Patent Application
20230123594
The disclosed inventive subject matter relates in general to diagnostic systems, devices, and methods for use in infectious disease, and more specifically to a rapid antimicrobial or antibiotic susceptibility test for directly detecting bacterial susceptibility to various antibiotics.
Selecting a proper antibiotic to treat a bacterial infection is typically accomplished through either polymerase chain reaction (PCR) identification of the bacteria and choosing a standard course of antibiotics or by directly testing antibiotic susceptibility to determine which antimicrobials will inhibit the growth of the bacteria causing a specific infection. Bacteria may be identified with PCR; however, PCR does not directly confirm the susceptibility of the identified bacteria to a standard treatment regimen. Ineffective or incomplete treatment with antibiotics can lead to the development of antibiotic resistant strains of bacteria, which is a widely recognized problem in healthcare. Direct antimicrobial susceptibility testing or antibiotic susceptibility testing (AST) may suggest a more successful treatment regimen, but such testing is much slower and more labor-intensive. Accordingly, there is an ongoing need for a high-throughput, rapid, reliable, and easy to use assay for directly determining bacterial susceptibility to a library of antibiotics.
The following provides a summary of certain example implementations 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 its scope. However, it is to be understood that the use of indefinite articles in the language used to describe and claim the disclosed inventive subject matter is not intended in any way to limit the described inventive subject matter. Rather the use of “a” or “an” should be interpreted to mean “at least one” or “one or more”.
One implementation of the disclosed technology provides a method for determining the susceptibility of a bacteria to an antibiotic, comprising obtaining a patient sample containing living bacterial cells; transferring one portion of the patient sample into a bacterial growth medium to create a control sample; transferring another portion of the patient sample in a bacterial grow medium to which a predetermined amount of antibiotic or predetermined amount of a library of antibiotics has been added to create a test sample; adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid incorporates into surface and/or internal proteins of the growing bacteria; reacting the alkyne-containing proteins with an azide-modified detection molecule using click-chemistry to label the living bacteria cells in a detectable manner; detecting the labeled bacterial cells using a method that generates a detectable signal; and comparing the signal generated by the control sample to the signal generated by the test sample, wherein a decrease in detectable signal between the control sample and the test sample is indicative of susceptibility of the living bacteria to the predetermined antibiotic or predetermined library of antibiotics.
The patient sample may be a biological sample derived from a bodily fluid or other bodily source. The antibiotic may be chloramphenicol or any other 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 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 fluorogenic azide probe. The method that generates a detectable signal may be fluorescence-based. The method that generates a detectable signal may be enzyme-linked immunosorbent assay (ELISA)-based. The method that generates a detectable signal may be P5G7 cell-based or P2D8 cell-based. The method that generates a detectable signal may be dot blot-based or microscopy-based. The signal may be quantifiable, and a predetermined amount of signal may be indicative of a minimal inhibitory concentration (minimal effective amount) of antibiotic. The method may be high-throughput method executed on a multi-well plate or microplate, wherein the type of multi-well plate or microplate may include filter plates, and wherein more than one type of antibiotic may be tested on the multi-well plate or microplate.
Another implementation of the disclosed technology provides a method for determining the susceptibility of a bacteria to an antibiotic, comprising obtaining a patient sample containing living bacterial cells, wherein the patient sample is a biological sample derived from a bodily fluid or other bodily source; transferring one portion of the patient sample into a bacterial growth medium to create a control sample; transferring another portion of the patient sample in a bacterial grow medium to which a predetermined amount of antibiotic or predetermined amount of a library of antibiotics has been added to create a test sample; adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid incorporates into surface and/or internal proteins of the growing bacteria, and wherein the alkyne-modified non-canonical amino acid is L-Homopropargylglycine; reacting the alkyne-containing proteins with an azide-modified detection molecule using click-chemistry to label the living bacteria cells in a detectable manner; detecting the labeled bacterial cells using a method that generates a detectable signal; and comparing the signal generated by the control sample to the signal generated by the test sample, wherein a decrease in detectable signal between the control sample and the test sample is indicative of susceptibility of the living bacteria to the predetermined antibiotic or predetermined library of antibiotics.
