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Cited by 187Bacterial Identification and Susceptibility TestingMicrofluidic and Bio-sensing TechnologiesAntimicrobial agents and applications

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A fast impedance-based antimicrobial susceptibility test

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Spencer, Daniel C., Paton, Teagan F., Mulroney, Kieran T., Inglis, Timothy J. J., Sutton, J. Mark, Morgan, Hywel·2020·Nature Communications
DOI 10.1038/s41467-020-18902-xPMC PMC7578651

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Abstract

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 10 5 cells are analysed one-by-one with microfluidic impedance cytometry for 2–3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.

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Fig. 1 Principle of rapid impedance-based antimicrobial susceptibility testing. a Multi-electrode microfluidic impedance chip. Cells flow one-by-one between sets of electrodes and are measured simultaneously at two frequencies using a lock-in-amplifier (LIA). b Equivalent electrical equivalent circuit model for a Gram-negative bacteria, and a simulated spectrum of the Real part of the impedance vs. frequency highlighting frequency windows where changes in cell properties become apparent. c Impedance scatter plot of bacteria (K. pneumoniae, 10,000 events) together with 1.5-μm diameter polystyrene beads (with doublets and triplets) that are used as reference particles. The x-axis is the cube root of the impedance (proportional to diameter) measured at a frequency of 5 MHz. The y-axis is the electrical opacity, a measure of membrane/cell wall properties normalised to cell volume. This is measured at 40 MHz where the electrical properties of the cell wall and membrane are most apparent (see b). Two data sets are pre- and post-exposure to Meropenem at the clinical breakpoint for 30 min at 37 °C. In the scatter plot, the red contour defines the initial cell population. The diagram illustrates the change in cell properties following exposure to a β-lactam antibiotic as the cell wall breaks down (reduction in opacity) and the bacteria swell (increase in volume). d Experimental methodology for the impedance-based Fast Antimicrobial Susceptibility Test (iFAST). An actively dividing culture is prepared and incubated for 30 min with antibiotics. Polystyrene beads are added and the sample is measured for 3 min to determine the electrical MIC (see Supplementary information for further details).
Fig. 1 Principle of rapid impedance-based antimicrobial susceptibility testing. a Multi-electrode microfluidic impedance chip. Cells flow one-by-one between sets of electrodes and are measured simultaneously at two frequencies using a lock-in-amplifier (LIA). b Equivalent electrical equivalent circuit model for a Gram-negative bacteria, and a simulated spectrum of the Real part of the impedance vs. frequency highlighting frequency windows where changes in cell properties become apparent. c Impedance scatter plot of bacteria (K. pneumoniae, 10,000 events) together with 1.5-μm diameter polystyrene beads (with doublets and triplets) that are used as reference particles. The x-axis is the cube root of the impedance (proportional to diameter) measured at a frequency of 5 MHz. The y-axis is the electrical opacity, a measure of membrane/cell wall properties normalised to cell volume. This is measured at 40 MHz where the electrical properties of the cell wall and membrane are most apparent (see b). Two data sets are pre- and post-exposure to Meropenem at the clinical breakpoint for 30 min at 37 °C. In the scatter plot, the red contour defines the initial cell population. The diagram illustrates the change in cell properties following exposure to a β-lactam antibiotic as the cell wall breaks down (reduction in opacity) and the bacteria swell (increase in volume). d Experimental methodology for the impedance-based Fast Antimicrobial Susceptibility Test (iFAST). An actively dividing culture is prepared and incubated for 30 min with antibiotics. Polystyrene beads are added and the sample is measured for 3 min to determine the electrical MIC (see Supplementary information for further details).CC BY · PubMed Central
Fig. 2 Time course of changes in electrical properties of K. pneumoniae following β-lactam exposure. A sample of a Meropenem-susceptible strain of K. pneumoniae was exposed to Meropenem at 2 mg/L. The sample was maintained at a temperature of 37 °C and measured continuously for 30 min. The data was segmented into 1 min intervals and plotted as a series of scatter plots. Cells measured in the first minute were used to define the reference contour shown in red. Note that the x- and y-axis limits are identical for all figures (not shown for clarity) and are 1.2–3.0 (x-axis) and 0.4–0.9 (y-axis). Source data are provided as a Source Data file.
Fig. 2 Time course of changes in electrical properties of K. pneumoniae following β-lactam exposure. A sample of a Meropenem-susceptible strain of K. pneumoniae was exposed to Meropenem at 2 mg/L. The sample was maintained at a temperature of 37 °C and measured continuously for 30 min. The data was segmented into 1 min intervals and plotted as a series of scatter plots. Cells measured in the first minute were used to define the reference contour shown in red. Note that the x- and y-axis limits are identical for all figures (not shown for clarity) and are 1.2–3.0 (x-axis) and 0.4–0.9 (y-axis). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 Electrical minimum inhibitory concentration (MIC). a Scatter plots showing electrical size vs. electrical opacity for three different strains of K. pneumoniae exposed to different concentrations of Meropenem ranging from 0 to 8 mg/L (see Supplementary Fig. 2, protocol 1) Top row: 18397 (susceptible), middle row: KS11 (susceptible, increased exposure), bottom row: K14 (resistant). No changes are observed across all concentrations for K14, whilst a small shift in population is observed for 18397 even at 0.25 mg/L. Broth micro-dilution (BMD) was used (same samples) to measure the MIC and to classify the strains (red = 128 mg/L, orange = 8 mg/L, and blue < 0.25 mg/L). b Electrical MIC for ten different strains of K. pneumoniae that have a range of different MICs (see Supplementary Table 1). The data is plotted as the % cells in a contour (or gate) defined by the unexposed population vs. antibiotic concentration for three biological replicates (mean ± SD). Source data are provided as a Source Data file.
