Rapid Single-Cell Antimicrobial Susceptibility Testing Workflow for Bloodstream Infections

“…A nanoarray digital polymerase chain reaction with high resolution melt curve analysis enables rapid broad bacteria identification and phenotypic AST [10,11]. Furthermore, single-cell microfluidic devices, along with molecular biosensors, allow the rapid classification of the pathogen, the detection of polymicrobial samples, the identification of bacterial species, and single-cell AST [12][13][14][15][16]. These platforms have been demonstrated for the rapid diagnosis of various common infections, such as urinary tract infections and wound infections.…”

A Cascaded Droplet Microfluidic Platform Enables High-throughput Single Cell Antibiotic Susceptibility Testing at Scale

“…Through the confinement of single bacteria within nano-, pico-, or even femtoliterscale channels, wells, or droplets, even a minute change in bacterial metabolism, genetic content, morphology or replication in response to antibiotic exposure can be detected. As a result, single-cell technologies have enabled some of the most rapid AST to date -some even in < 1 h. [17][18][19][20][21][22][23][24][25][26] The reported assay turnaround times of these single-cell AST technologies approach the required time scale for preventing empirical prescription of broad-spectrum antibiotics. Unfortunately, most rapid single-cell AST technologies can only test one [18,20,[22][23][24][25][26] to four [19] antibiotic conditions per device.…”

“…As a result, single-cell technologies have enabled some of the most rapid AST to date -some even in < 1 h. [17][18][19][20][21][22][23][24][25][26] The reported assay turnaround times of these single-cell AST technologies approach the required time scale for preventing empirical prescription of broad-spectrum antibiotics. Unfortunately, most rapid single-cell AST technologies can only test one [18,20,[22][23][24][25][26] to four [19] antibiotic conditions per device. They are effective for rapidly calling the susceptibility or resistance for one antibiotic, but are unable to provide additional AST information beyond this binary call, which is a critical limitation.…”