Sensitive and rapid determination of antimicrobial susceptibility

• US 8,895,255 B1
• Filed: 02/08/2013
• Issued: 11/25/2014
• Est. Priority Date: 07/12/2003
• Status: Active Grant

• Current Assignee: Accelerate Diagnostics, Inc.
• Original Assignee: Accelerate Diagnostics, Inc.
• Inventors: Goldberg, David A., Howson, David C., Metzger, Steven W., Buttry, Daniel A., Saavedra, Steven Scott
• Primary Examiner(s): LAM, ANN Y
• Application Number:
  US13/763,446
• Publication Number:
  US 20140363823A1
• Time in Patent Office: 655 Days
• Field of Search: None
• US Class Current: 435/7.2
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses

Abstract

The present invention relates to moving microorganisms to a surface, where they are grown in the presence and absence of antimicrobials, and by monitoring the growth of the microorganisms over time in the two conditions, their susceptibility to the antimicrobials can be determined. The microorganisms can be moved to the surface through electrophoresis, centrifugation or filtration. When the movement involves electrophoresis, the presence of oxidizing and reducing reagents lowers the voltage at which electrophoretic force can be generated and allows a broader range of means by which the target can be detected. Monitoring can comprise optical detection, and most conveniently includes the detection of individual microorganisms. The microorganisms can be stained in order to give information about their response to antimicrobials.

Claims

1. A method for the identification of microorganisms in a sample comprising:

contacting a sample with a device comprising a chamber and at least one capture surface;

capturing microorganisms on the capture surface, wherein an individual microorganism binds to the capture surface at a spatially discrete location;

introducing a first indicator to the device, wherein the first indicator is configured to bind to a first type of microorganism; and

detecting the presence of the first indicator.

2. The method of claim 1, wherein the capture surface comprises a nonspecific capture surface.

3. The method of claim 2, wherein the nonspecific capture surface comprises a polycationic polymer.

4. The method of claim 2, wherein the nonspecific capture surface comprises a polycationic polymer having amine moieties.

5. The method of claim 4, wherein the polycationic polymer comprises polyethylenimine or polylysine.

6. The method of claim 1, wherein the chamber comprises an optically transparent electrode disposed beneath the capture surface.

7. The method of claim 6, wherein the optically transparent electrode comprises an indium tin oxide electrode.

8. The method of claim 1, wherein the microorganisms are electrophoretically transported to the capture surface.

9. The method of claim 1, wherein the binding of the first indicator to the first type of microorganism is electrophoretically accelerated.

10. The method of claim 1, wherein the first indicator is detected with a method selected from the group consisting of fluorescence imaging, upconverting phosphor imaging, and chemiluminescence imaging.

11. The method of claim 1, wherein locations of the presence of the first indicator are registered by image analysis software and stored in a storage medium.

12. The method of claim 1, wherein the first indicator comprises a fluorophore.

13. The method of claim 1, wherein unbound first indicator is removed from the chamber following introduction and binding of the first indicator.

14. The method of claim 1, further comprising introducing a second indicator to the chamber and detecting the presence of the second indicator.

15. The method of claim 14, wherein the second indicator is introduced to the chamber following detection of the first indicator.

16. The method of claim 1, wherein a plurality of indicators are introduced to the chamber and detected.

17. The method of claim 16, wherein the plurality of indicators are introduced to the chamber simultaneously.

18. The method of claim 16, wherein the plurality of indicators are introduced into the chamber by fluid flow through an input port.

19. The method of claim 16, wherein a plurality of unbound indicators are washed from the chamber simultaneously.

20. The method of claim 19, wherein the plurality of unbound indicators are washed from the chamber by fluid flow through an input port and an output port.

21. The method of claim 16, wherein each of the plurality of indicators are detected serially.

22. The method of claim 16, wherein the plurality of indicators are detected concurrently.

23. The method of claim 16, wherein each of the plurality of indicators is a fluorophore.

24. The method of claim 23, wherein detection of each fluorophore is separable by excitation wavelength, emission wavelength, or both excitation and emission wavelengths.

25. The method of claim 1, further comprising detecting the presence of the individual microorganisms independently of the presence of an indicator.

26. The method of claim 25, wherein the individual microorganisms are detected with an optical detection method selected from the group consisting of light scattering imaging, brightfield imaging, darkfield imaging, and phase imaging.

27. The method of claim 25, wherein locations of the individual microorganisms captured on the capture surface are registered by image analysis software and stored in a storage medium.

28. The method of claim 25, wherein a software program automatically detects and stores a location of each individual microorganism.

29. The method of claim 1, wherein detection is performed automatically.

30. The method of claim 1, wherein multiple detection methods are utilized on the same sample.

31. The method of claim 1, further comprising allowing the individual microorganisms to grow for a first period of time prior to introduction of the first indicator.

32. The method of claim 1, wherein a vital stain, a mortal stain, or both a vital stain and a mortal stain are introduced to the chamber prior to introduction of the first indicator.