Method and kit for measuring antibacterial drug susceptibility

Abstract

The present disclosure relates to a method for measuring the antibiotic susceptibility of microorganisms in a sample. The method includes receiving a sample containing microorganisms, incubating at least one first portion of the sample with at least one antibacterial agent and incubating at least one second portion of the sample without the antibacterial agent, extracting nucleic acids associated with the microorganisms present in the sample from at least one first portion of the sample and at least one second portion of the sample, amplifying the nucleic acids extracted from at least one first portion of the sample and at least one second portion of the sample, and obtaining antibiotic susceptibility information associated with the microorganisms from the nucleic acids amplified from at least one first portion of the sample and at least one second portion of the sample.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the priority or benefit of Indian Patent Application 202241031205 filed on May 31, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the field of analysis of samples, and more particularly, to the field of measuring the antimicrobial susceptibility of microorganisms.

Background Art

[0003] The antimicrobial resistance rate of microorganisms is steadily increasing. This necessitates the administration of appropriate and sufficient antimicrobial treatment to patients. Current methods for measuring antimicrobial resistance are slow and do not provide complete information regarding antimicrobial susceptibility.

[0004] Conventionally, microorganism culture - based methods have been used to measure the susceptibility or resistance of antimicrobial agents, which take about 3 - 4 days to obtain clinically available results. Methods based on polymerase chain reaction (PCR) are also used when measuring antimicrobial resistance information. However, the turnaround time of such genotypic methods is about 2 - 3 hours, and the amount of antimicrobial resistance information obtained is limited. These tests require continuous progress and improvement to include changing genetic signatures from resistant microorganisms.

[0005] The related prior art includes Patent Documents 1 by Zhang et al., Patent Document 2, and Patent Document 3 by Varma et al. "Compositions, devices and methods for diagnosing and treating infectious disease" (all of which are incorporated herein by reference in their entirety).

[0006] From the above, there is a continuing need for an effective method of measuring the antimicrobial susceptibility of microorganisms that can assist physicians in making timely clinical decisions by providing complete information related to rapidity and antimicrobial resistance.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

[0008] A method, kit, and apparatus for measuring the antimicrobial susceptibility of one or more microorganisms are proposed herein. In one aspect, a method for measuring the antimicrobial susceptibility of one or more microorganisms in a sample, such as a biological sample, is disclosed.

[0009] In one aspect, the method of the present disclosure includes receiving a sample containing one or more microorganisms. The method then includes incubating at least one first portion of the sample with at least one antimicrobial agent and incubating at least one second portion of the sample without an antimicrobial agent. The method further includes extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample. The nucleic acids are related to the microorganisms present in the sample. In one aspect, the extracted nucleic acids are amplified, and antimicrobial susceptibility information related to the microorganisms is obtained from the difference between the presence and absence of an antimicrobial response observed in the results from the amplified nucleic acids.

[0010] In one aspect, the present disclosure includes a method for measuring the antimicrobial susceptibility of microorganisms in a sample. The method includes receiving a sample containing microorganisms, Incubating at least one first portion of the sample with at least one antibacterial agent and incubating at least one second portion of the sample without an antibacterial agent, Contacting the at least one first portion and / or the at least one second portion with one or more intercalating dyes, Extracting nucleic acids from the at least one first portion of the sample and the at least one second portion of the sample, the nucleic acids being related to the microorganisms present in the sample, Amplifying the nucleic acids extracted from the at least one first portion of the sample and the at least one second portion of the sample, Obtaining antibacterial susceptibility information related to the microorganisms from the amplified nucleic acids from the at least one first portion of the sample and the at least one second portion of the sample. In one aspect, the intercalating dye is ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, and the like.

[0011] In one aspect, the method of the present disclosure Incubating or culturing at least one first portion of a biological sample containing one or more microorganisms with at least one antibacterial agent and incubating or culturing at least one second portion of the biological sample without an antibacterial agent, Extracting nucleic acids from the at least one first portion of the biological sample and the at least one second portion of the biological sample, the nucleic acids being related to the microorganisms present in the biological sample, Amplifying the extracted nucleic acids and obtaining antibacterial susceptibility information related to the microorganisms from the difference between the presence and absence of an antibacterial reaction observed in the results from the amplified nucleic acids.

[0012] In one aspect, the method of the present disclosure Incubating or culturing at least one first portion of a biological sample containing one or more microorganisms with at least one antibacterial agent and incubating or culturing at least one second portion of the biological sample without an antibacterial agent. Extracting nucleic acids from at least one first portion of the biological sample and at least one second portion of the biological sample, the nucleic acids being related to the microorganisms present in the biological sample. Contacting the extracted nucleic acids with one or more intercalating dyes. Amplifying the extracted nucleic acids and obtaining antibacterial susceptibility information related to the microorganisms from the difference between the presence and absence of an antibacterial drug reaction observed in the results from the amplified nucleic acids. In one embodiment, contacting the extracted nucleic acids with one or more intercalating dyes further includes applying light (such as UV light) under conditions sufficient for the extracted nucleic acids and the intercalating dyes to associate with each other or form a nucleic acid / intercalating dye complex. For example, the intercalating dye is ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, etc.

[0013] In one aspect, the present disclosure includes a kit for measuring the antimicrobial susceptibility of microorganisms in a sample. In one aspect, the kit includes one or more antimicrobial agents. The antimicrobial agents are provided in a specified amount so as to obtain valid results. The kit further includes a reaction volume specific to the growth conditions of microorganisms that may be present in the sample. Also, the kit may include a medium for the growth of microorganisms present in the sample. The volume of the growth medium is defined so as to obtain optimal results for microorganism growth. Further, in one aspect, the kit includes nucleic acid extraction-based reagents and nucleic acid amplification-based reagents. Specifically, the nucleic acid amplification reagents include one or more primers targeting 16S rRNA and / or 16S rDNA of microorganisms in the sample. Additionally, the nucleic acid amplification kit may also include one or more TaqMan probes. In another aspect, the kit includes polymerase chain reaction-based melt analysis software. In yet another aspect, the kit includes at least one intercalating dye. For example, the dye may be ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, and the like.

[0014] In one aspect, the present disclosure includes a kit for measuring the antimicrobial susceptibility of microorganisms in a sample. The kit includes one or more antimicrobial agents, and a growth medium for microorganisms, and reagents related to nucleic acid extraction, and reagents related to nucleic acid amplification, and polymerase chain reaction-based melt analysis software. In one aspect, the kit includes instructions for using the kit and / or the software. In one aspect, the kit includes one or more intercalating dyes.

[0015] In one aspect, the present disclosure includes a product such as a system or its components including a non-transitory computer-readable medium containing encoded instructions. The instructions are configured to cause one or more processors to execute a method for measuring the antimicrobial susceptibility of microorganisms in a sample. This method includes Incubating at least one first portion of a sample containing a microorganism with at least one antibacterial agent and incubating at least one second portion of the sample without an antibacterial agent, Extracting nucleic acids from the at least one first portion of the sample and the at least one second portion of the sample, the nucleic acids being related to the microorganism present in the sample, Amplifying the nucleic acids extracted from the at least one first portion of the sample and the at least one second portion, Obtaining antibacterial susceptibility information related to the microorganism from the nucleic acids amplified from the at least one first portion of the sample and the at least one second portion, including. In one aspect, the method further includes contacting the sample, a portion of the sample, or the extracted nucleic acid with one or more intercalating dyes prior to amplification.

[0016] In one aspect, the present disclosure includes a non-transitory computer-readable medium containing encoded instructions. The instructions are configured to cause one or more processors to execute a method for measuring the antibacterial susceptibility of a microorganism in a sample. This method is Incubating at least one first portion of a sample containing a microorganism with at least one antibacterial agent and incubating at least one second portion of the sample without an antibacterial agent, Extracting nucleic acids from the at least one first portion of the sample and the at least one second portion of the sample, the nucleic acids being related to the microorganism present in the sample, Contacting the nucleic acid with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex, Amplifying the nucleic acids extracted from the at least one first portion of the sample and the at least one second portion, Obtaining antimicrobial susceptibility information related to the microorganism from the at least one first portion and the nucleic acid amplified from the at least one second portion of the sample.

[0017] The above summary is provided to briefly introduce options of the inventive concept detailed in the following description. It is not intended to identify features or essential features of the subject matter of the claims. Furthermore, the subject matter of the claims is not limited to embodiments that solve any or all of the disadvantages mentioned in a part of the present disclosure.

Brief Description of Drawings

[0018] Each aspect of the present disclosure outlined above and described in more detail below can be understood by referring to embodiments of the present disclosure illustrated in the accompanying drawings. However, the accompanying drawings only show typical embodiments of the present disclosure, that is, it should not be considered as limiting the scope allowing other equally effective embodiments of the present disclosure.

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[0019] For the sake of understanding, the same reference numerals as much as possible are used to indicate the same elements common to each drawing. The drawings are not drawn to an exact scale and may be simplified, considering clarity. The elements and features of one embodiment may be incorporated into other embodiments and enjoy the advantages thereof unless otherwise described.

