Antimicrobial susceptibility testing using microdroplets

Abstract

To provide compositions, methods, systems and / or kits for measuring microbial viability in a sample.SOLUTION: Compositions, methods, systems and / or kits for measuring microbial viability in a sample are provided herein. Certain embodiments of the present disclosure are related to detection tests comprising compositions, methods, systems and / or kits for measuring an antimicrobial minimum inhibitory concentration and for measuring microbial susceptibility to the antimicrobial. Certain embodiments of the present disclosure are related to detection tests comprising compositions, methods, systems and / or kits for assessing microbial proliferation in a sample.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 719,290, filed Aug. 17, 2018, which is incorporated by reference herein 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 measuring the minimum inhibitory concentration of an antimicrobial agent. [Background technology]

[0002] Microbial infections affect millions of people every year and are associated with millions of deaths per year. A precise diagnosis of the pathogen species often requires several days to obtain, even in state-of-the-art clinical microbiology laboratories. Furthermore, patients who receive inaccurate therapy early on exhibit lower survival rates than those who are treated with optimal therapy from early in the course of the disease. The rapidity of pathogen diagnosis in patients with microbial infections can have a significant impact on the prognosis. The current method for detecting microbial infections in blood is to culture the blood in hospital or commercial clinical microbiology laboratories. Liquid cultures can allow detection of the presence of certain growing organisms in the fluid within 4-30 hours. This assay is not quantitative, and without knowledge of the pathogen species and their specific antibiotic sensitivity, only broad-spectrum antibiotics can be administered at this time, which are suboptimal at best. To identify the specific species of pathogen and to perform sensitivity tests to determine their response to various potential antibiotic therapies, the pathogens growing in liquid medium must then be transferred to other growth media (e.g., agar plates). The total time for complete diagnostic and sensitivity testing is typically 3 to 7 days, and empirical antibiotic treatment based on clinical symptoms is initiated well before antibiotic sensitivity results are obtained, often within 1 to 3 hours after blood cultures are first drawn from the patient.

[0003] Many patients with microbial infections exhibit rapid decline within the first few hours of infection. Thus, rapid and reliable diagnostic and treatment methods are essential for effective patient care. Unfortunately, current antimicrobial susceptibility testing techniques as a whole require prior isolation of the microorganisms for culture (e.g., about 12 to about 48 hours) followed by processing requiring another about 6 to about 24 hours. For example, a definitive diagnosis regarding the type of infection traditionally requires microbiological analysis with seeding of blood cultures, incubation for 16 to 24 hours, plating of the causative microorganisms on solid media, another incubation period, and final identification after 1 to 2 days. Even with immediate aggressive treatment, some patients develop multiple organ dysfunction syndrome and ultimately die. Every hour lost before the correct treatment is administered can make a critical difference in patient outcome. It is therefore important for physicians to quickly determine whether an infection is present and, if so, which antimicrobials may be effective for treatment. Summary of the Invention

[0004] Described herein are compositions, methods, systems, and / or kits for determining microbial viability in a sample. Some embodiments provided herein relate to a method for evaluating the growth of microorganisms in a sample. In some embodiments, the method includes the steps of providing a sample containing microorganisms, separating the sample containing microorganisms into one or more portions of the sample containing microorganisms, forming one or more populations of microdroplets that encapsulate the microorganisms from the sample, contacting the one or more portions of the sample with an antimicrobial agent either before or after forming the one or more populations of microdroplets, and measuring the microbial viability of the microorganisms encapsulated in the microdroplets, thereby determining the susceptibility of the microorganisms to the antimicrobial agent. In some embodiments, the 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 the antimicrobial agent. In some embodiments, the step of measuring microbial viability includes obtaining a measure of microbial viability from a discrete subset of microdroplets from a first population of microdroplets from a first portion of the sample measured at a first time point, and obtaining a measure of microbial viability from a discrete subset of microdroplets from a second population of microdroplets from the first portion of the sample measured at a second time point. In some embodiments, the step of measuring microbial viability further includes comparing the measure of microbial viability from the discrete subset of microdroplets measured at the first time point to the measure of microbial viability from the discrete subset of microdroplets measured at the second time point for a plurality of subsets of microdroplets measured at the first and second time points. In some embodiments, the measuring step further includes obtaining a measure of microbial viability from a discrete subset of microdroplets in an additional population of microdroplets from the first portion of the sample measured at an additional time point. In some embodiments, an average of the microbial viability measurements from the multiple separate subsets of microdroplets measured at a first time point is compared to an average of the microbial viability measurements from the multiple separate subsets of microdroplets measured at a 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, one or more distinct subsets of microdroplets from the first population are absent in the second population, and one or more distinct subsets of microdroplets from the second population are absent in the first population. In some embodiments, the population of microdroplets is incubated for any one or more of 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 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 prior to measuring microbial viability. In some embodiments, the 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. In some embodiments, the step of measuring microbial viability further comprises obtaining a microbial viability measurement from a distinct subset of microdroplets in a first population of microdroplets from a first portion of the sample measured at a first time point, and obtaining a microbial viability measurement from a distinct subset of microdroplets in a second population of microdroplets from the first portion of the sample measured at a second time point. In some embodiments, the measuring step further comprises assigning the measurements obtained at the first and second time points to distinct subsets of microdroplets, at least some of the distinct subsets of microdroplets in the first population being the same distinct subsets of microdroplets in the second population such that a microbial viability measurement obtained for the distinct subset of microdroplets at the first time point can be compared to a microbial viability measurement obtained for that same distinct subset of microdroplets obtained at a second time point.

[0005] In some embodiments, the step of measuring microbial viability further comprises comparing a measurement of microbial viability obtained for a discrete subset of microdroplets at a first time point to a measurement of microbial viability obtained for the same discrete subset of microdroplets obtained at a second time point. 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. In some embodiments, the step of measuring further comprises obtaining a measurement of microbial viability from a discrete subset of microdroplets in an additional population of microdroplets measured at an additional time point. In some embodiments, the step of measuring microbial viability comprises obtaining a measurement of microbial viability from a discrete subset of microdroplets in a first population of microdroplets from a first portion of the sample measured at a first time point, and obtaining a measurement of microbial viability from a discrete subset of microdroplets in a second population of microdroplets from a first portion of the sample measured at a second time point. 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 measurement of microbial viability is the percentage of the multiple separate subsets of microdroplets measured at a time that exceed the threshold. In some embodiments, the preset threshold is exceeded if the indicator reaches the determined measurement of microbial viability. In some embodiments, the composite value of the measurement of microbial viability from the multiple separate subsets of microdroplets measured at a first time is compared to the composite value of the measurement of microbial viability from the multiple separate subsets of microdroplets measured at a second time. In some embodiments, the step of measuring microbial viability further includes comparing the measurement of microbial viability from the separate subsets of microdroplets obtained at the first time with the measurement of microbial viability from the separate subsets of microdroplets obtained at the second time for the multiple subsets of microdroplets measured at the first and second time points. In some embodiments, the measurements of microbial viability obtained at the first and second time points are not assigned to separate subsets of microdroplets.In some embodiments, the step of measuring microbial viability further comprises, for a plurality of subsets of microdroplets, comparing a measurement of microbial viability obtained for a separate subset of microdroplets at a first time point with a measurement of microbial viability obtained for the same separate subset of microdroplets obtained at a second time point. In some embodiments, one or more separate subsets of microdroplets from the first population are not present in the second population, and one or more separate subsets of microdroplets from the second population are not present in the first population. In some embodiments, if the indicator of microbial viability exceeds a preset threshold, the step of measuring further comprises obtaining a measurement of microbial viability from a separate subset of microdroplets in the additional population of microdroplets measured at an additional time point. In some embodiments, the method further comprises incubating one or more portions of the sample contacted with the antimicrobial agent for different periods of time before forming 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 period of time sufficient 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 incubated for any one or more of the following periods before forming microdroplets: 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 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.In some embodiments, the sensitivity of microorganisms to antibiotics is determined by measuring the survival rate of microorganisms in the presence of different concentrations of antibiotics.

