Antibiotic susceptibility testing

Abstract

11. A method for determining bacterial susceptibility to an antibiotic comprising: transferring a portion of a patient sample containing live bacterial cells to a bacterial growth medium to create a control sample; transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a predetermined amount of a library of antibiotics to create a test sample; adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, where the alkyne-modified non-canonical amino acid is incorporated into a surface protein, an internal protein, or both, of the growing bacteria; reacting an alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the live bacterial cells; detecting the labeled bacterial cells using a method that produces a detectable signal; and comparing the signal produced by the control sample with the signal produced by the test sample, where a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or the predetermined library of antibiotics.

Description

[Background technology]

[0001] The disclosed subject matter relates generally to diagnostic systems, devices, and methods for use in infectious diseases, and more specifically to rapid antibacterial or antibiotic susceptibility testing for directly detecting bacterial susceptibility to various antibiotics.

[0002] Selecting the appropriate antibiotic to treat a bacterial infection is usually accomplished by identifying the bacteria with polymerase chain reaction (PCR) and choosing a standard antibiotic course or by direct antibiotic susceptibility testing to determine which antimicrobials inhibit the growth of a particular infection-causing bacteria. Although bacteria can be identified by PCR, PCR does not directly confirm the susceptibility of the identified bacteria to standard treatment regimens. Ineffective or incomplete antibiotic treatment can lead to the development of antibiotic-resistant strains, which is a widely recognized problem in medicine. Although direct antimicrobial susceptibility testing or antibiotic susceptibility testing (AST) may suggest a more effective treatment regimen, such testing is much more time-consuming and labor-intensive. Thus, there is a need for a high-throughput, rapid, reliable, and easy-to-use assay to directly measure bacterial susceptibility to a library of antibiotics. Summary of the Invention

[0003] The following provides a summary of certain exemplary embodiments of the disclosed inventive subject matter. This summary is not an extensive overview and is not intended to identify key or critical aspects or elements of the disclosed inventive subject matter or to delineate the scope thereof. It should be understood, however, that the use of indefinite articles in the language used to describe and claim the disclosed inventive subject matter is not intended to limit the described inventive subject matter in any way. Rather, the use of "a" or "an" should be construed to mean "at least one" or "one or more."

[0004] One embodiment of the disclosed technology provides a method for determining susceptibility of bacteria to antibiotics, comprising the steps of obtaining a patient sample comprising live bacterial cells, transferring a portion of the patient sample comprising live bacterial cells to a bacterial growth medium to create a control sample, transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a library of predetermined amounts of antibiotics to create a test sample, and adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid is a polypeptide that is expressed by ... the alkyne-containing protein being incorporated into a surface and / or internal protein; reacting the alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the live bacterial cells; detecting the labeled bacterial cells using a method that generates a detectable signal; and comparing the signal generated by the control sample with the signal generated by the test sample, where a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or library of predetermined antibiotics.

[0005] The patient sample may be a biological sample derived from a bodily fluid or other bodily source. The antibiotic may be chloramphenicol or another antibiotic, or a combination of antibiotics. The non-canonical amino acid may be azide-modified rather than alkyne-modified, and the detection molecule may be alkyne-modified rather than azide-modified. The alkyne-modified non-canonical amino acid may be L-homopropargylglycine. The azide-modified detection molecule may be a biotinylated ligand. The azide-modified detection molecule may be a fluoroazide probe. The method for generating a detectable signal may be fluorescence-based. The method for generating a detectable signal may be enzyme-linked immunosorbent assay (ELISA)-based. The method for generating a detectable signal may be P5G7 cell-based or P2D8 cell-based. The method for generating a detectable signal may be dot blot-based or microscopy-based. The signal may be quantifiable, and a predetermined amount of signal may indicate a minimum inhibitory concentration (minimum effective amount) of the antibiotic. The method may be a high throughput method carried out on a multiwell plate or microplate, the type of multiwell plate or microplate may include a filter plate, and multiple types of antibiotics may be tested on the multiwell plate or microplate.

[0006] Another embodiment of the disclosed technology provides a method for determining susceptibility of bacteria to antibiotics, the method comprising obtaining a patient sample comprising live bacterial cells, the patient sample being a biological sample derived from a bodily fluid or other bodily source; transferring a portion of the patient sample to a bacterial growth medium to generate a control sample; transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a library of predetermined amounts of antibiotics to generate a test sample; and adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, the alkyne-modified non-canonical amino acid being capable of binding to a surface and / or internal protein of the growing bacteria. and the alkyne-modified non-canonical amino acid is L-homopropargylglycine; reacting the alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the living bacterial cells; detecting the labeled bacterial cells using a method for producing a detectable signal; and comparing the signal produced by the control sample and the signal produced by the test sample, where a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or the library of predetermined antibiotics.