Mode for Carrying Out the Invention

[0020] In exemplifying terms and results to explain in detail at least one embodiment of the present disclosure, it should be understood that the present disclosure is not limited to the details of the configurations and the arrangement of components mentioned in the following description regarding its application. The present disclosure can be other embodiments and can be implemented or carried out in various ways. Thus, the terms used herein are intended to be given the broadest scope and meaning possible, and the embodiments are intended to be illustrative and not exhaustive. Also, it should be understood that the expressions and phrases adopted herein are for the purpose of explanation and should not be regarded as limiting.

[0021] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular.

[0022] All patents, published patent applications, and non-patent literature referred to herein demonstrate the state of the art of one of ordinary skill in the art relevant to this disclosure. All patents, published patent applications, and non-patent literature referred to in each part of this application are hereby expressly incorporated by reference in their entirety to the same extent as if each individual patent or literature were specifically and individually indicated to be incorporated by reference.

[0023] All of the compositions, apparatus / devices, kits, and / or methods disclosed herein can be made and executed without undue experimentation based on this disclosure. Although the compositions, apparatus / devices, kits, and / or methods are described with respect to specific embodiments, it will be apparent to those of ordinary skill in the art that changes can be applied to the compositions, apparatus / devices, kits, and / or methods described herein, and to the process / steps of the method or the order of the process / steps, without departing from the spirit, gist, and scope of this disclosure. All such similar substitutions and modifications that are apparent to those of ordinary skill in the art are considered to be included within the spirit, scope, and gist of this disclosure as defined by the claims.

[0024] Embodiments of the present disclosure can provide robust and rapid drug susceptibility testing and drug screening and are beneficial. The various features and advantages that can be achieved by the present disclosure can be understood from the description herein and may include one or more of the following: (1) Compatibility for use with prokaryotic cells that may have insufficient growth by culture; (2) Use of a target drug concentration to inhibit the growth of organisms. In embodiments, susceptibility includes examples where an antibacterial drug has an inhibitory effect on the growth of a microorganism or a lethal effect on the microorganism. In embodiments, susceptibility includes examples where an antibacterial drug has a lethal effect on a microorganism. Susceptibility further includes the concept of the minimum inhibitory concentration (''MIC'') of an antibacterial drug as the concentration of the antibacterial drug that will stop the growth of a microorganism. The determination of susceptibility or lack of susceptibility, for example, using the systems and methods described herein, provides information useful to a clinician or healthcare decision maker in making decisions regarding antibacterial treatment for a patient in need of antibacterial treatment. In embodiments, a decreased growth or functional response in the presence of an antibacterial drug compared to a control group indicates that the microorganism is susceptible to the tested antibacterial drug.

[0025] [Definitions] When used in accordance with the present disclosure, the following terms should be understood to have the following meanings unless otherwise indicated.

[0026] The use of "a" or "an", when used in conjunction with "comprising" in the claims and / or the specification, has the meaning of "one", but is not inconsistent with the meanings of "one or more", "at least one", and "one or more than one". That is, "a", "an", and "the" include plural expressions unless the context clearly indicates otherwise. Thus, for example, the expression "a compound" can also refer to "one or more compounds", "two or more compounds", "three or more compounds", "four or more compounds", or "greater numbers of compounds". "Plurality" has the meaning of "two or more".

[0027] The use of "at least one" is understood to include, but is not particularly limited to, each quantity greater than one, such as 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc., when used in conjunction with "one". "At least one" extends up to 100 or 1000 or more depending on the term being modified, and additionally, the quantities of 100 / 1000 should not be considered limiting if higher limitations would also yield satisfactory results. Additionally, the use of "at least one of X, Y, and Z" is understood to include X alone, Y alone, Z alone, and the possible combinations of X, Y, and Z simultaneously.

[0028] The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is for the sole purpose of distinguishing two or more items and, unless otherwise expressly stated, does not imply any particular arrangement (connection) or order or importance of one item with respect to another item, or any additional order.

[0029] The use of "or" in the claims is used to mean the inclusive "and / or" unless the specification clearly indicates otherwise or the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or absent); A is false (or absent) and B is true (or present); both A and B are true (or present).

[0030] As used herein and in the claims, "comprising" (and its variants such as "comprise" and "comprises"), "having" (and its variants such as "have" and "has"), "including" (and its variants such as "includes" and "include"), or "containing" (and its variants such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherently present therein.

[0031] As used herein, "or combinations thereof" refers to all permutations and combinations of the items listed prior to this phrase. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular situation, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations including repetitions of one or more items or phrases, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are clearly included. Those of ordinary skill in the art will typically understand that there is no limit to the number of items or phrases in any combination, unless the context clearly dictates otherwise.

[0032] As used herein, the phrases "one embodiment", "an embodiment", "some embodiments", "one example", "for example", or "an example" mean that the individual elements, features, structures, or characteristics described in connection with a particular embodiment / example are included in at least one embodiment / example. The use of the phrases "in some embodiments" or "one example" in various places in this specification does not necessarily refer to the same embodiment / example, for instance. Further, all expressions of one or more embodiments or examples are to be construed as not limiting the claims.

[0033] Throughout this application, "about" is used to indicate that a value includes the inherent error range with respect to a composition / apparatus / device, the method used to determine that value, or the variations present in the subject of study. For example, by way of illustration and not limitation, when "about" is used, the specified value can vary by plus / minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent from the specified value, and such variations are consistent with performing the disclosed method and are understood by those of ordinary skill in the art.

[0034] "Analyte" is a nucleic acid polymer recognizable by an analyte-specific binding partner. In embodiments, the analyte refers to a nucleic acid polymer recognizable by an analyte-specific binding partner such as, for example (but not limited to), a DNA or RNA segment, strand or oligomer or a portion thereof. The binding partner is complementary or substantially complementary to the analyte so as to be able to bind to the analyte.

[0035] As used herein, "antibiotic" refers to a compound having the ability to kill bacteria or inhibit the growth of bacteria. In some embodiments, "antibiotic" refers to a compound having the ability to kill or inhibit the growth of bacteria selected from the genera including, but not limited to, Borrelia, Staphylococcus, Escherichia, Klebsiella, Acinetobacter, and Mycobacterium. In an embodiment, an antibacterial agent is an agent that kills or inhibits the growth of microorganisms. In an embodiment, antibacterial agents such as drugs can be classified according to the microorganisms on which they primarily act. For example, antibacterial agents are used against bacteria. As used herein, "β-lactam" or "β-lactam antibacterial agent" refers to an antibacterial agent having a β-lactam ring as part of its core structure, such as penicillin and penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. These antibacterial agents act by inhibiting bacterial cell wall biosynthesis, but other antibacterial agents such as protein synthesis inhibitors (tetracyclines, aminoglycosides, macrolides, etc.), nucleic acid synthesis inhibitors (fluoroquinolones, rifamycins, etc.) and antimetabolic sulfonamides can also be used in the antibacterial susceptibility tests by the methodology described herein.

[0036] As used herein, "bacterial infection" means the invasion, growth and / or presence of bacteria in a cell or subject (test subject).

[0037] As used herein, "cell culture" or "culture" includes the representation of a cultured cell population. In some embodiments, cell culture or culture can also refer to cells in a medium that promotes cell growth and, optionally, cells growing in the medium. Thus, in some embodiments, "bacterial culture" or "culture" can refer to bacteria growing or incubating in a medium that promotes bacterial growth. Bacterial cultures can be obtained in various types of containers such as flasks, test tubes, microwell plates, or arrays. Generally, bacteria have different growth phases. When a population of bacteria first enters a nutrient-rich environment that allows growth, the cells need to adapt to their new environment. The first phase of growth is the lag phase, a period of slow growth while the cells adapt to the nutrient-rich environment and prepare for rapid growth. The lag phase has a high biosynthetic rate as the proteins needed for rapid growth are produced. The second phase of growth is the log phase, also known as the logarithmic growth phase or exponential growth phase, during which the bacteria undergo rapid exponential growth. During the log phase, nutrients are metabolized at a maximum rate until one of the nutrients becomes depleted and growth begins to be limited. The third phase of growth is the stationary phase, which is caused by nutrient depletion. In some embodiments, a bacterial culture in the "stationary phase" means that the bacteria in the culture have approximately equal growth and death rates. As used herein, "growing forms" of bacteria generally refers to bacteria in the lag or log phase and not in the stationary phase. In some embodiments, a stationary-phase bacterial culture has been grown for about 7 days. In other embodiments, a stationary-phase bacterial culture contains non-replicating persister cells. "Non-replicating persister cells" means bacterial cells that enter a state where they stop replicating and can withstand antibacterial agents.

[0038] As used herein, "contacting" refers to any action that brings about at least one compound or component of the subject matter disclosed herein into physical contact with at least one cell (e.g., a bacterial cell) or an environment (e.g., a culture medium or a sample) in which at least one cell (e.g., a bacterial cell) is present.

[0039] As used herein, "disease" and "disorder" are used interchangeably to refer to a condition in a subject that involves a deleterious deviation from the normal structural or functional state of an organism. Non-limiting examples of diseases / disorders include a subject having one or more bacterial infections or sepsis.

[0040] Detection agent: As used herein, "detection agent" refers to any detectable element, molecule, functional group, compound, fragment, or moiety. In some embodiments, the detection agent is provided or utilized alone. In some embodiments, the detection agent is provided and / or utilized in combination with another agent (e.g., conjugated to another agent). Examples of detection agents include, but are not limited to, various ligands, fluorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins to which antiserum or monoclonal antibodies are available.

