JPWO2020037031A5 - - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62/719,290, filed August 17, 2018, which is incorporated herein by reference in its entirety.
The present disclosure generally relates to detection tests, including compositions, methods, systems, and/or kits, for determining the susceptibility of microorganisms in a sample to antibiotics. Certain embodiments of the present disclosure relate to detection tests including compositions, methods, systems, and/or kits for determining the minimum inhibitory concentration of antimicrobial agents.

Microbial infections affect millions of people each year and are associated with millions of deaths per year. Accurate diagnosis of pathogen species often requires several days to obtain, even in state-of-the-art clinical microbiology laboratories. Furthermore, patients who receive incorrect therapy early have lower survival rates than patients who are treated with optimal therapy early in the course of their disease. The rapidity of pathogen diagnosis in patients with microbial infections can have an important impact on prognosis. The current method for detecting microbial infections in blood is to culture the blood in a hospital or commercial clinical microbiology laboratory. Liquid culture can allow detection of the presence of certain growing organisms in the fluid within 4 to 30 hours. This assay is not quantitative, and without knowledge of the pathogen types and their specific antibiotic sensitivities, only suboptimal broad-spectrum antibiotics can be administered at this time. To identify the specific type of pathogen and to perform sensitivity tests to determine their response to various potential antibiotic therapies, pathogens grown in liquid media are then grown in other growth media. (e.g. agar plate). The total time for complete diagnosis and sensitivity testing is typically 3-7 days, and empiric antibiotic treatment based on clinical symptoms is often performed well before antibiotic sensitivity results are obtained. Blood cultures are initiated within 1-3 hours of being initially drawn from the patient.

Many patients with microbial infections exhibit rapid deterioration within the first few hours of infection. Therefore, rapid and reliable diagnostic and treatment methods are essential for effective patient care. Unfortunately, current antimicrobial susceptibility testing techniques generally require prior isolation of microorganisms per culture (e.g., about 12 to about 48 hours), followed by an additional about 6 to about 24 hours. processing. For example, a definitive diagnosis of the type of infection traditionally involves seeding a blood culture, incubation for 16-24 hours, plating the offending microorganism on solid media, another incubation period, and a final diagnosis after 1-2 days. Requires microbiological analysis with identification. Even with immediate aggressive treatment, some patients develop a multiple organ dysfunction syndrome and eventually die.
Every hour lost before the correct treatment is administered can make a critical difference in patient outcome. Therefore, it is important for physicians to quickly determine whether an infection is present and, if so, which antibiotics may be effective for treatment.

Compositions, methods, systems, and/or kits for measuring microbial viability in a sample are described herein.
Some embodiments provided herein relate to methods of assessing microbial growth in a sample. In some embodiments, the method includes the steps of: providing a sample containing the microorganism; separating the sample containing the microorganism into one or more portions of the sample containing the microorganism; forming one or more populations of droplets, contacting one or more portions of the sample with an antimicrobial agent either before or after forming one or more populations of microdroplets; and measuring the microbial viability of the microorganisms encapsulated within the microdroplets, thereby determining the susceptibility of the microorganisms to antimicrobial agents. In some embodiments, one or more populations of microdroplets are formed before or after separating the sample into one or more portions. In some embodiments, each of the one or more portions of the sample is contacted with a different concentration of an antimicrobial agent. In some embodiments, the step of measuring microbial viability comprises measuring a separate ( obtaining a measure of microbial viability from an individual, discrete subset, and measuring microbial viability from a second population of microdroplets from the first portion of the sample measured at a second time point; including obtaining measurements of microbial viability from separate subsets of droplets. In some embodiments, the step of measuring microbial viability comprises, with respect to the plurality of subsets of microdroplets measured at the first and second time points, The method further includes comparing the microbial viability measurements from the subset to microbial viability measurements from separate subsets of microdroplets measured at a second time point. In some embodiments, the step of measuring comprises measuring microbial viability from separate subsets of microdroplets in additional populations of microdroplets from the first portion of the sample, measured at additional time points. Further including obtaining. In some embodiments, the average of the microbial viability measurements from the plurality of separate subsets of microdroplets measured at the first time point is the average of the microbial viability measurements from the plurality of separate subsets of microdroplets measured at the second time point. compared to the average of microbial survival measurements from a subset of . In some embodiments, the microbial viability measurements taken at the first and second time points are not assigned to separate subsets of microdroplets. In some embodiments, one or more discrete subsets of microdroplets from the first population are absent from the second population, and one or more discrete subsets of microdroplets from the second population are absent from the second population. There are no separate subsets in the first population. In some embodiments, the population of microdroplets is collected for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, Incubate for any one or more of 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours. be done. In some embodiments, the microdroplets are applied within 1 second, 30 seconds, 1 minute, 15 minutes, 30 minutes, 1 hour, or 2 hours of contacting one or more portions of the sample with the antimicrobial agent. It is formed. In some embodiments, measuring microbial viability from a separate subset of microdroplets in the first population of microdroplets from the first portion of the sample measured at the first time point. obtaining a measurement of microbial viability from a separate subset of microdroplets in a second population of microdroplets from the first portion of the sample, measured at a second time point; further comprising obtaining a measurement of. In some embodiments, the step of measuring further comprises assigning the measurements taken at the first and second time points to separate subsets of microdroplets, wherein at least some of the subsets of microbial viability measurements obtained for the separate subset of microdroplets at the first time point are obtained for the same separate subset of microdroplets obtained at the second time point. The same discrete subset of microdroplets in a second population can be compared with microbial viability measurements taken.

In some embodiments, the step of measuring microbial viability comprises comparing the microbial viability measurements obtained for the separate subsets of microdroplets at the first time point to the microbial viability measurements obtained for the microdroplets obtained at the second time point. Further comprising comparing microbial viability measurements obtained for the same separate subset of droplets. In some embodiments, at least one discrete subset of microdroplets in the first population is absent from the second population, and at least one discrete subset of microdroplets in the second population is absent from the first population. does not exist in the group. In some embodiments, the step of measuring further comprises obtaining measurements of microbial viability from separate subsets of microdroplets in the additional population of microdroplets, measured at additional time points. In some embodiments, measuring microbial viability from a separate subset of microdroplets in the first population of microdroplets from the first portion of the sample measured at the first time point. obtaining a measurement of microbial viability from a separate subset of microdroplets in a second population of microdroplets from the first portion of the sample, measured at a second time point; including obtaining measurements of. In some embodiments, the measurement of microbial viability is whether the indicator of microbial viability exceeds a preset threshold. In some embodiments, the composite value of the microbial viability measurement is the percentage of multiple separate subsets of microdroplets measured at a certain point in time that exceed a threshold value. In some embodiments, the preset threshold is exceeded if the indicator reaches a determined measure of microbial viability. In some embodiments, the composite value of microbial viability measurements from multiple separate subsets of microdroplets measured at a first time point is a composite value of microbial viability measurements from multiple separate subsets of microdroplets measured at a second time point. Composite values of microbial survival measurements from separate subsets are compared. In some embodiments, the step of measuring microbial viability comprises determining microbial viability for multiple subsets of microdroplets obtained at the first and second time points. The method further includes comparing the microbial viability measurements from the subset to microbial viability measurements from separate subsets of microdroplets obtained at the second time point. In some embodiments, the microbial viability measurements taken at the first and second time points are not assigned to separate subsets of microdroplets. In some embodiments, the step of measuring microbial viability comprises, for the plurality of subsets of microdroplets, substituting the microbial viability measurements obtained for the separate subsets of microdroplets at a first point in time for a second time point. further comprising comparing microbial viability measurements obtained for the same separate subset of microdroplets obtained at the time point. In some embodiments, one or more distinct subsets of microdroplets from the first population are absent from the second population, and one or more distinct subsets of microdroplets from the second population are absent from the second population. There are no separate subsets in the first population. In some embodiments, if the indicator of microbial viability exceeds a preset threshold, measuring a separate subset of microdroplets in the additional population of microdroplets measured at an additional time point. further comprising obtaining a measurement of microbial viability from the method. In some embodiments, the method further comprises incubating the one or more portions of the sample contacted with the antimicrobial agent for different periods of time prior to forming the one or more populations of microdroplets. , whereby microdroplets are formed from each of one or more portions of the sample at different times. In some embodiments, one or more portions of the sample are incubated for a sufficient period of time to monitor microbial viability. In some embodiments, one or more portions of the sample are incubated for a sufficient period of time to allow quorum sensing of microorganisms. In some embodiments, one or more portions of the sample are removed for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, Any one of 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours, or Incubated for multiple periods. In some embodiments, the susceptibility of a microorganism to an antibiotic is determined by measuring the survival rate of the microorganism in the presence of different concentrations of the antibiotic.

In some embodiments, measuring microorganism viability in the droplet comprises determining bacterial concentration by post-lysis genetic analysis including qPCR or fluorescence in-situ hybridization (FISH). carried out using techniques that affect In some embodiments, measuring microbial viability in the droplet involves techniques that do not affect microbial viability, including measuring solution turbidity, pH, or fluorescence of metabolically active dyes. It is carried out using In some embodiments, measuring microbial viability comprises discrete subsets of microdroplets from a first population of microdroplets from a first portion of a sample measured at a first time point. obtaining a measurement of microbial viability from a separate subset of microdroplets from a second population of microdroplets from the first portion of the sample measured at a second time point; including obtaining measurements of viability. In some embodiments, the measurement of microbial viability is whether the indicator of microbial viability exceeds a preset threshold. In some embodiments, each subset of microdroplets includes one or more microdroplets. In some embodiments, one or more portions of the sample are cultured in a culture medium. In some embodiments, the culture medium is added before or during microdroplet formation. In some embodiments, the method further comprises immobilizing one or more populations of microdroplets encapsulating microorganisms in an indexed array. In some embodiments, the method further comprises passing the one or more populations of microdroplets encapsulating microorganisms through a high-throughput microdroplet reader. In some embodiments, the different concentrations of antimicrobial agent span a desired clinical range ranging from 0.002 mg/L to 500 mg/L. In some embodiments, measuring microbial viability includes measuring the fluorescent signal of the label. In some embodiments, fluorescence is measured using a fluorescence reader. In some embodiments, microbial viability is determined by measuring the absorbance or electrochemical properties of a viability indicator dye. In some embodiments, the viability indicator dye comprises resazurin, formazan, or an analog or salt thereof. In some embodiments, microbial viability is determined by measuring absorbance or electrochemical properties of a viability indicator, or by measuring pH or turbidity. In some embodiments, the average number of microorganisms per microdroplet is less than 2. In some embodiments, the average number of microorganisms per microdroplet is less than one. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacteria are E. coli, P. P. aeruginosa, S. S. aureus, S. aureus. S. epidermidis, E. E. faecalis, K. K. pneumoniae, E. Cloacae (E. cloacae), A. A. baumannii, S. marcescens (S. marcescens), or E. It is E. faecium. In some embodiments, the microorganism is a bacterium and the antimicrobial agent is an antibiotic. In some embodiments, the antibiotic is an aminocoumarin, aminoglycoside, ansamycin, carbacephem, carbapenem, cephalosporin, glycopeptide, lincosamide, lipopeptide, macrolide, monobactam, nitrofuran, penicillin, polypeptide, quinolone. , streptogramins, sulfonamides, or tetracyclines, or combinations thereof. In some embodiments, the antibiotic is ampicillin. In some embodiments, the microdroplets include an oil phase and a surfactant phase. In some embodiments, microdroplets are formulated by microfluidic channels, agitation, electrical force, or membrane filtration. In some embodiments, one or more populations of microdroplets are formulated as a stable water-in-oil emulsion. In some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent is accomplished in a shorter time than if microdroplets were not formed. In some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent comprises determining the susceptibility of a microorganism to an antimicrobial agent for 3 to 24 hours, 3 to 20 hours, 3 to 15 hours, 3 to 8 hours, 5 to 20 hours, 5 to 15 hours, or This is accomplished in a time range of 5 to 8 hours. In some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent is accomplished in 24 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 5 hours or less, 3 hours or less. . In some embodiments, the sample includes whole blood, positive blood culture, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax. , breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, gruel. chyle, bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal fluid, pancreatic juice, washings from the sinuses, bronchopulmonary aspirates or other washes. , blastocoel, cord blood, or maternal circulation.

