IP Library › Granted Patent US 12,448,655
Granted Patent B2
US 12,448,655 · App. 17/164,674 · Granted Oct 21, 2025

Antibiotic susceptibility of microorganisms and related methods and systems

Inventors: Rustem F. Ismagilov (Altadena, CA); Nathan G. Schoepp (Burlingame, CA); Emily S. Savela (Pasadena, CA); Eric J. Liaw (Pasadena, CA); Alexander Winnett (Pasadena, CA); Matthew M. Cooper (Pasadena, CA)
Assignee: CALIFORNIA INSTITUTE OF TECHNOLOGY
C12Q1/689
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Quick Facts
Patent No.
US 12,448,655
App. No.
17/164,674
Granted
Oct 21, 2025
Kind
B2
Abstract

Provided herein is an antibiotic susceptibility test and related compositions, methods and systems based on detection of a nucleic acid from a target microorganism in a sample in the presence or absence of a lysis treatment of the sample.

Claims (44)

1. A method to detect a nucleic acid of a microorganism in a plurality of samples of an isolate or specimen including the microorganism, the method comprising

contacting the plurality of samples with an antibiotic to provide a plurality of antibiotic-treated samples, and

quantitatively detecting in the plurality of antibiotic-treated samples an accessible nucleic acid of the microorganism, the quantitatively detecting independently performed

in the absence of a lysis treatment targeting the microorganism in an antibiotic-treated sample of the plurality of antibiotic-treated samples, and/or

in the presence of a lysis treatment in an antibiotic-treated sample of the plurality of antibiotic-treated samples, following pre-lysis mechanical separation of the microorganism from the nucleic acid in the antibiotic-treated sample of the plurality of antibiotic-treated samples subjected to lysis treatment to remove the inaccessible nucleic acids from the sample and

the contacting and quantitatively detecting performed to obtain a detected accessible antibiotic-treated nucleic acid concentration value of the microorganism for each antibiotic-treated sample of the plurality of antibiotic-treated samples.

2. The method of claim 1 , wherein the mechanical separation is performed by filtration or centrifugation.

3. The method of claim 1 , wherein the mechanical separation is performed by filtration and centrifugation.

4. The method of claim 1 , wherein contacting the plurality of samples with an antibiotic is performed by independently performing contacting the plurality of samples with an antibiotic for up to 90 minutes, or for up to 45 minutes, or for up to 30 minutes, or for up to 15 minutes, or for up to 5 minutes.

5. The method of claim 1 , wherein the quantitatively detecting is performed by independently performing in each antibiotic-treated sample of the plurality of samples quantitative real-time PCR (qPCR), digital PCR (dPCR), or droplet digital PCR (ddPCR).

6. The method of claim 1 , wherein the quantitatively detecting is performed by independently performing in each antibiotic-treated sample of the plurality of samples qLAMP, RPA, dLAMP, or dRPA.

7. The method of claim 1 , wherein the quantitatively detecting is performed by independently contacting the samples of the plurality of samples with a probe specific for a nucleic acid of the microorganism and/or for any nucleic acid complementary to the nucleic acid of the microorganism.

8. The method of claim 1 , wherein each antibiotic-treated sample of the plurality of antibiotic-treated samples is provided by independently contacting each antibiotic-treated sample for a set testing time, and the quantitatively detecting is independently performed following the contacting at a corresponding detection time.

9. The method of claim 1 , wherein the plurality of samples are aliquots provided by partitioning the isolate or specimen before or after the contacting.

10. The method of claim 1 , wherein the contacting is performed in parallel for each sample of the plurality of samples, and the detecting is performed in parallel for each antibiotic-treated sample of the plurality of the antibiotic-treated samples.

11. The method of claim 1 , wherein the method further comprises concurrently or after the contacting and before the detecting:

independently performing an enhancing treatment of an antibiotic-treated sample of the plurality of antibiotic-treated samples.

12. The method of claim 1 , the method further comprising

detecting an accessible nucleic acid concentration ratio in the each antibiotic-treated sample of the plurality of antibiotic-treated samples by comparing the detected accessible antibiotic-treated nucleic acid concentration value with a detected reference nucleic acid concentration value of a nucleic acid concentration parameter of the microorganism in the each antibiotic-treated sample of the plurality of antibiotic-treated samples.

13. The method of claim 12 , wherein the reference nucleic acid concentration value of the nucleic acid concentration parameter is an accessible control nucleic acid concentration value obtained by

detecting a nucleic acid concentration for the nucleic acid of the microorganism in a control sample of the isolate or specimen comprising the microorganism, the detecting in the control sample performed

in the absence of lysis treatment of the control sample for the antibiotic-treated sample when the quantitatively detecting is performed in absence of a lysis treatment of an antibiotic-treated sample of the plurality of antibiotic-treated samples and/or

in the presence of a lysis treatment of the control sample following mechanical pre-lysis separation of the microorganism from the nucleic acid in the control sample for the antibiotic-treated sample when the quantitatively detecting is performed in presence of a lysis treatment following mechanical separation of the microorganism from the nucleic acid to remove inaccessible nucleic acid in an antibiotic-treated sample of the plurality of antibiotic-treated samples subjected to lysis treatment,

the detecting a nucleic acid concentration in the control sample performed to provide the accessible control nucleic acid concentration value for the nucleic acid of the microorganism.