[0041] Diagnostic test: As used herein, "diagnostic test" is one step or a series of steps performed or completed to obtain information that is useful for determining whether a patient has a disease, disorder, or medical condition and / or for classifying the disease, disorder, or medical condition into a phenotypic category or any category that is relevant with respect to the prognosis of the disease, disorder, or medical condition or that will have a response to treatment (either general treatment or a particular treatment). Similarly, "diagnosis" refers to providing various diagnostic information, including but not limited to whether a subject has or is likely to develop a disease, disorder, or medical condition; the status, staging, or characteristics of a disease, disorder, or symptom that appears in a subject; information related to the nature or classification of a tumor; information related to prognosis; and / or information useful for selecting an appropriate treatment or additional diagnostic test. The choice of treatment can include the choice of a particular therapeutic agent or other treatment modality, such as surgery, radiation, etc., the choice of whether to defer or administer treatment, the choice regarding a dosing regimen (e.g., the frequency or level of one or more administrations of a particular therapeutic agent or combination of therapeutic agents), etc. The choice of additional diagnostic test can include more specialized tests for a specified disease, disorder, or medical condition.

[0042] Hybridization: "Hybridization" refers to the physical property of a single-stranded nucleic acid molecule (e.g., DNA or RNA) annealing to a complementary nucleic acid molecule. Hybridization can generally be assessed in a variety of contexts, including when interacting nucleic acid molecules are studied alone or in more complex systems (e.g., covalently or otherwise associated with carrier entities (transport proteins) and / or in biological systems or cells). Hybridization techniques and methods for assessing hybridization are well known in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY. Those of ordinary skill in the art will know how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a predetermined level of complementarity will stably hybridize, while sequences with less complementarity will not hybridize. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ.

[0043] "labeled" and "labeled with a detectable agent (or moiety)" are used interchangeably herein, which specifies that an entity (e.g., a target sequence) can be visualized, for example, directly or following hybridization with another entity that includes a detectable agent or moiety. In embodiments, the detectable agent or moiety is selected to generate a measurable signal whose intensity is related to (e.g., proportional to) the amount of the entity of interest (e.g., the target sequence). Methods for labeling nucleic acid molecules are well known in the art. In some embodiments, the labeled nucleic acid can be prepared by incorporation of a label or binding to a label that is directly or indirectly detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0044] In some embodiments, "oligonucleotide" is used herein to denote a polynucleotide comprising from about 5 to about 150 nucleotides, such as from about 10 to about 100 nucleotides, from about 15 to about 75 nucleotides, or from about 15 to about 50 nucleotides. Throughout this specification, when an oligonucleotide is represented by a character sequence (e.g., selected from the four base characters: A, C, G, and T, which represent adenosine, cytidine, guanosine, and thymidine, respectively), the nucleotides are presented in 5' to 3' order from left to right. "Polynucleotide sequence" refers to the sequence of nucleotide monomers along a polymer. Unless otherwise noted, when a polynucleotide sequence is represented, it is understood that the nucleotides are in the 5' to 3' direction from left to right.

[0045] As used herein, "nucleic acid" refers to a polynucleotide of nucleobases having a backbone of alternating sugar and phosphate units of DNA and RNA. In embodiments, "nucleic acid" and "polynucleotide" are considered equivalent and interchangeable. Nucleic acids are generally in the form of DNA or RNA. In some embodiments, "nucleic acid", "nucleic acid molecule", "polynucleotide" or "oligonucleotide" are used interchangeably herein. These refer to polymers of nucleotide monomers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) or analogs thereof. Nucleotides can be of genomic, synthetic or semi-synthetic origin. Unless otherwise specified, the term includes amplification products in addition to nucleic acid-like structures having a synthetic backbone. As would be understood by one of ordinary skill in the art, the length of these polymers (i.e., the number of nucleotides they contain) can often vary widely depending on their intended function or use. Polynucleotides can be linear, branched, or circular molecules. In embodiments, the polynucleotide is H + , NH4 + , trialkylammonium, Mg2 + , Na + and also has counterions such as. Polynucleotides can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or a chimeric mixture thereof. Polynucleotides can be composed of internucleotide nucleobases and sugar analogs.

[0046] As used herein, "substantially" means that the subsequently recited event or situation occurs completely, or that the subsequently recited event or situation occurs to a significant extent or degree. For example, "substantially" in relation to a particular event or situation means that the subsequently recited event or situation occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. When referring to "substantially adjacent", it can mean that two items are 100% adjacent to each other, or that two items are close to each other but not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is close to the other item.

[0047] As used herein, "associated with" includes both direct association of two parts with each other and indirect association of two parts with each other. Non-limiting examples of association include covalent bonding of one part to another part either by direct bonding or via a spacer group, non-covalent bonding of one part to another part either directly or by a specific binding pair element attached to the part, incorporation of one part into another part such as by dissolving one part into another part or by synthesis, and coating one part with another part.

[0048] As used herein, "biological fluid sample" is understood to include any liquid test sample obtained from a patient and available for use in accordance with the present disclosure. Examples of biological fluid samples that can be used include, but are not limited to, whole blood or any part thereof (i.e., plasma or serum), saliva, sputum, mucus, nasal, nasopharyngeal, anterior nasal, oropharyngeal, tracheal, bronchoalveolar, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, synovial fluid, sweat, interstitial fluid, tears, combinations thereof, and the like.

[0049] As used herein, "volume" with respect to a liquid test sample utilized in accordance with the present disclosure refers to the volume of the liquid test sample, for example, in the range of about 0.1 μl to about 100 μl, or in the range of about 1 μl to about 75 μl, or in the range of about 2 μl to about 60 μl, or a value of about 50 μl or less.

[0050] As used herein, "patient" includes human and veterinary subjects. In certain non-limiting embodiments, the patient is a mammal. In another certain non-limiting embodiment, the patient is a human. The "mammal" that is the subject of diagnosis / treatment refers to any animal classified as a mammal, including humans, domestic and breeding animals, non-human primates, and zoo, sports, or pet animals such as dogs, horses, cats, cows, etc.

[0051] "Health care provider" or "health care decision maker" includes an individual authorized to diagnose or treat a patient or to assist in the diagnosis or treatment of a patient. In the context of identifying a useful new drug for treating a disease, the health care provider can be an individual not authorized to diagnose or treat a patient or to assist in the diagnosis or treatment of a patient.

[0052] As used herein, "isolated" means a target, sample, polynucleotide, complex, nucleic acid, or oligonucleotide that is separated by origin or manipulation from at least some of the components that are naturally associated or that were associated when first obtained.

[0053] "Point of care testing" refers to real-time diagnostic tests that can be performed within a rapid time frame, such that the tests are performed more quickly than comparative tests that do not use the present system. Point of care testing can be rapidly performed in situ, such as in a hospital / clinic, at the bedside, in a stat laboratory, in an emergency room, or other such locations, particularly when rapid and accurate results are required. The patient may or may not be present. Point of care testing includes, but is not limited to, an emergency treatment room, an operating room, a hospital laboratory and other clinical laboratories, a hospital / clinic, the field, or situations where rapid and accurate results are desired.

[0054] Specifically (but not in a limiting sense), "specific binding partner" as used herein as a "target analyte-specific binding partner" is understood to refer to a molecule that can specifically associate with a target analyte. For example, but not by way of limitation, the binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix), combinations or derivatives thereof, and other molecules that can specifically bind to the target analyte.

[0055] As used herein, "inhibit", "inhibits", or "significant decrease" means, for example, reducing, suppressing, weakening, or stopping the growth and / or survival of cells (e.g., bacteria) in a culture or subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% as compared to an untreated control culture or subject. Inhibiting the "survival" of cells (e.g., bacteria) in this context means killing the cells or reducing the number of viable cells. In some embodiments, the growth of the cells is inhibited by more than about 50%. In other embodiments, the percentage of viable cells in the culture after treatment with the test compound is less than about 50% compared to the percentage of viable cells in a control under the same conditions but without the test compound. In still other embodiments, the stationary phase culture consists of non-growing cells such as non-replicating persister cells. Further, as used herein, "significant increase" means an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100%.

[0056] Sample: As used herein, "sample" refers to a biological sample obtained from or derived from a subject, as described herein. In some embodiments, the biological sample includes biological tissue or biological fluid. In some embodiments, the biological sample includes blood; blood cells; tissue or fine needle biopsy samples; cell-containing body fluids; free nucleic acids; cerebrospinal fluid; lymphatic fluid; tissue biopsy samples; surgical specimens; other body fluids, secretions, and / or excretions; and / or cells therefrom. In some embodiments, the biological sample includes cells obtained from an individual, e.g., a human or animal subject. In some embodiments, the obtained cells are cells derived from or include cells from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood). In some embodiments, the sample is heart tissue obtained from a subject. In some embodiments, as is apparent from the context, "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, filtration using a semipermeable membrane. As another example of sample processing, the sample is a plasma sample treated with an anticoagulant selected from the group consisting of EDTA, heparin, and citrate. As another example of sample processing, the sample is processed to isolate one or more proteins (e.g., by capturing the protein with one or more antibodies). A "processed sample" includes, for example, nucleic acids or polypeptides obtained by extracting from the sample or passing the primary sample through techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components.

