Rapid and Comprehensive Bacterial Identification and Antimicrobial Resistance Determination Using Microfluidic Enrichment and Whole-Genome Sequencing

Abstract

Sepsis and other severe bacterial infections affect over 750,000 Americans annually, resulting in a 28% mortality rate and $16 billion in health care costs due to delays in proper diagnosis. The current gold-standard method for determining the species identification (ID) and antimicrobial resistance (AMR) profile of a clinical pathogen relies on the century-old method of growing bacteria in culture media, which takes 2-5 days, and frequently fails to cultivate the causative pathogen. In the absence of microbiologic data, physicians are forced to treat suspected infections with broad-spectrum empiric antibiotics that can have significant toxicity to the patient, are not always effective due to the rise of drug resistant pathogens, and can be expensive. Here we describe a process to perform bacterial ID and AMR determination directly from patient samples with a novel enrichment process combined with downstream agnostic DNA sequencing and real-time analysis with a MinION sequencer. By combining a microfluidic cell sorter and immunomagnetic depletion, we achieve at least 2.5x10^4-fold depletion of leukocytes, 10^2-fold depletion of erythrocytes, and 10^2-fold depletion of platelets, while retaining an average of 75% of bacterial cells. With the MinION sequencer, we can achieve ten-fold genome coverage of a bacterial isolate in 30 minutes of sequencing time, even with sample multiplexing. Overall, we show that this approach is feasible when combined with additional human cell and DNA depletion steps, and has the potential to improve clinical care and antibiotic stewardship.