Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of D₂O Metabolic Incorporation in a Single Bacterium

Abstract

Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D 2 O) metabolism is reported. Metabolic incorporation of D 2 O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D 2 O medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D 2 O metabolic incorporation and SC-MIC determination after 1 h antibiotic treatment and 30 min mixture of D 2 O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.

Recent Development of Rapid Antimicrobial Susceptibility Testing Methods through Metabolic Profiling of Bacteria

“…In subsequent work, we used D 2 O to incubate bacteria and demonstrated a rapid phenotypic AST method in clinic-relevant environments with SRS [ 88 ]. The C−D vibrational band, which was generated in biomolecules after D 2 O incorporation, could be selectively detected with SRS ( Figure 4 ).…”

SRS-FISH: High-Throughput Platform Linking Microbiome Function to Identity at the Single Cell Level

“…Fixed cells were spotted onto a poly-L-lysine coated coverslip, and covered and sealed by another coverslip with a spacer in between 37 . Samples were prepared in this way to reduce cross-phase modulation signal while keeping the sample in the liquid environment that matches the refractive index of the water objective used for imaging.…”

“…Combined with stable isotope probing, SRS microscopy has allowed the tracing of glucose metabolism in eukaryotic cells 35,36 and the visualization of metabolic dynamics in living animals 24 . Recently, SRS was successfully applied to infer antibiotic resistance patterns of bacterial pure cultures and D2O metabolism 24,37 . Yet, SRS microscopy has not yet been adapted for studying functional properties of members of microbial communities through the integration with FISH analyses.…”

“…To retrieve information on the activity of single bacterial cells in culture or complex samples, we employed the D2O-based stable-isotope probing approach 10,37 . To this end, live cells present in simple (pure cultures) or complex (gut microbiome) samples were incubated in D2O containingmedia, to enable incorporation of D into biomolecules of metabolically-active cells (Fig.…”

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.…”