FIG. 2 shows a schematic diagram of an embodiment of a method for assessing microbial viability on separate subsets of microdroplets. 2 is a graphical illustration of an embodiment of the measurement of microbial viability of the method of FIG. 1 containing an antibiotic as compared to microdroplets containing an antibiotic at a concentration above the minimum inhibitory concentration (MIC) (dashed line); FIG. 3 shows a graphical illustration showing microdroplet fluorescence intensity as a function of time, including microdroplets containing no or less than MIC concentration of antibiotic (solid line). FIG. 3 shows the results of an embodiment of microdroplet antibiotic susceptibility testing using fluorescence viability indicators. Figure 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours in a sample without antibiotics (condition A) and in a sample containing twice the MIC of antibiotics (condition B). shows. FIG. 3B shows a micrograph of a microdroplet under the conditions of FIG. 3A. Figure 3C shows a photomicrograph of microdroplets containing E. coli without antibiotics. Figure 3D shows a photomicrograph of microdroplets containing E. coli and twice the MIC of antibiotic. FIG. 3 shows the results of an embodiment of microdroplet antibiotic susceptibility testing using fluorescence viability indicators. Figure 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours in a sample without antibiotics (condition A) and in a sample containing twice the MIC of antibiotics (condition B). shows. FIG. 3B shows a micrograph of a microdroplet under the conditions of FIG. 3A. Figure 3C shows a photomicrograph of microdroplets containing E. coli without antibiotics. Figure 3D shows a photomicrograph of microdroplets containing E. coli and twice the MIC of antibiotic. FIG. 3 shows the results of an embodiment of microdroplet antibiotic susceptibility testing using fluorescence viability indicators. Figure 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours in a sample without antibiotics (condition A) and in a sample containing twice the MIC of antibiotics (condition B). shows. FIG. 3B shows a micrograph of a microdroplet under the conditions of FIG. 3A. Figure 3C shows a photomicrograph of microdroplets containing E. coli without antibiotics. Figure 3D shows a photomicrograph of microdroplets containing E. coli and twice the MIC of antibiotic. FIG. 3 shows the results of an embodiment of microdroplet antibiotic susceptibility testing using fluorescence viability indicators. Figure 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours in a sample without antibiotics (condition A) and in a sample containing twice the MIC of antibiotics (condition B). shows. FIG. 3B shows a micrograph of a microdroplet under the conditions of FIG. 3A. Figure 3C shows a photomicrograph of microdroplets containing E. coli without antibiotics. Figure 3D shows a photomicrograph of microdroplets containing E. coli and twice the MIC of antibiotic. 1 shows a schematic diagram of an embodiment of a method for assessing microbial viability for a plurality of microdroplets. 1 shows a schematic diagram of an embodiment of a method for assessing microbial viability for microdroplets based on measuring microbial viability in microdroplets that exceeds a predetermined threshold. FIG. 6 is a graphical illustration of an embodiment of the measurement of microbial viability of the method of FIG. FIG. 2 shows a graphical diagram showing microbial survival in microdroplets above a threshold as a function of time, including microdroplets containing a concentration of antibiotic (solid line). Figure 2 shows a schematic diagram of an embodiment of a method for assessing microbial viability by incubating a sample in antibiotics and preparing microdroplets at each measurement point. 8 is a graphical illustration of an embodiment of the measurement of microbial viability of the method outlined in FIG. 7, comparing microdroplets containing an antibiotic at a concentration above the MIC (dashed line) with no antibiotic or FIG. 3 shows a graphical diagram showing microbial survival in microdroplets above a threshold as a function of time, including microdroplets containing antibiotics at concentrations below the MIC (solid line).

Rising antimicrobial resistance and shrinking antimicrobial pipelines are creating a global public health crisis in which an increasing number of patients become infected with antimicrobial-resistant bacteria. Appropriate antibiotic therapy, which can be given within hours of the onset of infection, can have a positive impact on patient outcome. However, current practices for identifying infectious diseases require excessive amounts of time. Therefore, there is a strong need for more rapid antimicrobial susceptibility tests, preferably those that can identify specific antimicrobial susceptibilities within hours after a blood sample is drawn. Therefore, this type of rapid testing allows physicians to start optimal drug therapy from the beginning, rather than starting with suboptimal or completely ineffective antibiotics, thus greatly increasing clinical responsiveness. It will improve. Major efforts to improve current antimicrobial susceptibility testing (AST) practices are aimed at shortening the target time to result (TTR).

In microdroplet-based microbial identification (ID) and antimicrobial susceptibility testing (AST), a clinical specimen is divided into small-volume microdroplets, each containing a small number of organisms, typically 1 per microdroplet. Contains ~5 creatures. This technique allows for high effective concentrations of organisms within each occupied microdroplet and also provides rapid time-to-results and It may enable direct specimen testing of specimens with polymicrobial infections.
A challenge with microdroplet-based ID/AST technology is that large numbers of microdroplets are generated, and many of these microdroplets may not contain any biological cells. Therefore, a large number of microdroplets must be evaluated to obtain clinically relevant results.

Accordingly, some embodiments provided herein provide a method for providing a sample having microorganisms, encapsulating the microorganisms from the sample within a microdroplet, wherein the sample forms a microdroplet. contacting each part of the sample with a different concentration of antimicrobial agent either before or after the formation of the microdroplet, and determining the microbial viability of the microorganisms within the microdroplet. This invention relates to a method for determining the susceptibility of microorganisms to antimicrobial agents by a measuring step. The methods described herein may be altered, modified, or modified according to the embodiments, methods, systems, and forms described in further detail herein.
Some of the embodiments, methods, and forms described herein may be useful, for example, in direct specimen methods without the need for specimen culture to obtain isolated colonies; The advantages include one or more advantages including: increased bacterial concentration due to partitioning of the sample in microdroplets, resulting in a starting concentration; and rapid growth detection where discrete growth events can be detected.

Without wishing to be bound by theory, embodiments of the antimicrobial susceptibility testing methods described herein may be used to detect microbial infections of samples caused by different pathogens (e.g., bacteremia, fungemia, etc.). , viremia) and provide antimicrobial sensitivity and resistance profiles of the causative agent (e.g., microbial pathogen).
Additionally, advantages of the microdroplet-based AST embodiments disclosed herein over existing AST methods include the possibility of handling specimen-direct (low titer) and/or polymicrobial (mixed infection) samples. Can be mentioned. Both of these advantages arise primarily because of the division of clinical specimens into microdroplets of very small volume, each of which can be tailored to contain no more than one organism. and can be treated individually. The embodiments of AST using microdroplets described herein are performed directly from clinical specimens (without the need for culturing in solid media) using microdroplets, and the results This can result in significant savings in time to obtain (TTR). Embodiments that partition bacteria into small volumes also allow for high effective concentrations of organisms, resulting in faster reaction kinetics, thereby further improving TTR.

An additional advantage of embodiments that perform AST using microdroplets is improved TTR for testing "late resistant" bacterial phenotypes. These bacterial drug combinations are particularly challenging with respect to both current and emerging AST technologies due to the very long TTRs required to accurately identify bacteria as resistant.
Accordingly, some embodiments provided herein relate to novel compositions, methods, systems, and/or kits for determining the minimum inhibitory concentration (MIC) of antimicrobial agents. In some embodiments, a method for determining an antimicrobial MIC comprises: exposing a sample having a microorganism to a concentration of an antimicrobial agent; encapsulating the microorganism within a microdroplet; This is carried out by measuring the microbial survival rate of microorganisms.

As described herein, method modifications may be performed based on the preparation of microdroplets, the measurement of microbial viability in the microdroplets, and/or the steps for measuring microbial viability. Some variations of the method are described herein as forms. Additional modifications and/or forms may be implemented, and in some embodiments, aspects of any given form may be exchanged, replaced, or replaced with aspects from a different form. It will be understood by those skilled in the art that this may be the case. Furthermore, in some embodiments, various aspects of any given form may be removed, added, altered, or otherwise modified.

<Form 1 - Measurement of microbial survival rate in a population of microdroplets>
Some embodiments provided herein relate to a first form for determining microbial viability. A first mode for determining microbial viability is shown schematically in FIG. 1. As described herein, a first form for determining microbial viability consists of providing a sample having microorganisms therein, dividing the sample into several parts, the parts being formed either before or after the step of preparing microdroplets encapsulating microorganisms from the sample (Figure 1 shows forming the part first), either before or after preparing the microdroplets. , by contacting each part with different concentrations of the antibiotic of interest (Figure 1 shows the addition of the antibiotic before forming microdroplets), and by measuring the microbial viability signal. including the step of measuring survival rate. Measuring the microbial viability signal is performed by obtaining microbial viability measurements from a discrete subset of microdroplets or from multiple discrete subsets of microdroplets from a population of microdroplets. obtain. Results for measurements from separate subsets of microdroplets at a first time point can then be combined to generate results for the portion of the sample for which the microdroplets were acquired at this first time point. This process is repeated at additional time points to determine microbial survival in the section over time, thereby determining the susceptibility of the microorganism to a given concentration of antimicrobial agent. Typically, exactly the same population of microdroplets is measured at the first and subsequent time points, as long as the population of microdroplets measured at each time point is representative of the portion of the sample from which the microdroplets are obtained. do not have to. The results from various parts exposed to different concentrations of antimicrobial agents can then be used to determine the susceptibility of the microorganism to the antimicrobial agent. Determination of antimicrobial susceptibility is the determination of microbial viability measurements over time that indicate that microbial growth does not occur over time when in contact with the concentrations of antimicrobials present in the part of the sample where the measurement was made. It can be done in deciding not to increase. Conversely, an increase in microbial viability measurements over time indicates microbial growth in the concentration of antimicrobial agent present in the portion of the sample where the measurements were made. The minimum inhibitory concentration can be determined using antimicrobial susceptibility and/or microbial growth results from several portions of a sample exposed to different concentrations of an antimicrobial agent.

In some embodiments, preparing microdroplets from each portion results in a population of microdroplets in each portion. In some embodiments, the size of the microdroplet is sufficient to encapsulate the microorganism of interest, such as from about 2 μm to about 500 μm, from 2 μm to 200 μm, from 2 μm to 50 μm, from 2 μm to 10 μm, from 10 μm to 200 μm, The size is within the range of 10 μm to 50 μm, 50 μm to 200 μm, or 50 μm to 100 μm. The size of microdroplets and methods for preparing them are described in further detail herein. For example, in some embodiments, a first portion of the sample is contacted with a first concentration of an antimicrobial agent and one or more populations of microdroplets are formed therefrom; a second concentration of the antimicrobial agent, and one or more populations of microdroplets are formed therefrom; similarly for any desired number of portions, each portion having a different concentration of the antimicrobial agent to be tested. be. Alternatively, microdroplets may be formed from the part prior to contacting the part with the antimicrobial agent. The concentration of the antimicrobial agent typically falls within a range of antimicrobial agents, including the concentration of the antimicrobial agent that is or is believed to be the minimum inhibitory concentration (MIC), as described in more detail herein. selected to include the concentration of the drug. In some embodiments, one or more portions of the sample are not exposed to any antimicrobial agent (antimicrobial concentration of 0), eg, for control purposes.

In some embodiments, the first form is a first form of microdroplets from a portion of the sample measured at a first time point to determine a change in microbial viability signal over time. obtaining measurements of microbial viability from separate subsets of microdroplets from a population and obtaining separate subsets of microdroplets from a second population of microdroplets from the same portion of the sample at a second time point; Measuring microbial viability includes obtaining a measurement of microbial viability from the subset. In some embodiments, measurements are taken from multiple separate subsets of microdroplets at first and/or second time points. The subset of microdroplets includes one or more than one microdroplet, for example, the subset includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or a range defined by any two of the preceding values, such as 1-5, 1-10, 5-10, 5-20, 10-50, 10-100, 50-100, comprises or at least comprises 50-500, 100-500, 100-1000, 500-1000, 500-5000, 1000-5000, 1000-10000, or 5000-10000 microdroplets. In some embodiments, the measurement of microbial viability is performed by measuring additional discrete subsets of microdroplets at additional time points, such as a third time point, a fourth time point, a fifth time point, etc. Similarly, for a given number of time points sufficient to determine microbial viability in microdroplets. For example, in some embodiments, microbial viability measurements are obtained from a third population of microdroplets obtained from the same portion of the sample at a third time point. Measurements of microbial viability for additional time points and populations of microdroplets may be performed, for example, from a fourth population of microdroplets at a fourth time point, from a fifth population of microdroplets at a fifth time point, from a fifth population of microdroplets at a fifth time point, from the sixth population of microdroplets at a time point 6; from the seventh population of microdroplets at a seventh time point; from the eighth population of microdroplets at a time point eight; from a ninth population of droplets, from a tenth population of microdroplets at a tenth time point, or more, etc., as required for any given assay. good. In some embodiments, the time point at which the measurement of microbial viability is selected is between time 0 (eg, the time at which microdroplets encapsulating the microorganisms are formed) to time 24 hours. Thus, in some embodiments, the time point is 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes, or 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12 , 15, 18, 21, or 24 hours, or an amount within the range defined by any two of the foregoing values. In some embodiments, the time points are 0-15 minutes, 0-10 minutes, 0-5 minutes, 0-1 minutes, 5-15 minutes, 5-10 minutes, or 10-15 minutes, 0-24 hours. , 0-21, 0-18, 0-15, 0-12, 0-10, 0-6, 0-5 hours, 0-4 hours, 0-3 hours, 0-2 hours, 0-1 hour, 0 to 0.5 hours, 0.25 to 6 hours, 0.25 to 5 hours, 0.25 to 4 hours, 0.25 to 3 hours, 0.25 to 2 hours, 0.25 to 1 hour, 0 .25 to 0.5 hours, 1 to 6 hours, 1 to 5 hours, 1 to 3 hours, or 1 to 2 hours, or any given number of measurements until determination of antimicrobial susceptibility can be achieved. include. In some embodiments, antimicrobial susceptibility is determined within 24, 20, 15, 10, 8, 5, 3, or 1 hour or less after contacting the sample with the antimicrobial agent and before making the antimicrobial susceptibility determination. or a sufficient amount of time to make a determination that the microorganism is susceptible and/or not susceptible to the antimicrobial agent tested. In some embodiments, antimicrobial susceptibility occurs within 24 hours or less, 15 hours or less between contacting the sample with the antimicrobial agent and making the antibiotic susceptibility determination; 12 hours or less, in some embodiments it is 10 hours or less, in some embodiments it is 8 hours or less, in some embodiments it is 5 hours or less, in some embodiments In some embodiments, it is less than 3 hours, and in some embodiments it is less than 1 hour. In some embodiments, a determination regarding whether a microorganism is susceptible and/or not susceptible to an antimicrobial agent is made using a microdroplet susceptibility test described herein. This can be accomplished in a shorter time than testing for antimicrobial susceptibility without testing. This process can be repeated for additional portions of the sample. In some embodiments, this process is repeated for second, third, fourth, fifth, or more portions of the sample.

