Method and apparatus for determining level of microorganisms using bacteriophage

Abstract

A predetermined amount of parent bacteriophage capable of infecting a target microorganism is added to a sample to create a bacteriophage-exposed sample; the sample is incubated for a defined incubation time and assayed to determine the level of a bacteriophage or bacterial marker in the sample; and if the measured marker level has increased, then the initial concentration of the microorganism exceeds a specific threshold value. An antibiotic in different concentrations is added to different and separate portions of the sample and tested to determine if the bacteriophage marker is present and thereby determine the Minimum Inhibitory Concentration (MIC) of a given antibiotic. The antibiotic preferably is an antibiotic that inhibits DNA replication or protein synthesis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a graph of bacteriophage concentration versus time in a sample that has an initial bacteria concentration of 10⁴ bacteria per milliliter illustrating how bacteriophage amplification can be used to determine the quantity of a microorganism as well as identify a microorganism;

FIG. 1 b is a graph of bacterial debris concentration versus time in the same sample illustrated in FIG. 1a;

FIG. 2 a is a graph of bacteriophage concentration versus time in a sample that has an initial bacteria concentration of 10⁶ bacteria per milliliter, but is otherwise identical to the sample of FIG. 1a;

FIG. 2 b is a graph of bacterial debris concentration versus time in the same sample illustrated in FIG. 2a;

FIG. 3 is a flow chart illustrating a preferred embodiment of the method according to the invention;

FIG. 4 is a flow chart illustrating another preferred embodiment of the method according to the invention;

FIG. 5 is a graph of bacteriophage concentration versus time that illustrates how bacteriophage amplification can be used to rapidly determine antibiotic susceptibility or resistance of a microorganism;

FIG. 6 is a graph showing how long it takes for a bacteriophage marker to exceed a threshold level with different bacterial strains as a function of antibiotic concentration;

FIG. 7 is an illustration of a bacteriophage;

FIG. 8 illustrates a typical phage reproduction process as a function of time; and

FIG. 9 shows a side plan view of a lateral flow microorganism detection device according to the invention.

Claims

1. A method of determining if a threshold concentration of a target microorganism is present in a sample to be tested, said method comprising: (a) combining with said sample a predetermined amount of parent bacteriophage capable of infecting said target microorganism to create a bacteriophage-exposed sample;(b) providing incubation conditions to said bacteriophage-exposed sample sufficient to allow said parent bacteriophage to infect said target microorganism,;(c) waiting a predetermined time period such that, if said target microorganism is present in said sample at or above a threshold concentration, a marker will be amplified in said sample; and(d) assaying said exposed sample to determine the level of said marker.

2. A method as in claim 1 wherein said target microorganism is a bacterium.

3. A method as in claim 1 wherein said parent bacteriophage has been genetically modified to add said marker.

4. A method as in claim 1 wherein said marker is a bacteriophage marker.

5. A method as in claim 4 wherein said bacteriophage marker comprises an element selected from the group consisting of said bacteriophage, bacteriophage nucleic acid, bacteriophage protein, and a portion of a bacteriophage nucleic acid or a bacteriophage protein.

6. A method as in claim 4 wherein said parent bacteriophage is combined in an amount below the detection limit of said bacteriophage marker.

7. A method as in claim 1 wherein said marker is a bacterial marker and comprises an element selected from the group consisting of: cell wall debris, bacterial nucleic acids, proteins, or enzymes that are released when a phage lyses the bacteria.

8. A method as in claim 1 wherein said assaying comprises a colorimetric test.

9. A method as in claim 1 wherein said assaying comprises one or more tests selected from the group consisting of immunoassay methods, nucleic acid amplification-based assays, DNA probe assays, aptamer-based assays, mass spectrometry, including MALDI, and flow cytometry.

10. A method as in claim 9 wherein said immunoassay methods are selected from the group consisting of ELISA, radioimmunoassay, immunoflouresence, lateral flow immunochromatography (LFI), flow-through assay, and a test using a SILAS surface.

11. A method of determining the initial quantity of a microorganism present in a sample, said method comprising: (a) combining with said sample a predetermined amount of parent bacteriophage capable of infecting said target microorganism to create a bacteriophage-exposed sample;(b) providing incubation conditions to said bacteriophage-exposed sample sufficient to allow said parent bacteriophage to infect said target microorganism and create an amplified marker in said bacteriophage-exposed sample;(c) assaying said marker in said exposed sample to determine a marker level in said sample;(d) measuring a reaction time associated with said marker level; and(e) determining said initial quantity of said microorganism present in said sample using said marker level and said measured reaction time.

12. A method as in claim 11 wherein said initial quantity comprises the concentration of said microorganism in said sample at the time of adding said parent bacteriophage.

13. A method as in claim 11 wherein said target microorganism is a bacterium

14. A method as in claim 11 wherein said parent bacteriophage has been genetically modified to add said marker.

15. A method as in claim 11 wherein said determining comprises: providing a table correlating said reaction time to said initial quantity; and selecting said initial quantity from said table.

