# US 10,501,772 B2 · Methods for rapid antimicrobial susceptibility testing

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- Number: US 10,501,772 B2
- Document ID: US10501772B2
- Status: Granted
- Kind: B2
- Application number: 15/717,569
- Filed: 2017-09-27
- Published: 2018-06-28
- Granted: 2019-12-10
- Earliest claimed: 2016-01-21
- Assignees: Selux Diagnostics, Inc.
- Applicants: SeLux Diagnostics, Inc.
- Inventors: Eric Stern; Aleksandar Vacic; Benjamin Spears; Alec Flyer; Kelly Flentie
- Web page: https://patentgrep.com/patent/US10501772B2

## CPC

- C12Q 1/18: Testing for antimicrobial activity of a material
- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- G01N 33/569: for microorganisms, e.g. protozoa, bacteria, viruses
- G01N 2333/195: from bacteria
- G01N 2458/00: Labels used in chemical analysis of biological material
- G01N 2500/10: involving cells
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates, in part, to methods and kits for rapidly determining antimicrobial susceptibility of microorganisms.

## Claims

### Claim 1

A method for determining antimicrobial susceptibility of microorganisms comprising:
incubating a liquid suspension of microorganisms in the presence of an antimicrobial under conditions that promote growth of the microorganisms,
adding a signaling agent that binds to a surface of the microorganisms;
separating the microorganisms bound by the signaling agent from unbound signaling agent; and
measuring signal levels associated with the microorganisms as compared to one or more controls, thereby measuring the antimicrobial susceptibility of the microorganisms;
wherein the signaling agent comprises a linker group L, and an amplifier group 104 comprises an Europium coordination complex; and wherein,
L forms a covalent bond to the amplifier group 104; or
L forms one or more non-covalent interactions with an amplifier group 104.

### Claim 2 (depends on 1)

The method of claim 1, wherein the antimicrobial susceptibility of the microorganisms is determined in less than 5 hours.

### Claim 3 (depends on 1)

The method of claim 1, wherein adding the signaling agent occurs during the incubating step.

### Claim 4 (depends on 1)

The method of claim 1, wherein adding the signaling agent occurs after the incubating step.

### Claim 5 (depends on 1)

The method of claim 1, wherein the linker group L comprises:
a microorganism binding chemical moiety 101, which forms a covalent bond or a non-covalent interaction with the surface of a microorganism;
a spacer moiety 102, covalently attached to the chemical moiety 101 and to another chemical moiety 103; and
the chemical moiety 103, which forms a covalent or non-covalent interaction with the amplifier group 104.

### Claim 6 (depends on 5)

The method of claim 5, wherein 101 forms covalent bond in the presence of one or more agents that promote coupling, selected from the group consisting of glutaraldehyde, formaldehyde, paraformaldehyde, EDC, DCC, CMC, DIC, HATU, Woodward's Reagent, N,N′-carbonyl diimidazole, acrylates, amides, imides, anhydrides, chlorotriazines, epoxides, isocyanates, isothiocyanates, organic acids, monomers, polymers, silanes, silcates, NHS, sulfo-NHS, and a combination thereof.

### Claim 7 (depends on 5)

The method of claim 5, wherein chemical moiety 101 forms a non-covalent interaction with the surface of a microorganism, wherein the non-covalent interaction comprises ionic interactions, van der Waals interactions, hydrophobic interactions, π-π interactions, or hydrogen bonding, or any combination thereof.

### Claim 8 (depends on 5)

The method of claim 5, wherein chemical moiety 101 comprises a nucleophilic functional group, wherein said nucleophilic functional group is amino, hydrazino, hydroxyamino, or thiol.

### Claim 9 (depends on 5)

The method of claim 5, wherein chemical moiety 101 comprises an electrophilic functional group, wherein said electrophilic functional group comprises an aldehyde, an α-halo ketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a tosylate ester, a glyoxal, an epoxide, an oxirane, a carbonate, an imidoester, an anhydride, a fluorophenyl ester, a hydroxymethyl phosphine derivative, a carbonate, a haloacetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, an acryloyl derivative, aldehyde, ketone, carboxylic acid, ester, acetyl chloride, or acetic anhydride.

### Claim 10 (depends on 5)

The method of claim 5, wherein spacer moiety 102 is hydrophobic.