Fig. 3 Electrical minimum inhibitory concentration (MIC). a Scatter plots showing electrical size vs. electrical opacity for three different strains of K. pneumoniae exposed to different concentrations of Meropenem ranging from 0 to 8 mg/L (see Supplementary Fig. 2, protocol 1) Top row: 18397 (susceptible), middle row: KS11 (susceptible, increased exposure), bottom row: K14 (resistant). No changes are observed across all concentrations for K14, whilst a small shift in population is observed for 18397 even at 0.25 mg/L. Broth micro-dilution (BMD) was used (same samples) to measure the MIC and to classify the strains (red = 128 mg/L, orange = 8 mg/L, and blue < 0.25 mg/L). b Electrical MIC for ten different strains of K. pneumoniae that have a range of different MICs (see Supplementary Table 1). The data is plotted as the % cells in a contour (or gate) defined by the unexposed population vs. antibiotic concentration for three biological replicates (mean ± SD). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Electrical breakpoint analysis for Meropenem. Eleven different strains of bacteria analysed by impedance cytometry after exposure to Meropenem at the clinical breakpoint. (KP K pneumoniae, EC E. coli, ACB A. baumannii, PAE P. aeruginosa) (see Supplementary Fig. 2, protocol 2). a, b Each bar is the percentage of cells in the unexposed contour (or gate) after exposure to antibiotics at the clinical breakpoint. The susceptible/susceptible, increased exposure(S/I) and susceptible/resistant (S/R) boundaries are 2 and 16 mg/L, respectively. The bars show the mean ± SD for three biological replicates (*p < 0.05; **p < 0.01; ***p < 0.001) with p values obtained using the Student’s t test for independent samples (one tailed). Red bars indicate resistant strains and blue bars indicate susceptible strains, as determined by broth micro-dilution. The orange bar (CNCR) is a carbapenemase negative strain that is carbapenem resistant. P values (from left to right) are as follows 1.04 × 10−4, 2.66 × 10−4, 7.14 × 10−4, 2.46 × 10−5, 1.43 × 10−2, 1.15 × 10−3, 1.97 × 10−3, 3.00 × 10−2, 2.19 × 10−4, 2.40 × 10−3, 6.13 × 10−4, 2.63 × 10−3. c Scatter plots for a CNCR strain of K. pneumoniae after exposure to Meropenem for 30 min at the S/NS breakpoint (2 mg/L) and the R/NR breakpoint (16 mg/L) concentrations. Source data are provided as a Source Data file.
Fig. 4 Electrical breakpoint analysis for Meropenem. Eleven different strains of bacteria analysed by impedance cytometry after exposure to Meropenem at the clinical breakpoint. (KP K pneumoniae, EC E. coli, ACB A. baumannii, PAE P. aeruginosa) (see Supplementary Fig. 2, protocol 2). a, b Each bar is the percentage of cells in the unexposed contour (or gate) after exposure to antibiotics at the clinical breakpoint. The susceptible/susceptible, increased exposure(S/I) and susceptible/resistant (S/R) boundaries are 2 and 16 mg/L, respectively. The bars show the mean ± SD for three biological replicates (*p < 0.05; **p < 0.01; ***p < 0.001) with p values obtained using the Student’s t test for independent samples (one tailed). Red bars indicate resistant strains and blue bars indicate susceptible strains, as determined by broth micro-dilution. The orange bar (CNCR) is a carbapenemase negative strain that is carbapenem resistant. P values (from left to right) are as follows 1.04 × 10−4, 2.66 × 10−4, 7.14 × 10−4, 2.46 × 10−5, 1.43 × 10−2, 1.15 × 10−3, 1.97 × 10−3, 3.00 × 10−2, 2.19 × 10−4, 2.40 × 10−3, 6.13 × 10−4, 2.63 × 10−3. c Scatter plots for a CNCR strain of K. pneumoniae after exposure to Meropenem for 30 min at the S/NS breakpoint (2 mg/L) and the R/NR breakpoint (16 mg/L) concentrations. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 5 Breakpoint analysis for different antibiotic mechanisms. Scatter plots for susceptible and resistant bacteria after exposure to different antibiotics (see inset labels) at the S/R clinical breakpoint. The figure shows K. pneumoniae (KP), E. coli (EC), and S. aureus (SA) exposed to antibiotics which have different modes of action. The bar chart shows the percentage of cells inside the unexposed contour (mean ± SD for three biological repeats, red bar: resistant strain, blue bar susceptible strain as determined by broth micro-dilution) *p < 0.05; **p < 0.01; ***p < 0.001, with p values obtained using the Student’s t test for independent samples (one tailed), from left to right as follows: 1.04 × 10−3, 1.83 × 10−3, 1.36 × 10−3, 1.53 × 10−3, 2.06 × 10−3, 1.96 × 10−6, 1.33 × 10−5. Source data are provided as a Source Data file.
Fig. 5 Breakpoint analysis for different antibiotic mechanisms. Scatter plots for susceptible and resistant bacteria after exposure to different antibiotics (see inset labels) at the S/R clinical breakpoint. The figure shows K. pneumoniae (KP), E. coli (EC), and S. aureus (SA) exposed to antibiotics which have different modes of action. The bar chart shows the percentage of cells inside the unexposed contour (mean ± SD for three biological repeats, red bar: resistant strain, blue bar susceptible strain as determined by broth micro-dilution) *p < 0.05; **p < 0.01; ***p < 0.001, with p values obtained using the Student’s t test for independent samples (one tailed), from left to right as follows: 1.04 × 10−3, 1.83 × 10−3, 1.36 × 10−3, 1.53 × 10−3, 2.06 × 10−3, 1.96 × 10−6, 1.33 × 10−5. Source data are provided as a Source Data file.CC BY · PubMed Central