The antibiotic may be chloramphenicol or any other 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 fluorogenic azide probe. The method that generates a detectable signal may be fluorescence-based. The method that generates a detectable signal may be enzyme-linked immunosorbent assay (ELISA)-based. The method that generates a detectable signal may be P5G7 cell-based or P2D8 cell-based. The method that generates a detectable signal may be dot blot-based or microscopy-based. The signal may be quantifiable, and a predetermined amount of signal may be indicative of a minimal inhibitory concentration (minimal effective amount) of antibiotic. The method may be high-throughput method executed on a multi-well plate or microplate, wherein the type of multi-well plate or microplate may include filter plates, and wherein more than one type of antibiotic may be tested on the multi-well plate or microplate.
Still another implementation of the disclosed technology provides a method for determining the susceptibility of a bacteria to an antibiotic, comprising obtaining a patient sample containing living bacterial cells, wherein the patient sample is a biological sample derived from a bodily fluid or other bodily source; transferring one portion of the patient sample into a bacterial growth medium to create a control sample; transferring another portion of the patient sample in a bacterial growth medium to which a predetermined amount of antibiotic or predetermined amount of a library of antibiotics has been added to create a test sample; adding an alkyne-modified non-canonical amino acid to the bacterial growth medium of both the control sample and test sample during bacterial growth, wherein the alkyne-modified non-canonical amino acid incorporates into surface and/or internal proteins of the growing bacteria, and wherein the alkyne-modified non-canonical amino acid is L-Homopropargylglycine; reacting the alkyne-containing proteins with an azide-modified detection molecule using click-chemistry to label the living bacteria cells in a detectable manner, wherein the azide-modified detection molecule is a biotinylated ligand or a fluorogenic azide probe; detecting the labeled bacterial cells using a method that generates a detectable signal; and comparing the signal generated by the control sample to the signal generated by the test sample, wherein a decrease in detectable signal between the control sample and the test sample is indicative of susceptibility of the living bacteria to the predetermined antibiotic or predetermined library of antibiotics.
The antibiotic may be chloramphenicol or any other 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 that generates a detectable signal may be fluorescence-based. The method that generates a detectable signal may be enzyme-linked immunosorbent assay (ELISA)-based. The method that generates a detectable signal may be P5G7 cell-based or P2D8 cell-based. The method that generates a detectable signal may be dot blot-based or microscopy-based. The signal may be quantifiable, and a predetermined amount of signal may be indicative of a minimal inhibitory concentration (minimal effective amount) of antibiotic. The method may be high-throughput method executed on a multi-well plate or microplate, wherein the type of multi-well plate or microplate may include filter plates, and wherein more than one type of antibiotic may be tested on the multi-well plate or microplate.
It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be implemented to achieve the benefits as described herein. Additional features and aspects of the disclosed system, devices, and methods will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the example implementations. As will be appreciated by the skilled artisan, further implementations are possible without departing from the scope and spirit of what is disclosed herein. Accordingly, the drawings and associated descriptions are to be regarded as illustrative and not restrictive in nature.
The accompanying drawings, which are incorporated into and form a part of the specification, schematically illustrate one or more example implementations of the disclosed inventive subject matter and, together with the general description given above and detailed description given below, serve to explain the principles of the disclosed subject matter, and wherein:
Example implementations are now described with reference to the Figures. Reference numerals are used throughout the detailed description to refer to the various elements and structures. Although the following detailed description contains many specifics for the purposes of illustration, a person of ordinary skill in the art will appreciate that many variations and alterations to the following details are within the scope of the disclosed inventive subject matter. Accordingly, the following implementations are set forth without any loss of generality to, and without imposing limitations upon, the claimed subject matter.
With reference to the Figures, the disclosed AST (which may be referred to as “BLAST”) determines antibiotic susceptibility by detecting changes in the number or amount of living bacteria after the bacteria have been incubated with a predetermined library of antibiotics. This assay exploits the fact that living bacteria have a significantly faster metabolism and protein production than dead or dying bacteria and will take up amino acids and incorporate them into newly formed proteins at a much faster rate. The assay replaces methionine in a bacterial media with a non-canonical amino acid (ncAA), which includes a specific reactive group, thereby enabling specific detection of living bacteria (see, for example, 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), which is incorporated by reference herein in its entirety). In one example implementation, the assay includes three important interactions for successfully detecting living 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 having an azide group, and (iii) detection of the newly biotinylated ligand using a predetermined type of cell, such as for example, a CytoSPAR™ P5G7 cell (see Kittle et al., Development of a Surface Programmable Activation Receptor system (SPAR): A living cell biosensor for rapid pathogen detection, bioRxiv 687426; doi: https://doi.org/10.1101/687426, which is incorporated by reference herein in its entirety for all purposes).
