Rapid, automated, and reliable antimicrobial susceptibility test from positive blood culture by CAST‐R

Abstract

Antimicrobial susceptibility tests (ASTs) are pivotal in combating multidrug resistant pathogens, yet they can be time‐consuming, labor‐intensive, and unstable. Using the AST of tigecycline for sepsis as the main model, here we establish an automated system of Clinical Antimicrobials Susceptibility Test Ramanometry (CAST‐R), based on D2O‐probed Raman microspectroscopy. Featuring a liquid robot for sample pretreatment and a machine learning‐based control scheme for data acquisition and quality control, the 3‐h, automated CAST‐R process accelerates AST by >10‐fold, processes 96 paralleled antibiotic‐exposure reactions, and produces high‐quality Raman spectra. The Expedited Minimal Inhibitory Concentration via Metabolic Activity is proposed as a quantitative and broadly applicable parameter for metabolism‐based AST, which shows 99% essential agreement and 93% categorical agreement with the broth microdilution method (BMD) when tested on 100 Acinetobacter baumannii isolates. Further tests on 26 clinically positive blood samples for eight antimicrobials, including tigecycline, meropenem, ceftazidime, ampicillin/sulbactam, oxacillin, clindamycin, vancomycin, and levofloxacin reveal 93% categorical agreement with BMD‐based results. The automation, speed, reliability, and general applicability of CAST‐R suggest its potential utility for guiding the clinical administration of antimicrobials.

Robust Spontaneous Raman Flow Cytometry for Single‐Cell Metabolic Phenome Profiling via pDEP‐DLD‐RFC

“…[ 10b ] The CD band and the “fingerprint” region have been used for AST and for revealing drug response mechanisms via RBCS, respectively, in bacteria. [ 5 , 24 ] The results produced via pDEP‐DLD‐RFC here are consistent with our past observations of cellular response after Amp treatment (i.e., the sharp decrease in intensity of nucleic acid‐related bands, while no change or an increase of lipid‐related bands, which is perhaps linked to the Amp associated inhibition of cell wall/membrane synthesis). [ 5 , 25 ] Previously, the acquisition of fs‐SCRS from drug‐exposed or D 2 O‐fed bacterial cells was typically conducted at a low throughput (≈3–5 cells min −1 ) using static cells on a substrate (e.g., CaF 2 ).…”

“…[ 5 , 25 ] Previously, the acquisition of fs‐SCRS from drug‐exposed or D 2 O‐fed bacterial cells was typically conducted at a low throughput (≈3–5 cells min −1 ) using static cells on a substrate (e.g., CaF 2 ). [ 5 , 24 ] An automated flow‐based Raman platform reported a similarly low throughput of 3.3–8.3 cells min −1 , due to the inability of optical tweezers to efficiently trap fast‐moving cells. [ 13 ] Notably, the pDEP‐DLD‐RFC greatly improved this rate to ≈30 events min −1 , representing a significant elevation in throughput.…”

“…[ 32 ] Moreover, the emergence of reference fs‐SCRS databases for specific organisms suggests the potential use of pDEP‐DLD‐RFC for direct profiling of microbial composition in a human, environmental, or industrial microbiota. [ 7 , 24 ]…”

Single cell metabolic phenome and genome via the ramanome technology platform: Precision medicine of infectious diseases at the ultimate precision?

“…To support the complete SCIVVS workflow and enable the plethora of circumstances in a clinical microbiology laboratory, a series of microbial single cell multiomics instruments have been developed and validated with clinical specimens (Figure 2). (a) The clinical antimicrobial susceptibility test ramanometry (CAST-R) instrument [38,41], which includes hardware devices for automated clinical sample pretreatment and intelligent SCRS acquisition and analysis in a parallel, 60-well platebased format, reports AST results for dozens of antimicrobials in each patient sample and processes dozens of patient samples in each run. (b) The flow-mode Ramanactivated cell sorter (FlowRACS) instrument [43,44], which is a microfluidics-based system, profiles the full spectrum spontaneous SCRS in a cell population with high speed (30 per min for Escherichia coli and 270 per min for budding yeasts) via its Raman flow cytometry mode, thereby providing a novel solution for automated, high-throughput SCRS-based ID and AST of pathogens [44].…”

“…Furthermore, this principle can be used for a cultureindependent AST of microbes [32] and cancer cells [34] by D 2 O-probed single cell Raman microspectroscopy based on quantitative inhibition of metabolic vitality of individual cells using a particular antimicrobial drug that results in either a reduction in the C-D band for susceptible cells or no change in the C-D band versus the drug-free control for resistant cells in each SCRS. Correspondingly, the new concept of minimal inhibitory concentration (MIC) via metabolic activity (MIC-MA) was introduced as a single cell resolution, quantitative parameter to measure antimicrobial susceptibility, which has general applicability to fast and slow growing pathogens [32,38,41]. Compared with the conventional, population average-based parameter of MIC, which is defined as the lowest drug dose at which cellular growth stops, MIC-MA offers important advantages.…”