14. The method of claim 13 , wherein the control sample is an aliquot of the antibiotic-treated sample of the plurality of antibiotic-treated samples, the aliquot obtained by partitioning the plurality of samples before the contacting.

15. The method of claim 12 , wherein the reference nucleic acid concentration value is a total nucleic acid concentration value obtained by

detecting the total nucleic acid concentration of the microorganism in a reference sample of an isolate or specimen comprising the microorganism and either treated or not treated with the antibiotic, the detecting performed to provide the total nucleic acid concentration value for the nucleic acid of the microorganism.

16. The method of claim 15 , wherein the reference sample is an aliquot of the antibiotic-treated sample of the plurality of antibiotic-treated samples, the aliquot obtained by partitioning the plurality of samples, before the contacting.

17. The method of claim 15 , wherein the reference sample is an aliquot of the antibiotic-treated sample of the plurality of antibiotic-treated samples, the aliquot obtained by partitioning the antibiotic-treated sample of the plurality of antibiotic-treated samples, after the contacting.

18. The method of claim 12 , wherein the reference nucleic acid concentration value of the nucleic acid concentration parameter of the antibiotic-treated sample of the plurality of antibiotic-treated samples is a control nucleic acid concentration and wherein the nucleic acid concentration ratio is more than 2 or less than 0.5.

19. The method of claim 12 , wherein the duration of the contacting of the plurality of samples with an antibiotic is independently performed in each sample of the plurality of samples for 20 minutes or less, and wherein the nucleic acid concentration ratio is more than 2 or less than 0.5; more than 3 or less than 0.33; more than 5 or less than 0.2; more than 10 or less than 0.1; more than 15 or less than 0.067; more than 20 or less than 0.05.

20. The method of claim 19 , wherein the nucleic acid is DNA and/or RNA.

21. The method of claim 12 , wherein the contacting of the plurality of samples with an antibiotic is independently performed for each sample of the plurality of samples for 30 minutes or less, and wherein the nucleic acid concentration ratio is more than 3 or less than 0.33; more than 5 or less than 0.2; more than 10 or less than 0.1; more than 15 or less than 0.067; more than 20 or less than 0.05.

22. The method of claim 21 wherein the nucleic acid is DNA and/or RNA.

23. The method of claim 12 , wherein the accessible nucleic acid concentration ratio of the each antibiotic-treated sample of the plurality of antibiotic-treated samples is indicative of resistance or susceptibility of the microorganism to the antibiotic.

24. The method of claim 23 , wherein the method further comprises establishing susceptibility of the microorganism to the antibiotic when the accessible nucleic acid concentration ratio in the each antibiotic-treated sample of the plurality of antibiotic-treated samples is above an accessible threshold of susceptibility.

25. The method of claim 23 , wherein the method further comprises establishing resistance of the microorganism to the antibiotic when the accessible nucleic acid concentration ratio in the each antibiotic-treated sample of the plurality of antibiotic-treated samples is below an accessible threshold of resistance.

26. The method of claim 1 , wherein the method further comprises purifying or extracting the nucleic acid from the plurality of antibiotic-treated samples before the detecting.

27. The method of claim 26 , wherein the quantitatively detecting is performed in the presence of a lysis treatment following mechanical separation of the microorganism from the nucleic acid to remove inaccessible nucleic acid in an antibiotic-treated sample of the plurality of antibiotic-treated samples and

wherein the method further comprises extracting the nucleic acid from the antibiotic-treated sample of the plurality of antibiotic-treated samples concurrently or after the lysis treatment and before the detecting.

28. The method of claim 1 , wherein the antibiotic is independently selected from one or more beta-lactams or one or more carbapenems.

29. The method of claim 1 , wherein the microorganism belongs to the family Enterobacteriaceae.

30. The method of claim 1 , wherein the sample is obtained from a specimen pretreated to enrich the sample with nucleic acids or with the target microorganism, and/or to remove human nucleic acid, or nucleic acid of other microorganisms.

31. The method of claim 1 , wherein the plurality of samples is a plurality of urine samples.

Assignments (3)
CONFIRMATORY LICENSE Recorded Nov 2, 2023
From: CALIFORNIA INSTITUTE OF TECHNOLOGY
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 065431/0190 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 8, 2021
From: ISMAGILOV, RUSTEM F.; SCHOEPP, NATHAN G.; SAVELA, EMILY S.; LIAW, ERIC J.
To: CALIFORNIA INSTITUTE OF TECHNOLOGY
Reel/Frame 057745/0254 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 8, 2021
From: WINNETT, ALEXANDER; COOPER, MATTHEW M.
To: CALIFORNIA INSTITUTE OF TECHNOLOGY
Reel/Frame 057745/0274 →
Continuity (5)
Continuation In Part 16158233 · Oct 11, 2018
Provisional Application 62968735 · Jan 31, 2020
Provisional Application 62722124 · Aug 23, 2018
Provisional Application 62571128 · Oct 11, 2017
Related Publication 20210301326A1 · Sep 30, 2021
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