Viability measurements are taken for a given portion of a sample (e.g., first, second, third portion, etc.) at the first, second, and subsequent time points, and the viability is calculated for each portion. can be compared to determine over time. In some embodiments, separate measurements at each of the various time points are aggregated such that the aggregate measurements at each time point can be compared to each other. For example, in one embodiment, the average of microbial viability measurements from multiple separate subsets of microdroplets measured at a first time point is the average of microbial viability measurements from multiple separate subsets of microdroplets measured at a second time point. compared to the average of microbial survival measurements from a subset of . In some embodiments, rather than aggregating data from several separate subsets of measurements before comparing between time points, separate data points from the subsets are compared over time and comparisons over time are aggregated to obtain a comparison. For example, in one embodiment, the measurement of microbial viability from separate subsets of microdroplets measured at a first time point is performed with respect to multiple subsets of microdroplets measured at first and second time points. , microbial viability measurements from separate subsets of microdroplets measured at a second time point are compared, and multiple separate comparisons are made to obtain a comparison between the first time point and the second time point. may be averaged to

In some embodiments, the individual microdroplets in the subset and/or first population measured at the first time point are the same as the individual microdroplets in the subset and/or second population measured during a second or subsequent time point. is not the same microdroplet in . overlap between individual microdroplets in the subset and/or population measured at a first time point and individual microdroplets in the subset and/or population measured at a second or subsequent time point; may be present, but it is not necessary that exactly the same individual microdroplets are measured at each time point. Thus, in some embodiments, one or more discrete subsets of microdroplets from the first population are absent from the second population, and one or more of the microdroplets from the second population Multiple separate subsets are not present in the first population, and the same is true for the third, fourth, fifth, and any subsequent populations and time points. In other embodiments, the same separate subsets of microdroplets are such that the separate subsets of microdroplets from the first population are the same separate subsets of microdroplets from the second and subsequent populations. , measured at the first and second time points, and any subsequent time points. In some embodiments, the microbial viability measurements taken at the first, second, and any subsequent time points are independent of whether the same separate subset of microdroplets are measured at each time point. , are not assigned to separate subsets of microdroplets.

As shown in the embodiment in FIG. 2, measurements of microbial viability can be made over time by determining fluorescence intensity, such as the fluorescence intensity of resazurin. Fluorescence intensity measurements are taken for microdroplets exposed to antibiotics below the minimum inhibitory concentration (MIC) or without added antibiotics (solid line), and for microdroplets exposed to antibiotics above the MIC. is shown for a microdroplet (dashed line). The solid line indicates increased fluorescence intensity due to increased microbial growth over time due to the absence of antimicrobial agent or the presence of antimicrobial agent at concentrations below the MIC. The dashed line indicates no or insignificant increase in fluorescence intensity due to no growth by microorganisms over time as a result of antibiotic concentrations above the MIC.

FIG. 3 illustrates the results of measuring microbial viability using certain embodiments of the method described in Form 1. Figure 3A shows microdroplet AST using fluorescence viability indicators for E. coli encapsulated within the microdroplet. Microdroplets encapsulating E. coli were placed in the first part of the sample that was not exposed to antibiotics (condition A) or in the second part of the sample that was exposed to a concentration of twice the MIC of antibiotics (condition B). ). The antibiotic used in this example was ampicillin. Microdroplets were formed and incubated for defined times including 1 hour (t1), 2 hours (t2), and 3 hours (t3). Figure 3A shows microdroplets (in this case single) from three populations of microdroplets at three time points (t1, t2, t3) obtained from two parts of the sample (conditions A or B). A plot of measurements from separate subsets of microdroplets (microdroplets) is shown. Triangles represent the mean of each subset, and error bars indicate one standard deviation of the mean. Figure 3B shows photomicrographs of representative microdroplets from each population at each time point from each of conditions A and B. At time 1 hour, fluorescence intensity remained low for microdroplets in both conditions A and B. At times 2 and 3 hours, the fluorescence intensity increased for the microdroplets in condition A, indicating E. coli growth due to the absence of antibiotics, whereas the fluorescence intensity remained low for the microdroplets in condition B. , indicating no E. coli growth due to the presence of ampicillin at twice the MIC. Figure 3C shows a micrograph of a microdroplet that has not been exposed to antibiotics and shows the presence of E. coli in the absence of antibiotics (Condition A). Figure 3D shows a photomicrograph of microdroplets exposed to twice the MIC of ampicillin (condition B), with no E. coli present.

Form 2 - Measuring microbial survival over time in separate subsets of microdroplets
Some embodiments provided herein relate to a second form for determining microbial viability, with respect to form 1 and other forms disclosed herein. Therefore, disclosures regarding other forms, including but not limited to form 1, are also applicable to form 2. An embodiment of Form 2 for determining microbial viability is shown schematically in FIG. 4. As described with respect to Form 1, a second form for determining microbial viability consists of providing a sample with microorganisms in it, dividing the sample into several parts, before the parts are formed or Either after the step of preparing microdroplets encapsulating microorganisms from the sample (Figure 4 shows forming the part first), either before or after preparing the microdroplets, each contacting the portions with different concentrations of the antimicrobial agent of interest (Figure 4 shows adding the antimicrobial agent prior to forming microdroplets), and contacting the microdroplets at the first, second, and optionally additional points. measuring microbial viability by measuring microbial viability signals in separate subsets of microdroplets from a population of droplets. As disclosed herein, time points can be selected over a variety of times and time ranges. Additionally, as disclosed herein, the time points are selected to allow sufficient time for determination that the microorganism is susceptible and/or not susceptible to the antimicrobial agent. In some embodiments, a determination regarding whether a microorganism is susceptible and/or not susceptible to an antimicrobial agent is made using a microdroplet susceptibility test described herein. (e.g., less than 24 hours, less than 15 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 5 hours, less than 3 hours, less than 1 hour) achieved. Embodiments of Form 2 provide that the results from a particular discrete subset of microdroplets (e.g., a single microdroplet) at a first point in time are the same across the population of microdroplets at a second point in time. Assign the microbial viability signal from a population of microdroplets to a separate subset of microdroplets so that results from that same separate subset of droplets (e.g. a single microdroplet) can be compared Including. Typically, for each portion of the sample exposed to different concentrations of the antimicrobial agent, measurements are made in sufficient numbers to ensure that a representative number of microdroplets are measured for each portion of the sample. obtained from separate subsets of microdroplets. The time-course results for each separate subset of microdroplets can then be combined to generate results for the portion of the sample from which the microdroplets were obtained, and for various samples exposed to different concentrations of antibiotics. The results from the section are used to determine the susceptibility of the microorganism to antimicrobial agents.

In certain embodiments of Form 2, the sample containing the microorganism is separated into one or more portions of the sample containing the microorganism, the one or more portions of the sample are contacted with the antimicrobial agent, and the sample containing the microorganism is Each of the parts comes into contact with a different concentration of antimicrobial agent. One or more populations of microdroplets encapsulating microorganisms are then formed from one or more portions of the sample. The survival rate of the microorganisms encapsulated within the microdroplets is then measured. Certain embodiments of Form 2 obtain measurements of microbial viability from separate subsets of microdroplets in a first population of microdroplets from a first portion of a sample, measured at a first time point. and obtaining measurements of microbial viability from separate subsets of microdroplets in a second population of microdroplets from the first portion of the sample, measured at a second time point. , further comprising assigning measurements taken at the first and second time points to separate subsets of microdroplets, at least a portion of the separate subsets of microdroplets in the first population The microbial viability measurements obtained for the separate subsets of microdroplets at a time point are compared to the microbial viability measurements obtained for the same separate subsets of microdroplets obtained at a second time point. The same separate subset of microdroplets in the second population, such that Some embodiments provide microbial viability measurements obtained for at least a plurality of subsets of microdroplets at a first time point and for a separate subset of microdroplets at a second time point (and any additional and actually performing a comparison with measurements of microbial viability obtained for that same separate subset of microdroplets obtained at time points). As described herein, a subset of microdroplets includes one or more than one microdroplet, e.g., the subset includes 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000 , 7000, 8000, 9000, or 10000 microdroplets, or a range defined by any two of the preceding values, such as 1-5, 1-10, 5-10, 5-20, 10-50 , 10-100, 50-100, 50-500, 100-500, 100-1000, 500-1000, 500-5000, 1000-5000, 1000-10000, or 5000-10000 microdroplets, or at least include.

As discussed herein with respect to Form 1, additional populations of microdroplets and subsets thereof are measured at additional time points (e.g., third, fourth, fifth, sixth, seventh, etc.) be able to. For some embodiments of Form 2, measurements taken at the first, second, and any subsequent time points are assigned to separate subsets of microdroplets, and the measurements taken at the first, second, and any subsequent time points are assigned to separate subsets of microdroplets in the first population. The at least one or more distinct subsets are configured such that the microbial viability measurements obtained for the distinct subsets of microdroplets (e.g., a single microdroplet) at the first time point are the same at a second and any subsequent time point. such that microbial viability measurements taken on that same separate subset of microdroplets taken at time points (e.g., 3rd, 4th, 5th, 6th, etc.) 2 and any subsequent population of the same discrete subset of microdroplets. In some embodiments, not all microdroplets in the first and second, or any subsequent populations, have to be utilized to make the comparison; At least one distinct subset of microdroplets is not present in the second population or any subsequent population, and at least one distinct subset of microdroplets in the second population or any subsequent population is present in the first population. does not exist.

In some embodiments, including but not limited to embodiments of Form 2, measuring separate subsets of microdroplets at a first time point and a subsequent time point includes measuring separate subsets of microdroplets at a first time point and a subsequent time point. May include indexing. Indexing can be used, for example, to assign microbial viability measurements to specific subsets of microdroplets, or to ensure that a representative subset of numbers of microdroplets is measured with respect to a portion of a sample. It can be used for. In some embodiments, the microdroplets are placed on an indexed array and the microbial viability measurements are performed on the indexed array. Indexing may be performed by methods known in the art. For example, indexing may be performed, in some embodiments, by incorporating optical and/or magnetic reporters (e.g., including organic fluorophores, quantum dots, SERS tags) into each sample microdroplet. .

<Form 3 - Measurement of microbial survival rate in microdroplets when the signal exceeds the threshold>
Some embodiments provided herein relate to a third form for determining microbial viability relative to other forms disclosed herein, such as forms 1 and 2. Accordingly, disclosures relating to other forms, including but not limited to forms 1 and 2, are also applicable to form 3. An embodiment of a third form for determining microbial viability is shown in FIG. As described with respect to Form 1, a third form for determining microbial viability consists of providing a sample with microorganisms in it, dividing the sample into several parts, before the parts are formed or Either after the step of preparing microdroplets encapsulating the microorganisms from the sample (Figure 5 shows forming the part first), either before or after preparing the microdroplets, each Microbial viability is determined by contacting the parts with different concentrations of the antibiotic of interest (Figure 5 shows adding the antibiotic before forming microdroplets) and measuring the microbial viability signal. including the step of measuring. Measuring the microbial viability signal is performed by measuring microbial viability in one or more separate subsets of microdroplets (e.g., single microdroplets) from a population of microdroplets. obtain. Embodiments of Form 3 differ from simply measuring the level or amount of a signal, in that the microbial viability signal exceeds a threshold value in distinct subsets of microdroplets (e.g., a single microdroplet). Including measuring whether or not. Therefore, in this sense, the measurement of microbial viability is digital - either a threshold is exceeded or it is not. This makes it possible to measure over time the change in the amount or number of a subset of microdroplets in the portion of the sample that exceeds the threshold. If the amount or number remains constant or does not increase with time, the microorganisms in that part are not viable and are therefore sensitive to the antibiotic concentration in that part of the sample. In contrast, if the number or amount of a subset of microdroplets that exceed a threshold increases over time and reaches a saturation level, this indicates that microorganisms are viable at the concentration of antimicrobial in that portion of the sample. shows.

In some embodiments, measuring microbial viability from a separate subset of microdroplets in the first population of microdroplets from the first portion of the sample measured at the first time point. obtaining a measurement of microbial viability from a separate subset of microdroplets in a second population of microdroplets from the first portion of the sample, measured at a second time point; The measurement of microbial viability is whether the indicator of microbial viability exceeds a preset threshold. In some embodiments, the preset threshold is exceeded if the measurement of microbial viability exceeds an amount determined to indicate that the microorganism is alive. For example, a threshold value may be the amount by which microbial growth, if exceeded, would indicate that the antimicrobial agent (and antimicrobial concentration) being tested is below the minimum inhibitory concentration (MIC). can be mentioned. In some embodiments, the threshold is exceeded when the indicator reaches a determined measurement of fluorescence. The subset of microdroplets includes one or more than one microdroplet, for example, the subset includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or a range defined by any two of the preceding values, such as 1-5, 1-10, 5-10, 5-20, 10-50, 10-100, 50-100, comprises or at least comprises 50-500, 100-500, 100-1000, 500-1000, 500-5000, 1000-5000, 1000-10000, or 5000-10000 microdroplets.

Comparison of multiple measurements taken at the first and second time points (as well as any subsequent time points) can take several forms. For example, the same subset of microdroplets may be monitored over time to determine whether the subset changes from not exceeding a threshold to exceeding a threshold. This can be done for multiple subsets. Alternatively, the number or percentage of subsets exceeding a threshold in the plurality of subsets may be monitored over time to determine whether the number or percentage is increasing. In this case, it is not necessary that exactly the same subset is monitored at the first, second, and any subsequent time points, but the monitored subset may be partially or completely the same subset. As disclosed herein, time points may be selected over a variety of times and time ranges. Additionally, as disclosed herein, the time point is sufficient for the determination that a microorganism is susceptible and/or not susceptible to an antimicrobial agent, for example, if the microbial viability signal exceeds a threshold. Selected to give time. In some embodiments, a determination regarding whether a microorganism is susceptible and/or not susceptible to an antimicrobial agent is made using a microdroplet susceptibility test described herein. (e.g., less than 24 hours, less than 15 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 5 hours, less than 3 hours, less than 1 hour) achieved. In some embodiments, the composite value of microbial viability measurements from multiple separate subsets of microdroplets measured at a first time point is a Composite values of microbial viability measurements from multiple separate subsets of drops are compared. In some embodiments, the composite value of the microbial viability measurement is the percentage of multiple separate subsets of microdroplets measured at a certain point in time that exceed a threshold value. Some embodiments include microorganisms from separate subsets of microdroplets obtained at a first time point, with respect to multiple subsets of microdroplets measured at a first, second, and any subsequent time point. and comparing the viability measurements with microbial viability measurements from separate subsets of microdroplets obtained at the second time point and any subsequent time points. In some embodiments, microbial viability measurements taken at the first, second, and any subsequent time points are not assigned to separate subsets of microdroplets. In some embodiments, they are assigned and in some embodiments microbial viability measurements taken on separate subsets of microdroplets at a first time point for multiple subsets of microdroplets. , comparing microbial viability measurements obtained for that same separate subset of microdroplets obtained at a second and any subsequent time points. In some embodiments, one or more distinct subsets of microdroplets from the first population are absent from the second population, and one or more distinct subsets of microdroplets from the second population are absent from the second population. There are no separate subsets in the first population, and the same for any additional populations.