16. A method as in claim 15 wherein said table also correlates said predetermined amount of parent bacteriophage to said initial quantity.

17. A method as in claim 11 wherein: said measuring comprises waiting a predetermined time;said assaying comprises establishing if said sample contains a detectable amount of said marker, andsaid determining comprises ascertaining that said initial quantity is below a threshold value.

18. A method as in claim 11 wherein said marker is a bacteriophage marker.

19. A method as in claim 18 wherein said bacteriophage marker comprises an element selected from the group consisting of said bacteriophage, bacteriophage nucleic acid, bacteriophage protein, and a portion of a bacteriophage nucleic acid or a bacteriophage protein.

20. A method as in claim 17 wherein said parent bacteriophage is added in an amount below the detection limit of said bacteriophage marker, and said marker level is at or near said detection limit.

21. A method as in claim 11 wherein said marker is a bacterial marker and comprises an element selected from the group consisting of: cell wall debris, bacterial nucleic acids, proteins, or enzymes that are released when a phage lyses the bacteria.

22. A method as in claim 11 wherein said assaying comprises a colorimetric test.

23. A method as in claim 11 wherein said assaying comprises one or more tests selected from the group consisting of immunoassay methods, nucleic acid amplification-based assays, DNA probe assays, aptamer-based assays, mass spectrometry, including MALDI, and flow cytometry.

24. A method as in claim 23 wherein said immunoassay methods are selected from the group consisting of ELISA, radioimmunoassay, immunoflouresence, lateral flow immunochromatography (LFI), flow-through assay, and a test using a SILAS surface.

25. A method of determining the susceptibility or resistance of a target microorganism to an antibiotic, said method comprising: (a) combining with said target microorganism and said antibiotic a predetermined amount of parent bacteriophage capable of infecting said target microorganism to create a bacteriophage-exposed sample;(b) providing incubation conditions to said bacteriophage-exposed sample sufficient to allow said parent bacteriophage to infect said target microorganism;(c) waiting a predetermined time period such that, if said target microorganism is not susceptible to said antibiotic, a bacteriophage marker will be amplified in said sample; and(d) assaying said exposed sample to determine the level of said bacteriophage marker as an indication of the susceptibility of said microorganism to said antibiotic.

26. A method as in claim 25 wherein said parent bacteriophage is combined in an amount below the detection limit of said bacteriophage marker.

27. A method as in claim 25 wherein said antibiotic inhibits nucleic acid replication.

28. A method as in claim 27 wherein said antibiotic is selected from the group consisting of: flouroquinilones, such as levofloxacin and ciprofloxacin, and rifampin.

29. A method as in claim 25 wherein said antibiotic inhibits protein synthesis.

30. A method as in claim 29 wherein said antibiotic is selected from the group consisting of: macrolides, aminoglycosides, tetracyclines, streptogramins, everninomycins, oxazolidinones, and lincosamides.

31. A method as in claim 25 wherein said assaying comprises a colorimetric test.

32. A method as in claim 25 wherein said assaying comprises one or more tests selected from the group consisting of immunoassay methods, nucleic acid amplification-based assays, DNA probe assays, aptamer-based assays, mass spectrometry, including MALDI, and flow cytometry.

33. A method as in claim 32 wherein said immunoassay methods are selected from the group consisting of ELISA, radioimmunoassay, immunoflouresence, lateral flow immunochromatography (LFI), flow-through assay, and a test using a SILAS surface.

34. A method as in claim 25 wherein said combining comprises diluting the concentration of said target microorganism to a level at which said bacteriophage infection will not occur immediately.

35. A method of determining the susceptibility or resistance of a target microorganism to an antibiotic, said method comprising: (a) combining said target microorganism, said antibiotic, and a predetermined amount of parent bacteriophage capable of infecting said target microorganism to create a bacteriophage-exposed sample;(b) providing incubation conditions to said bacteriophage-exposed sample sufficient to allow said parent bacteriophage to infect said target microorganism and create an amplified bacteriophage marker in said bacteriophage-exposed sample;(c) assaying said bacteriophage marker in said exposed sample to determine a marker level in said sample;(d) measuring a reaction time associated with said marker level; and(e) determining the susceptibility of said target microorganism to said antibiotic using said marker level and said measured reaction time.

36. A method as in claim 35 wherein said antibiotic inhibits nucleic acid synthesis.

37. A method as in claim 36 wherein said antibiotic is selected from the group consisting of: flouroquinilones, such as levofloxacin and ciprofloxacin, and rifampin.

38. A method as in claim 35 wherein said antibiotic inhibits protein synthesis.

39. A method as in claim 38 wherein said antibiotic is selected from the group consisting of: macrolides, aminoglycosides, tetracyclines, streptogramins, everninomycins, oxazolidinones, and lincosamides.