[0006] In some embodiments, measuring microbial viability in the droplets is performed using a technique that affects the viability of the microorganisms, including determining bacterial concentration by genetic analysis, including qPCR or fluorescent in-situ hybridization (FISH) after lysis. In some embodiments, measuring microbial viability in the droplets is performed using a technique that does not affect the viability of the microorganisms, including measuring solution turbidity, pH, or fluorescence of a metabolically active dye. In some embodiments, measuring microbial viability includes obtaining a measurement of 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, and 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. In some embodiments, the measurement of microbial viability is whether an indication 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 prior to or during the formation of the microdroplets. In some embodiments, the method further comprises immobilizing one or more populations of microdroplets encapsulating the microorganisms in an indexed array. In some embodiments, the method further comprises passing one or more populations of microdroplets encapsulating the 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 comprises measuring a fluorescent signal of the label. In some embodiments, the fluorescence is measured using a fluorescent 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 the 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 1. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium is E. coli, P. aeruginosa, S. aureus, S. epidermidis, E. faecalis, K. pneumoniae, E. cloacae, A. baumannii, S. marcescens, or 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, an aminoglycoside, ansamycin, a carbacephem, a carbapenem, a cephalosporin, a glycopeptide, a lincosamide, a lipopeptide, a macrolide, a monobactam, a nitrofuran, a penicillin, a polypeptide, a quinolone, a streptogramin, a sulfonamide, or a tetracycline, or a combination thereof. In some embodiments, the antibiotic is ampicillin. In some embodiments, the microdroplets include an oil phase and a surfactant phase. In some embodiments, the microdroplets are formulated by a microfluidic channel, stirring, electric force, or membrane filtration. In some embodiments, one or more populations of the microdroplets are formulated as a stable water-in-oil emulsion. In some embodiments, determining the susceptibility of the microorganism to an antimicrobial agent is accomplished in a shorter time than if the microdroplets were not formed. In some embodiments, determining the antimicrobial susceptibility of the microorganism is accomplished in a time period ranging from 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. In some embodiments, determining the antimicrobial susceptibility of the microorganism 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, or 3 hours or less.In some embodiments, the sample is whole blood, positive blood cultures, 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, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyle, chyle, bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool water, pancreatic juice, sinus lavage, bronchopulmonary aspirate or other lavage, blastocyst cavity, umbilical cord blood, or maternal circulation. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows a schematic diagram of one embodiment of a method for assessing microbial viability for discrete subsets of microdroplets. [Diagram 2] FIG. 2 shows a graphical representation of one embodiment of the method of FIG. 1 for measuring microbial viability, showing microdroplet fluorescence intensity as a function of time for microdroplets containing no antibiotic or a concentration of antibiotic below the minimum inhibitory concentration (MIC) (solid line) compared to microdroplets containing an antibiotic at a concentration above the MIC (dashed line). [Figure 3A] 3A shows the results of one embodiment of a microdroplet antibiotic susceptibility test using a fluorescent viability indicator. FIG. 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours for a sample without antibiotic (condition A) and a sample with antibiotic at twice the MIC (condition B). FIG. 3B shows micrographs of microdroplets under the conditions of FIG. 3A. FIG. 3C shows a micrograph of a microdroplet containing E. coli without antibiotic. FIG. 3D shows a micrograph of a microdroplet containing E. coli and antibiotic at twice the MIC. [Figure 3B]3A shows the results of one embodiment of a microdroplet antibiotic susceptibility test using a fluorescent viability indicator. FIG. 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours for a sample without antibiotic (condition A) and a sample with antibiotic at twice the MIC (condition B). FIG. 3B shows micrographs of microdroplets under the conditions of FIG. 3A. FIG. 3C shows a micrograph of a microdroplet containing E. coli without antibiotic. FIG. 3D shows a micrograph of a microdroplet containing E. coli and antibiotic at twice the MIC. [Figure 3C] 3A shows the results of one embodiment of a microdroplet antibiotic susceptibility test using a fluorescent viability indicator. FIG. 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours for a sample without antibiotic (condition A) and a sample with antibiotic at twice the MIC (condition B). FIG. 3B shows micrographs of microdroplets under the conditions of FIG. 3A. FIG. 3C shows a micrograph of a microdroplet containing E. coli without antibiotic. FIG. 3D shows a micrograph of a microdroplet containing E. coli and antibiotic at twice the MIC. [Figure 3D] 3A shows the results of one embodiment of a microdroplet antibiotic susceptibility test using a fluorescent viability indicator. FIG. 3A shows the change in fluorescence intensity of microdroplets at 1, 2, and 3 hours for a sample without antibiotic (condition A) and a sample with antibiotic at twice the MIC (condition B). FIG. 3B shows micrographs of microdroplets under the conditions of FIG. 3A. FIG. 3C shows a micrograph of a microdroplet containing E. coli without antibiotic. FIG. 3D shows a micrograph of a microdroplet containing E. coli and antibiotic at twice the MIC. [Figure 4] FIG. 1 shows a schematic diagram of one embodiment of a method for assessing microbial viability for multiple microdroplets. [Diagram 5] 1 shows a schematic diagram of one embodiment of a method for assessing microbial viability on a microdroplet based on measuring microbial viability in the microdroplet that exceeds a predetermined threshold. [Figure 6]FIG. 6 shows a graphical representation of one embodiment of the microbial viability measurement of the method of FIG. 5, illustrating microbial viability as a function of time in microdroplets that exceed a threshold, including microdroplets that contain no antibiotic or a concentration of antibiotic below the MIC (solid line), compared to microdroplets that contain an antibiotic at a concentration above the MIC (dashed line). [Figure 7] FIG. 1 shows a schematic diagram of one embodiment of a method for assessing microbial viability by incubating samples in antibiotics and preparing microdroplets at each measurement point. [Figure 8] FIG. 8 is a graphical representation of one embodiment of the microbial viability measurement of the method outlined in FIG. 7, showing microbial viability as a function of time in microdroplets that exceed a threshold, including microdroplets that contain no antibiotic or a concentration of antibiotic below the MIC (solid line), compared to microdroplets that contain an antibiotic at a concentration above the MIC (dashed line). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Rising antimicrobial resistance and a shrinking antimicrobial pipeline are creating a global public health crisis in which an increasing number of patients are infected with antimicrobial-resistant bacteria. Appropriate antimicrobial therapy that can be given within hours of the onset of infection can have a positive impact on patient outcomes. However, current practices for identifying infections require excessive time. Thus, there is a strong need for more rapid antimicrobial susceptibility testing, preferably one that can identify specific antimicrobial susceptibility within hours after a blood sample is drawn. Thus, this type of rapid testing would enable physicians to start optimal drug therapy from the beginning, rather than starting with suboptimal or completely ineffective antimicrobials, thus greatly improving clinical responsiveness. Major efforts to improve current antimicrobial susceptibility testing (AST) practices aim to shorten the target time to result (TTR).

[0009] In microdroplet-based microbial identification (ID) and antimicrobial susceptibility testing (AST), clinical specimens are divided into small-volume microdroplets, each of which contains a small number of organisms, typically 1-5 organisms per microdroplet. This approach allows for a high effective concentration of organisms within each occupied microdroplet, and because each organism is sequestered in an independent microdroplet, may allow for rapid time-to-result and direct-to-sample testing of specimens with polymicrobial infections. The challenge with microdroplet-based ID / AST techniques is that a large number 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.

[0010] Accordingly, some embodiments provided herein relate to a method for determining the susceptibility of a microorganism to an antimicrobial agent by providing a sample having a microorganism, encapsulating the microorganism from the sample in microdroplets, where the sample is divided into portions before or after forming the microdroplets, contacting each portion of the sample with different concentrations of an antimicrobial agent either before or after forming the microdroplets, and measuring the microbial viability of the microorganism within the microdroplets. The methods described herein may be modified, altered, or varied according to the embodiments, methods, systems, and configurations described in more detail herein. Some of the embodiments, methods, and configurations described herein include one or more advantages including, for example, a direct-to-sample method without the need for specimen culture to obtain isolated colonies; increased bacterial concentration due to partitioning of the sample in the microdroplets resulting in a high starting concentration in the occupied microdroplets; and rapid growth detection where separate growth events can be detected.

[0011] Without wishing to be bound by theory, embodiments of the antimicrobial susceptibility testing methods described herein can rapidly detect microbial infections (e.g., bacteremia, fungemia, viremia) in samples caused by different pathogens and provide antimicrobial sensitivity and resistance profiles of the causative agents (e.g., microbial pathogens). Further 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. Both of these advantages arise primarily due to the division of clinical specimens into very small volume microdroplets, each of which can be tailored to contain one or less organisms and can be handled individually. The microdroplet-based AST embodiments described herein can be performed using microdroplets directly from clinical specimens (without the need for culturing in solid media), resulting in significant savings in time to results (TTR). The embodiment of dividing bacteria into small volumes also allows for high effective concentrations of organisms, resulting in faster reaction kinetics, thereby further improving TTR.

[0012] An additional advantage of embodiments using microdroplets to perform AST is the improved TTR for testing "late-onset resistant" bacterial phenotypes. These bacterial drug combinations are particularly challenging for both current and emerging AST technologies due to the very long TTR required to accurately identify the 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 an antimicrobial. In some embodiments, the method for determining the antimicrobial MIC is performed by exposing a sample having a microorganism to a concentration of an antimicrobial, encapsulating the microorganism within a microdroplet, and measuring microbial viability of the microorganism within the microdroplet.

[0013] As described herein, modifications of the method may be implemented based on the preparation of the microdroplets, the measurement of microbial viability in the microdroplets, and / or the steps for measuring microbial viability. Some modifications of the method are described herein as forms. It will be understood by those skilled in the art that additional modifications and / or forms may be implemented, and that in some embodiments, aspects of any given form may be exchanged, replaced, or substituted with aspects from a different form. Furthermore, in some embodiments, various aspects of any given form may be removed, added, altered, or otherwise modified.

[0014] <Module 1 - Measurement of microbial viability in a population of microdroplets> Some embodiments provided herein relate to a first mode for determining microbial viability. The first mode for determining microbial viability is shown in FIG. 1. As described herein, the first mode for determining microbial viability includes the steps of providing a sample having microorganisms therein, dividing the sample into several portions, preparing microdroplets encapsulating the microorganisms from the sample either before or after the portions are formed (FIG. 1 shows the portions are first formed), contacting each portion with different concentrations of the antimicrobial agent of interest either before or after the microdroplets are prepared (FIG. 1 shows the antibiotic is added before forming the microdroplets), and measuring the viability of the microorganisms by measuring a signal of microbial viability. Measuring the signal of microbial viability can be performed by obtaining measurements of microbial viability from a separate subset of microdroplets from a population of microdroplets or from a plurality of separate subsets of microdroplets. The results of the measurements from the separate subsets of microdroplets at a first time point can then be combined to generate results for the portion of the sample from which the microdroplets were obtained at this first time point. This process can be repeated at additional time points to determine the microbial viability in the portion over time, thereby determining the susceptibility of the microorganism to a given concentration of antimicrobial. Typically, it is not necessary to measure the exact same population of microdroplets 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 derived. The results from the various portions exposed to different concentrations of antimicrobial can then be used to determine the susceptibility of the microorganism to the antimicrobial. The determination of antimicrobial susceptibility can be made upon determining that the measurements of microbial viability do not increase over time, indicating that no growth of the microorganism occurs over time when in contact with the concentration of antimicrobial present in the portion of the sample where the measurements were made. Conversely, an increase in the measurements of microbial viability over time indicates growth of the microorganism at the concentration of antimicrobial present in the portion of the sample where the measurements were made. The minimum inhibitory concentration can be determined utilizing the antimicrobial susceptibility and / or microbial growth results from several portions of the sample exposed to different concentrations of antimicrobial.

[0015] In some embodiments, the step of preparing microdroplets from each portion results in a population of microdroplets in each portion. In some embodiments, the size of the microdroplets is sufficient to encapsulate the microorganism of interest, for example, within a range of about 2 μm to about 500 μm, 2 μm to 200 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 and the method 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 antimicrobial agent and one or more populations of microdroplets are formed therefrom; a second portion of the sample is contacted with a second concentration of antimicrobial agent and one or more populations of microdroplets are formed therefrom; similarly for a desired number of portions, each portion having a different concentration of antimicrobial agent to be tested. Alternatively, microdroplets may be formed from the portions prior to contacting the portions with the antimicrobial agent. The concentration of the antimicrobial agent is typically selected to include a range of antimicrobial agent concentrations, including the concentration of the antimicrobial agent that is or is likely to be the minimum inhibitory concentration (MIC), as described in more detail herein. In some embodiments, one or more portions of the sample are not exposed to any antimicrobial agent (zero antimicrobial agent concentration), e.g., for control purposes.

