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US 12,618,096 B2Granted
Rapid screen for antibiotic resistance and treatment regimen
AssigneesSecretary of State for Health and Social Care, University of Southampton
InventorsJohn Mark Sutton, Charlotte Hind, Hywel Morgan, Daniel Spencer
Life of this patent
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Abstract
Methods are presented which use impedance flow cytometry for rapid susceptibility testing of antimicrobial agents including phage, antimicrobial peptides and rapid analysis of antimicrobial mediated serum bactericidal assays.
Claims21
A method of single-cell bacterial impedance flow cytometry comprising:
- flowing a sample of fluid comprising bacteria suspended in an electrolyte along a flow channel;
- and
- a) applying electrical signals to current paths through the fluid, the current paths comprising at least
- a first current path and a second current path produced by a first signal electrode and a second signal electrode, respectively, of a first electrode group, and
- a further first current path and a further second current path produced by a further first signal electrode and a further second signal electrode, respectively, of a second electrode group,
- wherein the electrical signals applied to the first current path and the further first current path have a frequency, magnitude and phase and the electrical signals applied to the second current path and the further second current path have substantially equal frequency and magnitude and opposite phase to the electrical signals applied to the first current path and the second current path,
- wherein the frequency of the electrical signals comprises at least two frequency components, in which a first frequency component comprises a low megahertz (MHz) frequency that does not penetrate into a bacterium and is diverted around it, and a second frequency component comprises a high MHz frequency, that is larger than the low MHz frequency, and that capacitively couples across the cell membrane of a bacterium;
- b) detecting current flow in the current paths as an individual bacterium passes through the current paths, wherein current flow in the first current path and the second current path are detected by a first measurement electrode and a second measurement electrode, respectively;
- and wherein the current flow in the further first current path and the further second current path are detected by a further first measurement electrode and a further second measurement electrode, respectively;
- c) producing a first summed signal representing the sum of the current flow detected in the first current path and the second current path, and a second summed signal representing the sum of the current flow detected in the further first current path and the further second current path;
- and
- d) obtaining a differential signal representing the difference between the first summed signal and the second summed signal, wherein each differential signal is for an individual bacterium;
- wherein said bacteria have been exposed to a phage and/or a phage-derived endolysin.
10 more elements
A method of single-cell bacterial impedance flow cytometry using an impedance flow cytometry apparatus, the method comprising:
- flowing a sample of fluid comprising bacteria suspended in an electrolyte along a flow channel;
- and
- a) applying electrical signals to current paths through the fluid, the current paths comprising at least
- a first current path and a second current path produced by a first signal electrode and a second signal electrode, respectively, of a first electrode group, and
- a further first current path and a further second current path produced by a further first signal electrode and a further second signal electrode, respectively, of a second electrode group,
- wherein the electrical signals applied to the first current path and the further first current path have a frequency, magnitude and phase and the electrical signals applied to the second current path and the further second current path have substantially equal frequency and magnitude and opposite phase to the electrical signals applied to the first current path and the second current path,
- wherein the frequency of the electrical signals comprises at least two frequency components, in which a first frequency component comprises a low megahertz (MHz) frequency that does not penetrate into a bacterium and is diverted around it, and a second frequency component comprises a high MHz frequency, that is larger than the low MHz frequency, and that capacitively couples across the cell membrane of a bacterium;
- and
- b) detecting current flow in the current paths as an individual bacterium passes through the current paths, wherein current flow in the first current path and the second current path are detected by a first measurement electrode and a second measurement electrode, respectively;
- and wherein the current flow in the further first current path and the further second current path are detected by a further first measurement electrode and a further second measurement electrode, respectively;
- wherein the first and second measurement electrodes are configured with circuitry that produces a first summed signal representing the sum of the current flow detected in the first current path and the second current path, and wherein the further first and further second measurement electrodes are configured with circuitry that produces a second summed signal representing the sum of the current flow detected in the further first current path and the further second current path;
- and
- wherein the impedance flow cytometry apparatus comprises circuitry configured to determine a differential signal representing the difference between the first summed signal and the second summed signal, wherein a differential signal is for an individual bacterium;
- wherein said bacteria have been exposed to a phage and/or a phage-derived endolysin.
11 more elements
A method of single-cell bacterial impedance flow cytometry comprising:
- flowing a sample of fluid comprising bacteria suspended in an electrolyte along a flow channel;
- and
- a) applying electrical signals to current paths through the fluid, the current paths comprising at least
- a first current path and a second current path produced by a first signal electrode and a second signal electrode, respectively, of a first electrode group, and
- a further first current path and a further second current path produced by a further first signal electrode and a further second signal electrode, respectively, of a second electrode group,
- wherein the electrical signals applied to the first current path and the further first current path have a frequency, magnitude and phase and the electrical signals applied to the second current path and the further second current path have substantially equal frequency and magnitude and opposite phase to the electrical signals applied to the first current path and the second current path,
- wherein the frequency of the electrical signals comprises at least two frequency components, in which a first frequency component comprises a low megahertz (MHz) frequency that does not penetrate into a bacterium and is diverted around it, and a second frequency component comprises a high MHz frequency, that is larger than the low MHz frequency, and that capacitively couples across the cell membrane of a bacterium;
- b) detecting current flow in the current paths as an individual bacterium passes through the current paths, wherein current flow in the first current path and the second current path are detected by a first measurement electrode and a second measurement electrode, respectively;
- and wherein the current flow in the further first current path and the further second current path are detected by a further first measurement electrode and a further second measurement electrode, respectively;
- wherein said bacteria have been exposed to a phage and/or a phage-derived endolysin.
7 more elements
Dependencies are inferred from the claim text.