[0007] The antibiotic may be chloramphenicol or another antibiotic or combination of antibiotics. The non-canonical amino acid may be azide-modified rather than alkyne-modified, and the detection molecule may be alkyne-modified rather than azide-modified. The azide-modified detection molecule may be a biotinylated ligand. The azide-modified detection molecule may be a fluoroazide probe. The method for generating a detectable signal may be fluorescence-based. The method for generating a detectable signal may be enzyme-linked immunosorbent assay (ELISA)-based. The method for generating a detectable signal may be P5G7 cell-based or P2D8 cell-based. The method for generating a detectable signal may be dot blot-based or microscopy-based. The signal may be quantifiable, and a predetermined amount of signal may indicate a minimum inhibitory concentration (minimum effective amount) of the antibiotic. The method may be a high-throughput method performed on a multiwell plate or microplate, and the type of multiwell plate or microplate may include a filter plate, and multiple types of antibiotics may be tested on the multiwell plate or microplate.

[0008] Yet another embodiment of the disclosed technology is a method of determining susceptibility of bacteria to antibiotics, the method comprising the steps of obtaining a patient sample comprising live bacterial cells, the patient sample being a biological sample derived from a bodily fluid or other bodily source; transferring a portion of the patient sample to a bacterial growth medium to create a control sample; transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a library of predetermined amounts of antibiotics to create a test sample; adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid is incorporated into a combination of surface and / or internal proteins of the growing bacteria; and the alkyne-modified non-canonical amino acid being L-homopropargylglycine; reacting the alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the living bacterial cells, the azide-modified detection molecule being a biotinylated ligand, a fluoroazide probe, or a fluorescent dye; detecting the labeled bacterial cells using a method that produces a detectable signal; and comparing the signal produced by the control sample with the signal produced by the test sample, where a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or library of predetermined antibiotics.

[0009] The antibiotic may be chloramphenicol or another antibiotic or combination of antibiotics. The non-canonical amino acid may be azide-modified rather than alkyne-modified, and the detection molecule may be alkyne-modified rather than azide-modified. The method of generating a detectable signal may be fluorescence-based. The method of generating a detectable signal may be enzyme-linked immunosorbent assay (ELISA)-based. The method of generating a detectable signal may be P5G7 cell-based or P2D8 cell-based. The method of generating a detectable signal may be dot blot-based or microscopy-based. The signal may be quantifiable, and a predetermined amount of signal may indicate a minimum inhibitory concentration (minimum effective amount) of the antibiotic. The method may be a high-throughput method performed on a multi-well plate or microplate, and the type of multi-well plate or microplate may include a filter plate, and multiple types of antibiotics may be tested on the multi-well plate or microplate.

[0010] It should be understood that all combinations of the foregoing concepts and additional concepts described in more detail below (provided that such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein and may be implemented to achieve the advantages as described herein. Additional features and aspects of the disclosed systems, apparatus, and methods will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of illustrative examples. As will be appreciated by those of ordinary skill in the art, further embodiments are possible without departing from the scope and spirit of what is disclosed herein. Accordingly, the drawings and associated description are to be regarded as illustrative and not restrictive in nature. [Brief description of the drawings]

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate generally one or more exemplary embodiments of the disclosed inventive subject matter and, together with the general description given above and the detailed description given below, serve to explain the principles of the disclosed subject matter, wherein:

[0012] [Figure 1] Figure 1 shows the click chemistry reaction scheme using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC); [Diagram 2] Figure 2 shows the general workflow of the disclosed antibiotic susceptibility testing (AST); [Diagram 3] Figure 3 is a series of images showing dot blots for bacterial biotinylation detection; [Figure 4] 4A-4C are a series of images showing bacterial biotinylation detection following non-canonical amino acid incorporation; [Diagram 5] Figures 5A-5C are a series of images showing biotinylated detection of bacteria after chloramphenicol treatment; [Figure 6] FIG. 6 shows the disclosed ELISA detection method for AST; and [Figure 7] FIG. 7 shows the AST response of E. coli to 100 μg / mL chloramphenicol and 200 μg / mL nitrofurantoin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Examples will now be described with reference to the figures. Reference numerals are used throughout the detailed description to refer to various elements and structures. Although the following detailed description contains many specific details for purposes of explanation, those skilled in the art will appreciate that many variations and modifications to the following details are within the scope of the disclosed inventive subject matter. Thus, the following embodiments are described without any loss of generality to, and without imposing limitations on, the claimed subject matter.

