Rapid antibiotic susceptibility testing

Patent metadata

Field	Value
Publication number	US-9632085-B2
Application number	US-201314382043-A
Country	US
Kind code	B2
Filing date	Feb 28, 2013
Priority date	Feb 29, 2012
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

Embodiments of various aspects described herein are directed to methods, compositions, kits and systems for rapid determination of antibiotic susceptibility of a microbe within hours after a sample is collected. In some embodiments, the methods, compositions, kits and systems described herein can allow determination of antibiotic susceptibility of a microbe based on a small number of microbes, e.g., as few as 5-10 microbes bound to a microbe-targeting substrate described herein.

First claim

What is claimed is:

1. A method for determining antibiotic susceptibility of a microbe, the method comprising:
(i) obtaining a sample comprising a microbe, wherein the microbe is bound to a microbe-targeting substrate, wherein the microbe-targeting substrate comprises a solid substrate and a microbe-binding molecule on a surface of the solid substrate, wherein the microbe-binding molecule comprises at least one carbohydrate recognition domain and N-terminus of the microbe-binding molecule is linked to the solid substrate;
(ii) incubating the solid substrate-bound microbe in the presence of at least one antibiotic agent for a pre-determined period of time; and
(iii) detecting the growth, viability, or functional response of the microbe to the antibiotic agent,
wherein reduced growth, viability, or function in the presence of the antibiotic agent relative to a reference or control sample indicates that the microbe is susceptible to the antibiotic agent.

2. The method of claim 1 , wherein the sample comprising the solid substrate-bound microbe is obtained by contacting a test sample suspected of comprising a microbe with the microbe-targeting substrate.

3. The method of claim 1 , wherein the solid substrate is selected from the group consisting of nucleic acid scaffolds, protein scaffolds, lipid scaffolds, dendrimers, nanoparticles, microparticles, microtiter plates, filters, fibers, screens, tubes, nanotubes, magnetic particles, microfluidic channels, membranes, microchips, filtration devices, diagnostic strips, dipsticks, extracorporeal devices, spiral mixers, hollow fibers, and any combination thereof.

4. The method of claim 3 , wherein the microbe-targeting substrate comprises magnetic particles.

5. The method of claim 1 , wherein the carbohydrate recognition domain comprises at least a microbial-binding portion of C-type lectins, collectins, ficolins, receptor-based lectins, lectins from the shrimp Marsupenaeus japonicas, non-C-type lectins, lipopolysaccharide (LPS)-binding proteins, endotoxin-binding proteins, peptidoglycan-binding proteins, or any combinations thereof.

6. The method of claim 5 , wherein the carbohydrate recognition domain comprises at least a microbial-binding portion of mannan-binding lectin (MBL), surfactant protein A, surfactant protein D, collectin 11, L-ficolin, ficolin A, DC-SIGN, DC-SIGNR, SIGNR1, macrophage mannose receptor 1, dectin-1, dectin-2, lectin A, lectin B, lectin C, wheat germ agglutinin, CD14, MD2, lipopolysaccharide-binding protein (LBP), limulus anti-LPS factor (LAL-F), mammalian peptidoglycan recognition protein-1 (PGRP-1), PGRP-2, PGRP-3, PGRP-4, or any combinations thereof.

7. The method of claim 1 , wherein the microbe-binding molecule is attached to the surface of the solid substrate through a linker.

8. The method of claim 7 , wherein N terminus of the linker comprises an amino acid sequence of AKT (alanine, lysine, threonine).

9. The method of claim 7 , wherein the linker comprises a Fc portion of an immunoglobulin.

10. The method of claim 1 , wherein the microbe-binding molecule is selected from the group consisting of MBL (mannose binding lectin), FcMBL (IgG Fc fused to mannose binding lectin), AKT-FcMBL (IgG Fc-fused to mannose binding lectin with the N-terminal amino acid tripeptide of sequence AKT (alanine, lysine, threonine)), and any combination thereof.

11. The method of claim 1 , wherein the microbe-binding molecule comprises an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and any combination thereof.

12. The method of claim 2 , wherein the test sample is a biological fluid obtained or derived from a subject, a fluid or specimen obtained from an environmental source, a fluid from a cell culture, a microbe colony, or any combinations thereof.