[0057] Subject: As used herein, "subject" refers to an organism, e.g., a mammal (e.g., a human). In some embodiments, the human subject (test subject) is an adult, adolescent, or pediatric subject. In some embodiments, the subject is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In some embodiments, the subject has a disease, disorder or medical condition, e.g., a disease, disorder or medical condition that can be treated as proposed herein. In some embodiments, the subject is susceptible to a disease, disorder, or medical condition, and in some embodiments, the susceptible subject is prone to and / or exhibits a high risk of a disease, disorder, or medical condition (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, disorder or medical condition. In some embodiments, the subject does not exhibit a specific symptom (e.g., a clinical sign of a disease) or characteristic of a disease, disorder, or medical condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or medical condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has received and / or is receiving a diagnosis and / or treatment.

[0058] Threshold value: As used herein, "threshold value" refers to one or more values used as a criterion for obtaining and / or classifying information regarding the result of a measurement, e.g., the result of a measurement obtained in an assay. The threshold value can be determined based on one or more control samples. The threshold value can be determined before, simultaneously with, or after the performance of the measurement of interest. In some embodiments, the threshold value can be a range of values. In some embodiments, the threshold value is a value (or range of values) reported in the relevant art (e.g., a value in a standard table).

[0059] As used herein, "therapeutically effective amount" means an amount of a compound that, when administered to a subject for treating or preventing a particular disorder, disease or medical condition, is sufficient to effect such treatment or prevention of the disorder, disease or medical condition. Dosage and therapeutically effective amounts vary according to a variety of factors including, for example, the activity of the particular agent employed, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and in the case of combination drugs, the nature of the drug combination, the effect the compound is believed to have on the subject and the nature of the compound (e.g., bioavailability, stability, efficacy, toxicity, etc.), and one or more specific disorders the subject is suffering from. Further, a therapeutically effective amount administered intravenously may depend on the subject's blood parameters such as lipid profile, insulin level, blood glucose or liver metabolism. A therapeutically effective amount may also vary according to disease state, organ function, or the severity of underlying disease or complications. Such appropriate dosages are determined using available assays. When one or more compounds or therapeutic agents are administered to humans, a physician, for example, initially prescribes relatively low dosages and then increases the dosage until an appropriate response is obtained.

[0060] The "treatment" or "treating" of a subject involves the application or administration of a compound to the subject with the aim of delaying, slowing, stabilizing, curing, healing, alleviating, removing, modifying, improving, reducing exacerbation, making better, effecting a remission, or acting, with respect to a disease or medical condition, the symptoms of a disease or medical condition, or the risk (or susceptibility) of a disease or medical condition. "Treating" refers to any sign of success in the treatment or improvement of an injury, pathology or medical condition, for example, reduction; remission; reduction in the rate of exacerbation; reduction in the severity of a disease; stabilization or reduction of symptoms or making an injury, pathology or medical condition more tolerable to the subject; slowing the rate of degeneration or decline; reducing debilitation at the end point of degeneration; or improving the physical or mental health of the subject, including objective or subjective parameters. In an embodiment, "treating" means reducing or improving the progression, severity, and / or duration of an infection. In an embodiment, "treating" means reducing or improving the progression, severity, and / or duration of a bacterial infection, or improving one or more symptoms of a bacterial infection by the implementation (administration) of one or more treatments (e.g., one or more therapeutic agents). In a particular embodiment, "treatment" means improving a measurable physical parameter of a bacterial infection. In an embodiment, "treating" means reducing or improving the progression, severity, and / or duration of sepsis, or improving one or more symptoms of sepsis by the implementation (administration) of one or more treatments (e.g., one or more therapeutic agents). In a particular embodiment, "treatment" means improving a measurable physical parameter of sepsis. In an embodiment, "treating" changes the natural or presenting state of the subject.

[0061] Hereinafter, embodiments for implementing the present invention will be described in detail. Although various embodiments are described with reference to the drawings, throughout, like reference numerals are used to refer to like elements. In the following description, for the purpose of explanation, many specific details are disclosed in order to provide a complete understanding of one or more embodiments. It is clear that these embodiments can be implemented without such specific details. In another example, well-known things and methods are not described in detail in order to avoid unnecessarily obscuring the embodiments of the present disclosure. The present disclosure is capable of various modifications and alternative forms, but specific embodiments thereof are illustrated in the drawings and described in detail herein. However, it should be understood that the intention is not to limit the present disclosure to the specific forms disclosed, but rather, on the contrary, the present disclosure covers all modifications, equivalents, and alternative forms that fall within the spirit and scope of the present disclosure. The disclosed embodiments provide a method and kit for measuring the antimicrobial susceptibility of microorganisms.

[0062] Figure 1 illustrates a flowchart of a method 100 for measuring antimicrobial susceptibility related to microorganisms according to a first embodiment. In an embodiment, a suitable microorganism includes a method for evaluating the drug susceptibility of bacterial cells, which are pathogenic bacteria as an option. The selected organism is a bacterium of a selected genus such as Staphylococcus, Escherichia, Klebsiella, Acinetobacter, or Mycobacterium, and the selected organism is, for example, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Mycobacterium tuberculosis. In some embodiments, the bacterium is a bacterial isolate or subspecies (e.g., from any of the aforementioned species). Optionally, when the cell is a bacterium or other microorganism or infectious agent, the cell is based on (including those derived from) a single individual or host or multiple individuals or hosts. In an embodiment, the bacterial cells are analyzed or characterized individually or collectively as a colony or group of bacterial cells.

[0063] In an embodiment, a cell culture containing the cells to be examined is obtained or established. The cells are isolated cells such as cells isolated from a selected organism (e.g., bacteria), and optionally may be from a selected species or genus. Thus, in one embodiment, the method is a method for evaluating the drug sensitivity of cells derived from a selected organism, and this method includes obtaining or establishing a culture containing cells isolated from the selected organism, such as bacterial cells such as the above-mentioned bacterial species and genera, or other bacterial cells as mentioned herein.

[0064] In an embodiment, in step 101, a sample containing microorganisms is obtained. The sample is, for example, a biological fluid sample, blood, sputum, urine, cerebrospinal fluid, etc. The sample may be divided so that a primary portion of the sample and a secondary portion of the sample are made. In step 102, the primary portion of the sample is processed to identify the types of microorganisms present in the sample. In one embodiment, when identifying the types of microorganisms, the microorganisms in the sample can be lysed. After lysis, nucleic acids are extracted from the lysed microorganisms. The nucleic acids are, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In another embodiment, the extracted nucleic acids are amplified using polymerase chain reaction (PCR). The polymerase chain reaction may be symmetric or asymmetric. TaqMan PCR can be performed after symmetric PCR. For example, TaqMan PCR includes the use of a TaqMan probe (oligonucleotide probe) that improves the specificity of quantitative PCR. The TaqMan probe binds or associates with a fluorophore at the 5' end of the oligonucleotide probe and a quencher at the 3' end of the oligonucleotide probe. The fluorophore is, for example, 6-carboxyfluorescein or tetrachlorofluorescein. The quencher is, for example, tetramethylrhodamine. The quencher molecule quenches the fluorescence emitted from the fluorophore excited by a light source. In the amplification process, Taq polymerase degrades the probe annealed to the nucleic acid related to the microorganism. After degradation, the fluorophore is released, increasing the distance from the quencher. Thereby, fluorescence occurs.

[0065] In an alternative embodiment, asymmetric PCR is performed, which can amplify nucleic acids related to microorganisms. Asymmetric PCR is followed by PCR melting curve analysis. The temperature at which 50% of the nucleic acid is denatured is identified as the melting temperature (Tm). Since the Tm for different microorganisms is different, effective identification of the types of microorganisms present in the sample is possible.

[0066] In step 103, the secondary portion of the sample is further divided into at least one first portion and at least one second portion. In step 104, at least one first portion of the sample is incubated with a plurality of antibacterial agents, and at least one second portion of the sample is incubated without antibacterial agents. Each portion incubated with and without antibacterial agents is of equal size. Thus, at least one second portion of the sample acts as a reference when measuring the antibacterial susceptibility of the microorganisms. In one embodiment, each of at least one first portion of the sample is incubated with a different antibacterial agent. In an alternative embodiment, the concentration of the antibacterial agent is varied for each sample. The microorganisms in the sample need to be grown / cultured in the shortest time possible to measure distinguishable nucleic acid differences in subsequent amplification reactions. For example, the microorganisms are cultured in the range of at least 1 - 3, 1 - 4, or 1 - 5 doubling cycles.

[0067] When the doubling cycle range is satisfied for at least one first and second portion of the sample, in step 105, the incubated sample is subjected to lysis. Based on the information obtained from step 102, only the portion incubated with the antibacterial agent related to the identified pathogen proceeds to the next treatment. The microorganisms in the sample can be lysed, for example, using an enzymatic lysis method or a chemical lysis method. In one embodiment, lysis of the microorganisms is performed only for at least one selected first portion of the sample. This is determined based on the type of microorganism determined in step 102. After lysis, in step 106, the nucleic acids related to the microorganisms are extracted. The nucleic acids are DNA or RNA.