As used herein, the term "allocate" or "assignment" refers to the attribution of a microbial viability measurement to a microdroplet or a separate population of microdroplets, where a microbial viability measurement is It may correspond to a specific microdroplet or a separate population of specific microdroplets.

Measurement of microbial viability may include, for example, but not limited to, measurements of microbial viability on populations of microdroplets and/or discrete subsets of microdroplets, as described herein, in Form 1 or It can be carried out as described for Form 2. Measurements of microbial viability may be performed continuously or periodically over time until the microbial viability signal exceeds a preset threshold. In some embodiments, for each separate subset or population of microdroplets, the number or percentage of a subset of microdroplets exhibiting a microbial viability measurement , such as a fluorescence intensity measurement , that exceeds a predetermined threshold. is determined. For example, the number of microdroplet subsets that exceed a predetermined threshold can be determined by using a high-throughput microdroplet analyzer to determine the number of microdroplets (e.g., single, two, three, or four or more per subset) that exceed a predetermined threshold. (microdroplets) and counting distinct detector events in which microbial viability measurements such as fluorescence intensity exceed and/or do not exceed preset thresholds. The number of microdroplet subsets that exceed a threshold value compared to the total number of microdroplet subsets evaluated is the microbial Provides percentage of survival rate. In some embodiments, determining the percentage of microbial survival for the population of microdroplets includes determining the percentage of microbial survival for the population of microdroplets as a function of time for each portion of the sample having the same concentration of antimicrobial agent. Iterated at various times to determine the Microbial viability is then assessed and the MIC determined from measurements across antibiotic concentrations.

An embodiment of measuring microbial viability by the method of Form 3 by determining microbial viability based on whether the measurement exceeds a threshold is shown in FIG. As shown in FIG. 6, microbial survival is measured as a function of time by measuring microbial survival and determining whether the microbial survival measurement exceeds a predetermined threshold. Microdroplets that are not exposed to antibiotics or are exposed to antibiotics below the MIC will increase their fluorescence intensity over time. When the fluorescence intensity signal exceeds a preset threshold, an indicator value for microbial survival is determined (solid line). In contrast, any signal that does not exceed the threshold indicates a survival rate of zero. For example, a microdroplet that has been exposed to an antimicrobial agent above the MIC does not exceed a preset threshold (dashed line) and is evaluated for a determination that microbial viability is zero.

<Formation 4 - Formation of microdroplets at the time of measurement>
Some embodiments provided herein relate to a fourth form for determining microbial viability with respect to other forms disclosed herein, such as forms 1, 2, and 3. Accordingly, disclosures related to other forms, including but not limited to forms 1, 2, and 3, are also applicable to form 4. An embodiment of the fourth form for determining microbial viability is shown in FIG. As described with respect to Form 1, the fourth form for determining microbial viability consists of preparing a sample with microorganisms in it, dividing the sample into several parts, and each part having a different concentration of interest. including exposure to antimicrobial agents. In a Form 4 embodiment, the microdroplets are not prepared immediately. Instead, the microorganisms in each portion of the sample with different concentrations of antimicrobial agent are incubated for a period of time, and microdroplets are prepared from each portion at various measurement points. Measurement of microbial viability of the microorganism is then performed on one of the microdroplets from the population of microdroplets, as described herein (including but not limited to embodiments 1-3). or by measuring the microbial viability signal of multiple separate subsets. Thus, in Form 4, portions of the sample are incubated for a period of time before forming microdroplets that are formed at various measurement time points. There may be several reasons for incubating portions of the sample and creating microdroplets at various measurement time points. For example, some microorganisms lose or gain resistance to antimicrobial agents depending on the density of the microorganisms in the culture (eg, due to quorum sensing). These effects may not be observed when microorganisms are cultured in a microdroplet environment. Another reason is that by maintaining the culture and making microdroplets from the culture as needed at various time points, the viability of microorganisms in the microdroplets is maintained after the microdroplets are prepared. It's not necessary. This may result in one or more advantages, such as increased flexibility in the materials and methods used to prepare the microdroplets, and/or improved ease of handling the microdroplets (e.g. temperature, (no need to maintain oxygen levels, etc.). In addition, because the viability of the microorganisms does not need to be maintained, the means used to assess viability can permit growth to be arrested or to kill the microorganisms; Alternatively, additional tests may be performed to kill the microorganism (eg, lysing the microorganism and performing genetic analysis). In Form 4, it may be possible to maintain the viability of microdroplets, and therefore the viability of microdroplets prepared after being cultured for various times can also be evaluated over time. In this regard, the features of Form 4 may be combined with the features of certain embodiments disclosed herein (e.g., embodiments of Form 2) in which microdroplet viability is assessed over time. can.

In some embodiments, a method for measuring microbial viability comprises exposing one or more portions of a sample that have been in contact with an antimicrobial agent for different periods of time prior to forming one or more populations of microdroplets. incubating, whereby microdroplets are formed from each of the one or more portions of the sample at different time points. In some embodiments, one or more portions of the sample are incubated for a sufficient period of time to produce any bacterial density-dependent phenomenon. In some embodiments, one or more portions of the sample are removed for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, Any one of 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 24 hours, or Incubated for multiple periods. In some embodiments, after microdroplet formation, the microdroplets are measured to determine microbial viability. In some embodiments, microdroplet measurements may be performed regardless of whether the microorganisms encapsulated within the microdroplet are alive or non-viable. In some embodiments, measuring microbial viability comprises discrete subsets of microdroplets from a first population of microdroplets from a first portion of a sample measured at a first time point. obtaining a measurement of microbial viability from a separate subset of microdroplets from a second population of microdroplets from the first portion of the sample measured at a second time point; including obtaining measurements of viability. In some embodiments, the measurement of microbial viability is whether the indicator of microbial viability exceeds a preset threshold. The subset of microdroplets includes one or more than one microdroplet, for example, the subset includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or a range defined by any two of the preceding values, such as 1-5, 1-10, 5-10, 5-20, 10-50, 10-100, 50-100, comprises or at least comprises 50-500, 100-500, 100-1000, 500-1000, 500-5000, 1000-5000, 1000-10000, or 5000-10000 microdroplets. Further details regarding measuring microbial viability in subsets of microdroplets that can be used in embodiments of Form 4 are provided herein, including but not limited to the disclosures regarding Forms 1, 2, and 3. Ru. In some embodiments, the method of measuring microbial viability using microdroplets formed at each time point includes, for example, an AST readout at the end point that allows for detection chemistries that affect viability, density-dependent Advantages include large-scale incubation of samples prior to microdroplet generation, and maximized microdroplet occupancy, allowing determination of resistance mechanisms.

In some embodiments, measuring microbial viability in the droplet is performed using a technique that affects microbial viability. Techniques that influence the viability of microorganisms include, for example, techniques using lysis of microorganisms, such as determination of bacterial concentration by genetic analysis techniques, which may include post-lysis qPCR or fluorescence in-situ hybridization (FISH). can be mentioned. In some embodiments, measuring microbial viability in the droplet is performed using a technique that does not affect microbial viability. Techniques that do not affect microorganism viability may include, for example, measuring solution turbidity, pH, or fluorescence of metabolically active dyes.

An embodiment of Form 4 is illustrated in FIG. 8, which shows measured microbial viability as a function of time in fluorescence intensity. Microdroplets that are not exposed to antibiotics or exposed to antibiotics below the MIC increase fluorescence intensity (solid line). In contrast, microdroplets exposed to antibiotics above the MIC do not exceed the preset threshold and thus no increase in fluorescence intensity is observed (dashed line).

The time point for incubating the portion of the sample containing the antimicrobial agent prior to microdroplet formation is selected to range from time 0 (eg, the time the antimicrobial agent contacts the portion of the sample) to time 24 hours. Thus, in some embodiments, the time point is 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes, or 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12 , 15, 18, 21, or 24 hours, or an amount within the range defined by any two of the foregoing values. In some embodiments, the time points are 0-15 minutes, 0-10 minutes, 0-5 minutes, 0-1 minutes, 5-15 minutes, 5-10 minutes, or 10-15 minutes, or 0-24 minutes. Time, 0-21, 0-18, 0-15, 0-12, 0-10, 0-6, 0-5 hours, 0-4 hours, 0-3 hours, 0-2 hours, 0-1 hour , 0 to 0.5 hours, 0.25 to 6 hours, 0.25 to 5 hours, 0.25 to 4 hours, 0.25 to 3 hours, 0.25 to 2 hours, 0.25 to 1 hour, This includes culturing for 0.25 to 0.5 hours, 1 to 6 hours, 1 to 5 hours, 1 to 3 hours, or 1 to 2 hours. After incubation for a given period of time, microdroplets are formed and measurements of microbial viability are typically determined as soon as possible after forming the microdroplets. Determination of microbial viability is performed to determine that microorganisms are susceptible and/or non-susceptible to antimicrobial agents. In some embodiments, a determination regarding whether a microorganism is susceptible and/or not susceptible to an antimicrobial agent is made using a microdroplet susceptibility test described herein. This can be accomplished in a shorter time than testing for antimicrobial susceptibility without testing.

Some embodiments of Form 4 form microdroplets at each time point and determine whether microbial survival exceeds a predetermined threshold for each population of microdroplets formed at each measurement point. measuring the microbial survival rate of the microdroplet. FIG. 7 illustrates a schematic diagram of Form 4. The sample is divided into portions and each portion is contacted with a different concentration of antimicrobial agent spanning the desired range. For each portion, microdroplets are prepared at each desired measurement time point. In some embodiments, a separate subset of microdroplets is measured at each time point, which may be after an additional period of culturing portions of the sample disclosed herein, and microbial viability is (eg, Form 1) or on the same subset of microdroplets (eg, Form 2). In some embodiments, the measurement of microbial viability is evaluated if the microbial viability signal exceeds a threshold (eg, Form 3). However, rather than forming a microdroplet at time 0 and then measuring the microbial viability of the microdroplet at a later time point, the microdroplet is measured when the portion of the sample that the microdroplet is formed from is measured. It is formed only at the time of measurement, such that it is incubated with the antimicrobial agent until the time of measurement. In some embodiments, measurements can be performed in any of the methods disclosed herein, including, for example, using a high-throughput microdroplet analyzer or an indexed array. In some embodiments, the number or percentage of microdroplets exceeding a threshold is determined as a function of time for each concentration of antimicrobial agent, and microbial viability and MIC are estimated from the measurements.

Although in each of the embodiments described above each step is described as a separate process, one or more of the steps may be implemented in a system. Thus, for example, one or more of the processes may be performed in a microfluidic device. Microfluidic devices may be used to automate processes and/or to allow simultaneous processing of multiple samples. Those skilled in the art are well aware of methods in the art for collecting, handling, and processing biological fluids that can be used in the practice of this disclosure. Additionally, microfluidic devices for various steps can be combined into one system for performing any of the forms described herein.

<Measurement of microbial survival rate>
As described, some embodiments provided herein relate to methods for antimicrobial susceptibility testing (AST). Any of the methods, embodiments, systems, or forms described herein may be interchangeable or modified to enable microbial AST by encapsulating microorganisms within microdroplets. or may be changed. Without wishing to be bound by theory, the method embodiments, systems, and forms described herein provide high sensitivity near the MIC, rapid determination of microbial viability, and/or or may have one or more advantages for measuring microbial viability, including large-scale determination of microbial viability over a wide range of antimicrobial concentrations.

In any of the embodiments, methods, systems, or forms described herein, microdroplets can be incubated at different antibiotic concentrations for any period of time. Growth by the microorganisms may be monitored during the incubation period, which can be continued until a sufficient difference in detection signal, eg, fluorescence or microbial number, is present between the microdroplets. For example, in some embodiments, incubation is for about 15, 30, or 45 seconds, about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 seconds, It can be 60, 90, 120, 150, 180, 210, 240, 300, or 360 minutes or more. In one embodiment, the incubation is for more than 2 hours, such as at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days. Depending on the proliferation and/or growth rate of the encapsulated microorganisms, one skilled in the art can determine the optimal incubation duration for subsequent analysis, such as cell viability analysis.

In any of the embodiments, methods, systems, or forms described herein, a discrete subset of microdroplets having microorganisms encapsulated therein determines the susceptibility of the microorganisms within the microdroplets to an antimicrobial agent. can be measured to In some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent is performed by measuring the fluorescence intensity of the microdroplet. In some embodiments, determining the susceptibility of the microorganism to an antimicrobial agent is performed during or after incubation of the microorganism. In some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent comprises continuously monitoring microbial viability continuously or monitoring microbial viability at one or more discrete time points. This will be carried out on a regular basis.