13. The method of claim 1 , wherein the test sample is a biological fluid selected from blood, plasma, serum, lactation products, amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, bronchial lavage aspirate fluid, perspiration, mucus, liquefied stool sample, synovial fluid, peritoneal fluid, pleural fluid, pericardial fluid, lymphatic fluid, tears, tracheal aspirate, a homogenate of a tissue specimen, or any mixtures thereof.

14. The method of claim 1 , wherein the test sample is a fluid or specimen obtained from an environmental source selected from a fluid or specimen obtained or derived from food products, food produce, poultry, meat, fish, beverages, dairy product, water (including wastewater), ponds, rivers, reservoirs, swimming pools, soils, food processing and/or packaging plants, agricultural places, hydrocultures (including hydroponic food farms), pharmaceutical manufacturing plants, animal colony facilities, or any combinations thereof.

15. The method of claim 1 , wherein said detecting the growth, viability, or functional response of the microbe is by an assay selected from the group consisting of cytolysis, membrane leakage, mitochondrial activity, caspase assays, Reactive Oxygen Species (ROS) production, ATP production, pH, genomic, metabolomic, transcriptomic, proteomic assays, and any combinations thereof.

16. The method of claim 1 , wherein said detecting the growth, viability, or functional response of the microbe comprises ELISA, optical or microscopic imaging, flow cytometry, mass spectrometry, PCR, luminescence or fluorescence labeling, or any combinations thereof.

17. The method of claim 1 , wherein the solid substrate-bound microbe from step (i) is immobilized in a gel matrix, prior to the incubation in the presence of at least one antibiotic agent.

18. The method of claim 17 , wherein the gel matrix is reactive to the growth, viability, or functional response of the microbe immobilized in the gel matrix.

19. The method of claim 17 , wherein the gel matrix comprises at least one detection agent to determine at least metabolism or viability of the microbe in the matrix.

20. The method of claim 19 , wherein said at least one detection agent is selected from the group consisting of resazurin or molecules derived from a nucleic acid binding agent, calcein AM, a tetrazolium salt, a protease marker, a pH indicator, an ATP indicator, a redox indicator, an esterase indicator, an ROS indicator, a cell-permeable dye, Carboxylic Acid Diacetate, Succinimidyl Ester, a cell-impermeable dye, cyanine, phenantridines, acridines, indoles, imidazoles, a nucleic acid stain, a cell permeant reactive tracer, intracellularly-activated fluorescent dyes, CMRA, CMF 2 HC (4-Chloromethyl-6,8-Difluoro-7-Hydroxycoumarin), CMFDA (5-Chloromethylfluorescein Diacetate), CMTMR (5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino)Tetramethylrhodamine), CMAC (7-Amino-4-Chloromethylcoumarin), CMHC (4-Chloromethyl-7-Hydroxycoumarin)), fluorescent DNA dyes, DAPI, Heochst family, SYBR family, SYTO family, SYTO 9, SYTOX family, SYTOX green, ethidium bromide, propidium iodide, acridines, chromogenic dyes, eosin, hematoxilin, methylene blue, azure, cytoplasma stain, calcofluor white, periodic acid-schiff stain, metabolic stains, any diacetate dye, rhodamine based-dye, fluorescin, resazurin/resorufin (alamar blue), ROS stains, DCFDA and related family, calcein-acetoxymethyl and related family, membrane stains, bodipy, FM 1-43, FM 4-64, Dil, DiO, DiA, biologic stains, labeled antibodies, labeled chitin-binding protein, and any combinations thereof.

21. The method of claim 17 , wherein the gel matrix is s

Assignees

• Harvard College

Inventors

• Super Michael
• Ingber Donald E
• Cartwright Mark J
• Watters Alexander
• Workman John Samuel
• Levner Daniel
• Rottman Martin Marcus

Classifications

• G01N2333/31

  from Staphylococcus (G) · CPC title

• G01N33/56916

  Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia · CPC title

• G01N2500/10

  involving cells · CPC title

• G01N2333/245

  Escherichia (G) · CPC title

• G01N33/56938 Primary

  Staphylococcus · CPC title