[0068] In step 107, the nucleic acid is amplified using PCR. In one embodiment, the primers used in the amplification process are specific to the 16srRNA fraction if the extracted nucleic acid is RNA, and specific to the 16srDNA fraction if the extracted nucleic acid is DNA. Alternatively, the primers are specific to 18srRNA, 18srDNA, 23srRNA, 23srDNA. Amplification using PCR enables the determination of the cycle threshold associated with the amplified nucleic acid. The cycle threshold is the number of amplification cycles required for the PCR result to reach the fluorescence level at which it changes from "undetectable" to "detectable". In step 108, a first cycle threshold associated with the amplified nucleic acid from at least one first portion of the sample is determined. In step 109, a second cycle threshold associated with the amplified nucleic acid from at least one second portion of the sample is determined. In step 110, the difference between the first cycle threshold and the second cycle threshold is calculated. If the first cycle threshold is greater than the second cycle threshold, the antimicrobial susceptibility of the microorganism is low. In one embodiment, a second PCR melting analysis is performed to identify the type of microorganism and provide species information, and to confirm that the pathogen detected from the 102 portions has grown in the presence of the antimicrobial agent, and thus the antimicrobial susceptibility can be clearly correlated with the identity of the pathogen. In the example, steps 101, 102, 103, 104, 105, 106 are performed in sequence.

[0069] Figure 2 illustrates a method for measuring the antimicrobial susceptibility of a microorganism according to another embodiment. In step 201, a sample containing the microorganism is received. In step 202, at least one first portion of the sample is incubated with an antimicrobial agent, and at least one second portion of the sample is incubated without the antimicrobial agent. The microorganism in the sample needs to be grown / cultured in the shortest time possible to measure the distinguishable nucleic acid differences between both portions. For example, the microorganism is cultured in the range of at least 1 to 5 doubling cycles. For example, the number of doubling cycles is determined in the development phase and may be fixed in the actual workflow. When the doubling cycles are completed for at least one first and second portion of the sample, in step 203, the incubated sample is lysed. The microorganism in the sample can be lysed, for example, using an enzymatic lysis method or a chemical lysis method. After lysis, in step 204, the nucleic acid associated with the microorganism is extracted and amplified. The nucleic acid is DNA or RNA. In one embodiment, the primer used in the amplification process is specific to the 16srRNA fraction if the extracted nucleic acid is RNA, and specific to the 16srDNA fraction if the extracted nucleic acid is DNA. Alternatively, the primer is specific to 18srRNA, 18srDNA, 23srRNA, 23srDNA.

[0070] In step 205, a first cycle threshold associated with the amplified nucleic acid from at least one first portion of the sample to which an antibacterial agent has been added is determined. In step 206, a second cycle threshold associated with the amplified nucleic acid from at least one second portion of the sample to which no antibacterial agent has been added is determined. In step 207, the difference between the first cycle threshold and the second cycle threshold is calculated. If the first cycle threshold is greater than the second cycle threshold, the microorganism is sensitive to the antibacterial agent in the first portion, and thus the threshold cycle is smaller than that in the second portion where no antibacterial agent is present. In the absence of an antibacterial agent, the microorganism doubles every cycle, and thus the threshold cycle should become smaller as the amount of DNA increases. If the difference between the threshold cycles is negligible or small relative to the set threshold, the microorganism incubated with the antibacterial agent in the first portion is resistant to the antibacterial agent. In step 208, PCR melting analysis is performed to confirm the type of microorganism. Pathogen identification can be achieved using pathogen-specific probes with various Tm values, each labeled. Each probe binds to a unique pathogen. The pathogen is identified by the combination of color and Tm. In the example, steps 201, 202, 203, 204, 205, 206, 207, 208 are executed in sequence.

[0071] Figure 3 illustrates a method for measuring the antimicrobial susceptibility of microorganisms according to another embodiment. In step 301, a sample containing microorganisms is received. In step 302, at least one first portion of the sample is incubated with an antimicrobial agent, and at least one second portion of the sample is incubated without an antimicrobial agent. The microorganisms in the sample need to be grown / cultured or incubated for the shortest time capable of measuring distinguishable nucleic acid differences. For example, the microorganisms are cultured or incubated within a range of at least 1 to 5 doubling cycles. Antimicrobial agents that are cell wall synthesis inhibitors (β-lactams) and cell membrane inhibitors (colistin) affect the integrity of the cell wall of the microorganisms. This facilitates the measurement of antimicrobial susceptibility. When the doubling cycle range is satisfied for at least one first and second portion of the sample, in step 303, an intercalating dye is introduced into at least one first portion of the sample and at least one second portion of the sample. After introducing the intercalating dye, the sample is incubated for at least 5 minutes (e.g., about 5 minutes, about 6 minutes, 5 to 10 minutes). For example, the intercalating dye is ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, etc. The intercalating dye is known to penetrate dead microorganisms and cross-link with the chromosomal nucleic acids of dead microorganisms. The intercalating dye does not act on living cells.

[0072] In step 304, the intercalating dye and the sample are exposed to visible light for a certain period using a light source. In the embodiment, the light source includes UV light. In the embodiment, the light source is applied for a duration sufficient for the intercalation of the dye and the nucleic acid to become irreversible. The light is intense light, and the light source emits light having a wavelength in the range of 465 nm to 475 nm. For example, the light source is a PMA-lite-LED photoreactor. In one embodiment, the sample is exposed to light for 15 to 20 minutes. The exposure is advantageous because it converts unbound dye into a compound that no longer binds to nucleic acids. In another embodiment, the sample is pelleted to collect the microorganisms and remove unbound dye.

[0073] In step 305, the sample is lysed. The microorganisms in the sample can be lysed, for example, using an enzymatic lysis method or a chemical lysis method. After lysis, in step 306, the nucleic acids associated with the microorganisms are extracted and amplified. Quantitative PCR is performed. In one embodiment, the primers used in the amplification process are specific to the 16srRNA fraction if the extracted nucleic acid is RNA, and specific to the 16srDNA fraction if the extracted nucleic acid is DNA. Alternatively, the primers are specific to 18srRNA, 18srDNA, 23srRNA, or 23srDNA.

[0074] In step 307, a first cycle threshold associated with the amplified nucleic acid from at least one first portion of the sample is determined. In step 308, a second cycle threshold associated with the amplified nucleic acid from at least one second portion of the sample is determined. In step 309, the difference between the first cycle threshold and the second cycle threshold is calculated. If the first cycle threshold is greater than the second cycle threshold, the antimicrobial susceptibility of the microorganism is low. The microorganisms exposed to the antimicrobial agent will either die (if susceptible) or continue to grow (if resistant). Intercalating dyes can penetrate the cell walls of susceptible microorganisms with damaged cell walls and bind to chromosomal DNA, making them PCR-incompatible. On the other hand, resistant microorganisms do not allow the penetration of intercalating dyes through their cell walls and thus continue to divide.

[0075] Normal human samples contain DNA from dead / dying cells, which distorts the results and makes it difficult to distinguish between susceptible and resistant microorganisms in the sample. Intercalating dyes are advantageous in that they can also bind to DNA from such dead / dying cells, enabling the detection of DNA only from living microorganisms by PCR. Furthermore, this makes it possible to make the difference between the cycle thresholds associated with at least one first portion and at least one second portion of the sample larger and faster.

[0076] Figure 4 provides a graphical representation 401, 402 of the difference in cycle threshold between a sample incubated with only an antibacterial agent and a sample incubated with a combination of an antibacterial agent and an intercalating dye. In this example, the antibacterial agent used was ampicillin (ampicillin / Amp), and the intercalating dye was ethidium monoazide (EMA) (EMA dye). Graph 401 specifically shows the difference in cycle threshold related to a first portion of the sample incubated with ampicillin (With Amp) and a second portion of the sample incubated without ampicillin (No Amp). Graph 402 specifically shows the difference in cycle threshold related to a first portion of the sample incubated with ampicillin and EMA (With Amp+EMA) and a second portion of the sample incubated without ampicillin or EMA (No Amp+EMA). It is observed that including EMA in the incubation process improves the difference in cycle threshold between the first and second portions of the sample. EMA penetrates the cell membrane of the microorganism treated with ampicillin, binds to the DNA, and renders it PCR-incompatible. On the other hand, the microorganism not treated with ampicillin remains intact, and the DNA is PCR-amplifiable.

[0077] In an example, the present disclosure includes a method for measuring the antibacterial susceptibility of microorganisms in a sample. The method comprises receiving a sample containing microorganisms, incubating at least one first portion of the sample with at least one antibacterial agent and at least one second portion of the sample without the antibacterial agent, contacting the sample, e.g., the at least one first portion and / or the at least one second portion, with one or more intercalating dyes, extracting nucleic acids related to the microorganisms present in the sample from the at least one first portion of the sample and the at least one second portion of the sample, amplifying the nucleic acid extracted from the at least one first portion and the at least one second portion of the sample; obtaining antimicrobial susceptibility information related to the microorganism from the nucleic acid amplified from the at least one first portion and the at least one second portion of the sample. In an embodiment, the contact with the intercalating dye is under conditions sufficient for the intercalating dye to form a complex with the nucleic acid in the sample or in the microorganism.