L-Homopropargylglycine (HPG) is an alkyne modified ncAA that mimics methionine during protein production. When HPG is present in bacterial growth media, bacteria growing in the media will incorporate HPG into newly synthesized proteins. Alkyne groups are not naturally found in bacterial cells and serve as a specific reactive group for bacteria undergoing active protein synthesis. HPG has been detected in bacteria after just a 30-minute incubation period, making the entire process comparatively fast.
The alkyne group is one component of the copper catalyzed alkyne-azide cycloaddition (CuAAC), more commonly known as click chemistry (see, for example, 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 herein in their entirety for all purposes). When a ligand with an azide group encounters an alkyne group, the reaction creates an irreversible ring structure (see
Multiple detection methods can be used with the disclosed assay, including fluorescence, cells, blotting, and ELISA-based methods depending on which detection molecule is chosen. To detect antibiotic susceptibility, the signal produced from a control sample (no antibiotic treatment) is compared with samples treated with antibiotics, thereby detecting changes in bacterial protein production that correlate to antibiotic susceptibility.
E. coli UTI Derived
S. saprophyticus UTI Derived
Streptococcus pyogenes
Pseudomonas aeruginosa
An example protocol for performing the disclosed assay includes performing the following assay methodology. Culture and resuspension volumes are held constant throughout the method (i.e., if a culture was 1 mL, then it was re-suspended in 1 mL of appropriate media in further steps). A high throughput microplate method is an aspect of the disclosed assay and is outlined below.
Media and Buffer Preparation
Bacteria Culture and Sample Preparation
Alkyne Labeling of Living Bacteria
Biotin and Fluorescent Labeling of Alkyne Modified Bacteria
Fluorescent and Biotinylation Detection Methods
Dot Blot Detection
ELISA Based Detection
Data Analysis
High Throughput Microplate Method
To better detect biotinylated bacteria, a fluorescent and P5G7 cell luminescent detection method was developed.
Chloramphenicol was chosen as a model antibiotic because it directly inhibits protein synthesis in bacteria.
Advantages of the disclosed technology include the following: the assay does not require plating samples; the assay does not require bacterial replication; the assay is rapid and can be completed in about 2-5 hours; assay sensitivity is within an appropriate range for urinary tract infections (UTIs); the assay does not require strict identification of bacteria and works in polymicrobial cultures; and the assay can be easily customized, can be automated, and may include a numerical readout. The filter plate-based assay was entirely performed on the same plate from beginning of the process to the end therefore, the process can be easily automated.
The methods and results disclosed herein are intended to be examples, and as will be appreciated by one of ordinary skill in the art, various substitutions and modifications are possible. For example, in one implementation 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 this azide-containing non-canonical amino acid is reacted with an alkyne-modified or alkyne-labeled detector molecule rather than an azide-modified detection molecule. Azides of amino acids can be labeled with terminal alkyne or strained alkyne (e.g., DBCO)-tagged reporter molecules by way of a Cu(I)-catalyzed Alkyne-Azide (CUAAC) or Cu(I)-free strain-promoted Alkyne-Azide Click-Chemistry (SPAAC) reaction, respectively. Certain cell-permeable click-functionalized amino acids are randomly incorporated instead of methionine during translation and are therefore suitable for residue selective protein synthesis monitoring. In another example implementation of the disclosed assay, the method that generates a detectable signal utilizes a polypeptide protein tag such as, for example, a FLAG-tag, and a target detector molecule that is specific for the tagged polypeptide. In another example implementation, the method that generates a detectable signal is P2D8 cell-based when a target detection molecule that includes streptavidin is used (see for example, U.S. patent application Ser. No. 16/353,337, which is incorporated herein in its entirety for all purposes).