[0016] In some embodiments, the first aspect includes measuring microbial viability including obtaining a measurement of microbial viability from a separate subset of microdroplets from a first population of microdroplets from a portion of the sample measured at a first time point, and obtaining a measurement of microbial viability from a separate subset of microdroplets from a second population of microdroplets from the same portion of the sample at a second time point to determine a change in microbial viability signal over time. In some embodiments, measurements are obtained from multiple separate subsets of microdroplets at the first and / or second time points. A subset of microdroplets includes one or more than one microdroplet, for example, a subset may include 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 1 0000 microdroplets, or a range defined by any two of the preceding values, e.g., 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. In some embodiments, the measurement of microbial viability may be performed by measuring additional separate subsets of the microdroplets at additional time points, e.g., a third time point, a fourth time point, a fifth time point, etc., as well as for a given number of time points sufficient to determine microbial viability in the microdroplets. For example, in some embodiments, the measurement of microbial viability is obtained from a third population of microdroplets obtained from the same portion of the sample at a third time point.Microbial viability measurements for additional time points and populations of microdroplets may be performed as required for any given assay, e.g., from the fourth population of microdroplets at a fourth time point, from the fifth population of microdroplets at a fifth time point, from the sixth population of microdroplets at a sixth time point, from the seventh population of microdroplets at a seventh time point, from the eighth population of microdroplets at an eighth time point, from the ninth population of microdroplets at a ninth time point, from the tenth population of microdroplets at a tenth time point, or more, etc. In some embodiments, microbial viability measurements are performed at time points selected in the range from time 0 (e.g., the time the microdroplets encapsulating the microorganisms are formed) to time 24 hours. Thus, in some embodiments, the time points include measurements at 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 a range defined by any two of the foregoing values. In some embodiments, the time points include 0-15 minutes, 0-10 minutes, 0-5 minutes, 0-1 minute, 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, 0-0.5 hours, 0.25-6 hours, 0.25-5 hours, 0.25-4 hours, 0.25-3 hours, 0.25-2 hours, 0.25-1 hour, 0.25-0.5 hours, 1-6 hours, 1-5 hours, 1-3 hours, or 1-2 hours, or any given number of measurements until a determination of antimicrobial susceptibility can be achieved. In some embodiments, antimicrobial susceptibility occurs within 24, 20, 15, 10, 8, 5, 3, or 1 hour or less from contacting the sample with the antimicrobial to making the antimicrobial susceptibility determination, or an amount of time sufficient to make a determination that the microorganism is susceptible and / or not susceptible to the antimicrobial tested.In some embodiments, the antimicrobial susceptibility occurs in 24 hours or less, 15 hours or less, in some embodiments, 12 hours or less, in some embodiments, 10 hours or less, in some embodiments, 8 hours or less, in some embodiments, 5 hours or less, in some embodiments, 3 hours or less, in some embodiments, 1 hour or less. In some embodiments, the determination of whether a microorganism is susceptible and / or not susceptible to an antimicrobial is achieved in less time using the microdroplet susceptibility tests described herein than when testing antimicrobial susceptibility when not formed in a microdroplet. This process can be repeated for additional portions of the sample. In some embodiments, this process is repeated for a second, third, fourth, fifth, or more portions of the sample.

[0017] Viability measurements, once taken at first, second, and subsequent time points for a given portion of the sample (e.g., first, second, third, etc. portions), can be compared to determine viability over time for each portion. In some embodiments, the separate measurements at each of the various time points are aggregated such that the aggregate measurements at each time point can be compared to one another. For example, in one embodiment, an average of the microbial viability measurements from a plurality of separate subsets of microdroplets measured at a first time point is compared to an average of the microbial viability measurements from a plurality of separate subsets of microdroplets measured at a second time point. In some embodiments, rather than aggregating data from measurements of several separate subsets before comparing between time points, separate data points from the subsets are compared over time, and the comparisons over time from the separate data points are aggregated to obtain a comparison. For example, in one embodiment, a microbial viability measurement from a separate subset of microdroplets measured at a first time point is compared to a microbial viability measurement from a separate subset of microdroplets measured at a second time point for multiple subsets of microdroplets measured at the first and second time points, and the multiple separate comparisons may be averaged to obtain a comparison between the first and second time points.

[0018] In some embodiments, the individual microdroplets in the subset and / or first population measured at a first time point are not the same microdroplets in the subset and / or second population measured during a second or subsequent time point. There may be an overlap between the individual microdroplets in the subset and / or first population measured at a first time point and the individual microdroplets in the subset and / or population measured at a second or subsequent time point, but it is not necessary that the exact same individual microdroplets are measured at each time point. Thus, in some embodiments, one or more separate subsets of microdroplets from the first population are not present in the second population, and one or more separate subsets of microdroplets from the second population are not present in the first population, as well as for the third, fourth, fifth, and any subsequent populations and time points. In other embodiments, the same separate subsets of microdroplets are measured at the first and second time points, and any subsequent time points, such that the separate subsets of microdroplets from the first population are the same separate subsets of microdroplets from the second and subsequent populations. In some embodiments, the microbial viability measurements taken at the first, second, and any subsequent time points are not assigned to separate subsets of microdroplets, even if the same separate subset of microdroplets is measured at each time point.

[0019] As shown in the embodiment in Figure 2, measurements of microbial viability can be made over time by determining fluorescence intensity, e.g., resazurin fluorescence intensity. Fluorescence intensity measurements are shown for microdroplets exposed to an antimicrobial below the minimum inhibitory concentration (MIC) or no antimicrobial added (solid lines), and for microdroplets exposed above the MIC of an antimicrobial (dashed lines). The solid lines show increased fluorescence intensity due to increased microbial growth over time due to the absence of an antimicrobial or the presence of an antimicrobial at a concentration below the MIC. The dashed lines show no or insignificant increase in fluorescence intensity due to no microbial growth over time as a result of an antimicrobial concentration above the MIC.

[0020] FIG. 3 illustrates the results of microbial viability measurements using an embodiment of the method described in form 1. FIG. 3A shows microdroplet AST using a fluorescent viability indicator for E. coli encapsulated in microdroplets. Microdroplets encapsulating E. coli were prepared from a first portion of the sample that was not exposed to antibiotics (condition A) or a second portion of the sample that was exposed to antibiotics at a concentration of twice the MIC (condition B). The antibiotic used in this example was ampicillin. The microdroplets were formed and incubated for a defined period of time, including 1 hour (t1), 2 hours (t2), and 3 hours (t3). FIG. 3A shows plots of measurements from separate subsets of microdroplets (in this case single microdroplets) from three populations of microdroplets at three time points (t1, t2, t3) obtained from two portions of the sample (conditions A or B). Triangles represent the average of each subset, and error bars indicate one standard deviation of the average. FIG. 3B shows micrographs of representative microdroplets from each population at each time point from each of conditions A and B. At time 1 hour, the fluorescence intensity remained low for microdroplets in both conditions A and B. At times 2 and 3 hours, the fluorescence intensity increased for microdroplets in condition A, indicating E. coli growth due to the absence of antibiotic, while the fluorescence intensity remained low for microdroplets in condition B, indicating no E. coli growth due to the presence of ampicillin at twice the MIC. FIG. 3C shows micrographs of microdroplets not exposed to antibiotics and showing the presence of E. coli in the absence of antibiotics (condition A). FIG. 3D shows micrographs of microdroplets exposed to ampicillin at twice the MIC (condition B), showing no E. coli presence.

[0021] <Module 2 - Measurement of microbial viability over time in separate subsets of microdroplets> Some embodiments provided herein relate to a second form for determining microbial viability, which relates to form 1 and other forms disclosed herein. Thus, the disclosures of other forms, including but not limited to form 1, are also applicable to form 2. One embodiment of form 2 for determining microbial viability is shown in FIG. 4. As described for form 1, the second form for determining microbial viability includes the steps of providing a sample having microorganisms therein, dividing the sample into several portions, preparing microdroplets encapsulating the microorganisms from the sample either before or after the portions are formed (FIG. 4 shows forming the portions first), contacting each portion with different concentrations of the antimicrobial agent of interest either before or after the microdroplets are prepared (FIG. 4 shows adding the antimicrobial agent before forming the microdroplets), and measuring the viability of the microorganisms by measuring the signal of microbial viability in a separate subset of microdroplets from the population of microdroplets at first, second, and optionally additional time points. As disclosed herein, the time points can be selected over a range of times and times. Furthermore, as disclosed herein, the time points are selected to provide sufficient time for the determination of the microorganism being susceptible and / or not susceptible to an antimicrobial agent. In some embodiments, the determination of the microorganism being susceptible and / or not susceptible to an antimicrobial agent is achieved in less time (e.g., 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 hour or less) using the microdroplet susceptibility test described herein than when testing the antimicrobial susceptibility when not formed in a microdroplet. An embodiment of form 2 includes allocating the microbial viability signal from the population of microdroplets to a separate subset of microdroplets such that the results from a particular separate subset of microdroplets (e.g., a single microdroplet) at a first time point can be compared to the results from that same separate subset of microdroplets (e.g., a single microdroplet) at a second time point across the population of microdroplets.Typically, for each portion of the sample exposed to different concentrations of the antimicrobial, measurements are obtained from a separate subset of microdroplets sufficient to ensure that a representative number of microdroplets are measured for each portion of the sample. The results over time for each separate subset of microdroplets can then be combined to generate a result for the portion of the sample from which the microdroplets were obtained, and the results from the various portions exposed to different concentrations of the antimicrobial are used to determine the susceptibility of the microorganism to the antimicrobial.

[0022] In one embodiment of form 2, a sample containing a microorganism is separated into one or more portions of the sample containing the microorganism, and the one or more portions of the sample are contacted with an antimicrobial agent, each of the one or more portions of the sample being contacted with a different concentration of the antimicrobial agent. One or more populations of microdroplets encapsulating the microorganism are then formed from the one or more portions of the sample. The viability of the microorganisms encapsulated within the microdroplets is then measured. An embodiment of form 2 includes obtaining microbial viability measurements from a separate subset of microdroplets in a first population of microdroplets from a first portion of the sample measured at a first time point, and obtaining microbial viability measurements 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, and further including assigning the measurements obtained at the first and second time points to separate subsets of microdroplets, at least some of the separate subsets of microdroplets in the first population being the same separate subsets of microdroplets in the second population such that the microbial viability measurements obtained for the separate subset of microdroplets at the first time point can be compared to the microbial viability measurements obtained for that same separate subset of microdroplets obtained at the second time point. Some embodiments include actually performing, for at least a plurality of subsets of microdroplets, a comparison of microbial viability measurements obtained for a separate subset of microdroplets at a first time point with microbial viability measurements obtained for that same separate subset of microdroplets obtained at a second time point (and any additional time points).As described herein, a subset of microdroplets includes one or more than one microdroplet, for example, a subset may include 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, 900 Including or at least including 0, or 10000 microdroplets, or a range defined by any two of the preceding values, e.g., 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.