[0014] Referring to the Figure, the disclosed AST (sometimes called "BLAST") determines antibiotic susceptibility by detecting changes in the number or amount of live bacteria after incubation of the bacteria with a defined library of antibiotics. The assay takes advantage of the fact that live bacteria have a significantly faster metabolism and protein production than dead bacteria, and can take up amino acids and incorporate them into newly formed proteins at a faster rate. The assay replaces methionine in bacterial media with a non-canonical amino acid (ncAA) that contains a specific reactive group, thereby allowing for the specific detection of live bacteria (see, e.g., Sherratt et al. Rapid Screening and Identification of Living Pathogenic Organisms via Optimized Bioorthogonal Non-canonical Amino Acid Tagging, Cell Chemical Biology 24, 1048-1055 (2017), incorporated herein by reference in its entirety). In one example embodiment, the assay involves three key interactions for successful detection of live bacteria: (i) bacterial incorporation of the ncAA, (ii) a click chemistry type reaction between the reactive group of the ncAA and a labeled (e.g., biotinylated) ligand bearing an azide group, and (iii) detection of the newly biotinylated ligand using a given type of cell, such as, for example, CytoSPAR™ P5G7 cells (Kittle et al., Development of a Surface Programmable Activation Receptor system (SPAR): A living cell activator that is capable of activating ncAA-binding domains in a cellular context, such as ... biosensor for rapid pathogen detection, bioRxiv 687426; doi: https: / / doi.org / 10.1101 / 687426, incorporated herein by reference in its entirety for all purposes.

[0015] L-Homopropargylglycine (HPG) is an alkyne-modified ncAA that mimics methionine during protein synthesis. When HPG is present in bacterial growth medium, bacteria growing in the medium will incorporate HPG into newly synthesized proteins. The alkyne group does not naturally occur in bacterial cells and serves as a specific reactive group for bacteria undergoing active protein synthesis. HPG can be detected in bacteria after an incubation period of only 30 minutes, making the entire process relatively fast.

[0016] The alkyne group is one component of the copper-catalyzed alkyne-azide cycloaddition reaction (CuAAC), more commonly known as click chemistry (see, e.g., Atwal et al., Clickable methionine as a universal probe for labelling intracellular bacteria, Journal of Microbiological Methods 169 (2020) 105182; and Li et al., Fluorogenic "click" reaction for labeling and detection of DNA in proliferating cells, BioTechniques 49:525-527 (July 2010), both of which are incorporated by reference in their entirety for all purposes). When a ligand bearing an azide group meets an alkyne group, the reaction forms an irreversible ring structure (see FIG. 1). After bacteria take up HPG, alkyne groups can be found on any protein that contains a methionine amino acid, including internal and surface proteins. Because the CuAAC reaction specifically labels live bacteria on their surfaces, in some embodiments, cell lysis can be eliminated as a necessary aspect of the assay. However, in other embodiments, cell lysis may be employed for assay optimization and to increase the amount of signal generated by the assay. The general workflow of the disclosed assay is shown in Figure 2. The assay has been demonstrated to detect antibiotic susceptibility in as little as 5 hours, depending on the patient sample.

[0017] The disclosed assays can use multiple detection methods, including fluorescent, cell, blotting, and ELISA-based methods, depending on which detection molecule is chosen. To detect antibiotic susceptibility, the signal generated from a control sample (no antibiotic treatment) is compared to a sample treated with antibiotic, thereby detecting changes in bacterial protein production that correlate with antibiotic susceptibility.

[0018] Materials and Methods [Table 1] TIFF2024537893000002.tif101149

[0019] An example of a protocol for carrying out the disclosed assay includes carrying out the following assay method: The culture and resuspension volume is kept constant throughout the method (i.e. if the culture was in 1 mL, it is resuspended in 1 mL of appropriate medium in a further step). A high throughput microplate method is one embodiment of the disclosed assay and is outlined below.

[0020] Preparation of media and buffers 1. Prepare supplemented M9 media by adding the following supplements to the specified final concentrations: 100 μM CaCl, 1 mM MgSO4, 16.65 mM glucose, 1X MEM vitamin mix. 2. Prepare methionine-inhibited growth medium (MIGM) by adding the following amino acids to supplemented M9 medium to the indicated final concentrations: L-lysine (100 μg / mL), L-threonine (100 μg / mL), L-phenylalanine (100 μg / mL), L-isoleucine (50 μg / mL), L-leucine (50 μg / mL), and L-valine (50 μg / mL). 3. Prepare 2.5 mg / mL stock solutions of L-homopropargylglycine and L-methionine in supplemented M9. This is a 50x stock solution of each amino acid to be added to the MIGM. 4. Prepare click chemistry buffer (CCB) by dissolving 100 µM CuSO4, 200 µM L-histidine, 2 mM sodium ascorbate, and 100 µM biotin azide (CCB-biotin) or 488-azide (CCB-488) in PBS (pH 7.5).

