Friday, May 9, 2025
5/9/2025
Rapid Antibiotic Susceptibility Testing via Selective Lysis and Nanogap Trapping - Project Summary The indiscriminate use of antibiotics has contributed to increased patient toxicity, poor clinical outcomes, excessive healthcare costs, and the widespread and increasing emergence of antibiotic resistant strains. Current clinical practice is driven by the limits of existing technologies for guiding rational treatment selection. Antibiotic susceptibility tests based on cell culture are too slow to support treatment selection during the critical early stages of infection, and although genotypic assays can be performed within several hours, genetic markers of resistance are not known for all mechanisms, nor is resistance assured when a particular sequence is present. Overcoming these limitations demands an alternate approach based on rapid phenotypic and culture-free susceptibility profiling, thereby allowing clinicians to move beyond the empiric antibiotic selection process by quickly identifying causative bacteria and evaluating their response to targeted antibiotics. Here we propose a unique platform to address this need based on a simple, inexpensive, and rapid microfluidic-enabled technology. The platform will isolate small numbers of bacteria directly from whole blood, identify bacterial pathogens, and evaluate their antibiotic response within 75 min from initial blood draw. These capabilities will be enabled by two complementary technologies. First, a novel porous silica monolith will be used to selectively lyse blood cells while passing bacteria in a high-throughput continuous-flow process, generating a solution of intact and viable bacteria directly from whole blood. Second, small numbers of bacteria present in the initial sample will be captured, purified, and concentrated within a passive nanogap trap capable of isolating bacteria at precisely defined locations. Each trap will consist of a narrow chamber terminating at a sharp tip, with a sub-micrometer gap surrounding the chamber periphery to allow blood lysate particles to be removed while concentrating bacteria at the chamber tip during sample perfusion. The nanogap chambers will constrain bacteria to defined regions with a total volume below 1 pL, enabling sensitive and rapid analysis of the immobilized bacteria by fiber optic Raman spectroscopy down to the level of single organisms, allowing for bacteria identification using a machine learning based classifier, followed by evaluation of pathogen response to antibiotic exposure to determine susceptibility to the selected drug. By taking advantage of the high concentration factor provided by the nanogap trap, the system will provide susceptibility testing within 75 min using blood samples with bacteria concentrations below 10 CFU/mL. System performance will be evaluated using wild type and antibiotic-resistant organisms, with bacteria response characterized for multiple classes of antibiotics using a low-cost fiber optic Raman spectrometer for detection, and validation of the resulting technology will be performed using whole blood specimens from patients treated for bacterial infections at the University of Maryland Medical Center.
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