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

Abstract

Bloodstream infections are a significant cause of morbidity and mortality worldwide. The rapid initiation of effective antibiotic treatment is critical for patients with bloodstream infections. However, the diagnosis of bloodborne pathogens is largely complicated by the matrix effect of blood and the lengthy blood tube culture procedure. Here we report a culture-free workflow for the rapid isolation and enrichment of bacterial pathogens from whole blood for single-cell antimicrobial susceptibility testing (AST). A dextran sedimentation step reduces the concentration of blood cells by 4 orders of magnitude in 20–30 min while maintaining the effective concentration of bacteria in the sample. Red blood cell depletion facilitates the downstream centrifugation-based enrichment step at a sepsis-relevant bacteria concentration. The workflow is compatible with common antibiotic-resistant bacteria and does not influence the minimum inhibitory concentrations. By applying a microfluidic single-cell trapping device, we demonstrate the workflow for the rapid determination of bacterial infection and antimicrobial susceptibility testing at the single-cell level. The entire workflow from blood to categorical AST result can be completed in less than two hours.

Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

“…Problematically, these strategies rely on knowledge about the diversity of mutants and tradeoffs that exist (or that can emerge) within an infectious population. While information about population heterogeneity, heteroresistance, and substructure is expensive and arduous to obtain (Andersson et al 2019b; Bottery et al 2021), new methods, in addition to the one presented in this study, are emerging (Kuchina et al 2021; Aissa et al 2021; Nagasawa et al 2021; Forsyth et al 2021; Hsieh et al 2022; Brettner et al 2022a). This type of richer data dovetails with emerging population genetic models that predict the likelihood of resistance to a given drug regimen (Read and Huijben 2009; Day et al 2015; Wilson et al 2016; Cannataro et al 2018; Somarelli et al 2020; Feder et al 2021; King et al 2022).…”

Microfluidic systems for infectious disease diagnostics

“…The workflow took less than 2 h and was performed with a clinically relevant pathogen concentration (10 CFU mL −1 ). 382…”

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

“…The incorporation of microfluidic devices in parallel with imaging provides new opportunities for pathogen identification and antibiotic susceptibility testing at the singlecell level, significantly decreasing the time from sample collection to diagnosis. After collecting a septic patient's blood sample, blood cells are removed by centrifugation and the supernatant-the bacteria-containing fraction-can be concentrated and loaded into a microfluidic device to isolate, visualize, and test individual bacteria [147]. A microfluidic device with adjustable channel heights can classify bacterial pathogens in a sample by their morphologies [148].…”

“…Bacterial cultures from septic patients can take anywhere from 5 to 7 days to analyze, costing precious time in which the patient's condition can rapidly deteriorate. Microfluidic apparatuses enable detection of antibiotic resistance in as little as 3 h [147].…”