Systems and Methods for Detecting Antibiotic Resistance

• US 20130157876A1
• Filed: 02/21/2013
• Published: 06/20/2013
• Est. Priority Date: 08/21/2010
• Status: Abandoned Application

• Current Assignee: TessArae, LLC, Regents of the University of California (University of California)
• Original Assignee: TessArae, LLC, Regents of the University of California (University of California)
• Inventors: Brodie, Eoin L., Karaoz, Ulas, Lynch, Susan V., Srinivasan, Ramya, Purkayastha, Anjan, Lorence, Matthew C., Tibbetts, Clark J.
• Application Number:
  US13/773,631
• Publication Number:
  US 20130157876A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A robust, automated computational pipeline was used to design a system comprising a microarray for the identification of microorganisms and their antibiotic resistance profiles. This system and methods will facilitate the study of the epidemiology and microbial ecology of antibiotic resistance and be an invaluable tool to rapidly and simultaneously identify organisms and their antimicrobial resistance elements in environmental, food and clinical samples.

Claims

1. (canceled)

2. (canceled)

3. A method for determining a disease condition of a subject, the method comprising the steps of:

(a) obtaining a sample from a patient;

(b) isolating nucleic acid material from said sample;

(c) amplifying a target locus from said nucleic acid material;

(d) contacting said target locus with a set of probes (resequencing probes), wherein said set comprising 8 probes (4 probes based on the sense strand and 4 probes based on the anti-sense strand) per nucleotide base interrogated in each target locus;

(e) determining hybridization signal strengths across the set of probes;

(f) determining hybridization signal strengths for a plurality of different interrogation probes, each of which is complementary to a section within said target locus;

(g) determining the sequence of the target locus by analysis of the hybridization signal strengths of the resequencing probes;

(h) comparing the target locus sequence with a set of known sequences to determine the presence and/or antibiotic resistance repertoire of one or more target organisms;

(j) defining therapeutic strategy for said patient based on the results of step (h);

(l) classifying, diagnosing, prognosing, and/or predicting an outcome of said condition based on the results of step (h).

4. A method for parallel detection and strain level-identification of a panel of more than 44 organisms, in parallel with antibiotic resistance profiling of said organisms comprising the steps of:

a) extraction of nucleic acids from a patient sample using a rapid optimized protocol;

b) amplifying target loci from that sample using multiplex polymerase chain reaction;

c) pooling target locus amplified products;

d) labeling pooled amplified products;

e) contacting the labeled amplified pool of products with a plurality of resequencing probes which target both the sense and anti-sense strands of the target loci represented in SEQ ID NOS;

1-1323;

f) determining hybridization signal strength for each of said probes, wherein said determination identifies the specific sequence of the target locus, providing either strain level organism identification in parallel with single nucleotide polymorphism resolution antibiotic resistance determinant sequence information.

5. (canceled)

6. An array system comprising:

a resequencing microarray configured to simultaneously detect a plurality of organisms and antibiotic resistance elements in a sample, wherein the microarray comprises resequencing probes for organism identification, antibiotic resistance element detection, and detection of polymorphisms related to said antibiotic resistance, whereby said resequencing probes for organism detection can provide strain-level detection and identification of a organism in a sample and whereby said resequencing probes for antibiotic resistance elements and said resequencing probes for antibiotic resistance related polymorphisms provide for detection of emerging antibiotic resistance of organisms in a sample.

7. The array system of claim 6, wherein the plurality of organisms comprise bacteria.

8. The array system of claim 6, wherein the fragments are about 25 nucleotides long.

9. The array system of claim 6, wherein the sample is an environmental sample.

10. The array system of claim 9, wherein the environmental sample comprises at least one of soil, water or atmosphere.

11. The array system of claim 6, wherein the sample is a clinical sample.

12. The array system of claim 11, wherein the clinical sample comprises at least one of tissue, skin, stool, bodily fluid or blood.

13. A method of simultaneously detecting an organism in a sample and its antibiotic resistance comprising the steps:

applying a sample comprising a plurality of organisms to the array system of claim 6; and

simultaneously identifying at least one organism in the sample and determining its antibiotic resistance.

14. The method of claim 13, wherein the plurality of organisms comprise bacteria.

15. The method of claim 13, wherein the fragments are about 25 nucleotides long.

16. The method of claim 13, wherein the antibiotic resistance elements detected represent new or emerging resistance in the organism or organisms detected in the sample.

17. A method of designing and fabricating an array system comprising:

identifying organism-specific and antibiotic resistance element sequences corresponding to a plurality of organisms and resistance elements of interest;

selecting target loci unique to each organism and resistance element;

selecting resequencing probes; and

fabricating said array system.

18. The method of claim 17, wherein the plurality of organisms comprise bacteria.

19. The method of claim 17, wherein each fragment has a corresponding set of 8 variant fragments per nucleotide base interrogated, 4 based on sense strand and 4 on anti-sense strand.

20. The method of claim 17, wherein the fragments are about 25 nucleotides long.

21. (canceled)

22. A computer-readable storage medium comprising a database of antibiotic resistance elements (BARChipDB) comprising 262 bacterial genera/species isolated polynucleotides (SEQ ID NOS:

971-1232) and 1,061 isolated polynucleotides comprising resistance determinants sequences (SEQ ID NOS;

1-970 and 1233-1323).