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Show all 45 references

Research works printed but not linked: 2

  1. O’Neill, J. (ed.) The Review on Antimicrobial Resistance. Tackling drug-resistant infections globally: final report and recommendations. 1–72 (amr-review.org, HM Government, 2016).
  2. Spencer, D. C., Paton, T., Inglis, T. J. J., Sutton, J. M., and Morgan, H. A fast impedance-based antimicrobial susceptibility test. Protoc. Exch. https://doi.org/10.21203/rs.3.pex-1088/v1 (2020). · DOI

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Metadata

Authors
Spencer, D. C., Paton, T. F., Mulroney, K. T., Inglis, T. J. J., Sutton, J. M., & Morgan, H.
Affiliations
  • University of Southampton (GB)
  • Pathwest Laboratory Medicine (AU)
  • The University of Western Australia (AU)
  • Public Health England (GB)
Corresponding author
Morgan, Hywel
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-10-21
Volume
11
Issue
1
Pages
5328-5328
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-18902-x
PMC
PMC7578651
OpenAlex
W3094320129
Cited by
183
References
45
Citations by year
2026: 31 · 2025: 26 · 2024: 33 · 2023: 39 · 2022: 29 · 2021: 24 · 2020: 1
FWCI
15.40
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ePrints Soton (University of Southampton) (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • UWA Profiles and Research Repository (UWA) (submittedVersion, cc-by)
Funders
  • National Institute for Health and Care Research (NIHR200968)
  • Royal Society
Topics
  • Health Sciences › Medicine › Microbiology (medical) › Bacterial Identification and Susceptibility Testing
  • Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Bio-sensing Technologies
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Antimicrobial agents and applications
Keywords
antimicrobial susceptibility testing, impedance cytometry, microfluidics, label-free detection, antibiotic mechanism of action, broth microdilution comparison, antibiotic prescribing
MeSH terms
Meropenem, Bacteria, Equipment Design, Humans, Klebsiella pneumoniae, Microbial Sensitivity Tests, Electric Impedance, Drug Resistance, Bacterial, Microfluidic Analytical Techniques, Biophysical Phenomena, Lab-On-A-Chip Devices

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