[0023] As discussed herein with respect to Form 1, additional populations of microdroplets and subsets thereof can be measured at additional time points (e.g., a third, fourth, fifth, sixth, seventh, etc.). 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 at least one or more separate subsets of microdroplets in the first population are the same separate subsets of microdroplets in the second and any subsequent populations such that microbial viability measurements taken for the separate subset of microdroplets (e.g., a single microdroplet) at the first time point can be compared to microbial viability measurements taken for that same separate subset of microdroplets taken at the second and any subsequent time points (e.g., a third, fourth, fifth, sixth, etc.). In some embodiments, not all microdroplets in the first and second, or any subsequent populations, must be utilized to make the comparison, such that at least a distinct subset of the microdroplets in the first population are not present in the second population or any subsequent population, and at least a distinct subset of the microdroplets in the second population or any subsequent population are not present in the first population.

[0024] In some embodiments, including but not limited to those of form 2, measuring the separate subsets of microdroplets at the first and subsequent time points may include indexing the separate subsets of microdroplets. Indexing can be used, for example, to assign a measurement of microbial viability to a particular subset of microdroplets or to ensure that a representative number of subsets of microdroplets are measured for a portion of the sample. In some embodiments, the microdroplets are placed in an indexed array and the measurement of microbial viability is performed in the indexed array. Indexing can be performed by methods known in the art. For example, indexing can be performed in some embodiments by incorporating optical and / or magnetic reporters (including, for example, organic fluorophores, quantum dots, SERS tags) into each sample microdroplet.

[0025] <Mode 3 - Measurement of microbial viability in microdroplets when the signal exceeds a threshold> Some embodiments provided herein relate to a third form for determining microbial viability, which relates to other forms disclosed herein, such as forms 1 and 2. Thus, disclosures related to other forms, including but not limited to forms 1 and 2, are also applicable to form 3. An embodiment of the third form for determining microbial viability is shown in FIG. 5. As described with respect to form 1, the third form for determining microbial viability includes the steps of providing a sample having microorganisms therein, dividing the sample into several portions, preparing microdroplets encapsulating the microorganisms from the sample either before or after the portions are formed (FIG. 5 shows forming the portions first), contacting each portion with different concentrations of the antimicrobial agent of interest either before or after preparing the microdroplets (FIG. 5 shows adding the antibiotic before forming the microdroplets), and measuring the viability of the microorganisms by measuring a signal of microbial viability. Measuring the signal of microbial viability can be performed by measuring the microbial viability in one or more separate subsets of microdroplets (e.g., single microdroplets) from a population of microdroplets. The embodiment of form 3 includes measuring whether the signal of microbial viability exceeds a threshold value in a separate subset of microdroplets (e.g., a single microdroplet), as opposed to simply measuring the level or amount of the signal. Thus, in this sense, the measurement of microbial viability is digital - either exceeding the threshold or not. This allows the change in the amount or number of the subset of microdroplets in the portion of the sample that exceeds the threshold to be measured over time. If the amount or number remains constant or does not increase with time, the microorganisms in that portion are not viable and therefore sensitive to the antimicrobial concentration in that portion of the sample. In contrast, if the number or amount of the subset of microdroplets that exceed the threshold increases over time to a saturation level, this indicates that the microorganisms are viable at the concentration of antimicrobial in that portion of the sample.

[0026] In some embodiments, the step of measuring microbial viability includes obtaining a measurement of microbial viability from a separate subset of microdroplets in a first population of microdroplets from a first portion of the sample measured at a first time point, and 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, and the measurement of microbial viability is whether an indicator of microbial viability exceeds a preset threshold. In some embodiments, the preset threshold is exceeded if the measurement of microbial viability exceeds a determined amount to indicate that the microorganism is viable. For example, the threshold may include a value that, if exceeded, causes microbial growth to exceed an amount that may indicate that the antimicrobial (and antimicrobial concentration) being tested is below the minimum inhibitory concentration (MIC). In some embodiments, the threshold is exceeded if the indicator reaches a determined measurement of fluorescence. A subset of microdroplets includes one or more than one microdroplet, for example, a subset may include 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 1 0000 microdroplets, or a range defined by any two of the preceding values, e.g., 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.

[0027] The comparison of the 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 can be monitored over time to determine whether the subset changes from not exceeding the threshold to exceeding the threshold. This can be done for multiple subsets. Alternatively, the number or percentage of the subsets that exceed the threshold in the multiple subsets can be monitored over time to determine whether the number or percentage is increasing. In this case, it is not necessary that the exact 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, the time points can be selected at various times and over a range of times. Furthermore, as disclosed herein, the time points are selected to allow sufficient time for the determination that the microorganism is susceptible and / or not susceptible to an antimicrobial agent, for example, when the signal of microbial viability exceeds the threshold. In some embodiments, a determination regarding whether a microorganism is susceptible and / or not susceptible to an antimicrobial agent is achieved using the microdroplet susceptibility tests described herein in less time (e.g., 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 hour or less) than when testing for antimicrobial susceptibility when not formed in a microdroplet. In some embodiments, a composite value of microbial viability measurements from a plurality of separate subsets of microdroplets measured at a first time point is compared to a composite value of microbial viability measurements from a plurality of separate subsets of microdroplets measured at a second and / or subsequent time point. In some embodiments, the composite value of microbial viability measurements is the percentage of a plurality of separate subsets of microdroplets measured at a time point that exceed a threshold value.Some embodiments include comparing the microbial viability measurements from the separate subsets of microdroplets taken at the first time point with the microbial viability measurements from the separate subsets of microdroplets taken at the second time point and any subsequent time point for the multiple subsets of microdroplets measured at the first, second, and any subsequent time points. In some embodiments, the microbial viability measurements taken at the first, second, and any subsequent time points are not assigned to the separate subsets of microdroplets. In some embodiments, they are assigned, and some embodiments include comparing the microbial viability measurements taken for the separate subsets of microdroplets at the first time point with the microbial viability measurements taken for that same separate subset of microdroplets taken at the second and any subsequent time points for the multiple subsets of microdroplets. In some embodiments, one or more separate subsets of microdroplets from the first population are not present in the second population, and one or more separate subsets of microdroplets from the second population are not present in the first population, and similarly for any additional populations.

[0028] As used herein, the term "assign" or "assignment" refers to the attribution of a measurement of microbial viability to a microdroplet or a discrete population of microdroplets, where the measurement of microbial viability may correspond to a particular microdroplet or a discrete population of particular microdroplets.

[0029] The measurement of microbial viability may be performed as described herein, including, but not limited to, the measurement of microbial viability for a population of microdroplets and / or a separate subset of microdroplets, as described for Form 1 or Form 2. The measurement of microbial viability may be performed continuously or periodically over time until the signal of microbial viability exceeds a preset threshold. In some embodiments, for each separate subset of microdroplets or population of microdroplets, the number or percentage of the subset of microdroplets that exhibit a measurement of microbial viability, e.g., a measurement of fluorescence intensity, that exceeds a predetermined threshold is determined. For example, the number of subsets of microdroplets that exceed a predetermined threshold can be determined by scanning the subsets of microdroplets (e.g., a single, two, three, or four or more microdroplets per subset) with a high-throughput microdroplet analyzer and counting distinct detector events in which the measurement of microbial viability, e.g., fluorescence intensity, exceeds a preset threshold and / or does not. The number of microdroplet subsets that exceed the threshold value compared to the total number of microdroplet subsets evaluated provides a percentage of microbial viability for a given population of microdroplets (e.g., a given concentration of antimicrobial). In some embodiments, determining the percentage of microbial viability for the population of microdroplets is repeated at various time points to determine microbial viability for the population of microdroplets as a function of time for each portion of the sample having the same concentration of antimicrobial. Microbial viability is then evaluated and a MIC is determined from the measurements across the antimicrobial concentrations.

[0030] 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. 6. As shown in FIG. 6, microbial viability is measured as a function of time by measuring microbial viability and determining whether the measurement of microbial viability exceeds a predetermined threshold. Microdroplets that are not exposed to an antimicrobial or are exposed to an antimicrobial below the MIC increase in fluorescence intensity over time. When the fluorescence intensity signal exceeds a preset threshold, an indication of microbial viability is determined (solid line). In contrast, any signal that does not exceed the threshold indicates a viability of zero. For example, microdroplets exposed to an antimicrobial above the MIC do not exceed the preset threshold (dashed line) and a determination that microbial viability is zero is evaluated.

[0031] <Formation of microdroplets at the time of measurement> Some embodiments provided herein relate to a fourth form for determining microbial viability, which relates to other forms disclosed herein, such as forms 1, 2, and 3. Thus, disclosures relating 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. 7. As described with respect to form 1, the fourth form for determining microbial viability includes providing a sample having microorganisms therein, dividing the sample into several portions, and exposing each portion to different concentrations of the antimicrobial agent of interest. In an embodiment of form 4, the microdroplets are not prepared immediately. Instead, the microorganisms in each portion of the sample with different concentrations of the antimicrobial agent are cultured for a period of time, and microdroplets are prepared from each portion at various measurement time points. Measurement of the microbial viability of the microorganism is then performed by measuring the signal of microbial viability of one or more separate subsets of microdroplets from the population of microdroplets, as described herein (including but not limited to embodiments of forms 1-3). Thus, in form 4, a portion of the sample is cultured for a period of time before forming the microdroplets that are formed at various measurement times. There may be several reasons for culturing a portion of the sample and creating microdroplets at various measurement times. For example, some microorganisms lose or acquire resistance to antimicrobials (e.g., due to quorum sensing) depending on the density of the microorganism in the culture. These effects may not be observed when the microorganism is cultured in a microdroplet environment. Another reason is that by maintaining the culture and creating microdroplets from the culture as needed at various time points, the viability of the microorganism in the microdroplet does not need to be maintained after the microdroplet is prepared. This may have one or more advantages, such as increased versatility in the materials and methods used to prepare the microdroplets, and / or increased ease of handling the microdroplets (e.g., no need to maintain temperature, oxygen level, etc.).In addition, since the viability of the microorganism does not need to be maintained, the means used to assess the viability may allow for growth inhibition or killing of the microorganism, and additional tests to inhibit growth or kill the microorganism may be performed (e.g., lysing the microorganism and performing genetic analysis). Form 4 may allow for the viability of the microdroplets to be maintained, and therefore the viability of the microdroplets prepared after incubation for various times may also be assessed over time. In this respect, the features of Form 4 may be combined with the features of certain embodiments disclosed herein (e.g., the embodiment of Form 2) in which the viability of the microdroplets is assessed over time.