[0078] Referring to FIG. 6, an EMA workflow suitable for use in accordance with the present disclosure is shown. In this example, the intercalating dye EMA can be used to look for early changes in the cycle threshold difference in cells exposed to a beta-lactam antimicrobial such as ampicillin. FIG. 6 presents EMA-ethidium monobromide azide used to improve the differentiation between susceptible and resistant strains to beta-lactam antimicrobials. The use of EMA results in improved early [Delta]Ct values (cycle threshold) between with-antimicrobial and no-antimicrobial aliquot of the beta-lactam antimicrobial.

[0079] Referring to FIG. 5, a block diagram of a system 1000 in which an embodiment is implemented is shown. For example, system 1000 is configured to measure the antimicrobial susceptibility of a microorganism and execute the processes described herein. In FIG. 5, system 1000 includes a processing unit 1010, a memory 1020, a storage unit 1030, an input unit 1040, a bus 1060, an output unit 1050, and a network interface 1070.

[0080] When used herein, the processing unit 1010 means various computer calculation circuits. For example, without limitation and by way of enumeration, a microprocessor, a microcontroller, a complex instruction set computing microprocessor, a reduced instruction set computing microprocessor, a very long instruction word microprocessor, an explicitly parallel instruction computing microprocessor, a graphics processor, a digital signal processor, or other various processing circuits. The processing unit 1010 may also include a general-purpose or programmable logic device or array, an application-specific integrated circuit, an embedded controller such as a single-chip computer, etc.

[0081] The memory 1020 can be a volatile memory and a non-volatile memory. The memory 1020 is connected to communicate with the processing unit 1010. The processing unit 1010 executes instructions and / or code stored in the memory 1020. Various computer-readable storage media are stored in the memory 1020 and accessed from the memory 020. The memory 1020 includes a read-only memory, a random access memory, an erasable programmable read-only memory, an electrically erasable programmable read-only memory, a hard drive, and appropriate elements for storing data and machine-readable instructions, such as a removable media drive that handles compact discs, digital video discs, floppy disks, magnetic tape cartridges, memory cards, etc. In this embodiment, the memory 1020 includes a sensitivity module 1100 stored in the form of machine-readable instructions in any of the above storage media, communicates with the processor 1010, and is executed by the processor 1010. In the embodiment, the method steps executed by the processor 1010 to achieve the above functions are described in detail in FIG. 1, FIG. 2, and / or FIG. 3.

[0082] The storage unit 1030 is a non - volatile storage medium that stores the medical database 1120 and other information. The medical database 1120 is a repository of patient data, including blood parameters, maintained by a healthcare service provider. The input unit 1040 includes input means such as a keypad, a touch - sensitive display, a camera (such as a camera that receives gesture - based input) that can receive input signals. The bus 106 functions as an interconnection between the processing unit 1010, the memory 1020, the storage unit 1030, the input unit 1040, the output unit 1050, and the network interface 1070.

[0083] Those of ordinary skill in the art will understand that the hardware shown in FIG. 5 can be varied for a specific implementation. For example, other peripheral devices such as an optical disk drive, a local area network (LAN) / wide area network (WAN) / wireless (e.g., Wi - Fi) adapter, a graphics adapter, a disk controller, an input / output (I / O) adapter can also be used in addition to or instead of the illustrated hardware. The illustrated example is provided for illustrative purposes only and is not meant to imply architectural limitations regarding the present disclosure.

[0084] System 1000 according to an embodiment of the present disclosure includes an operating system that uses a graphical user interface. This operating system enables multiple display windows to be presented simultaneously in the graphical user interface, and each display window provides an interface to a separate application or a separate instance of one application. The cursor within the graphical user interface is manipulated by the user via a pointing device. When the position of the cursor is changed and / or an event such as a mouse button click occurs, a desired response is initiated. Any of various commercially available operating systems, such as a version of Microsoft Windows (trademark) of Microsoft Corporation in Redmond, Washington, can be used if appropriately modified. The operating system is modified or created according to the present disclosure to be described.

[0085] Also provided is a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the methods presented herein. In an embodiment, the present disclosure includes a system or a product such as a component thereof that includes a non-transitory computer-readable medium containing encoded instructions that are configured to cause one or more processors to perform a method including measuring the antimicrobial susceptibility of a microorganism according to the present disclosure. In an embodiment, the present disclosure includes a non-transitory computer-readable medium containing encoded instructions that are configured to cause one or more processors to perform a method including measuring the antimicrobial susceptibility of a microorganism according to the present disclosure.

[0086] In an embodiment, the present disclosure includes a product such as a system or a component thereof that includes a non-transitory computer-readable medium containing encoded instructions, the instructions being configured to cause one or more processors to perform a method including measuring the antimicrobial susceptibility of a microorganism in a sample, the method including a non-transitory computer-readable medium containing encoded instructions, the instructions being configured to cause one or more processors to perform a method (such as method 100, method 200, or method 300) for measuring the antimicrobial susceptibility of a microorganism.

[0087] In an embodiment, the present disclosure includes a product such as a system or a component thereof that includes a non-transitory computer-readable medium containing encoded instructions, the instructions being configured to cause one or more processors to perform a method for measuring the antimicrobial susceptibility of a microorganism in a sample. The method includes incubating at least one first portion of a sample containing the microorganism with at least one antimicrobial agent and at least one second portion of the sample without the antimicrobial agent, extracting nucleic acid associated with the microorganism present in the sample from the at least one first portion of the sample and the at least one second portion of the sample, amplifying the nucleic acid extracted from the at least one first portion of the sample and the at least one second portion of the sample, obtaining antimicrobial susceptibility information associated with the microorganism from the nucleic acid amplified from the at least one first portion of the sample and the at least one second portion of the sample. In an embodiment, the method further includes contacting the nucleic acid with one or more intercalating dyes before amplifying the nucleic acid or before extracting the nucleic acid. In an embodiment, the method further includes contacting the nucleic acid with one or more intercalating dyes before extracting the nucleic acid.

[0088] In an embodiment, the present disclosure includes a non-transitory computer-readable medium including encoded instructions that are configured to cause one or more processors to perform a method for measuring the antimicrobial susceptibility of microorganisms in a sample. The method includes incubating at least one first portion of a sample containing microorganisms with at least one antimicrobial agent and incubating at least one second portion of the sample without an antimicrobial agent, extracting nucleic acid associated with the microorganisms present in the sample from the at least one first portion of the sample and the at least one second portion of the sample, contacting the nucleic acid with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex, amplifying the nucleic acid extracted from the at least one first portion of the sample and the at least one second portion of the sample, obtaining antimicrobial susceptibility information associated with the microorganisms from the amplified nucleic acid from the at least one first portion of the sample and the at least one second portion of the sample. In an embodiment, contacting the nucleic acid with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex is performed prior to extraction. In an embodiment, contacting the nucleic acid with one or more intercalating dyes under conditions for forming a nucleic acid / intercalating dye complex is performed prior to amplification.

[0089] Referring to FIG. 7, a method for measuring the antimicrobial susceptibility of microorganisms according to another embodiment is shown. Here, a culturing step is shown, followed by extraction and amplification according to the present disclosure. AST is shown disassembled, and the pathogen ID is disassembled using Tm information and color-coded identification.

[0090] Referring to FIG. 8, a method for measuring the antimicrobial susceptibility of microorganisms according to another embodiment is shown. Here, the duration of the workflow is shortened by obtaining bacterial identification information in advance according to FIG. 8.

[0091] FIG. 9 shows a method for measuring the antimicrobial susceptibility of microorganisms according to another embodiment.

[0092] FIG. 15 shows the antimicrobial incubation time according to an embodiment of the present disclosure. FIG. 15 shows that an incubation time of about 30 minutes is sufficient to reveal antimicrobial susceptibility in E. coli.

[0093] -Treatment- In an embodiment, the present disclosure includes a method of treating a subject in need of treatment by determining that the subject is a subject in need of treatment (e.g., having a pathogenic bacterial infection or sepsis) and subsequently treating the subject. In an embodiment, the methods, systems, and kits described herein are suitable for use by a healthcare provider in a point-of-care environment. For example, the methods of the present disclosure are applicable as a diagnostic test to diagnose a patient or subject as being positive for a pathogenic bacterial infection or sepsis, and subsequently administering a therapeutic agent or compound in a therapeutically effective amount to a subject in need thereof. For example, a physician can administer a therapeutically effective amount of a drug, therapeutic agent, or biologic suitable for treating a bacterial infection or disease state after a diagnosis according to the present disclosure. One non-limiting example of an agent suitable for treating a bacterial infection includes one or more antimicrobial agents according to the present disclosure. The therapeutically effective amount of the agent provided is determined by the physician based on the response of the subject, comorbidities, etc. In an embodiment, the therapeutic compound is administered in an effective amount to a subject with a disease state. In an embodiment, a compound such as a therapeutically effective amount of an antimicrobial agent is administered to a subject in need thereof in an amount sufficient to change the subject's natural or presenting state.

[0094] In certain embodiments, the disease treated according to the methods described herein is a disease caused by a bacterial infection. Non-limiting examples of bacteria that cause the disease include Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jirovecii, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis, and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and Treponema pallidum.

[0095] In certain embodiments, a composition such as an antibacterial agent is administered to a patient diagnosed with a disease caused by a bacterial infection. For example, the patient is infected with Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Bordetella bronchiseptica, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jirovecii, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis, and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and / or Treponema pallidum.

