US-12618096-B2 - Rapid screen for antibiotic resistance and treatment regimen

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.

Inventors

• John Mark Sutton
• Charlotte HIND
• Hywel Morgan
• Daniel Spencer

Assignees

• Secretary of State for Health and Social Care
• THE UNIVERSITY OF SOUTHAMPTON

Dates

Publication Date

20260505

Application Date

20210319

Priority Date

20200319

Claims (20)

1. 1 . 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.
2. 2 . A method according to claim 1 , further comprising calculating from the differential signal a first impedance signal representing one or more components of impedance values of the bacteria, wherein the one or more components of the impedance values comprise a magnitude of the impedance values at the first frequency component and a magnitude of the impedance values at the second frequency component.
3. 3 . A method according to claim 2 , further comprising plotting the one or more components of impedance values of the bacteria on a graph to show a distribution of a population of bacteria; and further comprising establishing a contour on the graph that indicates a boundary of the distribution of the population, wherein the boundary encloses a central proportion of the data points wherein the proportion is 99%, 95%, 90%, 75%, or 50% such that outlying measurements are excluded.
4. 4 . A method according to claim 3 , further comprising obtaining a differential signal and calculating an impedance signal for a further sample of fluid to plot a graph of impedance values for bacteria in the further sample, and comparing the distribution of the population of bacteria in the further sample with the contour to identify any difference between the bacteria in the sample and the bacteria in the further sample.
5. 5 . A method according to claim 4 , in which the bacteria in the sample and the bacteria in the further sample are two groups of a same bacteria, the bacteria in the sample being unexposed to antimicrobial agents and the bacteria in the further sample having been exposed to an antimicrobial agent, wherein the identification of a difference between the bacteria in the sample and the bacteria in the second sample indicates a susceptibility of the bacteria to the antimicrobial agent, or in which the bacteria in the sample and the bacteria in the further sample are bacteria in sub-samples of a same sample, the sample including an antimicrobial agent to which the bacteria are exposed, wherein the differential signal is obtained for two or more time intervals while the sample flows continuously along the flow channel, each time interval corresponding to a different sub-sample, and a first time interval covering a time immediately following exposure of the bacteria to the antimicrobial agent is designated as corresponding to a sub-sample for which the bacteria are not affected by the antimicrobial agent.
6. 6 . A method according to claim 5 , further comprising obtaining a differential signal and calculating an impedance signal for additional further samples, wherein each further sample comprises a group of the same bacteria exposed to either (a) to a different concentration of the same antimicrobial agent, so that the identification of a difference indicates a minimum concentration of the antimicrobial agent at which the bacteria are susceptible or (b) a different antimicrobial agent.
7. 7 . A method according to claim 1 , in which the first frequency component is a frequency at or below 10 MHz and the second frequency component is a frequency at or above 10 MHz.
8. 8 . A method according to claim 2 , in which identifying any difference comprises identifying a change in the distribution for impedance values at the low frequency, indicating a change in bacteria size, or in which identifying any difference comprises identifying a change in the distribution for impedance values at the high frequency, indicating a change in bacteria morphology.
9. 9 . A method according to claim 1 , further comprising analysing the differential signal, or the impedance signal if calculated, to identify a pattern or patterns known to be caused by the presence of a bacteria flowing through the current paths, and counting number of occurrences of the pattern or patterns to determine a number of bacteria in the sample, or further comprising measuring a magnitude of the differential signal, or of the impedance signal if calculated, and calculating a size of the bacteria from the measured magnitude.
10. 10 . A method according to claim 1 , in which the current paths are substantially transverse to a direction of flow of the sample of fluid along the flow channel, or in which the current paths are substantially along a direction of flow of the sample of fluid along the flow channel, or in which one of the first current path and the second current path and one of the further first current path and the further second current path are substantially transverse to a direction of flow of the sample of fluid along the flow channel, and the other of the first current path and the second current path and the other of the further first current path and the further second current path are substantially along a direction of flow of the sample of fluid along the flow channel.
11. 11 . The method according to claim 1 , wherein the bacteria are selected from i) members of the ESKAPEE group selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and members of the Enterobacteriaceae family, ii) drug resistant Neisseria gonorrhoea, iii) Stenotophomonas maltophilia , or iv) Burkholderia cepacia/cenocepacia complex.
12. 12 . The method according to claim 11 , wherein the bacteria have been exposed to lytic phage.
13. 13 . The method according to claim 1 , wherein the bacteria have been exposed to a phage and the method further comprises identifying a bacterial species in the sample by detecting phage lysis with a defined phage or species-specific phage cocktail.
14. 14 . The method according to claim 1 , wherein the bacteria have been exposed to a phage or phage cocktail specific for Enterobacteriaceae Spp and the method comprises detecting the presence of phage lysis in an isolated patient sample with said phage or phage cocktail, wherein the presence of phage lysis indicates that patient has a urinary tract infection, pyelonephritis, or catheter-associated urinary tract infection (CAUTI).