[0032] In some embodiments, the method for measuring microbial viability includes incubating one or more portions of a sample contacted with an antimicrobial agent for different time periods before forming one or more populations of microdroplets, whereby microdroplets are formed from each of one or more portions of the sample at different time points. In some embodiments, one or more portions of a sample are incubated for a period sufficient to cause any bacterial density-dependent phenomenon to occur. In some embodiments, one or more portions of a sample are incubated for any one or more of the following periods before forming the microdroplets: 0 hours, 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 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. In some embodiments, after forming the microdroplets, the microdroplets are measured to determine microbial viability. In some embodiments, the measurement of the microdroplets can be performed regardless of whether the microorganisms encapsulated within the microdroplets are viable or non-viable. In some embodiments, the step of measuring microbial viability includes obtaining a measurement of 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, and 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. In some embodiments, the measurement of microbial viability is whether an indication of microbial viability exceeds a preset threshold.A subset of microdroplets includes one or more than one microdroplet, for example, a subset may include 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 1 0000 microdroplets, or a range defined by any two of the preceding values, e.g., 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. Further details regarding the measurement of microbial viability in a subset of microdroplets that can be used in embodiments of Form 4 are provided herein, including but not limited to the disclosure regarding Forms 1, 2, and 3. In some embodiments, methods of measuring microbial viability using microdroplets formed at each time point provide advantages including, for example, end-point AST readouts that allow for detection chemistries that affect viability, bulk incubation of samples prior to microdroplet generation that allows for determination of density-dependent resistance mechanisms, and maximized microdroplet occupancy.

[0033] In some embodiments, the step of measuring the microbial viability in the droplet is performed using a technique that affects the viability of the microorganism. Techniques that affect the viability of the microorganism may include, for example, techniques that use lysis of the microorganism, such as determining the bacterial concentration by genetic analysis techniques that may include qPCR or fluorescent in-situ hybridization (FISH) after lysis. In some embodiments, the step of measuring the microbial viability in the droplet is performed using a technique that does not affect the viability of the microorganism. Techniques that do not affect the viability of the microorganism may include, for example, measuring solution turbidity, pH, or fluorescence of metabolically active dyes.

[0034] One embodiment of Form 4 is shown in Figure 8, which shows the measured microbial viability as a function of time in terms of fluorescence intensity. Microdroplets that are not exposed to an antimicrobial agent or that are exposed to an antimicrobial agent below the MIC increase in fluorescence intensity (solid line). In contrast, microdroplets that are exposed to an antimicrobial agent above the MIC do not exceed the preset threshold and therefore no increase in fluorescence intensity is observed (dashed line).

[0035] The time point for incubating the portion of the sample containing the antimicrobial agent prior to forming the microdroplets is selected in the range of time 0 (e.g., the time the antimicrobial agent contacts the portion of the sample) to time 24 hours. Thus, in some embodiments, the time point includes incubating for 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 a range defined by any two of the foregoing values. In some embodiments, the time points include culturing for 0-15 minutes, 0-10 minutes, 0-5 minutes, 0-1 minute, 5-15 minutes, 5-10 minutes, or 10-15 minutes, or for 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, 0-0.5 hours, 0.25-6 hours, 0.25-5 hours, 0.25-4 hours, 0.25-3 hours, 0.25-2 hours, 0.25-1 hour, 0.25-0.5 hours, 1-6 hours, 1-5 hours, 1-3 hours, or 1-2 hours. After culturing for a given period of time, the microdroplets are formed and a measurement of microbial viability is typically determined as soon as possible after forming the microdroplets. The determination of microbial viability is performed to determine whether a microorganism is susceptible and / or not susceptible to an antimicrobial agent. In some embodiments, the determination regarding whether a microorganism is susceptible and / or not susceptible to an antimicrobial agent is achieved in less time using the microdroplet susceptibility tests described herein than when testing for antimicrobial susceptibility when not formed in a microdroplet.

[0036] Some embodiments of Form 4 include forming microdroplets at each time point and measuring the microbial viability of the microdroplets by determining whether the microbial viability exceeds a predetermined threshold for each population of microdroplets formed at each measurement point. Figure 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 a desired range. For each portion, a microdroplet is prepared at each desired measurement time point. In some embodiments, a separate subset of the microdroplets is measured at each time point, which may be after an additional period of culturing the portion of the sample as disclosed herein, and the microbial viability is measured for a separate subset of the microdroplets (e.g. Form 1) or for the same subset of the microdroplets (e.g. Form 2). In some embodiments, the measurement of microbial viability is evaluated when the microbial viability signal exceeds a threshold (e.g. Form 3). However, rather than forming microdroplets at time 0 and then measuring the microbial viability of the microdroplets at subsequent time points, microdroplets are formed only at the measurement time point such that a portion of the sample is incubated with the antimicrobial until the measurement time point at which the microdroplets are formed and measured. In some embodiments, the measurement 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 that exceed a threshold value is determined as a function of time for each concentration of antimicrobial, and the microbial viability and MIC are assessed from the measurements.

[0037] In each of the above-described embodiments, each step is described as a separate process, but one or more of these steps may be implemented in a system. Thus, for example, one or more of the processes may be implemented in a microfluidic device. The microfluidic device may be used to automate the process and / or allow for the simultaneous processing of multiple samples. Those skilled in the art are well aware of the methods in the art for collecting, handling, and processing biological fluids that can be used in the practice of the present disclosure. In addition, the microfluidic devices for various steps may be combined into one system for carrying out any of the embodiments described herein.

[0038] <Measurement of microbial viability> As described, some embodiments provided herein relate to methods for antimicrobial susceptibility testing (AST). Any of the methods, embodiments, systems, or configurations described herein may be interchangeable, modified, or altered to allow microbial AST by encapsulating microorganisms in microdroplets. Without wishing to be bound by theory, the method embodiments, systems, and configurations described herein may have one or more advantages for measuring microbial viability, including high sensitivity near MIC, rapid determination of microbial viability, and / or large-scale determination of microbial viability over a wide range of antimicrobial concentrations.

[0039] In any of the embodiments, methods, systems, or configurations described herein, the microdroplets can be incubated at different antimicrobial concentrations for any period of time. Microbial growth can be monitored during the incubation period, and the incubation period can continue until there is a sufficient difference in detection signal, such as fluorescence or microbial count, between the microdroplets. For example, in some embodiments, the incubation can be about 15, 30, or 45 seconds, about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210, 240, 300, or 360 minutes or more. In one embodiment, the incubation is more than 2 hours, for example, 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, the skilled artisan can determine the optimal incubation duration for subsequent analyses, e.g. cell viability analyses.

[0040] In any of the embodiments, methods, systems, or configurations described herein, a separate subset of the microdroplets with encapsulated microorganisms therein may be measured to determine the susceptibility of the microorganisms in the microdroplets to an antimicrobial agent. In some embodiments, determining the susceptibility of the microorganisms to an antimicrobial agent is performed by measuring the fluorescence intensity of the microdroplets. In some embodiments, determining the susceptibility of the microorganisms to an antimicrobial agent is performed during or after incubation of the microorganisms. In some embodiments, determining the susceptibility of the microorganisms to an antimicrobial agent is performed continuously by continuously monitoring microbial viability or periodically by monitoring microbial viability at one or more separate time points.

[0041] In any of the embodiments, methods, systems, or configurations described herein, the sample may be divided into sample portions, and each portion of the sample may be contacted with a different concentration of antimicrobial agent. In some embodiments, for each portion of the sample, measurements of microbial viability are measured for a separate subset of microdroplets as a function of time, either by arranging the microdroplets in an indexed array and interrogating them or by using a high-throughput microdroplet reader. Measurements may be made, for example, by measuring the fluorescence intensity of the separate subset of microdroplets at an early time point (e.g., a first time point) and at a subsequent time point (e.g., a second, third, fourth, etc.). The measured fluorescence intensity for the separate subset of microdroplets may be used to assess bacterial viability at each antimicrobial agent concentration. In some embodiments, measurements are made at multiple time points during incubation, for example, a first time point and then a second time point. In some embodiments, measurements of microbial viability for separate subsets of microdroplets (e.g., a single microdroplet) are measured at time 0, 1 hour, 2 hours, 3 hours, or later. In some embodiments, the measurements are performed at any number of desired time points within a time window from time 0 to time 24 hours. As disclosed herein, the time points can be selected over a range of times and times. Additionally, as disclosed herein, the time points are selected to allow sufficient time for a determination that the microorganism is susceptible and / or not susceptible to an antibiotic, for example, when the microbial viability signal exceeds a threshold value. In some embodiments, the determination that the microorganism is susceptible and / or not susceptible to an antimicrobial is achieved in less time using the microdroplet susceptibility tests described herein than when testing antimicrobial susceptibility when not formed in a microdroplet. Thus, in some embodiments, determining the susceptibility of the microorganism to an antimicrobial is achieved within a time range of 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, or within an amount of time within a range defined by any two of the aforementioned values.In some embodiments, determining the antimicrobial susceptibility of the microorganism 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).

[0042] In some embodiments, the measurement of microbial viability is at a time point selected in the range from time 0 (e.g., the time the microdroplets encapsulating the microorganisms are formed) to time 24 hours. Thus, in some embodiments, the time points include measurements at 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 a range defined by any two of the foregoing values. In some embodiments, the time points include 0-15 minutes, 0-10 minutes, 0-5 minutes, 0-1 minute, 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, 0-0.5 hours, 0.25-6 hours, 0.25-5 hours, 0.25-4 hours, 0.25-3 hours, 0.25-2 hours, 0.25-1 hour, 0.25-0.5 hours, 1-6 hours, 1-5 hours, 1-3 hours, or 1-2 hours, or any given number of measurements until a determination of antimicrobial susceptibility can be achieved. In some embodiments, antimicrobial susceptibility occurs within 24, 20, 15, 10, 8, 5, or 3 hours or less from contacting the sample with the antimicrobial to making the antimicrobial susceptibility determination, or an amount of time sufficient to make a determination that the microorganism is susceptible and / or not susceptible to the antimicrobial tested.

