Single-cell electro-phenotyping for rapid assessment of Clostridium difficile heterogeneity under vancomycin treatment at sub-MIC (minimum inhibitory concentration) levels

[Display omitted] •Microbial-culture independent antibiotic susceptibility assessment using electrophysiology.•Susceptibility to MIC vancomycin levels identified by cytoplasmic conductivity alteration.•Electro-phenotyping based antibiotic susceptibility benchmarked by broth dilution assay.•Rapid identification (< 4 h) of live subpopulation after sub-MIC level vancomycin.•Single-cell phenotyping to quantify live subpopulation after sub-MIC level vancomycin. Current methods for measurement of antibiotic susceptibility of pathogenic bacteria are highly reliant on microbial culture, which is time consuming (requires > 16 h), especially at near minimum inhibitory concentration (MIC) levels of the antibiotic. We present the use of single-cell electrophysiology-based microbiological analysis for rapid phenotypic identification of antibiotic susceptibility at near-MIC levels, without the need for microbial culture. Clostridium difficile (C. difficile) is the single most common cause of antibiotic-induced enteric infection and disease recurrence is common after antibiotic treatments to suppress the pathogen. Herein, we show that de-activation of C. difficile after MIC-level vancomycin treatment, as validated by microbiological growth assays, can be ascertained rapidly by measuring alterations to the microbial cytoplasmic conductivity that is gauged by the level of positive dielectrophoresis (pDEP) and the frequency spectra for co-field electro-rotation (ROT). Furthermore, this single-cell electrophysiology technique can rapidly identify and quantify the live C. difficile subpopulation after vancomycin treatment at sub-MIC levels, whereas methods based on measurement of the secreted metabolite toxin or the microbiological growth rate can identify this persistent C. difficile subpopulation only after 24 h of microbial culture, without any ability to quantify the subpopulation. The application of multiplexed versions of this technique is envisioned for antibiotic susceptibility screening.