[0021] Bacterial culture and sample preparation 1. Patient sample preparation is based on E. coli preparation. 2. Place the E. coli on the agar plate overnight at 37°C. 3. Select a single colony and inoculate LB overnight at 37°C with gentle agitation. 4. Add the LB culture to supplemented M9 (LB:M9 = 1:4) and incubate at 37°C with gentle agitation for 2 hours.

[0022] Alkyne labeling of live bacteria 1. This method involves culturing bacteria under six different experimental conditions, as shown in Table 2 below. In a typical assay, patient samples are treated with HPG / no detection azide (negative control), HPG / detection azide (positive control), and HPG / selection agent / detection azide. 2. Pellet the LB / M9 culture and reconstitute with supplemented M9. 3. Split the culture into three equal portions, pellet and reconstitute in MIGM. 4. Prepare drug stock solutions in 200 proof ethanol. In this study, the drug chloramphenicol was used as a model antibiotic. Because of its low water solubility, a 50mg / mL stock solution was made in absolute ethanol and then diluted to 50μg / mL in MIGM. This resulted in a 0.1% ethanol solution. 5. Control ethanol is added to two cultures and drug stock solution is added to one culture. 6. Incubate at 37°C for 30 minutes with gentle agitation. 7. Add stock solutions of HPG or methionine to the appropriate cultures as outlined in Table 2. 8. Incubate under the same conditions for an additional 2 hours. 9. Pellet the bacteria and wash 3 times with PBS.

[0023] [Table 2]

[0024] Biotin and fluorescent labeling of alkyne-modified bacteria 1. Resuspend the pellet in PBS and split each culture into two equal volumes, one receiving 488-azide and the other receiving biotin azide. 2. Pellet the bacteria and resuspend in CCB-biotin buffer or CCB-488 buffer. 3. Incubate at 37°C for 30 minutes with gentle agitation. 4. Pellet the bacteria and rinse 3 times with PBS.

[0025] Fluorescence and biotinylation detection methods 1. While the bacteria are performing the click chemistry reaction, allow the P5G7 cells to thaw at room temperature for 30 minutes. 2. Add anti-biotin antibody (final concentration 5 μg / mL) to the cells and further incubate at room temperature for 30 minutes. 3. Resuspend the bacterial pellet in PBS and reserve 300 μL for fluorescence detection. 4. Pellet the bacteria and resuspend in DMEM. 5. While the bacteria are pelleting, prepare a black plate with 100 μL of each experimental condition in triplicate. Read this on a TopRead fluorescent plate reader (Ex / Em: 500 / 521 optimal, 1 second integration time). 6. Add 30 μL of each experimental condition mixture in duplicate to a white plate. 7. Prepare the luminometer and read the plate with a 1 second integration time and a 20 minute kinetic read. 8. Pipette 90 μL of the P5G7 cell / antibody mixture into each well and read immediately.

[0026] Dot Blot Detection 1. Prepare the PVDF membrane by soaking it in methanol for at least 5 minutes. 2. Remove excess methanol and immediately add 2 μL of each condition mix to the activated membrane. 3. Allow the membrane to dry completely. 4. Reactivate the membrane with methanol for an additional 5 minutes. 5. Remove excess methanol and cover membrane with blocking buffer and gently rock at room temperature for 1 hour. 6. Immerse the membrane in avidin-HRP solution (1 / 2000 dilution in PBS-T) and gently rock at room temperature for 1 hour. 7. Rinse the membrane three times with PBS-T (10 min each). 8. Soak the membrane in ECL for 5 minutes and image the blot.

[0027] ELISA-based detection 1. Add 200 μL of streptavidin coating solution (5 μg / mL in PBS) to each well of a high-binding 96-well microplate, cover and incubate overnight at 4°C. 2. Wash the plate once with wash buffer (PBS-T, PBS with 0.05% tween-20). 3. Place bacteria from the AST into the wells of a streptavidin-coated plate and allow to adsorb for 1 hour at room temperature. 4. Rinse the plate 3 times with wash buffer. 5. Block the plate with blocking buffer for 1 hour at room temperature. 6. Dilute Avidin-HRP 1 / 500 in blocking buffer and add to the wells. Incubate the microplate at room temperature for 1 hour. 7. Rinse the plate 3 times with wash buffer. 8. Add 100 μL of TMB solution to each well and incubate at room temperature for 30 minutes. 9. Add 100 μL of stop solution (1N HCL) to each well. 10. Measure the absorbance of each well at 450 nm.

[0028] Data analysis 1. For P5G7 cell-based detection, the area under the curve from 180 seconds to 540 seconds is calculated by summing all RLU measurements between these time points and multiplying by the time between measurements. Compare AUC values ​​between groups to determine detection of biotinylation. 2. For fluorescent detection, compare RFU measurements between each group to determine detection of the fluorescent dye. 3. Finally, antibiotic susceptibility is detected when the control conditions (HPG containing azide-biotin or azide-488) show a greater signal than the conditions containing the drug.

