Impedance flow cytometry methods

Patent metadata

Field	Value
Publication number	US-12072274-B2
Application number	US-201917276717-A
Country	US
Kind code	B2
Filing date	Sep 16, 2019
Priority date	Sep 17, 2018
Publication date	Aug 27, 2024
Grant date	Aug 27, 2024

Abstract

A method of antimicrobial susceptibility testing comprises: preparing samples of microorganisms suspended in an electrolyte, comprising a first sample of the microorganisms unexposed to antimicrobial agents and a second sample of the microorganisms exposed to an antimicrobial agent; passing the first sample through an impedance flow cytometer to obtain a first impedance signal representing one or more components of impedance values of the unexposed microorganisms; passing the second sample through the impedance flow cytometer to obtain a second impedance signal representing one or more components of the impedance values of the exposed microorganisms; comparing the first impedance signal and the second impedance signal; and determining a susceptibility of the microorganisms to the antimicrobial agent based on any differences between the first impedance signal and second impedance signal.

First claim

The invention claimed is:

1. A method of antimicrobial agent susceptibility testing comprising:
preparing samples of microorganisms suspended in an electrolyte, comprising a first sample of the microorganisms unexposed to antimicrobial agents and a second sample of the microorganisms exposed to an antimicrobial agent;
adding non-biological particles to each sample;
passing the first sample through an impedance flow cytometer to obtain a first impedance signal representing one or more components of impedance values of the unexposed microorganisms;
passing the second sample through the impedance flow cytometer to obtain a second impedance signal representing one or more components of impedance values of the microorganisms exposed to the antimicrobial agent;
comparing the first impedance signal and the second impedance signal; and
determining a susceptibility of the microorganisms to the antimicrobial agent based on any differences between the first impedance signal and second impedance signal.

2. A method according to claim 1 , further comprising preparing additional samples of the microorganisms each exposed to a different concentration of the antimicrobial agent;
passing each additional sample through the impedance flow cytometer to obtain a corresponding additional impedance signal;
comparing each additional impedance signal with the first impedance signal; and
determining a minimum concentration of the antimicrobial agent to which the microorganisms are susceptible based on any differences between the first impedance signal and the second and additional impedance signals.

3. A method according to claim 1 , in which preparing the samples of microorganisms comprises including the microorganisms for the first sample, the second sample and any additional samples in a volume of electrolyte to produce a single sample and adding the antimicrobial agent to the single sample; and the method comprising:
passing the single sample through the impedance flow cytometer;
obtaining impedance signals for the single sample at two or more time intervals during passage of the single sample through the impedance flow cytometer, wherein an impedance signal at a first time interval is the first impedance signal, an impedance signal at a second time interval is the second impedance signal; and impedance signals at later time intervals are additional impedance signals; and
including the additional impedance signals in the comparing and the determining, such that the determining is based on any differences between the first impedance signal, the second impedance signal and the additional impedance signals.

4. A method according to claim 1 , in which preparing the samples of microorganisms comprises including the microorganisms for the first sample, the second sample and any additional samples in a volume of electrolyte to produce a single sample; and the method comprising:
passing the single sample through the impedance flow cytometer;
obtaining the first impedance signal in a first time interval during passage of the single sample through the impedance flow cytometer;
adding an antimicrobial agent to the single sample after the first time interval; and
obtaining further impedance signals in one or more additional time intervals during passage of the single sample through the impedance flow cytometer, wherein an impedance signal in a second time interval after the first time interval is the second impedance signal and impedance signals in later time intervals are additional impedance signals; and
including the additional impedance signals in the comparing and the determining, such that the determining is based on any differences between the first impedance signal, the second impedance signal and the additional impedance signals.

5. A method according to claim 1 , in which the second sample comprises the microorganisms exposed to a concentration of the antimicrobial agent predefined for use in breakpoint analysis of the microorganisms, and the determining a susceptibility comprises classifying the microorganisms as susceptible, not susceptible, resistant, or not resistant to the antimicrobial agent.

6. A method according to claim 5 , in which the concentration of the antimicrobial agent in the second sample is a concentration predefined to indicate that the microorganisms are susceptible or not susceptible to the antimicrobial agent, and the method further comprises:
preparing a third sample of the microorganisms exposed to the antimicrobial agent at a higher concentration than the second sample which is predefined to indicate that the microorganisms are resistant or not resistant to the antimicrobial agent;
passing the third sample through the impedance flow cytometer to obtain a third impedance signal representing one or more components of impedance values of the microorganisms exposed to the antimicrobial agent;
comparing the first impedance signal and the third impedance signal; and
the determining a susceptibility comprises classifying the microorganisms as susceptible or not susceptible based on any differences between the first impedance signal and the second impedance signal, and classifying the microorganisms as resistant or not resistant based on any differences between the first impedance signal and the third impedance signal.

7. A method according to claim 1 , in which the components of the impedance values of the unexposed microorganisms and/or of the microorganisms exposed to the antimicrobial agent indicate one or more of microorganism size, microorganism shape, microorganism cell wall structure, cell membrane properties and microorganism internal structure.

8. A method according to claim 1 , comprising obtaining the impedance signals using current paths arranged along more than one direction relative to a direction of passage of the sample through the impedance flow cytometer so that the impedance values of the unexposed microorganisms and/or of the microorganisms exposed to the antimicrobial agent indicate a shape of the microorganisms.

9. A method according to claim 1 , comprising obtaining the impedance signals using current paths arranged along more than one direction relative to a direction of sample passage through the impedance flow cytometer, and further comprising deducing a stiffness of the microorganisms by comparing impedance signals obtained using current paths with different directions.

10. A method according to claim 1 , comprising obtaining the impedance signals at more than one frequency.

11. A method according to claim 1 , in which the non-biological particles comprise reference beads.

12. A method according to claim 11 , in which the reference beads are of known size and/or concentration.

13. A method according to claim 11 , in which the reference beads provide reference information in the impedance signals for use in compensating fluctuations in the samples and/or in operation of the impedance flow cytometer.

14. A method according to claim 13 , comprising determining phase and/or magnitude of the impedance signals in order to distinguish between the microorganisms and the non-biological particles within the impedance signals, and/or between the microorganisms and any different microorganism species in the samples.

15. A method according to claim 14 , further comprising using the phase and/or magnitude to improve the resolution of impedance signals obtained from the impedance flow cytometer.

Assignees

• Univ Southampton
• Univ Western Australia

Inventors

• Morgan Hywel
• Spencer Daniel
• Inglis Timothy John Jay
• Paton Teagan
• Mulroney Kieran

Classifications

• G01N2015/103

  Particle shape · CPC title

• G01N15/1031

  by measuring electrical or magnetic effects · CPC title

• G01N15/1023

  Microstructural devices for non-optical measurement · CPC title

• G01N15/0227

  using imaging; using holography · CPC title

• G01N2015/0294

  Particle shape · CPC title