[0096] In certain embodiments, the disease treated according to the methods described herein is a disease caused by a bacterial infection. Non-limiting examples of bacteria that cause the disease include Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jirovecii, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis, and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and Treponema pallidum.

[0097] In certain embodiments, the composition is administered to a patient diagnosed with a disease caused by a bacterial infection. For example, the patient is infected with Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Bordetella bronchiseptica, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jirovecii, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis, and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and / or Treponema pallidum.

[0098] -Exemplification of numbered embodiments- ·Example 1 A method for measuring the antimicrobial susceptibility of microorganisms in a sample, the method comprising: Receiving a sample containing microorganisms; Incubating at least one first portion of the sample with at least one antimicrobial agent and at least one second portion of the sample without an antimicrobial agent; Extracting nucleic acid associated with the microorganisms present in the sample from the at least one first portion of the sample and the at least one second portion of the sample; Amplifying the nucleic acid extracted from the at least one first portion of the sample and the at least one second portion of the sample; Obtaining antimicrobial susceptibility information associated with the microorganisms from the amplified nucleic acid from the at least one first portion of the sample and the at least one second portion of the sample.

[0099] ·Example 2 The method of Example 1, further comprising identifying the type of microorganism present in the sample.

[0100] ·Example 3 Lysing the microorganisms present in the sample; Extracting nucleic acid associated with the microorganisms; Amplifying the extracted nucleic acid using polymerase chain reaction; further comprising identifying the type of the microorganism present in the sample based on the amplified nucleic acid, The method according to Example 1 or 2, wherein polymerase chain reaction-based melting analysis is applied to the amplified nucleic acid.

[0101] · Example 4 lysing the microorganism present in the sample, extracting nucleic acid related to the microorganism, amplifying the extracted nucleic acid using polymerase chain reaction, further comprising identifying the type of the microorganism present in the sample based on the amplified nucleic acid, the nucleic acid is amplified using a probe that binds to a fluorophore, the probe is complementary to a target nucleic acid sequence related to the microorganism, The method according to any one of Examples 1 to 3, wherein the type of the microorganism is identified based on the color emitted from the fluorophore and the Tm of the target having the binding probe. The probe may further have various Tm values.

[0102] · Example 5 incubating the at least one first portion of the sample and the at least one second portion of the sample with an intercalating dye, The method according to any one of Examples 1 to 4, further comprising exposing the incubated sample to visible light using a light source.

[0103] · Example 6 The method according to any one of Examples 1 to 5, wherein the intercalating dye is at least one of ethidium monoazide, ethidium monoazide bromide, and propidium monoazide.

[0104] · Example 7 obtaining the antimicrobial susceptibility information related to the microorganism from the amplified nucleic acid, Determining a first cycle threshold associated with the nucleic acid amplified from the at least one first portion of the sample; Determining a second cycle threshold associated with the nucleic acid amplified from the at least one second portion of the sample; Calculating a difference between the first cycle threshold and the second cycle threshold; Determining the antimicrobial susceptibility information based on the cycle threshold associated with the amplified nucleic acid based on the difference between the first cycle threshold and the second cycle threshold, wherein when the cycle threshold is high, the antimicrobial susceptibility of the microorganism is low, according to the method of any one of Examples 1 to 6.

[0105] · Example 9 Further comprising determining the identity of the microorganism after obtaining the antimicrobial susceptibility information associated with the microorganism, according to the method of any one of Examples 1 to 8.

[0106] · Example 10 wherein the at least one first portion of the sample is incubated with more than one type of antimicrobial agent, according to the method of any one of Examples 1 to 9.

[0107] · Example 11 wherein the at least one first portion of the sample is incubated with more than one type of antimicrobial agent, and the antimicrobial agents are present at various concentrations, according to the method of any one of Examples 1 to 9.

[0108] · Example 12 A kit for measuring the antimicrobial susceptibility of a microorganism in a sample, comprising the components of Examples 1 to 11.

[0109] · Example 13 A kit for measuring the antimicrobial susceptibility of a microorganism in a sample, comprising one or more antimicrobial agents, a growth medium for the microorganism, reagents related to nucleic acid extraction, reagents related to nucleic acid amplification, A kit comprising polymerase chain reaction-based melting analysis software.

[0110] · Example 14 The kit of Example 12 or 13, further comprising one or more intercalating dyes.

[0111] · Example 15 A method of treating a subject in need thereof, comprising measuring the antimicrobial susceptibility of a microorganism according to an embodiment of the present disclosure and subsequently treating the subject in need thereof. In an example, treating comprises administering to the subject a therapeutically effective amount of a composition, such as one or more antimicrobial agents.

[0112] · Example 16 A non-transitory computer-readable medium comprising encoded instructions, the instructions being configured to cause one or more processors to perform a method of measuring the antimicrobial susceptibility of a microorganism in a sample, the method comprising: incubating at least one first portion of a sample containing a microorganism with at least one antimicrobial agent and at least one second portion of the sample without an antimicrobial agent; extracting nucleic acid associated with the microorganism present in the sample from the at least one first portion of the sample and the at least one second portion of the sample; contacting the nucleic acid with one or more intercalating dyes under conditions for forming a nucleic acid / intercalating dye complex; amplifying the nucleic acid extracted from the at least one first portion of the sample and the at least one second portion of the sample; obtaining antimicrobial susceptibility information associated with the microorganism from the amplified nucleic acid from the at least one first portion of the sample and the at least one second portion of the sample.

[0113] · Example 17 A method of treating a subject in need thereof, comprising measuring the antimicrobial susceptibility of microorganisms in a biological sample from the subject, receiving the sample containing the microorganisms, incubating at least one first portion of the sample with at least one antimicrobial agent and at least one second portion of the sample without an antimicrobial agent, extracting nucleic acid associated with the microorganisms present in the sample from the at least one first portion of the sample and the at least one second portion of the sample, amplifying the nucleic acid extracted from the at least one first portion of the sample and the at least one second portion of the sample, obtaining antimicrobial susceptibility information associated with the microorganisms from the amplified nucleic acid from the at least one first portion of the sample and the at least one second portion of the sample, and then treating the subject in need thereof. A method comprising. In an embodiment, treating comprises administering to the subject a therapeutically effective amount of a composition. In an embodiment, the disease is characterized as a bacterial disease or the like.

[0114] "Treatment" or "treating" with respect to a disease or medical condition, a symptom of a disease or medical condition, or a risk (or susceptibility) of a disease or medical condition, includes the application or administration of a compound to a subject for the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, removing, modifying, improving, reducing worsening, making better, effecting a remission, or acting. "Treating" refers to any sign of success in the treatment or improvement of an injury, pathology or medical condition, for example, reduction; remission; reduction in the rate of worsening; reduction in the severity of a disease; stabilization or reduction of symptoms or making an injury, pathology or medical condition more tolerable to the subject; slowing the rate of degeneration or decline; reducing debilitation at the end point of degeneration; or improving the physical or mental health of the subject, including objective or subjective parameters. In an example, "treating" means reducing or improving the progression, severity, and / or duration of a bacterial infection, or improving one or more symptoms of a bacterial infection by the administration (application) of one or more treatment methods (for example, one or more therapeutic agents). In a particular example, "treatment" means improving a measurable physical parameter of a bacterial infection. In an example, "treating" means reducing or improving the progression, severity, and / or duration of a bacterial infection, or improving one or more symptoms of a bacterial infection by the administration (application) of one or more treatment methods (for example, one or more therapeutic agents). In a particular example, "treatment" means improving a measurable physical parameter of a bacterial infection. In an example, "treating" means reducing or improving the progression, severity, and / or duration of sepsis, or improving one or more symptoms of sepsis by the administration (application) of one or more treatment methods (for example, one or more therapeutic agents). In a particular example, "treatment" means improving a measurable physical parameter of sepsis. In an example, "treating" changes the natural or presenting state of the subject.

[0115] As used herein, "therapeutically effective amount" means an amount of a compound that, when administered to a subject for treating or preventing a particular disorder, disease or medical condition, is sufficient to effect such treatment or prevention of the disorder, disease or medical condition. Dosage amount and therapeutically effective amount may vary according to various factors, including, for example, the activity of the particular agent used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and in the case of combination drugs, the nature of the combination, the effect the compound is believed to have on the subject and the properties of the compound (e.g., bioavailability, stability, potency, toxicity, etc.), and one or more specific disorders the subject is suffering from. Further, the therapeutically effective amount for intravenous administration may depend on the blood parameters of the subject, such as lipid profile, insulin level, blood glucose or liver metabolism. The therapeutically effective amount may also vary according to the disease state, organ function, or severity of underlying disease or complication. Such appropriate dosages are determined using available assays. When one or more compounds or therapeutic agents are administered to a human, a physician, for example, initially prescribes a relatively low dosage and then increases the dosage until an appropriate response is obtained.

[0116] -Specific examples- Figure 9 shows the experimental process flow outlining the goals of the experiments described below, the bacterial tests, and the antibacterial agents tested according to the present disclosure.