15. 15 . The method according to claim 14 , wherein said Enterobacteriaceae is selected from E. coli, K. pneumoniae , and Proteus mirabilis.
16. 16 . The method according to claim 1 , wherein the method further comprising providing a graph that plots one or more components of impedance values for a control sample comprising bacteria that have not been exposed to the phage, wherein a contour is established on said graph to enclose 50% of the bacteria in the control sample, and determining the number of bacteria in the sample that sit within said contour.
17. 17 . The method according to claim 1 , further comprising determining a cell count for the number of bacteria in the sample.
18. 18 . The method according to claim 1 , wherein said antimicrobial peptides are membrane penetrating peptide, membrane disrupting peptide or pore-forming peptide antimicrobials.
19. 19 . 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.
20. 20 . 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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is the U.S. national phase of PCT/GB2021/050694, filed Mar. 19, 2021, which claims priority to United Kingdom Patent Application No. 2004021.8, filed Mar. 19, 2020, each expressly incorporated herein by reference its entirety. BACKGROUND OF THE INVENTION The present invention relates to methods of impedance flow cytometry, for example the use of impedance flow cytometry to determine antimicrobial susceptibility. Antimicrobial resistance (AMR) is the ability of a microorganism to avoid, modify or adapt to the adverse effects of an antimicrobial agent used against it. Of particular concern is a rise in, and global spread of, resistant bacteria, which is now recognised as a leading threat to the health and wealth of the world's population. When an infection is suspected, a doctor often immediately prescribes antimicrobial agents with the aim of promptly providing effective treatment. However, in many cases the antimicrobial agents are either not needed, or are inappropriate for the particular infection-causing organism. One notable problem is treatment of bacterial infections with antimicrobial agents which are ineffective owing to the presence of resistance mechanisms. This can mean the infection may persist, increase in severity and possibly spread to other patients. A leading reason for the rapid prescribing of potentially ineffective antimicrobial agents is that laboratory tests for checking antimicrobial resistance traits are far too slow to be useful in informing antimicrobial prescribing when the patient initially presents for treatment. Typically, antimicrobial susceptibility tests measure microbial growth in the presence of antimicrobial agents in liquid cultures or on solid agar plates. A common test known as a disk diffusion test (or quantitative variations of this test principle called Etests) requires that a microbial culture be grown overnight to obtain a sample which is then placed on an agar plate. Discs or strips containing known concentrations of antimicrobial agents are placed on the agar plate, and the inhibition of microbial growth close to the discs or strips containing antimicrobial agents is measured after a long incubation period. The broth microdilution method measures the growth of microorganisms in liquid cultures with different concentrations of antimicrobial agents to determine the antimicrobial concentration at which microbial growth is inhibited (known as the minimum inhibitory concentration, or MIC). The broth microdilution MIC method may be performed using automated laboratory equipment. These conventional assays measure the growth of populations of microorganisms over time and take many hours to perform. The results cannot therefore be used to inform or guide prescription in the early stages of infection, when that guidance is most important. As an alternative to these conventional tests which measure populations of microorganisms, analysing the optical properties of single microorganisms exposed to antimicrobial agents has been demonstrated to correlate closely with the antimicrobial susceptibility measured with the conventional tests, but within a shorter time window of less than one hour (WO 2012/164547). Commonly, a population of microorganisms is exposed to an antimicrobial agent for a fixed duration of time, typically thirty minutes. The microorganisms are then washed by centrifugation to remove the antimicrobial agent, and stained with a specific membrane-permeable fluorescent dye that can be used to indicate susceptibility to the agent. The optical properties of the microorganisms are measured with an optical flow cytometer, which detects light scattered from the microorganisms in a forward direction that indicates particle size, and a fluorescence signal corresponding to microorganisms. The optical data are compared with data obtained from a population of the same sample of microorganism, also stained with dye but which has not been exposed to the antimicrobial agent. Differences in the data from the two samples indicate whether the microorganisms are susceptible to the agent. Additionally, exposure of samples to a series of different antimicrobial concentrations is used to determine a minimum dose of antimicrobial agent required to effectively inhibit the microbial growth. Optical cytometry has a number of drawbacks. The use of dyes typically requires one or more wash steps in the procedure, which limits the scope for miniaturising and automating the test procedure. Removal of the antimicrobial agent by washing prior to addition of the dye suspends the antimicrobial treatment at that moment, so preventing continuous measurements of antimicrobial effects on a single sample over time. Optical cytometers are bulky and very costly, and require manipulation techniques such as hydrodynamic and/or acoustic focusing to correctly position the microorganisms within an optical analysis zone. The fluorescent dyes are also