In any of the embodiments, methods, systems, or forms described herein, a sample may be divided into portions of the sample, and each portion of the sample may be contacted with a different concentration of an antimicrobial agent. In some embodiments, for each portion of the sample, microbial viability measurements are made by either interrogating microdroplets by placing them in an indexed array or by using a high-throughput microdroplet reader. Measured as a function of time for separate subsets of microdroplets. Measurements may be performed, for example, by measuring the fluorescence intensity of separate subsets of microdroplets at an initial time point (e.g., first time point) and at subsequent time points (e.g., second, third, fourth, etc.). Can be done. Fluorescence intensities measured on separate subsets of microdroplets can be used to assess bacterial viability at each antibiotic concentration. In some embodiments, measurements are performed at multiple time points during incubation, eg, a first time point and then a second time point. In some embodiments, microbial viability measurements for separate subsets of microdroplets (e.g., a single microdroplet) are made at times 0, 1 hour, 2 hours, 3 hours, or later. be measured. In some embodiments, measurements are performed at any number of desired time points within a time frame of time 0 to time 24 hours. As disclosed herein, time points can be selected over a variety of times and time ranges. Additionally, as disclosed herein, the time point is sufficient for the determination that a microorganism is susceptible and/or not susceptible to an antibiotic, e.g., if the microbial viability signal exceeds a threshold. Selected to give time. In some embodiments, a determination regarding whether a microorganism is susceptible and/or not susceptible to an antimicrobial agent is made using a microdroplet susceptibility test described herein. This can be accomplished in a shorter time than testing for antimicrobial susceptibility without testing. Accordingly, in some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent may be performed for 3-24 hours, 3-20 hours, 3-15 hours, 3-8 hours, 5-20 hours, 5-15 hours. , or within a period of time ranging from 5 to 8 hours, or within an amount of time defined by any two of the foregoing values. In some embodiments, determining the susceptibility of a microorganism to an antimicrobial agent is accomplished in 24 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 5 hours or less, 3 hours or less. ).

In some embodiments, the time point at which the measurement of microbial viability is selected is between time 0 (eg, the time at which microdroplets encapsulating the microorganisms are formed) to time 24 hours. Thus, in some embodiments, the time point is 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes, or 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12 , 15, 18, 21, or 24 hours, or an amount within the range defined by any two of the foregoing values. In some embodiments, the time points are 0-15 minutes, 0-10 minutes, 0-5 minutes, 0-1 minutes, 5-15 minutes, 5-10 minutes, or 10-15 minutes, 0-24 hours. , 0-21, 0-18, 0-15, 0-12, 0-10, 0-6, 0-5 hours, 0-4 hours, 0-3 hours, 0-2 hours, 0-1 hour, 0 to 0.5 hours, 0.25 to 6 hours, 0.25 to 5 hours, 0.25 to 4 hours, 0.25 to 3 hours, 0.25 to 2 hours, 0.25 to 1 hour, 0 .25 to 0.5 hours, 1 to 6 hours, 1 to 5 hours, 1 to 3 hours, or 1 to 2 hours, or any given number of measurements until determination of antimicrobial susceptibility can be achieved. include. In some embodiments, the antimicrobial susceptibility is determined by the time between contacting the sample with the antimicrobial and making the antimicrobial susceptibility determination, which is 24, 20, 15, 10, 8, 5, or 3 hours or less; or occurs in a sufficient amount of time to make a determination that the microorganism is susceptible and/or not susceptible to the antimicrobial agent tested.

In any of the embodiments, methods, systems, or forms described herein, the measurement of microbial viability is performed from a first portion exposed to a first concentration of an antimicrobial agent at a first time point. Microbial viability measurements may be obtained from separate subsets of microdroplets from the first portion exposed to the first concentration of the antimicrobial agent at a second time point. Retrieved from In some embodiments, the average measurement of microbial survival from the separate subsets of microdroplets at the first time point is the average measurement of microbial survival from the separate subsets of microdroplets measured at the second time point. compared to the average measured value. Microorganisms can be tested, for example, by growth in the presence of antimicrobials (to determine the resistance of bacteria to a particular antimicrobial agent), cell death (to determine bactericidal activity), and/or growth inhibition (to determine bacteriostatic activity). (to do so) can be observed. For example, microbial growth and/or cell death can be measured by (i) counting the number of microorganisms in a subset of microdroplets compared to a control or standard; (ii) counting the number of microorganisms in a subset of microdroplets compared to a control or standard; (iii) the proportion of cells expressing at least one microbial marker in the subset of microdroplets compared to a control or reference; (iv) the proportion of cells expressing at least one microbial marker in the subset of microdroplets compared to a control or reference; It can be assessed by relative metabolite levels in a subset, or (v) any combination thereof. In some embodiments, growth by the microorganism or functional response of the microorganism can be determined or monitored in real time, eg, by microscopy or flow cytometry.

Any method known in the art for determining the viability of microorganisms in a sample measures the viability of microorganisms encapsulated within microdroplets over time and when exposed to different concentrations of antimicrobial agents. can be used to compare and determine the growth of encapsulated microorganisms. Overall, cell viability can be determined by cell lysis or membrane leakage assays (e.g. lactate dehydrogenase assay), mitochondrial activity or caspase assays (e.g. resazurin and formazan (MTT/XTT) assay), reactive oxygen species (ROS) production assays, It can be assayed using functional assays, or genomic and proteomic assays. Exemplary methods include, but are not limited to, ATP testing, ROS testing, calcein AM, pH sensitive dyes, clonogenic assays, ethidium homodimer assays, Evans blue, fluorescein diacetate hydrolysis/propidium iodide staining (FDA /PI staining), flow cytometry, formazan-based assay (MTT/XTT), green fluorescent protein, lactate dehydrogenase (LDH), methyl violet, propidium iodide, can identify necrotic, apoptotic, and normal cells. DNA stains, resazurin, trypan blue (live cell exclusion dye (the dye crosses only the cell membrane of dead cells)), 7-aminoactinomycin D, TUNEL assay, cell labeling or staining (e.g. cell permeable dyes (e.g. carboxylic acid)), 7-aminoactinomycin D, TUNEL assay, diacetate, succinimidyl ester (carboxy-DFFDA, SE)), cell-impermeable dyes, cyanine, phenantridine, acridine, indole, imidazole, nucleic acid stains, cell-permeable reactive tracers (e.g. intracellular activity The fluorescent dyes CMRA, CMF2HC (4-chloromethyl-6,8-difluoro-7-hydroxycoumarin), CMFDA (5-chloromethylfluorescein diacetate), CMTMR (5-(and -6)-((( (4-chloromethyl)benzoyl)amino)tetramethylrhodamine), CMAC (7-amino-4-chloromethylcoumarin), CMHC (4-chloromethyl-7-hydroxycoumarin)), or any combination thereof), fluorescence DNA dyes (e.g. DAPI, Heochst family, SYBR family, SYTO family (e.g. SYTO9), SYTOX family (e.g. SYTOX Green), ethidium bromide, propidium iodide, acridine, or any combination thereof); color development sexual dyes (e.g. eosin, hematoxylin, methylene blue, azure, or any combination thereof); cytoplasmic stains (e.g. calcofluor white, periodic acid Schiff stain, or any combination thereof); metabolic stains (e.g. Any metabolic stain described in the specification; any diacetate dye (including rhodamine-based dyes, fluorescin, or any combination thereof); resazurin/resorufin (Alamar Blue); ROS stains (e.g. Any of the ROS stains described herein, DCFDA and related families, calcein acetoxymethyl and related families); membrane stains (e.g., Bodipy, FM1-43, FM4-64, and their functional equivalents, CellMask ( Trademark staining, Dil, DiO, DiA); biological staining (e.g., labeled antibodies, labeled chitin-binding proteins), optical imaging, microscopic imaging after staining, ELISA, mass spectrometry (e.g., peptides, proteins, glycopeptides); , lipopeptides, carbohydrates, and/or metabolites), modification of metabolomic fingerprints, degradation of RNA or protein content, and the like. In some embodiments, detection of microbial growth or functional response to antimicrobial agents can be performed using solid phase, microfluidic, or microdroplet-based assays. In some embodiments, detecting microbial growth or functional response to an antimicrobial agent can include the use of mass spectrometry. In some embodiments, detecting microbial growth or functional response to an antimicrobial agent can include detecting at least one metabolite, or metabolic profile. In some embodiments, detecting microbial growth or functional response to an antimicrobial agent can include detecting transcriptional changes. In some embodiments, microbial survival in microdroplets may be determined by performing imaging of microorganisms in microdroplets to collect both growth and morphology data. In some embodiments, the methods of analysis disclosed herein may be applied to microbial count data to determine the MIC. In some embodiments, measuring microbial viability involves distributing discrete subsets of microdroplets in indexed arrays or by using a high speed scanning device.

In any of the embodiments, methods, systems, or forms described herein, microbial viability measurements may be performed by measuring fluorescent dyes in microdroplets. In some embodiments, the fluorescent dye is a viability indicator dye. In some embodiments, the fluorescent dye is resazurin. Resazurin is reduced to resorufin as a result of bacterial growth. Resorufin has a higher fluorescence quantum yield compared to resazurin, resulting in increased fluorescence intensity as bacteria grow within the microdroplet. In some embodiments, microbial viability measurements can be assessed from measurements of microdroplet fluorescence, and MICs can be determined therefrom. In some embodiments, algorithms for MIC determination are developed and used.

<Micro droplets>
Microdroplets provided herein and prepared in any of the forms described herein for measuring microbial viability can be used in microfluidic devices or other devices for microdroplet production. can be manufactured using In some embodiments, the microdroplet is of sufficient size to encapsulate the microorganism of interest. For example, the microdroplet has a diameter of 2 μm to about 500 μm, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, Diameters such as 70, 80, 90, 100, 110, 120, 130, 10, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 μm, or any of the values listed above. or two. In some embodiments, the microdroplets range from 2 μm to 500 μm, 2 μm to 50 μm, 2 μm to 10 μm, 10 μm to 200 μm, 10 μm to 50 μm, 50 μm to 200 μm, or 50 μm to 100 μm. The size of the microdroplets may be based on the mode of microdroplet preparation or the specific desired size range. Those skilled in the art will appreciate that microdroplet sizes can be modified in size to suit the particular microorganism of interest or the particular assay used. In some embodiments, the microdroplets have volumes in the range of picoliters, e.g., 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 pl, or by any two of the aforementioned values. has a volume within the defined range. In some embodiments, the microdroplets have a volume of 0.001 pl to 1000 pl, 0.001 pl to 100 pl, 100 pl to 1000 pl, 100 pl to 500 pl, or 500 pl to 1000 pl.

In any of the embodiments, methods, systems, or forms described herein, microdroplets may be formed after contacting a sample with an antimicrobial agent. In some embodiments, the microdroplets are released within 1, 2, 3, 4, 5, 10, 15, 30, 45, or 60 seconds after contacting the sample with the antibiotic, or 1, 2, within 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or 1, 2, 3, 4, 5, 6, 8, 10, 12, Formed within 14, 16, 18, 20, 22, or 24 hours, or within a time frame defined by any two of the aforementioned values. In some embodiments, the microdroplets are applied within 1 second, 30 seconds, 1 minute, 15 minutes, 30 minutes, 1 hour, or 2 hours of contacting one or more portions of the sample with the antimicrobial agent. It is formed.

In any of the embodiments, methods, systems, or forms described herein, microdroplets disperse a sample containing microorganisms of interest into a continuous hydrophobic oil phase containing a surfactant. It can be a stable water-in-oil emulsion made by. Examples of surfactants include, but are not limited to, sulfonates, alkyl sulfates, monoesters of polyalkoxylated sorbitans, polyester polyols, fatty alcohol esters, aromatic alcohol esters, tall oil fatty acid diethanolamides, polyoxyethylene ( 5) Sorbitan monooleate, ammonium salt of polyacrylic acid, ammonium salt of 2-acrylamido-2-methylpropanesulfonic acid/acrylic acid copolymer, alkyl sulfonate, alkylaryl sulfonate, α-olefin sulfonate, diphenyl ether sulfonate, sorbitan monooleate ate, α-sulfofatty acid methyl ester. Combinations of surfactants may also be used.

Other methods of microdroplet production may include, for example, membrane emulsification, external forces such as mechanical shear (impeller driven microdroplet generator), or electrical forces such as dielectrophoretically controlled microdroplet generators. .

In any of the embodiments, methods, systems, or forms described herein, the microdroplet size, surfactant, and oil are molecules from the droplet to the microdroplet or from the oil to the microdroplet. can be optimized to reduce or eliminate diffusion of (eg, assay reagents, dyes, antimicrobials, nutrients, metabolites). In some embodiments, the microdroplet size, surfactant, and oil are optimized to enable or enhance gas diffusion from the oil phase to the microdroplets. In some embodiments, microdroplet size, surfactants, and oils are optimized to improve microdroplet stability and suppress undesirable microdroplet coalescence or fusion. In some embodiments, the microdroplet composition is optimized to facilitate AST reactions occurring in a clinical specimen matrix.

In any of the embodiments, methods, systems, or forms described herein, production of microdroplets may be performed in the presence of a sample containing microorganisms. In some embodiments, production of microdroplets in the presence of a microorganism results in encapsulation of a single microorganism within the microdroplet. The occupancy and distribution of microorganisms within the microdroplets can be altered by adjusting the concentration of microorganisms in the sample. Antibiotics and viability indicator dyes at predetermined concentrations can also be incorporated into the microdroplets at the point where they are formed or at a later point using techniques such as microinjection or microdroplet coalescence. It can also be added to each microdroplet. These microdroplets can then be collected in vials, incubated in appropriate bacterial growth conditions, and monitored at regular intervals.

The methods disclosed herein for performing AST using microdroplets can be applied to any embodiment or instrument capable of measuring microdroplet properties of interest over time. can. These methods are independent of the embodiment used to generate and investigate microdroplets, but may benefit from certain microdroplet characteristics. Some of these attributes are the size/volume of the microdroplets, the stability of the microdroplets over the duration of AST, and the composition of oil and water systems that support bacterial growth. Systems that can be used to monitor the fluorescence intensity of microdroplets include (a) indexed arrays for the placement of microdroplets and their subsequent fluorescence readout, or (b) Instruments that enable high-throughput investigations are mentioned.

