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Impedance Spectroscopy Measures Antimicrobial Susceptibility in Real Time

Jun 02, 2022

Kathy Liszewski
©: jarun011 /Getty Images

Antimicrobial drugs save millions of lives each year. However, in 2019, antibiotic-resistant microorganisms were associated with nearly 5 million deaths worldwide1. The alarming trend of bacterial antimicrobial resistance may further worsen as many COVID-19 patients are administered broad spectrum antibiotics.2

Antibiotic susceptibility tests (AST) determine the appropriate antibiotic therapy and thus help in formulating the best therapeutic management. Clearly, a rapid and efficient AST would play a critical role in treating bacterial infections. According to current recommendations, testing begins with overnight incubation of a bacterial culture with various concentrations of tested antibiotics.3 The readout is the ‘minimal inhibitory concentration (MIC)’ or the dose of antibiotic needed to kill the bacteria. 

 “This is an intrinsically slow process limited by the slow growth rate of bacteria,” says Shalini Gupta, PhD, associate professor, Chemical Engineering, Indian Institute of Technology Delhi. Gupta and colleagues recently developed a rapid impedance spectroscopy method for AST.4 She reports, “We use an approach based on the measurements of bulk ionic conductivity changes in bacterial suspensions when the cells are exposed to antibiotic. As a result, the bacteria do not need to be immobilized on any surface unlike in previous studies. We simply mix the cells and drug together in solution and start measuring. This makes the technique truly label-free.”

The impedance spectroscopy experimental setup is simple. It consists of a standard micro-interdigitated electrode chip connected to an impedance analyzer. The method does not measure changes in cell number, rather it measures the ions released by the cells in response to an antibiotic. Gupta estimates, “Based on back-of-the-envelope stoichiometric calculations, one cell releases up to one million ions. These ions can only be measured once they are pushed out of the cell into the bulk.”

Gupta says this method is very quick. “As the antibiotic starts to penetrate the cell (this happens in minutes), cytoplasmic ions from the cells are released into the surrounding medium causing a shift in the overall conductivity of the suspension. If the drug cannot penetrate the cell wall, as happens in many resistant cells, the cytoplasmic ions are not released (or are released to a lower extent) and thus there is little change in signal. All this can be measured in real-time.”

One of the advances Gupta made came as a result of changing the buffer systems. 5 Measuring conductivity changes requires that a large amount of ions be released, meaning many cells need to die prior to detecting a signal change. Reported times in the literature are 8 to 10 hours for 107 to 109 cells/ml. She says, “The reason why we can pick up changes in signal very early--within minutes--is because we use a low conductivity zwitterionic buffer that is highly sensitive to changes in conductivity.”

In principle, the test can also measure changes in ionic conductivity during cell growth as cells release ionic metabolites upon consuming sugars. Extending the concept, she says, “Even differences in cell growth can be measured in the presence of varying antibiotic concentrations. This is similar to a typical MIC experiment except that it is much faster given the high sensitivity of our system. We can pick up changes in just 2-3 doubling cycles as opposed to 30-40 reported in the literature.” 

While the traditional MIC-based AST is performed in PBS (phosphate buffered saline), tris-HCl or culture media, Gupta’s group prepares their own low-conductivity growth buffer using a special mix of glucose, sodium chloride, and HEPES buffer. 

Gupta and colleagues have so far established the proof-of-concept of their technology for bacterial AST which they call iQAST-Z (impedance-based quick AST using zwitterionic buffers). She says, theoretically the test can be extended to any microorganism that releases conductive metabolites in response to drug action.”

Gupta will be working to increase the sensitivity and limit of detection, examine more bacterial strains, and improve instrumentation in future studies. “We would like to develop a low-cost portable impedance analyzer with miniaturized components for fluidic handling and automatic data acquisition. This would allow rapid AST to be performed in a clinical setting with minimal infrastructure and manpower training and have tremendous impact on guiding targeted antibiotic therapy at an early stage to rule out the burden of antimicrobial resistance.”


1.         Murray C. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. Jan 18 2022;doi:10.1016/S0140-6736(21)02724-0

2.         Pelfrene E, Botgros R, Cavaleri M. Antimicrobial multidrug resistance in the era of COVID-19: a forgotten plight? Antimicrob Resist Infect Control. Jan 29 2021;10(1):21. doi:10.1186/s13756-021-00893-z

3.         Vasala A, Hytonen VP, Laitinen OH. Modern Tools for Rapid Diagnostics of Antimicrobial Resistance. Front Cell Infect Microbiol. 2020;10:308. doi:10.3389/fcimb.2020.00308

4.         Swami P, Verma G, Holani A, et al. Rapid antimicrobial susceptibility profiling using impedance spectroscopy. Biosens Bioelectron. Mar 15 2022;200:113876. doi:10.1016/j.bios.2021.113876

5.         Anand S, Swami P, Goel G, Gupta S. Zwitterions for impedance spectroscopy: The new buffers in town. Anal Chim Acta. Jun 29 2021;1166:338547. doi:10.1016/j.aca.2021.338547



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