[0029] High-throughput microplate analysis A. The filter plate method can be used as a high-throughput method. B. All steps, including bacterial growth, are carried out on plates, but the working volume is reduced to 300 μL. C. Add patient samples to multiple wells of a plate and filter the media through the plate rather than pelleting the bacteria: 1. Measure the OD of overnight cultures using a spectrophotometer 600 Measure. 2. Inoculate M9 medium with the overnight culture at a ratio of 1:32 (overnight culture:supplemented M9 medium). Add 0.3 mL of the inoculated supplemented M9 medium to each well of a 96-well filter plate (skip wells if using translucent plates). 3. Incubate the filter plate at 37°C with shaking at 250 rpm for 2 hours. 4. Filter the plates to remove the medium and wash once with PBS. Resuspend the pellet in MIGM medium containing different antibiotics as described in Table 3. 5. Incubate the filter plate at 37°C with shaking at 250 rpm for 30 minutes. 6. Filter the plate to remove the medium and resuspend the pellet in MIGM medium containing different antibiotics and each amino acid as listed in Table 3. a. Prepare 16 (0.3 mL / well) culture conditions as follows: 4. Culture in MIGM containing L-methionine (50 μg / mL) 4. Culture in MIGM containing L-homopropargylglycine (50 μg / mL) 4. Culture in MIGM containing dL-homopropargylglycine (50 μg / mL); chloramphenicol (100 μg / mL) 4. Culture in MIGM containing L-homopropargylglycine (50 μg / mL); nitrofurantoin (200 μg / mL) 7. Incubate at 37°C for 2 hours with shaking at 250 rpm. 8. Filter the plate and rinse 3 times with PBS. Reconstitute the pellet in Click Chemistry Buffer (CCB) containing 488-azide (50 μM) or buffer alone (Table 3). a. Test conditions are as follows: iL-Methionine / 488-Azide ii. L-methionine alone iii.HPG / 488-Azide iv. HPG / 488-azide / chloramphenicol v.HPG / 488-Azide / Nitrofurantoin vi. HPG alone 9. Mix thoroughly by pipetting and incubate at 37°C for 30 minutes. 10. Rinse the plate 3 times with PBS to remove all buffer. 11. Read signal on a fluorescent plate reader at excitation 484, emission 524.

[0030] result Figure 1 shows a click chemistry reaction scheme using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Figure 2 shows the general workflow of the disclosed antibiotic susceptibility testing (AST).

[0031] Figure 3 is a series of images showing dot blots for bacterial biotinylation detection, where 2 μL of bacteria from the disclosed AST were placed on a PVDF membrane and detected with avidin-HRP / ECL. Control biotinylated bacteria were generated by reacting E. coli with NHS-biotin for 30 min at room temperature. Dot blots are a simple method for detecting biotinylation using avidin-HRP. Figure 3 shows dot blots with control bacteria (biotinylated E. coli labeled by NHS-biotin reaction) and only bacteria that underwent HPG, demonstrating the success of the click chemistry reaction under these conditions.

[0032] To better detect biotinylated bacteria, a fluorescent and P5G7 cytoluminescent detection method was developed. Figure 4A-4C is a series of images showing biotinylated detection of bacteria after ncAA incorporation. Fluorescence (Figure 4A) or P5G7-based detection (Figure 4B-4C) of bacteria that received either HPG or methionine control and azide-conjugated detection molecule are shown. Fluorescence was measured on a Victor X5 fluorescent plate reader using a fluorescence filter set. Figure 4B represents a kinetic readout of luminescence over 20 min. The positive control showed a peak at 2.8 million RLU, but is not shown in the figure. Figure 4C is the area under the curve (AUC) calculated from the data in the middle panel. Figure 4A-4C show the success of both methods to specifically detect bacteria that have incorporated the ncAA, HPG. Bacteria that received methionine and 488-azide showed a higher fluorescent signal than bacteria that did not receive the fluoroazide, suggesting that some off-target reaction is occurring. However, this signal is small compared to the positive signal from the HPG / 488-azide group.