In any of the embodiments, methods, systems, or forms described herein, a sample may be divided into portions, and each portion may be exposed to a different concentration of an antimicrobial agent. In some embodiments, the microorganisms in the sample are first encapsulated within microdroplets and then exposed to an antimicrobial agent. In some embodiments, the microorganism is encapsulated within the microdroplet upon exposure to the antimicrobial agent. Thus, a sample containing microorganisms can be exposed to an antimicrobial agent before, during, or after encapsulation within a microdroplet. The microorganisms in one or more portions can be incubated with at least one antimicrobial agent, including, but not limited to, two, three, or more antimicrobial agents, for example, to determine the effectiveness of a combination therapy. At least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) portions are of any antimicrobial agent to serve as a control. can be incubated without the addition of Alternatively or additionally, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) portions serve as a positive control. may be incubated with a broad-spectrum antimicrobial agent.

In any of the embodiments, methods, systems, or forms described herein, portions of a sample containing different concentrations of an antimicrobial agent may be introduced into a microdroplet generator, and the microdroplets may be Produced to encapsulate one microorganism, or on average less than one microorganism per microdroplet. In some embodiments, the microdroplets are generated concurrently with exposure to the antimicrobial agent. In some embodiments, microdroplets are generated at time zero from a sample containing different concentrations of added antimicrobial agents spanning a desired clinical range. In some embodiments, microdroplets are measured to determine microbial viability. Measuring microdroplets may include measuring a subset of microdroplets. The subset of microdroplets includes one or more than one microdroplet, for example, the subset includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or a range defined by any two of the preceding values, such as 1-5, 1-10, 5-10, 5-20, 10-50, 10-100, 50-100, comprises or at least comprises 50-500, 100-500, 100-1000, 500-1000, 500-5000, 1000-5000, 1000-10000, or 5000-10000 microdroplets.

Microdroplets encapsulating microorganisms with varying concentrations of antimicrobial agents can be incubated under any conditions suitable for growth by the microorganisms. Those skilled in the art will appreciate the optimum culture conditions for microbial growth (e.g. bacterial growth), e.g. culturing them at a suitable temperature and atmosphere, e.g. ℃ to about 45℃ can be easily determined. In some embodiments, the incubation is from about 25°C to about 40°C, or from about 30°C to about 42°C, or from about 35°C to about 40°C. In one embodiment, the incubation is at about 37°C.

<Antibacterial agent>
Any of the embodiments, methods, systems, or forms described herein may include contacting the sample with an antimicrobial agent. In any of the embodiments or forms described herein, the antimicrobial agent can be incorporated into or exposed to the microdroplets. In some embodiments, the antimicrobial agent is dried in the device at a defined concentration and/or amount and forms droplets after being reconstituted by the portion of the sample containing the microorganism. The amount of dried antimicrobial agent, when reconstituted by a portion of the sample, can be adjusted so that the resulting concentration is within the desired range. In some embodiments, the antimicrobial agent is added to the microdroplet after the microdroplet is prepared, rather than being added to the sample before the microdroplet is formed.

In any of the embodiments, methods, systems, or forms described herein, the antimicrobial agent inhibits the growth of microorganisms (e.g., bacteria, fungi, viruses, parasites, and microbial spores), thereby The agents can be naturally occurring, semi-synthetic, or fully synthetic, preventing their occurrence and microbial or pathogenic effects. Antimicrobial agents are known to those skilled in the art. However, by way of example, antimicrobial agents can be used as organic or inorganic small molecules; saccharin; oligosaccharides; polysaccharides; biopolymers such as peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen-binding fragments thereof; ; nucleic acid analogs and derivatives; glycogen or other sugars; immunogens; antigens; extracts made from biological materials such as bacterial, plant, fungal, or animal cells; animal tissues; naturally occurring compositions or synthetics; composition; as well as any combination thereof. In some embodiments, antimicrobial agents include antibacterial agents (or antibiotics), antifungal agents, antiprotozoal agents, antiviral agents, and mixtures thereof.

Non-limiting examples of antibiotics include, for example, aminoglycosides (including, e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin), ansamycins (e.g., geldanamycin, herb. mycin, rifaximin), carbacephems (including e.g. loracarbef), carbapenems (e.g. including ertapenem, antipseudomonal, doripenem, imipenem, cilastatin, meropenem, biapenem, panipenem), cephalosporins (e.g. cefazolin, cephalexin, Cefdroxil, cefapirin, cefazedone, cefazaflu, cefradine, cefuroxazine, ceftezol, cefaloglycine, cefacetril, cephalonium, cephaloridine, cephalothin, cefatridine, cefaclor, cefotetan, cefamycin, cefoxitin, cefprozil, cefuroxime, cefuroxime axetil, cefamandole , cefminox, cefonicid, cefolanid, cefothiam, cefbuperazone, cefzonam, cefmetazole, carbacefem, loracarbef, cefixime, ceftriaxone, antipseudomonas, ceftazidime, cefoperazone, cefdinir, cefcapene, cefdaroxime, ceftizoxime, cefmenoxime, cefotaxime, cefpiramide, cef Podoxime, ceftibuten , cefditoren (defditoren), ceftameth, cefodidime, cefpimizole, cefsulodine, cefteram, cefthiolene, oxacefem, flomoxef, latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftaroline fosamil, ceftolozane, ceftobiprole, ceftiofur, cefki Nomu, cefovecin glycopeptides (including, for example, vancomycin, oritavancin, telavancin, teicoplanin, dalbavancin, ramoplanin), lincosamides (including, for example, clindamycin, lincomycin), lipopeptides (including, for example, daptomycin), macrolides (including, for example, daptomycin), For example, azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin), monobactams (e.g., aztreonam, tigemonam, carmonam, nocardicin A), nitrofurans (e.g., furazolidone, nitrofurantoin) ), oxazolidonones (including, for example, linezolid, posizolid, radezolid, trezolid), penicillins (for example, penams, beta-lactams, benzylpenicillin, benzathine benzylpenicillin, procaine benzylpenicillin, phenoxymethylpenicillin, propicillin, Fenethicillin, azidocillin, clomethocillin, penamecillin, cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, methicillin, amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, ticarcillin, carbenicillin, calindacillin, temoci phosphorus, piperacillin, azlocillin, mezlocillin, mecillinam, pivmecillinam, sulbenicillin), penems (e.g., faropenem or litipenem), polypeptides (e.g., bacitracin, colistin, polymyxin B), quinolones (e.g., ciprofloxacin, enoxaxin ), gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin), sulfonamides (e.g. , mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimde, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, cotrimoxazole, (including sulfonamide chrysoidine), tetracyclines (including, for example, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline); Namin, aspoxycillin, azidocillin, azithromycin, aztreonam, valofloxacin, biapenem, brodimoprim, capreomycin, cefaclor, cefadroxil, cefatridine, cefcapene, cefdinir, cefetameth, cefoxitin, cefprozil, cefuroxazine, ceftarolin, ceftazidime, ceftib ten, cefmetazole (ceftmetazole) , ceftobiprole, cefuroxime, cephalexin, cephalonium, cephaloridine, cephamandole, cefazolin, cefrazine, chloramphenicol, chlorquinaldol, chlortetracycline, cyclacilline, cino Kisacin , ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, clofazimine, clofazimine, cloxacillin, colistin, cycloserine, dalfopristin, danofloxacin, dapsone, daptomycin, demeclocycline, dicloxacillin, difloxacin, doripenem, doxycycline, enoxacin , enrofloxacin, erythromycin, ethambutol, ethionamide, fleroxacin, flomoxef, flucloxacillin, flumequin, fosfomycin, fusidic acid, gentamycin, imipenem, isoniazid, kanamycin, LC benzylpenicillin, levofloxacin, linezolid, mandelic acid, Mecillinam, meropenem, metronidazole, minocycline, moxalactam, mupirocin, nadifloxacin, nalidixic acid, netilmycin, netilmycin, nifurtoinol, nitrofurantoin, nitroxoline, norfloxacin, ofloxacin, oxytetracycline, panipenem, peflox sacin, phenoxymethylpenicillin, Pipemidic acid, pyromidic acid, pivampicillin, pivmecillinum, platensimycin, prulifloxacin, pyrazinamide, quinupristin, rifabutin, rifampicin, rifapentine, rufloxacin, sparfloxacin, streptomycin, sulbactam, sulfabenzamide, sulfacytin, sulfamethopyrazine , sulfacetamide, sulfadiazine, sulfadimidine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasomidine, sulfathiazole, Teicoplanin, teixobactin, temafloxacin ), tetracycline, tetroxoprim, thiamphenicol, tigecyclin, tinidazole, tobramycin, tosufloxacin, trimethoprim, or vancomycin, and pharmaceutically acceptable salts or esters thereof.

Exemplary antifungal agents include, but are not limited to, 5-flucytosine, aminocandin, amphotericin B, anidulafungin, bifonazole, butoconazole, caspofungin, chlordantoin, chlorphenesin, ciclopirox olamine, trimazole, eberconazole, econazole, fluconazole, flutrimazole, isavuconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, nifuroxime, posaconazole, ravuconazole, tioconazole, terconazole, undecenoic acid, and pharmaceutically acceptable salts thereof or ester.

Exemplary antiprotozoal agents include, but are not limited to, acetalzole, azanidazole, chloroquine, metronidazole, nifratel, nimorazole, omidazole, propenidazole, secnidazole, sinefungin, tenonitrozole, temidazole, tinidazole, and their pharmaceutical Examples include acceptable salts or esters.
Exemplary antiviral agents include, but are not limited to, acyclovir, brivudine, cidofovir, curcumin, desciclovir, 1-docosanol, edoxudine, gQ Fameyclovir, fiacitabine, ivacitabine, imiquimod, lamivudine, penciclovir, valacyclovir. , valganciclovir, and pharmaceutically acceptable salts or esters thereof.

In any of the embodiments, methods, systems, or forms described herein, the antimicrobial agent may include amoxicillin/clavulanate, amikacin, ampicillin, aztreonam, ceftazidime, cephalothin, chloramphenicol, Profloxacin, clindamycin, ceftriaxone, cefotaxime, cefuroxime, erythromycin, cefepime, gentamicin, imipenem, levofloxacin, linezolid, meropenem, minocycline, nitrofurantoin, oxacillin, penicillin, piperacillin, ampicillin/sulbactam, trimethoprim/sulfa It may be selected from the group consisting of methoxazole or cotrimoxazole, tetracycline, tobramycin, vancomycin, or any combination thereof.

In any of the embodiments, methods, systems, or forms described herein, a newly developed antimicrobial agent is used to determine the effectiveness of a newly developed antimicrobial agent, or to determine the effectiveness of a newly developed antimicrobial agent, Can be incorporated into or exposed to microdroplets for determination of susceptibility to developed antimicrobial agents.

In any of the embodiments, methods, systems, or forms described herein, the concentration of antimicrobial agent in the AST provided herein may be provided at various concentrations to determine the range of susceptibility. . In some embodiments, the concentration of the antimicrobial agent is provided at various concentrations to determine the minimum inhibitory concentration (MIC). The broad range of antimicrobial agents provided herein may be provided in varying concentrations and have varying efficacies. Therefore, the concentration of antimicrobial agent can be varied depending on the antimicrobial agent selected. The MIC of any given antimicrobial agent is known to those skilled in the art, including the range of any given antimicrobial agent that may be useful for antimicrobial susceptibility testing. The concentration of the antimicrobial agent includes a range of concentrations of the antimicrobial agent, including the concentration of the antimicrobial agent that is or is believed to be the minimum inhibitory concentration (MIC), as described in more detail herein. selected to include. In some other embodiments, the antimicrobial agent concentration range covers a clinically or physiologically relevant concentration range, but may not include concentrations that may allow determination of the MIC. In some embodiments, one or more portions of the sample are not exposed to any antimicrobial agent (antimicrobial concentration of 0), eg, for control purposes. Thus, for example, when a microdroplet is exposed to an antimicrobial agent (or when an antimicrobial agent is incorporated into the microdroplet during its formation), the concentration of the antimicrobial agent may range from about 0.001 μg/ml to about 5000 μg /ml, such as 0.001, 0.01, 0.1, 1, 10, 100, 1000, or 5000 μg/ml, or within an amount defined by any of the aforementioned values. In some embodiments, the antibiotic concentration is between 0.001 μg/ml and 5000 μg/ml, between 0.001 μg/ml and 1000 μg/ml, between 0.001 μg/ml and 100 μg/ml, and between 0.001 μg/ml and 10 μg/ml. ml, 10 μg/ml to 5000 μg/ml, 10 μg/ml to 1000 μg/ml, 10 μg/ml to 100 μg/ml, 100 μg/ml to 5000 μg/ml, 100 μg/ml to 1000 μg/ml, or 1000 μg/ml to 5000 μg/ml It is. In some embodiments, the antibiotic concentration is a serial dilution to determine the MIC. Serial dilutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, The dilution can be in an amount of 150, 200, 300, 400, 500, 1000, or 10,000 times dilution, or within a range defined by any of the aforementioned values. In some embodiments, the dilution is 1-10000x, 1-1000x, 1-100x, 1-10x, 10-10000x, 10-1000x, 10-100x, 100-10000x, Or 100 to 1000 times the amount. In some embodiments, the concentration of antimicrobial agent is zero (eg, no antimicrobial agent is present), thereby providing a control showing normal microbial growth in the absence of any antimicrobial agent.

In some embodiments, a sample of microdroplets encapsulating microorganisms can be divided into one or more parts of the microdroplet, a first part can be exposed to a first concentration of an antimicrobial agent, and a first part can be exposed to a first concentration of an antimicrobial agent; two portions may be exposed to a second concentration of the antimicrobial agent and a third portion may be exposed to a third concentration of the antimicrobial agent, with respect to a desired number of portions exposed to a desired number of antimicrobial agent concentrations. The same is true. Thus, for example, in a sample of microdroplets, a first concentration of antimicrobial agent exposed to a first portion of the microdroplet may be 0.001 μg/ml, and a first concentration of antimicrobial agent exposed to a second portion of the microdroplet. The second concentration of antimicrobial agent exposed to the third portion of the microdroplet may be 0.01 μg/ml, and the third concentration of antimicrobial agent exposed to the third portion of the microdroplet may be 0.01 μg/ml, with the desired The same is true for any desired number of moieties exposed to different concentrations of a particular antimicrobial agent or multiple antimicrobial agents. In some embodiments, each portion may be exposed to serial dilutions of the antimicrobial agent instead of the desired number of dilutions of the antimicrobial agent. The range of concentrations and the number of microdroplet portions are determined based on the antibiotic being tested, its clinical or physiological can be confirmed based on the relevant concentration range, the suspected microorganism, or the specific assay being performed.