[0043] In any of the embodiments, methods, systems, or configurations described herein, a measurement of microbial viability may be obtained from a separate subset of microdroplets from a first portion exposed to a first concentration of an antimicrobial at a first time point, and a measurement of microbial viability is obtained from a separate subset of microdroplets from a first portion exposed to a first concentration of an antimicrobial at a second time point. In some embodiments, the average measurement of microbial viability from the separate subset of microdroplets at the first time point is compared to the average measurement of microbial viability from the separate subset of microdroplets measured at the second time point. Microorganisms can be observed, for example, for growth in the presence of an antimicrobial (to determine resistance of the bacteria to a particular antimicrobial), cell death (to determine bactericidal activity), and / or inhibition of growth (to determine bacteriostatic activity). For example, microbial growth and / or cell death can be assessed by (i) counting the number of microorganisms in a subset of microdroplets compared to a control or standard, (ii) the total amount of microorganisms in a subset of microdroplets compared to a control or standard, (iii) the ratio of cells expressing at least one microbial marker in a subset of microdroplets compared to a control or standard, (iv) relative metabolite levels in a subset of microdroplets compared to a control or standard, or (v) any combination thereof. In some embodiments, microbial growth or a functional response of a microorganism can be determined or monitored in real time, for example, by microscopy or flow cytometry.

[0044] Any method known in the art for determining the viability of a microorganism in a sample can be used to determine the viability of the microorganism encapsulated in the microdroplets over time and in comparison to the growth of the encapsulated microorganism exposed to different concentrations of the antimicrobial agent. Overall, cell viability can be assayed using 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, 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 assays (MTT / XTT), green fluorescent protein, lactate dehydrogenase (LDH), methyl violet, propidium iodide, a DNA stain capable of discriminating between necrotic, apoptotic, and normal cells, resazurin, trypan blue (a live cell exclusion dye (the dye only crosses the cell membrane of dead cells)), 7-aminoactinomycin D, TUNEL assays, cell labeling or staining (e.g., cell permeable dyes (e.g., carboxyl diacetate, succinimidyl ester (carboxy-D FFDA, SE), cell impermeable dyes, cyanines, phenanthridines, acridines, indoles, imidazoles, nucleic acid stains, cell permeable reactive tracers (e.g., intracellularly activated 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), fluorescent 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; chromogenic 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 herein, any diacetate dye (including rhodamine-based dyes, fluorescin, or any combination thereof), resazurin / resorufin (alamar blue); R OS staining (e.g., any of the ROS stains described herein, DCFDA and related family, calcein acetoxymethyl and related family); membrane staining (e.g., Bodipy, FM1-43, FM4-64, and their functional equivalents, CellMask™ stains, Dil, DiO, DiA); biological staining (e.g., labeled antibodies, labeled chitin-binding proteins), optical imaging, microscopic imaging after staining, ELISA, mass spectrometry (e.g., of peptides, proteins, glycopeptides, lipopeptides, carbohydrates, and / or metabolites), metabolomic fingerprinting In some embodiments, the detection of microbial growth or functional response to an antimicrobial agent may be performed using solid-phase, microfluidic, or microdroplet-based assays. In some embodiments, the detection of microbial growth or functional response to an antimicrobial agent may include the use of a mass spectrometer. In some embodiments, the detection of microbial growth or functional response to an antimicrobial agent may include the detection of at least one metabolite, or metabolic profile. In some embodiments, the detection of microbial growth or functional response to an antimicrobial agent may include the detection of transcriptional changes. In some embodiments, the microbial viability in the microdroplet may be determined by performing imaging of the microorganisms in the microdroplet to collect both growth and morphology data. In some embodiments, the methods of analysis disclosed herein may be applied to microbial enumeration data to determine MIC. In some embodiments, the measurement of microbial viability may include:Distributing separate subsets of microdroplets in an indexed array or by using a high speed scanning device.

[0045] In any of the embodiments, methods, systems, or configurations described herein, the measurement of microbial viability may be performed by measuring a fluorescent dye in the 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 an increasing fluorescence intensity as bacteria grow in the microdroplets. In some embodiments, the measurement of microbial viability may be evaluated from measurements of microdroplet fluorescence, and the MIC may be determined therefrom. In some embodiments, an algorithm for MIC determination is developed and used.

[0046] <Micro droplets> The microdroplets provided herein and prepared in any of the forms described herein for measuring microbial viability can be manufactured using a microfluidic device or other device for microdroplet generation. In some embodiments, the microdroplets are of a size sufficient to encapsulate the microorganism of interest. For example, the microdroplets can be in a range of sizes, such as diameters of 2 μm to about 500 μm, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 10, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 μm, or diameters within a range defined by any two of the aforementioned values. In some embodiments, the microdroplets are in the range of 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 form of preparation of the microdroplets or the specific desired size range. One skilled in the art will understand that the microdroplet size can be modified in size to suit a specific microorganism of interest or a particular assay to be used. In some embodiments, the microdroplets have a volume in the picoliter range, 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 a volume within a range defined by any two of the aforementioned values. 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.

[0047] In any of the embodiments, methods, systems, or configurations described herein, the microdroplets may be formed after contacting the sample with the antimicrobial agent. In some embodiments, the microdroplets are formed within 1, 2, 3, 4, 5, 10, 15, 30, 45, or 60 seconds, or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or within 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or within a time frame defined by any two of the aforementioned values, after contacting one or more portions of the sample with the antimicrobial agent. In some embodiments, the microdroplets are formed within 1 second, 30 seconds, 1 minute, 15 minutes, 30 minutes, 1 hour, or 2 hours after contacting one or more portions of the sample with the antimicrobial agent.

[0048] In any of the embodiments, methods, systems, or configurations described herein, the microdroplets may be stable water-in-oil emulsions made by dispersing a sample containing the microorganism of interest in a continuous hydrophobic oil phase containing a surfactant. Examples of surfactants may include, but are not limited to, sulfonates, alkyl sulfates, monoesters of polyalkoxylated sorbitan, polyester polyols, aliphatic alcohol esters, aromatic alcohol esters, tall oil fatty acid diethanolamides, polyoxyethylene (5) sorbitan monooleate, ammonium salts of polyacrylic acid, ammonium salts of 2-acrylamido-2-methylpropanesulfonic acid / acrylic acid copolymers, alkyl sulfonates, alkylaryl sulfonates, α-olefin sulfonates, diphenyl ether sulfonates, sorbitan monooleate, α-sulfo fatty acid methyl esters. Combinations of surfactants may also be used.

[0049] Other methods of microdroplet production can include, for example, film emulsification, external forces such as mechanical shear (impeller-driven microdroplet generators), or electrical forces such as dielectrophoretic controlled microdroplet generators.

[0050] In any of the embodiments, methods, systems, or configurations described herein, the microdroplet size, surfactant, and oil may be optimized to inhibit or eliminate diffusion of molecules (e.g., assay reagents, dyes, antimicrobials, nutrients, metabolites) from the droplets to the microdroplets or from the oil to the microdroplets. In some embodiments, the microdroplet size, surfactant, and oil are optimized to enable or enhance gas diffusion from the oil phase into the microdroplets. In some embodiments, the microdroplet size, surfactant, and oil are optimized to improve microdroplet stability and inhibit undesired microdroplet coalescence or fusion. In some embodiments, the microdroplet composition is optimized to facilitate AST reactions occurring in clinical specimen matrices.

[0051] In any of the embodiments, methods, systems, or configurations described herein, the production of microdroplets can be performed in the presence of a sample containing a microorganism. In some embodiments, the production of microdroplets in the presence of a microorganism results in the encapsulation of a single microorganism in the microdroplet. The occupancy and distribution of the microorganism in the microdroplet can be altered by adjusting the concentration of the microorganism in the sample. Antimicrobial agents and viability indicator dyes at predetermined concentrations can also be incorporated into the microdroplets at the point at which they are formed, or can be added to each microdroplet at a later time point using techniques such as microinjection or microdroplet coalescence. These microdroplets can then be collected in a vial, incubated in appropriate bacterial growth conditions, and monitored at regular intervals.

[0052] The methods disclosed herein for performing AST using microdroplets can be applied to any embodiment or instrument that can measure the microdroplet characteristics of interest over time. These methods are independent of the embodiment used to generate and interrogate the microdroplets, but may benefit from certain microdroplet characteristics. Some of these characteristics are the size / volume of the microdroplets, the stability of the microdroplets over the duration of the AST, and the composition of the 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 allow high-throughput interrogation of microdroplets.

[0053] In any of the embodiments, methods, systems, or configurations described herein, the sample can be divided into portions, and each portion can be exposed to different concentrations of antimicrobial agents. In some embodiments, the microorganisms in the sample are first encapsulated in microdroplets and then exposed to the antimicrobial agents. In some embodiments, the microorganisms are encapsulated in microdroplets simultaneously with exposure to the antimicrobial agents. Thus, the sample containing the microorganisms can be exposed to the antimicrobial agents before, during, or after encapsulation in the microdroplets. Without being limited thereto, the microorganisms in one or more portions can be incubated with at least one antimicrobial agent, including two, three, or four or more antimicrobial agents, for example, to determine the effectiveness of combination therapy. At least one portion (e.g., one, two, three, four, five, six, seven, eight, nine, or ten or more) can be incubated without the addition of any antimicrobial agent to serve as a control. Alternatively or in addition, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) portion may be incubated with a broad-spectrum antimicrobial agent to serve as a positive control.

[0054] In any of the embodiments, methods, systems, or configurations described herein, portions of a sample containing different concentrations of antimicrobial agent may be introduced into a microdroplet generating device, and microdroplets are generated to encapsulate a single microorganism, or an average of less than one microorganism per microdroplet. In some embodiments, the microdroplets are generated simultaneously with exposure to the antimicrobial agent. In some embodiments, the microdroplets are generated at time zero from a sample containing different concentrations of added antimicrobial agent spanning a desired clinical range. In some embodiments, the microdroplets are measured to determine microbial viability. Measuring the microdroplets may include measuring a subset of the microdroplets. A subset of microdroplets includes one or more than one microdroplet, for example, a subset may include 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 1 0000 microdroplets, or a range defined by any two of the preceding values, e.g., 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.