[0033] Chloramphenicol was chosen as a model antibiotic since it directly inhibits bacterial protein synthesis. Figures 5A-5C are a series of images showing biotinylated detection of bacteria after chloramphenicol treatment. Shown is the fluorescent (Figure 5A) or P5G7 cell-based detection (Figures 5B-5C) detection of bacteria that received either HPG, chloramphenicol or control ethanol, and an azide-conjugated detection molecule. Fluorescence was measured on a Victor X5 fluorescent plate reader using a fluorescent filter set. Figure 5B shows the kinetic readout of luminescence over 20 minutes. The positive control showed a peak at 2.8 million RLU, but is not shown in the figure. Figure 5C is the area under the curve (AUC) calculated from the data in the middle panel. Figures 5A-5C show that bacteria that received antibiotics prior to incubation with HPG showed a reduced signal in both detection methods, thereby indicating that the disclosed assay is effective for detecting antibiotic susceptibility. Results from the modified sandwich ELISA method also confirmed these findings. FIG. 6 shows the disclosed ELISA detection method for AST, in which the bacteria used in FIGS. 5A-5C were captured on a streptavidin-coated ELISA plate and detected with avidin-HRP / TMB.

[0034] FIG. 7 shows the results for the AST response of E. coli when treated with either 100 μg / mL chloramphenicol or 200 μg / mL nitrofurantoin. The starting overnight bacterial culture was diluted 1:32 (overnight LB culture: supplemented M9 medium). Detection was performed using an azide-conjugated fluorescent tag. Bacterial samples were treated with either: HPG alone; HPG + chloramphenicol; HPG + nitrofurantoin; or methionine alone. Each sample was then reacted with an azide-conjugated detection molecule. Fluorescence was measured on a Molecular Devices Spectramax M2 at excitation 484 and emission 524. The results show that samples treated with chloramphenicol or nitrofurantoin show a reduced signal compared to the untreated sample (HPG alone). This indicates that the disclosed assay is effective for detecting antibiotic susceptibility.

[0035] Advantages of the disclosed technology include: the assay does not require plating of samples; the assay does not require bacterial replication; the assay is rapid and can be completed in approximately 2-5 hours; the assay sensitivity is within the appropriate range for urinary tract infections (UTIs); the assay does not require strict identification of the bacteria and works with polymicrobial cultures; and, the assay can be easily customized, can be automated, and can include a numerical readout. Assays using filter plates facilitate automation of the process because everything from start to finish is done on the same plate.

[0036] The methods and results disclosed herein are intended to be exemplary, and various substitutions and modifications are possible, as will be appreciated by those of skill in the art. For example, in one embodiment of the disclosed assay, an azide-containing non-canonical amino acid is used in the assay, rather than an alkyne-modified non-canonical amino acid, and the azide-containing non-canonical amino acid is reacted with an alkyne-modified or alkyne-labeled detection molecule, rather than an azide-modified detection molecule. The azide of the amino acid can be labeled with a terminal alkyne or strained alkyne (e.g., DBCO)-tagged reporter molecule by Cu(I)-catalyzed alkyne-azide (CUAAC) or Cu(I)-free strain-alkyne-azide click chemistry (SPAAC) reaction, respectively. Certain cell-permeable click-functionalized amino acids are suitable for residue-selective protein synthesis monitoring, as they are randomly incorporated in place of methionine during translation. In another example of the disclosed assay, the method of generating a detectable signal utilizes a polypeptide protein tag, such as a FLAG tag, and a targeted detector molecule specific for the tagged polypeptide. In another example, the method for generating a detectable signal is P2D8 cell-based when a target detection molecule comprising streptavidin is used (see, e.g., U.S. Patent Application No. 16 / 353,337, which is incorporated herein in its entirety for all purposes).

[0037] In some embodiments of the disclosed assays, the growth medium contains the desired amino acid analogs, simply allowing the bacterial cells to metabolize or at least undergo protein synthesis. In some embodiments, the growth medium contains only the amino acid analogs and a buffer. While the disclosed methods can be used to incorporate alkyne-modified non-canonical amino acids into proteins of growing bacterial cells, the bacterial cells can also be lysed using alkaline buffers, for example, to increase the detectable signal by including internal bacterial proteins in the detection method. Alternatively, the cells may be permeabilized or fixed. Some embodiments include the use of fluoroazide tags, which increase brightness (quantum yield) when reacted with alkyne groups on the labeled protein, thereby lowering the background from unreacted tags. Other embodiments include the use of fluorescent dyes as detection molecules. See, e.g., Beatty et al., Selective Dye-Labeling of Newly Synthesized Proteins in Bacterial Cells, J. Am. Chem. Soc. 127: 14150-14151 (2005); and Shieh et al., Fluorogenic Azidofluoresceins for Biological Imaging, J. Am. Chem. Soc. 134(42): 17428-17431 (2012), both of which are incorporated by reference in their entireties for all purposes.

[0038] As discussed herein, the disclosed assays can include the use of multi-well plates or microplates. In some embodiments, the plates are pre-treated to selectively bind the bacteria of interest, thereby improving the signal-to-noise ratio and selectivity of the process. Filter plates can also be used to significantly improve processing speed and efficiency. Such an embodiment would be useful in tests such as tuberculosis, where non-pathogenic bacterial contaminants may overwhelm the signal from slow-growing pathogens. In some embodiments, multiple antibiotics are tested on a single plate. Table 3 below is an example of a plate layout for labeling and tagging according to the disclosed method.