<Microbes>
Embodiments provided herein relate to a process for measuring microbial viability. In any of the embodiments, methods, systems, or forms described herein, microorganisms may be encapsulated within the microdroplet during microdroplet formation. For example, in some embodiments, a sample containing microorganisms is dispersed in a continuous hydrophobic oil phase containing a surfactant at conditions that produce water-in-oil microdroplets. In some embodiments, the sample containing microorganisms is a processed clinical sample. In some embodiments, the sample is peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid. , semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, Interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal fluid, pancreatic juice, washings from sinuses, bronchopulmonary aspirates or other washes, blastocoel, umbilical cord blood, or the maternal circulation, which may be of fetal or maternal origin. In some embodiments, the sample is collected from a human, one or more companion animals, or one or more commercially important animals. In some embodiments, the human, one or more companion animals, or one or more commercially important animals have a microbial infection, such as a bacterial infection.

In any of the embodiments, methods, systems, or forms described herein, the sample can be a clinical sample, which can be obtained from a human subject, and the microbial population of interest is extracted from one or more components of the sample. can be processed for isolation. Samples include, for example, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen (prostatic fluid), etc. Cowper's or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstruation secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal fluids, pancreatic fluid, sinus lavages, bronchopulmonary aspirates or other lavage fluids, blastocoel, umbilical cord blood, or of fetal origin. It can also be obtained as maternal circulation, which may be of maternal origin.

In any of the embodiments, methods, systems, or forms described herein, the sample can be a fluid or analyte obtained from an environmental source. For example, fluids or specimens obtained from environmental sources may include foodstuffs, food, poultry, meat, fish, beverages, dairy products, water (including wastewater), ponds, rivers, reservoirs, swimming pools, soil, food It may be obtained from or derived from processing and/or packaging plants, agricultural fields, hydroponics (including hydroponic food farms), pharmaceutical manufacturing plants, animal colony facilities, or any combination thereof. In some embodiments, the sample is a fluid or specimen collected from or derived from a cell culture or microbial colony.

In any of the embodiments, methods, systems, or forms described herein, it may be necessary or desirable to process the sample prior to encapsulation of the microorganism into the microdroplet. Even if treatment is not necessary, it may be performed for convenience (eg, as part of a regimen on a commercial platform). The processing reagent can be any reagent suitable for use in the methods described herein. The sample processing step may include, for example, adding one or more reagents to the sample. This treatment can serve several different purposes including, but not limited to, hemolyzing cells such as blood cells, diluting samples, and the like. Processing reagents include, but are not limited to, surfactants and detergents, salts, cell lysis reagents, anticoagulants, degradative enzymes (e.g., proteases, lipases, nucleases, lipases, collagenases, cellulases, amylases, etc.); Solvents such as buffer solutions may be mentioned. In some embodiments, the processing reagent is a surfactant or detergent. In some embodiments, the sample is a clinical sample obtained directly from the subject, and the sample includes removing one or more components of the clinical sample and/or adding one or more drugs to the clinical sample. It is treated by adding For example, a sample may be filtered, purified, cleaned, decontaminated, centrifuged, or otherwise processed to isolate a microorganism or population of microorganisms within the sample from one or more components of the sample.

In any of the embodiments, methods, systems, or forms described herein, the sample includes: adding one or more processing reagents to the sample to degrade unwanted molecules present in the sample; /or the sample may be further processed by diluting it for further processing. These processing reagents include, but are not limited to, surfactants and detergents, salts, cell lysis reagents, anticoagulants, degradative enzymes (e.g., proteases, lipases, nucleases, lipases, collagenases, cellulases, amylases, heparinases). etc.), and solvents such as buffer solutions. The amount of processing reagent added may depend on the particular sample being analyzed, the time required for sample analysis, the identity of the microorganisms detected, or the amount of microorganisms present in the sample being analyzed. .
Although not required, if one or more reagents are to be added, the reagents can be presented in a mixture at an appropriate concentration (eg, in a solution, ie, a "processing buffer"). The amounts of the various components of the processing buffer can be varied depending on the sample, the microorganisms being detected, the concentration of microorganisms in the sample, or the time constraints for the analysis.

Processing buffers can be made up in any suitable buffer solution known to those skilled in the art. Such buffer solutions include, but are not limited to, TBS, PBS, BIS-TRIS, BIS-TRIS propane, HEPES, HEPES sodium salt, MES, MES sodium salt, MOPS, MOPS sodium salt, sodium chloride, ammonium acetate. solution, ammonium formate solution, ammonium dihydrogen phosphate solution, diammonium tartrate solution, BICINE buffer solution, bicarbonate buffer solution, citrate concentrated solution, formic acid solution, imidazole buffer solution, MES solution, magnesium acetate solution, magnesium formate solution, acetic acid Potassium solution, potassium acetate solution, potassium acetate solution, tripotassium citrate solution, potassium formate solution, dipotassium phosphate solution, dipotassium phosphate solution, sodium potassium tartrate solution, propionic acid solution, STE buffer solution, STET buffer solution, Sodium acetate solution, sodium formate solution, sodium dihydrogen phosphate solution, sodium monohydrogen phosphate solution, sodium tartrate dihydrate solution, TNT buffer solution, TRIS glycine buffer solution, TRIS acetate-EDTA buffer solution, triethylammonium phosphate solutions, trimethylammonium acetate solution, trimethylammonium phosphate solution, Tris-EDTA buffer solution, TRIZMA® base, and TRIZMA® HCL. Alternatively, the processing buffer may be made in water.

After addition of the processing reagents, the sample can be incubated for a period of time, eg, at least 1, 2, 3, 4, 5, 10, 15, 30, 45, or 60 minutes. Such incubation may be carried out at any suitable temperature, for example, about 16°C to about 30°C, room temperature (e.g., about 20°C to about 25°C), low temperature (e.g., about 0°C to about 16°C), or elevated temperature (e.g. (about 30°C to about 95°C). In some embodiments, the sample is incubated at room temperature for about 15 minutes.

In any of the embodiments, methods, systems, or forms described herein, the microorganism can be a bacterium, fungus, virus, parasite, protozoa, or microbial spore. In some embodiments, the bacteria belong to the following phyla: Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldisericum. Phylum Caldiserica, Chlamydiae, Chlorobium, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferiba cteres), Deinococcus , Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes ), Porta Lentisfaera (Lentisphaerae), Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Teneric Phylum Tenericutes, Thermodesulfobacteria , Thermomicrobia, Thermotogae, or Verrucomicrobia, or any one of the microbial species, such as genes and/or They are derived from mutants and derivatives produced by recombinant techniques.

In any of the embodiments, methods, systems, or forms described herein, the bacteria can be Gram-positive or Gram-negative bacteria. In some embodiments, the bacteria are aerobic or anaerobic. In some embodiments, the bacteria are autotrophic or heterotrophic. In some embodiments, the bacteria are mesophiles, trophophiles, extremophiles, acidophiles, alkalophiles, thermophiles, psychrophiles, halophiles, or sacophiles.

In any of the embodiments, methods, systems, or forms described herein, the bacteria may include Bacillus anthracis, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus ( Staphylococcus, Streptococcus, or Clostridium tetani. In some embodiments, the bacteria are mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (Staphylococcus aureus) (MRSA), or Clostridium difficile. In some embodiments, the bacterium can be Mycobacterium tuberculosis. In some embodiments, the bacteria are E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus fae calis), Klebsiella pneumoniae ), Enterobacter cloacae, Acinetobacter baumannii, Serratia marcescens, or Enterococcus fae cium).

In any of the embodiments, methods, systems, or forms described herein, the microorganism is a protozoan that causes diseases such as malaria, sleeping sickness, or toxoplasmosis; It can be a disease-causing fungus. The term "microbe" or "microbes" can also encompass non-pathogenic microorganisms, such as microorganisms used in industrial applications.

As used herein, section headings are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, specialist texts, and Internet web pages, are hereby incorporated by reference in their entirety for all purposes. Included explicitly. If the definition of a term in an incorporated reference differs from the definition provided in the present teachings, the definition provided in the present teachings shall control. It is understood that there is an implied "about" before any temperature, concentration, time, etc. discussed in the present teachings, such that minor and insubstantial deviations are within the scope of the present teachings herein. will be done.

In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, "comprise", "comprises", "comprising", "contain", "contains", "containing", "including" The use of the words "include", "includes", and "including" are not intended to be limiting.
As used in this specification and the claims, the singular forms "a,""an," and "the" do not clearly dictate otherwise. contains multiple referents.

Although the invention has been disclosed in the context of certain specific embodiments and examples, those skilled in the art will appreciate that the invention extends beyond the specifically disclosed embodiments and/or to other alternative embodiments and/or examples of the invention. or the use thereof, as well as obvious modifications and equivalents thereof. Additionally, while several variations of this invention have been shown and described in detail, other modifications that are within the scope of this invention will be readily apparent to those skilled in the art based on this disclosure. . It is also contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the invention. It should be understood that various features and aspects of the disclosed embodiments can be combined or substituted with each other to form various forms or embodiments of the disclosed invention. Therefore, it is intended that the scope of the invention herein disclosed should not be limited by the particular disclosed embodiments described above.
However, this detailed description, while indicating preferred embodiments of the invention, is given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. It should be understood that