[0055] The microdroplets encapsulating the microorganisms with various concentrations of antimicrobial agents can be incubated under any conditions suitable for microbial growth. One skilled in the art can easily determine optimal culture conditions for microbial growth (e.g., bacterial growth), such as incubating them at a suitable temperature and atmosphere, e.g., at about 20°C to about 45°C, with or without appropriate levels of oxygen and / or carbon dioxide. In some embodiments, incubation is at about 25°C to about 40°C, or about 30°C to about 42°C, or about 35°C to about 40°C. In one embodiment, incubation is at about 37°C.

[0056] <Antibacterial agent> Any of the embodiments, methods, systems, or configurations described herein may include contacting the sample with an antimicrobial agent. In any of the embodiments or configurations described herein, the antimicrobial agent may be incorporated into the microdroplets or the antimicrobial agent may be exposed to the microdroplets. In some embodiments, the antimicrobial agent is dried in the device at a defined concentration and / or amount and forms the droplets after being reconstituted with a portion of the sample containing the microorganism. The amount of the dried antimicrobial agent can be adjusted so that the resulting concentration is within a desired range when reconstituted with a portion of the sample. In some embodiments, the antimicrobial agent is added to the microdroplets after they are prepared, rather than being added to the sample before the formation of the microdroplets.

[0057] In any of the embodiments, methods, systems, or forms described herein, the antimicrobial agent may be a naturally occurring, semi-synthetic, or fully synthetic agent that inhibits the growth of microorganisms (e.g., bacteria, fungi, viruses, parasites, and microbial spores), thereby preventing their development and microbial or pathogenic action. Antimicrobial agents are known to those skilled in the art. However, by way of example, the antimicrobial agent may be selected from the group consisting of 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 acids; nucleic acid analogs and derivatives; glycogen or other sugars; immunogens; antigens; extracts made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combination thereof. In some embodiments, the antimicrobial agent includes antibacterial agents (or antibiotics), antifungal agents, antiprotozoal agents, antiviral agents, and mixtures thereof.

[0058] Non-limiting examples of antibiotics include, for example, aminoglycosides (including, for example, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin), ansamycins (including, for example, geldanamycin, herbimycin, rifaximin), carbacephems (including, for example, loracarbef), carbapenems (including, for example, ertapenem, antipseudomonas, doripenem, imipenem, cilastatin, meropenem, biapenem, panipenem), cephalosporins, (e.g., cefazolin, cephalexin, cefadroxil, cephapirin, cephazedone, cefazaflur, cephradine, cefroxadine, ceftezole, cephaloglycin, cephacetrile, cephalonium, cephaloridine, cephalothin, cefatrizine, cefaclor, cefotetan, cephamycin, cefoxitin, cefprozil, cefuroxime, cefuroxime axetil, cefamandole, cefminox, cefonicid, ceforanide, cefotiam, cefbuperazone, cefuzonam, cefmetazole, carbacephem, loracarbef , cefixime, ceftriaxone, antipseudomonas, ceftazidime, cefoperazone, cefdinir, cefcapene, cefdaloxime, ceftizoxime, cefmenoxime, cefotaxime, cefpiramide, cefpodoxime, ceftibuten, cefditoren, cefetamet, cefodizime, cefpimizole, cefsulodin, cefteram, ceftiolene, oxacephem, flomoxef, latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftaroline fosamil, ceftolozane, ceftobiprole, cefthio fluoxetine, ceftriaxone, and cefovecin), glycopeptides (including, for example, vancomycin, oritavancin, telavancin, teicoplanin, dalbavancin, and ramoplanin), lincosamides (including, for example, clindamycin and lincomycin), lipopeptides (including, for example, daptomycin), macrolides (including, for example, azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin), monobactams (including, for example, aztreonam, tigemonam, carumonam, and nocardicin A),Nitrofurans (including, for example, furazolidone, nitrofurantoin), oxazolidonones (including, for example, linezolid, posizolid, radezolid, torezolid), penicillins (including, for example, penams, β-lactams, benzylpenicillin, benzathine benzylpenicillin, procaine benzylpenicillin, phenoxymethylpenicillin, propicillin, phenethicillin, azidocillin, clomethocillin, penamecillin, cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, methicillin, amoxacillin, including cyclosporine, ampicillin, pivampicillin, hetacillin, bacampicillin, methampicillin, talampicillin, epicillin, ticarcillin, carbenicillin, carindacillin, temocillin, piperacillin, azlocillin, mezlocillin, mecillinam, pivmecillinam, sulbenicillin), penems (including, for example, faropenem or ritipenem), polypeptides (including, for example, bacitracin, colistin, polymyxin B), quinolones (including, for example, ciprofloxacin, enocaxin, 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, cotoxacin, rimoxazole, sulfonamide chrysoidine), tetracyclines (including, for example, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline), or, for example, aclosoxacin, amifloxacin, amikacin, amoxycillin, ampicillin, arsphenamine, aspoxicillin, azidocillin, azithromycin, aztreonam, balofloxacin, biapenem, brodimoprim, capreomycin, cefaclor, cefadroxil,Cefatrizine, cefcapene, cefdinir, cefetamet, cefoxitin, cefprozil, cefroxadine, ceftaroline, ceftazidime, ceftibuten, cefmetazole, ceftobiprole, cefuroxime, cephalexin, cephalonium, cephaloridine, cephamandole, cefazolin, cephradine, chloramphenicol, chlorquinaldol, chlortetracycline cyclosporine, cyclacillin, cinoxacin, 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, flumequine, fosfomycin, fucidin acid, gentamycin, imipenem, isoniazid, kanamycin, lc benzylpenicillin, levofloxacin, linezolid, mandelic acid, mecillinam, meropenem, metronidazole, minocycline, moxalactam, mupirocin, nadifloxacin, nalidixic acid, netilmycin, netromycin, nifuirtoinol, nitrofurantoin, nitroxoline, norfloxacin, ofloxacin, oxytetracycline, panipenem, pefloxacin, pheno Noxymethylpenicillin, pipemidic acid, piromidic acid, pivampicillin, pivmecillinam, platensimycin, prulifloxacin, pyrazinamide, quinupristin, rifabutin, rifampicin, rifapentine, rufloxacin, sparfloxacin, streptomycin, sulbactam, sulfabenzamide, sulfacytine, sulfamethpyrazine, sulfacetamide, sulfadiazine, sulfadimidine, sulfamethizole,Sulphamethoxazole, sulfanilamide, sulfasomidine, sulfathiazole, teicoplanin, teixobactin, temafioxacin, tetracycline, tetroxoprim, thiamphenicol, tigecycline, tinidazole, tobramycin, tosufloxacin, trimethoprim, or vancomycin, and pharma- ceutically acceptable salts or esters thereof.

[0059] Exemplary antifungal agents include, but are not limited to, 5-flucytosine, aminocandin, amphotericin B, anidulafungin, bifonazole, butoconazole, caspofungin, chlordantoin, chlorphenesin, ciclopirox olamine, clotrimazole, eberconazole, econazole, fluconazole, flutrimazole, isavuconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, nifuroxime, posaconazole, ravuconazole, tioconazole, terconazole, undecenoic acid, and pharmaceutically acceptable salts or esters thereof.

[0060] Exemplary antiprotozoal agents include, but are not limited to, acetarzole, azanidazole, chloroquine, metronidazole, nifuratel, nimorazole, omidazole, propenidazole, secnidazole, sinefungin, tenonitrozole, temidazole, tinidazole, and pharmaceutically acceptable salts or esters thereof. Exemplary antiviral agents include, but are not limited to, acyclovir, brivudine, cidofovir, curcumin, desciclovir, 1-docosanol, edoxudine, gQ Fameyclovir, fiacitabine, ibacitabine, imiquimod, lamivudine, penciclovir, valacyclovir, valganciclovir, and pharmaceutically acceptable salts or esters thereof.

[0061] In any of the embodiments, methods, systems, or configurations described herein, the antibacterial agent may be selected from the group consisting of amoxicillin / clavulanate, amikacin, ampicillin, aztreonam, ceftrazidime, cephalothin, chloramphenicol, ciprofloxacin, clindamycin, ceftriaxone, cefotaxime, cefuroxime, erythromycin, cefepime, gentamicin, imipenem, levofloxacin, linezolid, meropenem, minocycline, nitrofurantoin, oxacillin, penicillin, piperacillin, ampicillin / sulbactam, trimethoprim / sulfamethoxazole or cotrimoxazole, tetracycline, tobramycin, vancomycin, or any combination thereof.

[0062] In any of the embodiments, methods, systems, or configurations described herein, the newly developed antimicrobial agent may be incorporated into or exposed to microdroplets to determine the effectiveness of the newly developed antimicrobial agent or to determine the susceptibility of a microorganism to the newly developed antimicrobial agent.

[0063] In any of the embodiments, methods, systems, or configurations described herein, the concentration of the antimicrobial agent in the AST provided herein may be provided at various concentrations to determine the susceptibility range. 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 at various concentrations and have various efficacies. Thus, the concentration of the antimicrobial agent may vary 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 is selected to include a range of concentrations of the antimicrobial agent, including the concentration of the antimicrobial agent that is or would be the minimum inhibitory concentration (MIC), as described in more detail herein. In some other embodiments, the concentration range of the antimicrobial agent may cover a clinically or physiologically relevant concentration range, but not include a concentration that may allow for the determination of the MIC. In some embodiments, one or more portions of the sample are not exposed to any antimicrobial agent (0 antimicrobial concentration), for example, for control purposes. Thus, for example, when the microdroplets are exposed to an antimicrobial (or when the antimicrobial is incorporated into the microdroplets upon formation), the concentration of the antimicrobial can range from about 0.001 μg / ml to about 5000 μg / ml, e.g., 0.001, 0.01, 0.1, 1, 10, 100, 1000, or 5000 μg / ml, or within an amount defined by any of the values ​​set forth above. In some embodiments, the antimicrobial concentration is 0.001 μg / ml to 5000 μg / ml, 0.001 μg / ml to 1000 μg / ml, 0.001 μg / ml to 100 μg / ml, 0.001 μg / ml to 10 μg / 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. In some embodiments, the antimicrobial concentration is a serial dilution to determine the MIC.The serial dilutions can be in the amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1000, or 10000 dilutions, or dilutions within a range defined by any of the aforementioned values. In some embodiments, the dilutions are in the amount of 1-10000, 1-1000, 1-100, 1-10, 10-10000, 10-100, 100-10000, or 100-1000. In some embodiments, the concentration of antimicrobial agent is 0 (e.g., no antimicrobial agent is present), thereby providing a control indicative of normal microbial growth in the absence of any antimicrobial agent.