[0039] [Table 3]

[0040] Some embodiments of the disclosed assays utilize variations of click chemistry that do not involve copper catalysis, such as the use of strained azides or strained alkynes, which are highly reactive and do not require catalysis. See, for example, Friscourt et al., A Fluorogenic Probe for the Catalyst-Free Detection of Azide-Tagged Molecules, J Am Chem Soc. 134(45): 18809-18815 (November 14, 2012), the entire contents of which are incorporated by reference herein for all purposes. In addition to ELISA-based detection methods, fluorescence microscopy is used in pathology laboratories, which has the advantage of being able to distinguish which bacteria in a mixed culture are resistant to antibiotics. Such complex image-based methods are made possible by the use of image analysis techniques and artificial intelligence (AI)-based software to determine the results.

[0041] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or conflicts with this application, including but not limited to defined terms, term usage, techniques described, etc., this application controls.

[0042] As mentioned above, and as used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, implied elements or method steps. Although many methods and materials similar or equivalent to those described herein can be used, certain preferred methods and materials are described herein. Unless the context dictates otherwise, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range. Furthermore, references to "one embodiment" are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, unless expressly stated to the contrary, an embodiment that "comprises" or "has" an element or elements having a particular characteristic can include additional elements whether or not they have that characteristic.

[0043] The terms "substantially" and "about" as used throughout this specification or in use account for and take into account minor variations, such as those due to process variations. For example, these terms can refer to ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less, and / or 0%.

[0044] Underlined and / or italic headings and subheadings are used for convenience only, are not intended to limit the disclosed subject matter, and are not to be referenced in connection with interpreting the description of the disclosed subject matter. All structural and functional equivalents to the elements of the various embodiments described throughout this disclosure that are known or that later become known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the disclosed subject matter. Furthermore, nothing disclosed herein is intended to be generally exclusive, regardless of whether such disclosure is expressly set forth in the description above.

[0045] There may be many alternative ways of implementing the disclosed technology. The various functions and elements described herein may be divided differently than shown without departing from the scope of the disclosed technology. The general principles defined herein may be applied to other implementations. Different numbers of a given module or unit may be employed, different types or varieties of a given module or unit may be employed, given modules or units may be added, and given modules or units may be omitted.

[0046] In the context of this disclosure, the term "plurality" refers to two or more than two. Unless otherwise clearly defined, the orientation or positional relationship indicated by terms such as "upper" and "lower" is based on the orientation or positional relationship shown in the figures only to facilitate and simplify the description of the disclosed technology, and does not indicate or imply that the referenced device or element must be in a particular orientation or configured or operated in a particular orientation, and therefore should not be construed as limiting the disclosed technology. Terms such as "connected," "attached," and "fixed" should be understood in a broad sense. For example, "connected" may be a fixed connection, a detachable connection, or an integral connection, a direct connection, or an indirect connection through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in the disclosed technology may be understood according to the specific situation.

[0047] It should be understood that all combinations of the above concepts and additional concepts discussed in more detail herein (provided such concepts are not mutually inconsistent) are contemplated as part of the disclosed technology. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the disclosed technology. The disclosed technology has been illustrated by the description of exemplary embodiments, and although the exemplary embodiments have been described in certain details, there is no intention to limit the scope of the appended claims to such details, and is not intended to limit them in any manner. Additional advantages and modifications will readily occur to those skilled in the art. Thus, the disclosed technology, in its broader aspects, is not limited to any of the specific details, representative apparatus and methods, and / or illustrative examples shown and described. Thus, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

Claims

1. 1. A method for determining bacterial susceptibility to an antibiotic, comprising: (a) transferring a portion of the patient sample containing viable bacterial cells to a bacterial growth medium to create a control sample; (b) transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a library of predetermined amounts of antibiotics to create a test sample; (c) adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid is incorporated into a surface protein, an internal protein, or a combination of a surface protein and an internal protein of the growing bacteria; (d) reacting the alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the live bacterial cells; (e) detecting the labeled bacterial cells using a method that produces a detectable signal; and (f) comparing the signal produced by the control sample with the signal produced by the test sample, wherein a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or library of predetermined antibiotics; A method comprising:

2. 10. The method of claim 1, wherein the patient sample is a biological sample derived from a bodily fluid or other bodily source.

3. 10. The method of claim 1, wherein the antibiotic is chloramphenicol, or another antibiotic, or a combination of other antibiotics.

4. 10. The method of claim 1, wherein the non-canonical amino acid is azide-modified rather than alkyne-modified, and the detection molecule is alkyne-modified rather than azide-modified.