### Claim 11 (depends on 5)

The method of claim 5, wherein spacer moiety 102 is hydrophilic.

### Claim 12 (depends on 5)

The method of claim 5, wherein spacer moiety 102 is oligomeric or polymeric, derived from peptide linkages, or comprised of inorganic linkages.

### Claim 13 (depends on 5)

The method of claim 5, wherein spacer moiety 102 comprises a repeating group that is:
wherein,
each of n, m, o, p, and q independently is an integer of 1 to 300.

### Claim 14 (depends on 5)

The method of claim 5, wherein spacer moiety 102 is

### Claim 15 (depends on 5)

The method of claim 5, wherein chemical moiety 103 comprises a nucleophilic group.

### Claim 16 (depends on 5)

The method of claim 5, wherein chemical moiety 103 comprises an electrophilic group.

### Claim 17 (depends on 5)

The method of claim 5, wherein chemical moiety 103 comprises a group that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, or biotinyl.

### Claim 18 (depends on 5)

The method of claim 5, wherein chemical moiety 103 comprises a group that is

### Claim 19 (depends on 5)

The method of claim 5, wherein chemical moiety 103 is formed from a chemical structure comprising a group that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotinyl, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate.

### Claim 20 (depends on 19)

The method of claim 19, wherein said group that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, or biotinyl forms a covalent bond to the amplifier group 104, or forms a non-covalent bond to the amplifier group 104.

### Claim 21 (depends on 1)

The method of claim 1, wherein linker group L has the following structure or is formed from the following structure,
wherein
X is
R is
Y is
each of j and k independently is an integer of 0 to 100; and
each of n, m, o, p, and q independently is an integer of 1 to 100.

### Claim 22 (depends on 21)

The method of claim 21, wherein X forms a covalent bond or a non-covalent interaction with the surface of a microorganism;
and/or
Y forms a covalent bond to an amplifier group 104 that is a chemical or biochemical amplifier.

### Claim 23 (depends on 1)

The method of claim 1, wherein said Europium coordination complex comprises a structure that is:

### Claim 24 (depends on 23)

The method of claim 23, wherein the signaling agent comprises or is formed from a structure selected from the group consisting of:

### Claim 25 (depends on 1)

The method of claim 1, wherein the signaling agent comprises:
an antibody, lectin, natural peptide, synthetic peptides, bacteriophage, synthetic and/or natural ligands, synthetic and/or natural polymers, synthetic and/or natural glycopolymers, carbohydrate-binding proteins and/or polymers, glycoprotein-binding proteins and/or polymers, charged small molecules, other proteins, bacteriophages, and/or aptamers.

### Claim 26 (depends on 25)

The method of claim 25, wherein the ligand and/or peptide is selected from the group consisting of bis(zinc-dipicolylamine), TAT peptide, serine proteases, cathelicidins, cationic dextrins, cationic cyclodextrins, salicylic acid, lysine, and combinations thereof.

### Claim 27 (depends on 25)

The method of claim 25, wherein the natural and/or synthetic polymer is linear or branched and selected from the group consisting of amylopectin, Poly(N-[3-(dimethylamino)propyl] methacrylamide), poly(ethyleneimine), poly-L-lysine, poly[2-(N,N-dimethylamino)ethyl methacrylate], and combinations thereof.

### Claim 28 (depends on 25)

The method of claim 25, wherein the natural and/or synthetic polymer and/or glycopolymer comprises moieties including, but not limited to, chitosan, gelatin, dextran, trehalose, cellulose, mannose, cationic dextrans and cyclodextrans, quaternary amines, pyridinium tribromides, histidine, lysine, cysteine, arginine, sulfoniums, phosphoniums, or combinations thereof including, but not limited to, co-block, graft, and alternating polymers.

### Claim 29 (depends on 1)

The method of claim 1, wherein multiple antimicrobials are tested in parallel.

### Claim 30 (depends on 1)

The method of claim 1, wherein the determining signal levels comprises measuring the signal levels associated with intact microorganisms.

### Claim 31 (depends on 1)

The method of claim 1, wherein the method further comprises a step of determining whether a microorganism is resistant, intermediately resistant or susceptible to one or more antimicrobials and/or determining one or more antimicrobial minimum inhibitory concentrations (MIC) based upon the signal levels associated with intact microorganisms.