[0117] ·Example 1: Detection of bacterial susceptibility to ampicillin An overnight Escherichia coli culture sensitive to ampicillin was subcultured for an additional 2 - 3 hours until the logarithmic phase. The cells during this culture were quantified using OD600nm. The culture was diluted to 1×106 cells / mL and 10 mL was dispensed into two 50 mL Erlenmeyer flasks. 100 μg / mL of ampicillin was added to one of the Erlenmeyer flasks. The two flasks, with and without this antibacterial drug, were incubated at 37°C and two 50 μL aliquots were taken periodically (0, 30, 40, 60 minutes) from each flask. Nucleic acid (NA) was extracted from all sample aliquots using the VERSANT® brand Sample Preparation 1.0 Reagents Kit. Next, the NA samples were processed by PCR and RT - PCR (reverse transcription - PCR) using 16S rDNA - specific primers (each DNA extraction sample was divided into two replicates for PCR). As seen in Table - 1 of Figure 10, RNA levels were detectable regarding ampicillin sensitivity at 60 minutes, but DNA levels did not reach a point where the samples with the antibacterial drug could be distinguished from the samples without the antibacterial drug even after 60 minutes of incubation (Table - 2 of Figure 10). In this test, all RNA samples were treated with DNase at 37°C for 60 minutes before RT - PCR. In subsequent tests, it was shown that treatment for up to 5 minutes was sufficient to remove DNA from the samples.

[0118] ·Example 2: Use of EMA to Accelerate Detection of Bacterial Sensitivity to Ampicillin

[0119] An overnight culture of Escherichia coli sensitive to ampicillin was subcultured for an additional 2 - 3 hours until the logarithmic phase. The cells during this culture were quantified using OD600nm. The culture was diluted to 1×106 cells / mL, and 10 mL was dispensed into two 50 - mL Erlenmeyer flasks. 100 μg / mL of ampicillin was added to one of the Erlenmeyer flasks. The two flasks, one with and one without this antibacterial agent, were incubated at 37°C, and four 50 - μL aliquots were taken periodically (0, 20, 30 minutes) from each flask. 25 μL of 2 μM EMA (ethidium monoazide made in 20% DMSO) was added to two of the four aliquots from each sampling. After EMA addition, these samples were vortex - mixed, incubated in the dark for 5 minutes, transferred to 1.5 - mL Eppendorf tubes, and exposed to blue LED light for 15 minutes so that a photodegradation reaction occurred. DNA was extracted from all sample aliquots using Versant® Sample Preparation Reagents. Next, the DNA samples were processed by PCR using 16S rDNA - specific primers (each DNA extraction sample was divided into two replicates). As seen in Table - 1 of Figure 14, the samples exposed to EMA showed a large difference in Ct between ampicillin - exposed and non - exposed within 30 minutes. The same samples without exposure to EMA + blue light showed no difference between ampicillin - exposed and non - exposed samples (Table - 2 of Figure 14).

[0120] - Additional Example - The same experiment as above was conducted.

[0121] Figure 11 shows data regarding Escherichia coli and ciprofloxacin obtained according to an embodiment of the present disclosure.

[0122] Figure 12 shows data regarding Escherichia coli and ciprofloxacin obtained according to an embodiment of the present disclosure.

[0123] Figure 13 shows data regarding Pseudomonas using ciprofloxacin and chloramphenicol obtained according to an embodiment of the present disclosure.

[0124] The above embodiments are provided for illustrative purposes only and should not be construed as limiting the invention disclosed herein. Although the invention has been described with reference to various embodiments, it is understood that the terms used herein are not limiting terms but terms of description and illustration. Further, although the invention has been described herein with reference to specific means, materials, and embodiments, the invention is not intended to be limited to the details disclosed herein. Rather, the invention extends to all functionally equivalent structures, methods, and uses that fall within the scope of the claims. Those of ordinary skill in the art who benefit from the teachings herein can make numerous modifications and variations in each aspect of the invention without departing from the scope and spirit of the invention.

Claims

1. A method for measuring the antimicrobial susceptibility of microorganisms in a sample, comprising: receiving a sample containing microorganisms; incubating at least one first portion of the sample with at least one antimicrobial agent and at least one second portion of the sample without an antimicrobial agent; extracting nucleic acids related to the microorganisms present in the sample from the at least one first portion and the at least one second portion of the sample; amplifying the nucleic acids extracted from the at least one first portion and the at least one second portion of the sample; obtaining antimicrobial susceptibility information related to the microorganisms from the amplified nucleic acids from the at least one first portion and the at least one second portion of the sample.

2. The method according to claim 1, further comprising identifying the type of microorganism present in the sample.

3. lysing the microorganisms present in the sample; extracting nucleic acids related to the microorganisms; amplifying the extracted nucleic acids using polymerase chain reaction; further comprising identifying the type of microorganism present in the sample based on the amplified nucleic acids; The method according to claim 2, wherein polymerase chain reaction-based melting analysis is applied to the amplified nucleic acids.

4. lysing the microorganisms present in the sample; extracting nucleic acids related to the microorganisms; amplifying the extracted nucleic acids using polymerase chain reaction; further comprising identifying the type of microorganism present in the sample based on the amplified nucleic acids; The nucleic acid is amplified using one or more probes that bind to a fluorophore, The one or more probes are complementary to a target nucleic acid sequence associated with the microorganism, The method according to claim 2, wherein the type of the microorganism is identified based on the color emitted from the fluorophore and the Tm of the target having the binding probe. **Claim 5** The method according to claim 4, wherein the one or more probes have various Tm values. **Claim 6** Incubating the at least one first portion of the sample and the at least one second portion of the sample with an intercalating dye, Exposing the incubated sample to visible light using a light source, the method according to claim 1, further comprising. **Claim 7** The method according to claim 6, wherein the intercalating dye is at least one of ethidium monoazide, ethidium monoazide bromide, propidium monoazide, and combinations thereof. **Claim 8** Obtaining the antimicrobial susceptibility information related to the microorganism from the amplified nucleic acid is, Determining a first cycle threshold related to the nucleic acid amplified from the at least one first portion of the sample, Determining a second cycle threshold related to the nucleic acid amplified from the at least one second portion of the sample, Calculating a difference between the first cycle threshold and the second cycle threshold, Determining the antimicrobial susceptibility information based on a cycle threshold related to the amplified nucleic acid based on the difference between the first cycle threshold and the second cycle threshold, including, When the cycle threshold is high, the antimicrobial susceptibility of the microorganism is low, the method according to claim 1. **Claim 9** The method according to claim 1, further comprising determining the identity of the microorganism after obtaining the antimicrobial susceptibility information related to the microorganism.

10. The method according to claim 1, wherein the at least one first portion of the sample is incubated with more than one type of antimicrobial agent.

11. The method according to claim 1, wherein the at least one first portion of the sample is incubated with more than one type of antimicrobial agent, and the antimicrobial agent is present at various concentrations.

12. A kit for measuring the antimicrobial susceptibility of microorganisms in a sample, comprising: one or more antimicrobial agents; a growth medium for microorganisms; reagents related to nucleic acid extraction; reagents related to nucleic acid amplification; a polymerase chain reaction-based melting analysis software.

13. The kit according to claim 12, further comprising one or more intercalating dyes.

14. A product such as a system or a component thereof comprising a non-transitory computer-readable medium encoding instructions, wherein the instructions are configured to cause one or more processors to execute a method for measuring the antimicrobial susceptibility of microorganisms in a sample, the method comprising: incubating at least one first portion of a sample containing microorganisms with at least one antimicrobial agent and at least one second portion of the sample without an antimicrobial agent; extracting nucleic acids related to the microorganisms present in the sample from the at least one first portion and the at least one second portion of the sample; amplifying the nucleic acids extracted from the at least one first portion and the at least one second portion of the sample; Obtaining antibacterial susceptibility information related to the microorganism from the at least one first portion and the nucleic acid amplified from the at least one second portion of the sample, a product comprising the same. **Claim 15** A non-transitory computer-readable medium including encoded instructions, wherein the instructions are configured to cause one or more processors to execute a method for measuring the antibacterial susceptibility of a microorganism in a sample, the method comprising: Incubating at least one first portion of a sample containing a microorganism with at least one antibacterial agent and at least one second portion of the sample without an antibacterial agent; Extracting nucleic acid related to the microorganism present in the sample from the at least one first portion and the at least one second portion of the sample; Contacting the nucleic acid with one or more intercalating dyes under conditions for forming a nucleic acid / intercalating dye complex; Amplifying the nucleic acid extracted from the at least one first portion and the at least one second portion of the sample; Obtaining antibacterial susceptibility information related to the microorganism from the nucleic acid amplified from the at least one first portion and the at least one second portion of the sample, a non-transitory computer-readable medium including the same. **Claim 16** A non-transitory computer-readable medium including encoded instructions, wherein the instructions are configured to cause one or more processors to execute a method for measuring the antibacterial susceptibility of a microorganism in a sample, the method comprising: Incubating at least one first portion of a sample containing a microorganism with at least one antibacterial agent and at least one second portion of the sample without an antibacterial agent; Contacting the sample or the portion thereof with one or more intercalating dyes; Extracting nucleic acids related to the microorganisms present in the sample from the at least one first portion of the sample and the at least one second portion of the sample; Amplifying the nucleic acids extracted from the at least one first portion of the sample and the at least one second portion of the sample; Obtaining antimicrobial susceptibility information related to the microorganisms from the nucleic acids amplified from the at least one first portion of the sample and the at least one second portion of the sample, a non-transitory computer-readable medium comprising the same.

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