5. 2. The method of claim 1, wherein the alkyne-modified non-canonical amino acid is L-homopropargylglycine.

6. 10. The method of claim 1, wherein the azide-modified detection molecule is a biotinylated ligand, a fluoroazide probe, or a fluorescent dye.

7. 2. The method of claim 1, wherein the method for generating the detectable signal is fluorescence-based, enzyme-linked immunosorbent assay (ELISA)-based, cell-based including P5G7 cells or P2D8 cells, dot blot-based, or microscopy-based.

8. 10. The method of claim 1, wherein the signal is quantifiable and a predetermined amount of signal indicates a minimum inhibitory concentration of the antibiotic.

9. 10. The method of claim 1, wherein the method is a high-throughput method performed on a multiwell plate or microplate, the type of multiwell plate or microplate includes a filter plate, and the multiwell plate or microplate can test multiple types of antibiotics.

10. 1. A method for determining bacterial susceptibility to an antibiotic, comprising: (a) transferring a portion of a patient sample to a bacterial growth medium to create a control sample, said patient sample comprising viable bacterial cells and being a biological sample derived from a bodily fluid or other bodily source of said patient; (b) transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a library of predetermined amounts of antibiotics to create a test sample; (c) adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid is incorporated into a surface protein, an internal protein, or a combination of a surface protein and an internal protein of the growing bacteria, and the alkyne-modified non-canonical amino acid is L-homopropargylglycine; (d) reacting the alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the live bacterial cells; (e) detecting the labeled bacterial cells using a method that produces a detectable signal; and (f) comparing the signal produced by the control sample with the signal produced by the test sample, wherein a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or library of predetermined antibiotics; A method comprising:

11. 11. The method of claim 10, wherein the antibiotic is chloramphenicol, or another antibiotic, or a combination of other antibiotics.

12. 11. The method of claim 10, wherein the non-canonical amino acid is azide-modified rather than alkyne-modified, and the detection molecule is alkyne-modified rather than azide-modified.

13. 11. The method of claim 10, wherein the azide-modified detection molecule is a biotinylated ligand, a fluoroazide probe, or a fluorescent dye.

14. 11. The method of claim 10, wherein the method for generating the detectable signal is fluorescence-based, enzyme-linked immunosorbent assay (ELISA)-based, cell-based including P5G7 cells or P2D8 cells, dot blot-based, or microscopy-based.

15. 11. The method of claim 10, wherein the signal is quantifiable and a predetermined amount of signal indicates a minimum inhibitory concentration of the antibiotic.

16. 11. The method of claim 10, wherein the method is a high-throughput method performed on a multiwell plate or microplate, the type of multiwell plate or microplate includes a filter plate, and the multiwell plate or microplate can test multiple types of antibiotics.

17. 1. A method for determining bacterial susceptibility to an antibiotic, comprising: (a) transferring a portion of a patient sample to a bacterial growth medium to create a control sample, said patient sample comprising viable bacterial cells and being a biological sample derived from a bodily fluid or other bodily source of said patient; (b) transferring another portion of the patient sample to a bacterial growth medium supplemented with a predetermined amount of an antibiotic or a library of predetermined amounts of antibiotics to create a test sample; (c) adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and the test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid is incorporated into a surface protein, an internal protein, or a combination of a surface protein and an internal protein of the growing bacteria, and the alkyne-modified non-canonical amino acid is L-homopropargylglycine; (d) reacting the alkyne-containing protein with an azide-modified detection molecule using click chemistry to detectably label the live bacterial cells, wherein the azide-modified detection molecule is a biotinylated ligand, a fluoroazide probe, or a fluorescent dye; (e) detecting the labeled bacterial cells using a method that produces a detectable signal; and (f) comparing the signal produced by the control sample with the signal produced by the test sample, wherein a decrease in detectable signal between the control sample and the test sample indicates susceptibility to the predetermined antibiotic or library of predetermined antibiotics; A method comprising:

18. 18. The method of claim 17, wherein the antibiotic is chloramphenicol, or another antibiotic, or a combination of other antibiotics, and the method of generating the detectable signal is fluorescence-based, enzyme-linked immunosorbent assay (ELISA)-based, cell-based including P5G7 cells or P2D8 cells, dot blot-based, or microscopy-based.

19. 18. The method of claim 17, wherein the non-canonical amino acid is azide-modified rather than alkyne-modified, and the detection molecule is alkyne-modified rather than azide-modified.

20. 18. The method of claim 17, wherein the signal is quantifiable and a predetermined amount of signal indicates a minimum inhibitory concentration of the antibiotic, the method is a high-throughput method performed on a multiwell plate or microplate, the type of multiwell plate or microplate includes a filter plate, and the multiwell plate or microplate can test multiple types of antibiotics.

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