In some implementations of the disclosed assay, the growth medium contains a desired amino acid analog and simply allows the bacterial cells to metabolize or at least undergo protein synthesis. In some implementations, the growth medium contains only the amino-acid analog and a buffer. While alkyne-modified non-canonical amino acids can be incorporated into proteins of growing bacterial cells using the disclosed methods, the bacterial cells may also be lysed using an alkaline buffer, for example, to increase the detectable signal by including internal bacterial proteins in the detection method. Alternately, the cells may be permeabilized or fixed. Some implementations include the use of fluorogenic azide tags that increase in brightness (quantum yield) when reacted with the alkyne group on the labeled protein, thereby resulting in lower background from unreacted tag. Other implementations include the use of fluorogenic dyes as the detection molecule. See, for example, 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 herein in their entirety for all purposes.
As discussed herein, the disclosed assay may involve the use of a multi-well plate or microplate. In some implementations, the plate is pretreated to selectively bind bacteria of interest, thereby improving processing signal to noise and selectivity. A filter plate may also be used for significantly improving processing speed and efficiency. Such implementations may be useful in tests against, by way of example, Tuberculosis, wherein non-pathogenic bacterial contaminants may potentially overwhelm the signal from a slow growing pathogen. In some implementations, multiple antibiotics may be tested on a single plate. Table 3, below, provides an example plate layout for labeling and tagging in accordance with the disclosed methods.
Some implementations of the disclosed assay utilize variations of click chemistry that do not involve copper catalysis such as, for example, the use of a strained azide or strained alkyne that is highly reactive and does 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 (Nov. 14, 2012), which is incorporated by reference herein, in its entirety, for all purposes. In addition to ELISA-based detection methods, fluorescent microscopy in a pathology lab is used to provide the advantage of being able to distinguish which bacteria in a mixed culture is defeating the antibiotic. Such complex image-based methods are enabled by using image analysis techniques and artificial intelligence (AI)-based software for determining results.
All literature and similar material 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. Should one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
As previously stated and as used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. Unless context indicates otherwise, the recitations of numerical ranges by endpoints include all numbers subsumed within that range. Furthermore, references to “one implementation” are not intended to be interpreted as excluding the existence of additional implementations that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, implementations “comprising” or “having” an element or a plurality of elements having a particular property may include additional elements whether or not they have that property.
The terms “substantially” and “about”, if or when used throughout this specification describe and account for small fluctuations, such as due to variations in processing. For example, these terms can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%, and/or 0%.
Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the disclosed subject matter, and are not referred to in connection with the interpretation of the description of the disclosed subject matter. All structural and functional equivalents to the elements of the various implementations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the disclosed subject matter. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description.
There may be many alternate ways to implement the disclosed technology. Various functions and elements described herein may be partitioned differently from those shown without departing from the scope of the disclosed technology. Generic principles defined herein may be applied to other implementations. Different numbers of a given module or unit may be employed, a different type or types of a given module or unit may be employed, a given module or unit may be added, or a given module or unit may be omitted.
Regarding this disclosure, the term “a plurality of” refers to two or more than two. Unless otherwise clearly defined, orientation or positional relations indicated by terms such as “upper” and “lower” are based on the orientation or positional relations as shown in the Figures, only for facilitating description of the disclosed technology and simplifying the description, rather than indicating or implying that the referred devices or elements must be in a particular orientation or constructed or operated in the particular orientation, and therefore they should not be construed as limiting the disclosed technology. The terms “connected”, “mounted”, “fixed”, etc. 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 an ordinary skilled in the art, the specific meaning of the above terms in the disclosed technology may be understood according to specific circumstances.
It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the disclosed technology. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the technology disclosed herein. While the disclosed technology has been illustrated by the description of example implementations, and while the example implementations have been described in certain detail, there is no intention to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the disclosed technology in its broader aspects is not limited to any of the specific details, representative devices and methods, and/or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.
This patent application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/256,059 filed on Oct. 15, 2021, and entitled “Antibiotic Susceptibility Test”, the disclosure of which is hereby incorporated by reference herein in its entirety and made part of the present U.S. utility patent application for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63256059 | Oct 2021 | US |
