Single-cell microfluidic impedance cytometry to study antimicrobial susceptibility

Abstract

Multidrug-resistant (MDR) infections are rapidly increasing, with deaths predicted to reach 10 million per year by 2050. Patients with rapidly progressing, life-threatening infections are often left waiting for 24-48 hours for Antimicrobial Susceptibility Test (AST) results. The development of a novel, rapid AST would enable faster prescribing for the patient, reducing the misuse and overuse of broad-spectrum antibiotics. A new electrical impedance-based rapid AST, the bacterial impedance cytometer (BIC), has been developed that measures the properties of single cells. Thousands of individual bacteria are measured as they flow through the channel of a microfluidic chip. The electrodes are driven by an alternating current (AC) signal of multiple frequencies and when a cell flows along the channel it perturbs the AC current. This is interpreted to extract electrical diameter (a measure of cell volume) and electrical opacity (the capacitance of the cell wall and membrane). The phenotypic response of bacteria cells to antibiotic treatment can be analysed after only 2 hours exposure.This thesis used the BIC to understand how changes in the electrical impedance of cells are linked to cell biophysical properties following exposure to different forms of treatment. The electrical properties of Escherichia coli and Staphylococcus aureus were measured after heat treatment and live and dead cells were successfully characterised. The impedance spectrum of the individual cells was measured across a range of frequencies, to extract the dielectric parameters of the cells. The technology was used to rapidly distinguish methicillin-resistant from methicillin-susceptible S. aureus (MRSA/MSSA) using a well characterised panel of bacterial isolates. The isolates were exposed to the β-lactam antibiotic cefoxitin for 2 hours, the optimum time that could clearly differentiate MRSA from MSSA. A threshold that allowed discrimination of resistant and susceptible was experimentally determined. This threshold was then applied to an uncharacterised clinical panel within the diagnostic laboratory at the University Hospital Southampton giving 100% concordance with standard disk diffusion results. Impedance data showed a decrease in electrical diameter and total cell count in MSSA isolates of S. aureus and an increase in electrical diameter and a decrease in total cell count in MRSA isolates of S. aureus when exposed to breakpoint concentrations of cefoxitin (8µg/ml). The increase in electrical diameter was shown to correlate with an increase in cell volume. This was confirmed with a strain of S. aureus that had the cell division protein FtsZ inhibited which showed a similar increase in electrical diameter compared to the control when tested on the BIC and the size change was confirmed by fluorescence microscopy. Population dynamics of E. coli were examined using the BIC following treatment with the fluoroquinolone antibiotic ciprofloxacin (CIP) which induces a stress response. A CIP-induced stressed cell population was identified as a sub-population with a larger electrical diameter compared to the position of the control population. This was confirmed through the addition of a stress response inhibitor or adjuvant, indicating that the technology could be used to identify novel stress response inhibitors as new antimicrobial therapies.In summary, the impedance response of cells following different treatment was measured using single cell impedance cytometry in bacteria. Its effectiveness as a rapid AST was confirmed in a clinical laboratory demonstrating 100% concordance. The technology can also identify different types of bacterial response to antibiotics or other drugs and can provide evidence of bacterial stress responses in sub-populations of bacteria.

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ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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