The terms used in the description provided herein are not intended to be construed in any limiting or restrictive manner. Rather, the terms are merely utilized in conjunction with detailed descriptions of embodiments of systems, methods, and related components. Additionally, embodiments may include a number of novel features, none of which alone are responsible for desirable attributes or which are considered essential to practicing the invention herein as described. It's not like I'm doing it.
Preferred embodiments of the present invention are as follows.
[1] A method for evaluating the growth of microorganisms in a sample, comprising:
a) preparing a sample containing microorganisms;
b) separating the sample containing the microorganism into one or more parts of the sample containing the microorganism;
c) forming one or more populations of microdroplets encapsulating microorganisms from the sample, where the one or more populations of microdroplets separate the sample into one or more parts; formed before or after
d) contacting one or more portions of the sample with an antimicrobial agent, either before or after forming one or more populations of microdroplets, wherein the one or more portions of the sample each of the microorganisms is contacted with a different concentration of an antimicrobial agent, and e) measuring the microbial viability of the microorganisms encapsulated within the microdroplets, thereby determining the susceptibility of the microorganisms to the antimicrobial agent. .
[2] The step of measuring microbial viability comprises microbial microorganisms from separate subsets of microdroplets from a first population of microdroplets from a first portion of the sample measured at a first time point. obtaining measurements of viability, and measuring microbial viability from separate subsets of microdroplets from a second population of microdroplets from the first portion of the sample, measured at a second time point; The method according to [1] above, comprising obtaining a measurement .
[3] The average of microbial viability measurements from multiple separate subsets of microdroplets measured at a first time point is equal to the average of microbial viability measurements from multiple separate subsets of microdroplets measured at a second time point. The method according to [2] above, wherein the microbial survival rate is compared with an average measurement .
[4] The step of measuring the microbial survival rate may include, with respect to the plurality of subsets of microdroplets measured at the first and second time points, 2. The method of item 2, further comprising comparing the microbial viability measurements with microbial viability measurements from separate subsets of microdroplets measured at the second time point.
[5] The method according to any one of [2] to [4] above, wherein the microbial viability measurements obtained at the first and second time points are not assigned to separate subsets of microdroplets. .
[6] The one or more distinct subsets of microdroplets from the first population are not present in the second population, and the one or more distinct subsets of microdroplets from the second population are absent from the second population; The method according to any one of [2] to [5] above, which does not exist in the first population.
[7] The step of measuring obtains measurements of microbial viability from separate subsets of microdroplets in additional populations of microdroplets from the first portion of the sample, measured at additional time points. The method according to any one of [2] to [6] above, further comprising:
[8] The population of microdroplets was collected for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours before measuring the microbial survival rate. 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours. The method according to any one of [2] to [7].
[9] Microdroplets are formed within 1 second, 30 seconds, 1 minute, 15 minutes, 30 minutes, 1 hour, or 2 hours of contacting one or more portions of the sample with the antimicrobial agent; The method according to any one of [2] to [8] above.
[10] The step of measuring microbial viability comprises determining microbial survival from separate subsets of microdroplets in a first population of microdroplets from a first portion of a sample, measured at a first time point. and obtaining measurements of microbial viability from separate subsets of microdroplets in a second population of microdroplets from the first portion of the sample, measured at a second time point. and further comprising assigning measurements taken at the first and second time points to separate subsets of microdroplets, at least one of the separate subsets of microdroplets in the first population. wherein the microbial viability measurements obtained on a separate subset of microdroplets at a first time point are microbial viability measurements obtained on that same separate subset of microdroplets obtained at a second time point. The method according to [1] above, wherein the same discrete subset of microdroplets in the second population can be compared with the measured values of the microdroplets in the second population.
[11] The step of measuring microbial viability comprises combining the microbial viability measurements obtained for the separate subsets of microdroplets at the first time point with those of the microbial droplets obtained at the second time point. The method according to [10] above, further comprising comparing microbial survival measurements obtained for the same separate subset.
[12] at least one distinct subset of microdroplets in the first population is absent from the second population, and at least one distinct subset of microdroplets in the second population is present in the first population; The method according to [10] above.
[13] [10] above, wherein the step of measuring further comprises obtaining measurements of microbial viability from separate subsets of microdroplets in the additional population of microdroplets, measured at additional time points. ~ The method according to any one of [12].
[14] The population of microdroplets was collected for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours before measuring the microbial survival rate. incubated for any one or more of 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours; The method according to any one of [10] to [13] above.
[15] Microdroplets are formed within 1 second, 30 seconds, 1 minute, 15 minutes, 30 minutes, 1 hour, or 2 hours of contacting one or more portions of the sample with the antimicrobial agent; The method according to any one of [10] to [14] above.
[16] The step of measuring microbial viability comprises measuring microbial viability from separate subsets of microdroplets in the first population of microdroplets from the first portion of the sample, measured at the first time point. and obtaining measurements of microbial viability from separate subsets of microdroplets in a second population of microdroplets from the first portion of the sample, measured at a second time point. The method according to [1] above, wherein the measurement of the microbial survival rate is whether an index of the microbial survival rate exceeds a preset threshold.
[17] A composite value of microbial viability measurements from multiple separate subsets of microdroplets measured at a first time point is a composite value of microbial viability measurements from multiple separate subsets of microdroplets measured at a second time point. The method according to [16] above, wherein the method is compared with a composite value of measurements of microbial survival rate.
[18] The method of [17] above, wherein the composite value of the measurement of microbial viability is the percentage of a plurality of separate subsets of microdroplets measured at a certain point in time that exceed a threshold value.
[19] The step of measuring microbial viability comprises, with respect to the plurality of subsets of microdroplets measured at the first and second time points, 17. The method of claim 16, further comprising comparing the microbial viability measurements with microbial viability measurements from separate subsets of microdroplets obtained at the second time point.
[20] The method according to any one of [16] to [19] above, wherein the microbial viability measurements obtained at the first and second time points are not assigned to separate subsets of microdroplets. .
[21] The step of measuring microbial viability comprises, for a plurality of subsets of microdroplets, a measurement of microbial viability obtained for a separate subset of microdroplets at a first time point, at a second time point. The method of [16] above, further comprising comparing microbial viability measurements obtained for the same separate subset of microdroplets obtained.
[22] The one or more distinct subsets of microdroplets from the first population are not present in the second population, and the one or more distinct subsets of microdroplets from the second population are The method according to any one of [16] to [21] above, which is not present in the first population.
[23] The method according to any one of [16] to [22], wherein the preset threshold is exceeded if the indicator reaches a determined measurement of microbial viability.
[24] If the indicator of microbial viability exceeds a preset threshold, the step of measuring microbial survival from separate subsets of microdroplets in additional populations of microdroplets measured at additional time points The method according to any one of [16] to [23] above, further comprising obtaining a measurement of the ratio.
[25] The population of microdroplets was incubated for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours before measuring the microbial survival rate. incubated for any one or more of 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours; The method according to any one of [16] to [22] above.
[26] Microdroplets are formed within 1 second, 30 seconds, 1 minute, 15 minutes, 30 minutes, 1 hour, or 2 hours of contacting one or more portions of the sample with the antimicrobial agent. The method according to any one of [16] to [23] above.
[27] Prior to forming the one or more populations of microdroplets, the step further comprises incubating the one or more portions of the sample contacted with the antimicrobial agent for different periods of time, thereby causing the microdroplets to form The method according to [1] above, wherein the method is formed from one or more portions of the sample at different times.
[28] The method of [27] above, wherein one or more portions of the sample are incubated for a period sufficient to monitor microbial viability.
[29] The method of [28] above, wherein the one or more portions of the sample are incubated for a period sufficient to enable quorum sensing of the microorganism.
[30] One or more portions of the sample are present for 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours before forming microdroplets. for any one or more of the following: hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours. The method according to any one of [27] to [29] above, wherein the method is incubated.
[31] The susceptibility of microorganisms to antibiotics is determined by measuring the survival rate of microorganisms in the presence of different concentrations of antibiotics, according to any one of [2] to [30] above. the method of.
[32] Measuring microbial viability in the droplet affects microbial viability, including determining bacterial concentration by genetic analysis including post-lysis qPCR or fluorescence in-situ hybridization (FISH) The method according to any one of [2] to [31] above, which is carried out using a technique.
[33] Measuring microbial viability in the droplet using a technique that does not affect microbial viability, including measurement of solution turbidity, pH, or fluorescence of metabolically active dyes. The method according to any one of [2] to [31], which is carried out.
[34] The step of measuring microbial viability comprises measuring microbial viability from a separate subset of microdroplets from a first population of microdroplets from a first portion of the sample, measured at a first time point. obtaining measurements of viability, and measuring microbial viability from separate subsets of microdroplets from a second population of microdroplets from the first portion of the sample, measured at a second time point; The method according to any one of [27] to [33] above, comprising obtaining a measurement .
[35] The method according to [34], wherein the measurement of microbial survival rate is whether an index of microbial survival rate exceeds a preset threshold.
[36] The method according to any one of [2] to [35] above, wherein each subset of microdroplets includes one or more microdroplets.
[37] The method according to any one of [1] to [36] above, wherein one or more portions of the sample are cultured in a culture medium.
[38] The method according to [37] above, wherein the culture medium is added before or during the formation of the microdroplets.
[39] The method according to any one of [1] to [38] above, further comprising the step of immobilizing one or more populations of microdroplets encapsulating microorganisms on an indexed array.
[40] The method according to any one of [1] to [39] above, further comprising the step of passing one or more populations of microdroplets encapsulating microorganisms through a high-throughput microdroplet reader.
[41] The method according to any one of [1] to [40] above, wherein the different concentrations of the antimicrobial agent span a desired clinical range of 0.002 mg/L to 500 mg/L.
[42] The method according to any one of [1] to [41] above, wherein the step of measuring microbial survival rate includes measuring a fluorescent signal of a label.
[43] The method according to [42] above, wherein the fluorescence is measured using a fluorescence reader.
[44] The method according to [42] or [43], wherein the microorganism survival rate is determined by measuring the absorbance or electrochemical properties of a viability indicator dye.
[45] The method according to [44] above, wherein the viability indicator dye contains resazurin, formazan, or an analog or salt thereof.
[46] Any one of [1] to [41] above, wherein the microbial survival rate is determined by measuring the absorbance or electrochemical properties of a survival rate indicator, or by measuring pH or turbidity. The method described.
[47] The method according to any one of [1] to [46] above, wherein the average number of microorganisms per microdroplet is less than 2.
[48] The method according to any one of [1] to [47] above, wherein the average number of microorganisms per microdroplet is less than 1.
[49] The method according to any one of [1] to [48] above, wherein the microorganism is a bacterium.
[50] The bacteria are E. coli, P. coli, and P. coli. P. aeruginosa, S. S. aureus, S. aureus. S. epidermidis, E. E. faecalis, K. K. pneumoniae, E. Cloacae (E. cloacae), A. A. baumannii, S. marcescens (S. marcescens), or E. The method according to [49] above, which is E. faecium.
[51] The method according to any one of [1] to [50] above, wherein the microorganism is a bacterium and the antibacterial agent is an antibiotic.
[52] The antibiotic is aminocoumarin, aminoglycoside, ansamycin, carbacephem, carbapenem, cephalosporin, glycopeptide, lincosamide, lipopeptide, macrolide, monobactam, nitrofuran, penicillin, polypeptide, quinolone, streptogramin, The method according to [51] above, which is a sulfonamide, a tetracycline, or a combination thereof.
[53] The method according to [51] or [52] above, wherein the antibiotic is ampicillin.
[54] The method according to any one of [1] to [53] above, wherein the microdroplets contain an oil phase and a surfactant phase.
[55] The method according to any one of [1] to [54] above, wherein the microdroplets are formulated by microfluidic channels, stirring, electric force, or membrane filtration.
[56] The method according to any one of [1] to [55] above, wherein the one or more populations of microdroplets are formulated as a stable water-in-oil emulsion.
[57] The method according to any one of [1] to [56], wherein determining the susceptibility of a microorganism to an antimicrobial agent is achieved in a shorter time than when no microdroplets are formed.
[58] Determining the susceptibility of a microorganism to an antimicrobial agent may be performed for 3 to 24 hours, 3 to 20 hours, 3 to 15 hours, 3 to 8 hours, 5 to 20 hours, 5 to 15 hours, or 5 to 8 hours. The method according to any one of [1] to [57] above, which is achieved in a time within the range of.
[59] Determining the susceptibility of the microorganism to an antimicrobial agent is accomplished in 24 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 5 hours or less, 3 hours or less, [1] ] to [58].
[60] If the sample is whole blood, positive blood culture, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchus Alveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle , bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal fluid, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate or other lavage fluid, blastocoel, The method according to any one of [1] to [59] above, which is umbilical cord blood or maternal circulation.

Claims (20)

A method of assessing microbial growth in a sample, comprising:
a) providing a sample containing microorganisms,
b) separating the sample containing the microorganisms into one or more portions of the sample containing the microorganisms;
c) forming one or more populations of microdroplets encapsulating microorganisms from the sample, wherein the one or more populations of microdroplets separate the sample into one or more portions formed before or after,
d) contacting one or more portions of the sample with an antimicrobial agent, either before or after forming one or more populations of microdroplets, wherein one or more portions of the sample; is contacted with a different concentration of the antimicrobial agent; and e) measuring the microbial viability of the microbes encapsulated within the microdroplets, thereby determining the susceptibility of the microbes to the antimicrobial agent. . The step of measuring microbial viability comprises measuring microbial viability from separate subsets of microdroplets from the first population of microdroplets from the first portion of the sample at a first time point. obtaining a measurement and measuring microbial viability from a separate subset of microdroplets from a second population of microdroplets from the first portion of the sample at a second time point; 2. The method of claim 1, comprising obtaining a . The average of the microbial viability measurements from the plurality of separate subsets of microdroplets measured at the first time point is the microbial viability from the plurality of separate subsets of microdroplets measured at the second time point. 3. The method of claim 2, wherein the ratio is compared to an average of the ratio measurements. The step of measuring microbial viability comprises microbial viability from separate subsets of microdroplets measured at the first time point with respect to the plurality of subsets of microdroplets measured at the first and second time points. 3. The method of claim 2, further comprising comparing the measurement of , with measurements of microbial viability from separate subsets of microdroplets measured at a second time point. 5. The method of any one of claims 2-4, wherein the microbial viability measurements obtained at the first and second time points are not assigned to separate subsets of microdroplets. One or more separate subsets of microdroplets from the first population are absent from the second population, and one or more separate subsets of microdroplets from the second population are absent from the first population A method according to any one of claims 2 to 5, which is absent from the population. The measuring step further obtains measurements of microbial viability from separate subsets of microdroplets in additional populations of microdroplets from the first portion of the sample measured at additional time points. A method according to any one of claims 2 to 6, comprising measuring microbial viability from separate subsets of microdroplets in the first population of microdroplets from the first portion of the sample, wherein said measuring microbial viability is measured at a first time point; obtaining values and obtaining measurements of microbial viability from separate subsets of microdroplets in a second population of microdroplets from the first portion of the sample measured at a second time point; and assigning the measurements obtained at the first and second time points to separate subsets of microdroplets, wherein at least some of the separate subsets of microdroplets in the first population is the same separate subset of microdroplets in the second population, and the microbial viability measurements obtained for the separate subset of microdroplets at the first time point are the microdroplets obtained at the second time point 2. The method of claim 1 , wherein the microbial viability measurements obtained for that same separate subset of droplets can be compared. The step of measuring microbial viability comprises measurements of microbial viability from separate subsets of microdroplets in the first population of microdroplets from the first portion of the sample measured at the first time point. and obtaining measurements of microbial viability from separate subsets of microdroplets in a second population of microdroplets from the first portion of the sample measured at a second time point 2. The method of claim 1, wherein the measure of microbial viability is whether the microbial viability indicator exceeds a preset threshold. further comprising incubating the one or more portions of the sample in contact with the antimicrobial agent for different periods of time prior to forming the one or more populations of microdroplets, whereby the microdroplets are at different time points; 2. The method of claim 1, formed from each of one or more portions of the sample. 11. The method of claim 10 , wherein one or more portions of the sample are incubated for a period of time sufficient to monitor microbial viability. 12. The method of claim 11 , wherein one or more portions of the sample are incubated for a period of time sufficient to allow quorum sensing of the microorganisms. A method according to any one of claims 2 to 12 , wherein the susceptibility of micro-organisms to antibiotics is determined by measuring the viability of the micro-organisms in the presence of different concentrations of the antibiotic. The step of measuring microbial viability in droplets uses techniques that affect microbial viability, including determination of bacterial concentration by genetic analysis, including qPCR or fluorescence in-situ hybridization (FISH) after lysis. A method according to any one of claims 2 to 13 , wherein the method is performed as The step of measuring microbial viability in the droplets is performed using techniques that do not affect microbial viability, including measuring solution turbidity, pH, or fluorescence of metabolically active dyes. , the method according to any one of claims 2 to 13 . The step of measuring microbial viability comprises measuring microbial viability from separate subsets of microdroplets from the first population of microdroplets from the first portion of the sample at a first time point. obtaining measurements and microbial viability measurements from separate subsets of microdroplets from a second population of microdroplets from the first portion of the sample, measured at a second time point; A method according to any one of claims 10 to 15 , comprising obtaining 17. The method of claim 16 , wherein the measure of microbial viability is whether the microbial viability indicator exceeds a preset threshold. A method according to any one of claims 2 to 17 , wherein each subset of microdroplets comprises one or more microdroplets. Within 3-24 hours, 3-20 hours, 3-15 hours, 3-8 hours, 5-20 hours, 5-15 hours, or 5-8 hours to determine the susceptibility of the microorganism to the antimicrobial agent A method according to any one of claims 1 to 18 , wherein the method is achieved in a time of Determining the susceptibility of a microorganism to an antimicrobial agent is accomplished in 24 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 5 hours or less, 3 hours or less. A method according to any one of paragraphs.

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