[0064] In some embodiments, a sample of microdroplets encapsulating microorganisms is divided into one or more portions of the microdroplets, and a first portion may be exposed to a first concentration of antimicrobial, a second portion may be exposed to a second concentration of antimicrobial, and a third portion may be exposed to a third concentration of antimicrobial, and so on for a desired number of portions exposed to a desired number of antimicrobial concentrations. Thus, for example, in a sample of microdroplets, a first concentration of antimicrobial exposed to a first portion of the microdroplets may be 0.001 μg / ml, a second concentration of antimicrobial exposed to a second portion of the microdroplets may be 0.005 μg / ml, and a third concentration of antimicrobial exposed to a third portion of the microdroplets may be 0.01 μg / ml, and so on for a desired number of portions exposed to a desired number of different concentrations of a particular antimicrobial or antimicrobials. In some embodiments, each portion may be exposed to a serial dilution of the antimicrobial instead of a desired number of dilutions of the antimicrobial. The range of concentrations and number of microdroplet portions may be ascertained based on the antimicrobial being tested, its clinically or physiologically relevant concentration range, the microorganism suspected, or the particular assay being performed, to cover a range of antimicrobial concentrations, including, for example, the minimum inhibitory concentration of the antimicrobial.

[0065] <microbes> The embodiments provided herein relate to measuring microbial viability. In any of the embodiments, methods, systems, or configurations described herein, microorganisms can be encapsulated in microdroplets during the formation of the microdroplets. For example, in some embodiments, the sample containing microorganisms is dispersed in a continuous hydrophobic oil phase containing a surfactant under conditions that generate water-in-oil microdroplets. In some embodiments, the sample containing microorganisms is a processed clinical sample. In some embodiments, the sample comprises one or more of 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, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage, bronchopulmonary aspirate or other lavage, blastocyst cavity, umbilical cord blood, or 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, a human, one or more companion animals, or one or more commercially important animals has a microbial infection, such as a bacterial infection.

[0066] In any of the embodiments, methods, systems, or configurations described herein, the sample may be a clinical sample that can be obtained from a human subject and processed to isolate the microbial population of interest from one or more components of the sample.The sample may be obtained, for example, as 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, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate or other lavage fluid, blastocyst cavity, umbilical cord blood, or maternal circulation that may be of fetal or maternal origin.

[0067] In any of the embodiments, methods, systems, or configurations described herein, the sample may be a fluid or specimen obtained from an environmental source. For example, the fluid or specimen obtained from an environmental source may be obtained or derived from foodstuffs, food, poultry, meat, fish, beverages, dairy products, water (including wastewater), ponds, rivers, reservoirs, swimming pools, soil, food processing and / or packaging plants, agricultural land, 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 a microbial colony.

[0068] In any of the embodiments, methods, systems, or configurations described herein, it may be necessary or desirable to process the sample prior to encapsulation of microorganisms in microdroplets. Even if processing is not required, processing may be performed for convenience (e.g., as part of a regimen for a commercial platform). The processing reagent may 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 processing may serve several different purposes, including, but not limited to, lysing cells such as blood cells, diluting the sample, and the like. Processing reagents may 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.), and solvents such as buffer solutions. In some embodiments, the processing reagent is a surfactant or detergent. In some embodiments, the sample is a clinical sample obtained directly from a subject, and the sample has been treated by removing one or more components of the clinical sample and / or adding one or more agents to the clinical sample. For example, the sample may be filtered, purified, cleaned, decontaminated, centrifuged, or otherwise treated to isolate a microorganism or population of microorganisms within the sample from one or more components of the sample.

[0069] In any of the embodiments, methods, systems, or configurations described herein, the sample may be further processed by adding one or more processing reagents to the sample to degrade undesired molecules present in the sample and / or dilute the sample 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 microorganism being detected, or the amount of microorganism 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 (e.g., in a solution, i.e., a "processing buffer") at an appropriate concentration. The amounts of the various components of the processing buffer can be varied depending on the sample, the microorganism to be detected, the concentration of the microorganism in the sample, or the time constraints for the analysis.

[0070] The treatment buffer can be made in any suitable buffer solution known to those skilled in the art, including but 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, potassium acetate solution, potassium acetate solution, potassium acetate solution, tripotassium citrate solution. , potassium formate solution, dipotassium phosphate solution, dipotassium phosphate solution, potassium sodium 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 solution, trimethylammonium acetate solution, trimethylammonium phosphate solution, TRIS-EDTA buffer solution, TRIZMA® Base, and TRIZMA® HCL. Alternatively, the processing buffer may be made up in water.

[0071] After addition of the treatment reagent, the sample can be incubated for a period of time, e.g., at least 1, 2, 3, 4, 5, 10, 15, 30, 45, or 60 minutes. Such incubation can be at any suitable temperature, e.g., from about 16° C. to about 30° C., room temperature (e.g., from about 20° C. to about 25° C.), low temperature (e.g., from about 0° C. to about 16° C.), or high temperature (e.g., from about 30° C. to about 95° C.). In some embodiments, the sample is incubated at room temperature for about 15 minutes.

[0072] In any of the embodiments, methods, systems, or configurations described herein, the microorganism may be a bacterium, a fungus, a virus, a parasite, a protozoa, or a microbial spore. In some embodiments, the bacteria are from the following phyla: Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, or a combination of both phyla. s), Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermomicrobia, Thermotogae, or Verrucomicrobia, or any mutants and derivatives of the microbial species, such as mutants and derivatives made by genetic and / or recombinant techniques.

[0073] In any of the embodiments, methods, systems, or configurations described herein, the bacteria can be gram-positive or gram-negative bacteria. In some embodiments, the bacteria is an aerobic or anaerobic bacteria. In some embodiments, the bacteria is an autotrophic or heterotrophic bacteria. In some embodiments, the bacteria is a mesophile, a mesophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, a halophile, or an osmophile.

[0074] In any of the embodiments, methods, systems, or configurations described herein, the bacteria may be anthrax, antibiotic resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. In some embodiments, the bacteria may be mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, Methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. In some embodiments, the bacterium can be Mycobacterium tuberculosis. In some embodiments, the bacterium is Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Serratia marcescens, or Enterococcus faecium.

[0075] In any of the embodiments, methods, systems, or configurations described herein, the microorganism may be a protozoan causing a disease such as malaria, sleeping sickness, or toxoplasmosis; a fungus causing a disease such as ringworm, candidiasis, or histoplasmosis. The term "microbe" or "microbes" can also encompass non-pathogenic microorganisms, such as those used in industrial applications.

[0076] As used herein, section headings are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, treatises, and Internet web pages, are expressly incorporated by reference in their entirety for all purposes. In the event that 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 prevail. It will be understood that there is an implied "about" before temperatures, concentrations, times, etc. discussed in the present teachings, such that minor and insubstantial deviations are within the scope of the present teachings herein.

[0077] In this application, the use of the singular includes the plural unless specifically stated otherwise, and the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0078] Although the present invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the invention, as well as obvious modifications and equivalents thereof. In addition, while certain variations of the present invention have been shown and described in detail, other modifications that are within the scope of the present 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 present invention. It should be understood that the various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form various forms or embodiments of the disclosed invention. It is therefore intended that the scope of the invention disclosed herein should not be limited by the specific disclosed embodiments described above. It should be understood, however, that this detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.

[0079] The terms used in the description presented herein are not intended to be interpreted 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. Furthermore, although the embodiments may include several novel features, none of them are solely responsible for desirable attributes or are considered essential to practicing the inventions herein described.

Claims

1. 1. A method for assessing microbial growth in a sample, comprising: a) providing a sample containing a microorganism; b) separating the sample containing the microorganism into one or more portions 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 are formed before or after separating the sample into one or more portions; d) contacting one or more portions of the sample with an antimicrobial agent, either before or after forming the one or more populations of microdroplets, where each of the 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 microorganisms encapsulated in the microdroplets Including, thereby determining the susceptibility of a microorganism to an antimicrobial agent, the step of measuring microbial viability includes obtaining microbial viability measurements 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, and obtaining microbial viability measurements 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; and The method, wherein one or more distinct subsets of microdroplets from the first population are not present in the second population, and one or more distinct subsets of microdroplets from the second population are not present in the first population.

2. 10. The method of claim 1, wherein an average of the microbial viability measurements from a plurality of separate subsets of microdroplets measured at a first time point is compared to an average of the microbial viability measurements from a plurality of separate subsets of microdroplets measured at a second time point.

3. 3. The method of claim 2, wherein the step of measuring microbial viability further comprises, for a plurality of subsets of microdroplets measured at the first and second time points, comparing a measurement of microbial viability from a separate subset of microdroplets measured at a first time point to a measurement of microbial viability from a separate subset of microdroplets measured at a second time point.

4. The method of claim 2 or 3, wherein the microbial viability measurements taken at the first and second time points are not assigned to separate subsets of microdroplets.

5. 5. The method of any one of claims 2 to 4, wherein the measuring step further comprises obtaining 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.

6. The method of claim 1, further comprising assigning the measurements taken at the first and second time points to separate subsets of the microdroplets, wherein at least a portion of the separate subsets of the microdroplets in the first population are the same separate subsets of the microdroplets in the second population such that the microbial viability measurements taken for the separate subset of microdroplets at the first time point can be compared to the microbial viability measurements taken for that same separate subset of the microdroplets taken at the second time point.

7. The method according to any one of claims 1 to 6, wherein the susceptibility of the microorganisms to antibiotics is determined by measuring the survival rate of the microorganisms in the presence of different concentrations of the antibiotic.

8. The method according to any one of claims 1 to 7, wherein the step of measuring microbial viability in the droplet is carried out using a technique that affects the viability of microorganisms, including determining the bacterial concentration by genetic analysis including qPCR or fluorescent in-situ hybridization (FISH) after lysis.

9. 8. The method of any one of claims 1 to 7, wherein the step of measuring microbial viability in the droplet is performed using a technique that does not affect the viability of the microorganisms, including measuring solution turbidity, pH, or fluorescence of a metabolically active dye.

10. The method according to any one of claims 1 to 9, wherein the average number of microorganisms per microdroplet is less than one.

11. The method of any one of claims 1 to 10, wherein determining the antimicrobial susceptibility of a microorganism is accomplished in 3 hours or less.

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