### Claim 32 (depends on 1)

The method of claim 1 wherein the microorganisms are bacteria, fungi, protozoa, or archaea.

### Claim 33 (depends on 1)

The method of claim 1, wherein the microorganisms are obtained from a biological sample from a subject having an infection of the microorganisms and/or are obtained from a culture derived from the biological sample.

### Claim 34 (depends on 33)

The method of claim 33, wherein the biological sample is selected from the group consisting of blood or blood components, bronchoalveolar lavage, cerebrospinal fluid, nasal swabs, sputum, stool, throat swabs, vaginal swabs, urine, and wound swabs, or a combination thereof.

### Claim 35 (depends on 1)

The method of claim 1, wherein the one or more controls comprise a positive control measured from microorganisms under otherwise identical conditions but without antimicrobials or with one or more antimicrobials for which the microorganisms are not susceptible.

### Claim 36 (depends on 1)

The method of claim 1, wherein the one or more controls comprise a control measured from microorganisms under otherwise identical conditions but without nutrients.

### Claim 37 (depends on 1)

The method of claim 1, wherein the one or more controls comprise a control measured from microorganisms under otherwise identical conditions with one or more toxins known to inhibit growth of the microorganisms.

### Claim 38 (depends on 1)

The method of claim 1, wherein the separating the microorganisms is performed by centrifugation, magnetic separation, filtration, electrophoresis, dielectrophoresis, precipitation, agglutination, or a combination thereof.

### Claim 39 (depends on 1)

The method of claim 1, wherein the signal is an optical signal, an electrical signal, a fluorescent, time-resolved fluorescent, absorbent, or luminescent signal.

### Claim 40

A method for determining antimicrobial susceptibility of microorganisms comprising:
incubating a liquid suspension of microorganisms in a cartridge comprising a plurality of chambers, each chamber containing one or more antimicrobials, under conditions that promote growth of the microorganisms;
adding a signaling agent to the plurality of chambers, wherein the signaling agent binds to a surface of the microorganisms
removing unbound signaling agent; and
measuring signaling levels in the plurality of chambers as compared to one or more controls, thereby determining the susceptibility of microorganisms to the one or more antimicrobials; wherein the signaling agent comprises a linker group L, and an amplifier group 104 comprises an Europium coordination complex; and wherein,
L forms a covalent bond to the amplifier group 104; or
L forms one or more non-covalent interactions with an amplifier group 104.

### Claim 41 (depends on 40)

The method of claim 40, wherein the antimicrobial susceptibility of the microorganisms is determined in less than 5 hours.

### Claim 42 (depends on 40)

The method of claim 40, wherein the one or more controls comprise a positive control measured from microorganisms under otherwise identical conditions but without antimicrobials or with one or more antimicrobials for which the microorganisms are not susceptible.

### Claim 43 (depends on 40)

The method of claim 40, wherein the linker group L comprises:
a microorganism binding chemical moiety 101, which forms a covalent bond or a non-covalent interaction with the surface of a microorganism;
a spacer moiety 102, covalently attached to the chemical moiety 101 and to another chemical moiety 103; and
the chemical moiety 103, which forms a covalent or non-covalent interaction with the amplifier group 104.

### Claim 44 (depends on 40)

The method of claim 40, wherein the signaling agent forms covalent bond with the surface of the microorganism in the presence of one or more agents that promote coupling, selected from the group consisting of glutaraldehyde, formaldehyde, paraformaldehyde, EDC, DCC, CMC, DIC, HATU, Woodward's Reagent, N,N′-carbonyl diimidazole, acrylates, amides, imides, anhydrides, chlorotriazines, epoxides, isocyanates, isothiocyanates, organic acids, monomers, polymers, silanes, silcates, NHS, sulfo-NHS, and a combination thereof.

### Claim 45 (depends on 40)

The method of claim 40, wherein the microorganisms are obtained from a biological sample from a subject having an infection of the microorganisms and/or obtained from a culture derived from the biological sample; and wherein the biological sample is selected from the group consisting of blood or blood components, bronchoalveolar lavage, cerebrospinal fluid, nasal swabs, sputum, stool, throat swabs, vaginal swabs, urine, and wound swabs, or a combination thereof.