Compositions, methods, and systems for detecting methicillin-resistant Staphylococcus aureus

Abstract

This invention provides compositions, methods, apparatus, systems, and kits for detecting nasal cavity colonization of MRSA. [Solution] In a particular embodiment, the method uses bacteriophage-based amplification of signals in the detection of bacteria and other microorganisms to detect MRSA. The method for detecting MRSA may include preparing an assay comprising a selectant and a cocktail comprising at least two different types of recombinant bacteriophages, incubating a sample in the assay, capturing an indicator protein product, and detecting the indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates the presence of MRSA in the sample.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority and the benefit of U.S. Provisional Application No. 63 / 018,081, filed Apr. 30, 2020, which is hereby incorporated by reference in its entirety. Field

[0002] The present disclosure relates to compositions, methods, and systems for detecting methicillin - resistant Staphylococcus aureus (MRSA) using infectious agents.

Background Art

[0003] Background There is a strong interest in detecting bacteria and other microorganisms that can cause various forms of debilitating and lethal infections. Bacterial pathogens can not only cause significant pathological conditions among humans and livestock, but also cause substantial economic losses. Specifically, methicillin - resistant Staphylococcus aureus (MRSA) is a highly important human pathogen with the ability to cause fatal infections. MRSA is a major cause of surgical site infections in hospitals, accompanied by prolonged hospital stays, increased readmission rates, decreased survival rates, and economic losses. Due to the significant clinical and financial burden on the healthcare industry, substantial efforts have been made to understand and control the causes of MRSA - related infections. Nasal carriage of MRSA has been found to be a major risk factor for subsequent disease, and most Staphylococcus aureus infections can be attributed to endogenous colonizing strains. Removal of this risk factor through decolonization of MRSA nasal carriers has proven to be a successful strategy for reducing surgical site infections.

[0004] Traditional microbiological tests for detecting MRSA rely on non-selective and selective enrichment cultures, subsequent plating onto selective media, and further testing to identify suspicious colonies from patient nasal swab specimens. Culture-based detection methods may involve the use of chromogenic and selective agar and can often demonstrate robust performance in terms of sensitivity and specificity. While often considerably less expensive than some other methods, one major drawback of culture-based methods is that results typically require 18–24 hours of incubation before detection.

[0005] To shorten testing time, various rapid methods have been studied and implemented. However, these methods also have drawbacks. For example, techniques involving immunoassays or gene probes generally require an enrichment step to obtain sufficient sensitivity. Polymerase chain reaction (PCR) testing also includes an amplification step and therefore allows for both extremely high sensitivity and selectivity. Detection of MRSA-specific DNA sequences by real-time PCR has demonstrated excellent sensitivity and specificity, rapid time to results, and overall clinical efficacy. Although real-time PCR has yielded promising results, this method also has drawbacks. Firstly, since some assays for detecting novel MRSA strains have failed with previous PCRs, new generations of real-time PCR must be constantly developed to adapt to the changing genetic landscape of MRSA resistance. Secondly, the higher cost of real-time PCR compared to culture-based alternatives has made cost-effectiveness uncertain, especially in areas with low regional carrier rates.

[0006] Therefore, there is a need for faster, simpler, and more sensitive detection and identification of MRSA. [Overview of the Initiative] [Means for solving the problem]

[0007] Brief summary Embodiments of this disclosure include compositions, methods, apparatus, systems, and kits for detecting nasal cavity colonization of MRSA. This disclosure can be implemented in various forms.

[0008] In some embodiments, the present disclosure provides a method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample. The method comprises obtaining a sample, adding a selectant to the sample, and contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene that is specific to Staphylococcus aureus, the indicator gene encodes an indicator protein product, captures the indicator protein product, and detects a signal generated by the indicator protein product, the detection of which is used to determine the presence of MRSA in the sample.

[0009] In some embodiments, the present disclosure provides a method for detecting microorganisms in a sample. The method includes: obtaining a sample; contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene that is specific to a microorganism, the indicator gene encoding an indicator protein product; contacting the indicator protein product with a surface comprising an immobilized binding partner for capturing the indicator protein product; and detecting a signal generated by the indicator protein product, the detection of which is used to determine the presence of the microorganism in the sample.

[0010] In some embodiments, the Disclosure utilizes novel recombinant bacteriophages to detect MRSA from nasal swab specimens. In some embodiments, the novel recombinant bacteriophages are specific to Staphylococcus aureus. Novel diagnostic screenings utilize assays comprising recombinant bacteriophages containing a luciferase reporter capable of recognizing Staphylococcus aureus, while relying on antibiotics to limit the growth of non-MRSA stains. Various MRSA strains can be detected using the methods described herein.

[0011] In some embodiments, the Disclosure provides a method for detecting MRSA from a sample, comprising: (a) contacting the sample with a selector; (b) contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene that is specific to Staphylococcus aureus, the indicator gene encoding an indicator protein product; and (c) detecting a signal produced by the indicator protein product, the detection of which is used to determine the concentration of MRSA in the sample. In some embodiments, the selector comprises an antibiotic (e.g., cefoxitin). In some embodiments, the sample is derived from a nasal swab.

[0012] In some embodiments, the infectious agent is a specific recombinant phage specific to Staphylococcus aureus. In further embodiments, the indicator gene encodes an indicator protein product that generates a unique signal or an enzyme that generates a signal when it reacts with a substrate.

[0013] In some embodiments, the present disclosure provides a method for detecting MRSA from a sample, comprising: contacting the sample with a selectant, wherein the sample is derived from a nasal swab; and contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene that is specific to Staphylococcus aureus, the indicator gene encodes an indicator protein product; and detecting a signal produced by the indicator protein product, wherein the detection of the signal is used to determine the presence of MRSA in the sample.

[0014] In some embodiments, the disclosure provides kits and systems for detecting nasal swabs containing MRSA, as well as assays comprising recombinant bacteriophages and antibiotic solutions specific to Staphylococcus aureus. In some embodiments, the disclosure provides kits and systems for detecting nasal swabs containing microorganisms, assays comprising recombinant bacteriophages specific to microorganisms and optionally comprising antibiotics, and surfaces for capturing indicator protein products.

[0015] Certain embodiments of this disclosure utilize methods and constructs described in U.S. Patent Application Publication 2015 / 0218616, which is incorporated herein in its entirety by reference. [Modes for carrying out the invention]

[0016] Detailed explanation Compositions, methods, and systems exhibiting remarkable sensitivity for detecting various strains of methicillin-resistant Staphylococcus aureus (MRSA) in test samples (e.g., biological samples) within shorter timeframes than conventional methods. The compositions, methods, and systems disclosed herein can detect MRSA within shorter timeframes than previously thought possible by using genetically modified infectious bacteriophages with reduced culture time for enrichment, or, in some embodiments, genetically modified infectious bacteriophages with minimal incubation time during which MRSA can potentially grow. Remarkably, assays using one or more recombinant bacteriophages in the presence of an antibiotic (e.g., cefoxitin) for incubation with test samples detect various MRSA strains at concentrations that produce extremely low colony-forming units (CFUs). Such low CFU concentrations were previously said to be detectable only after using culture-based methods requiring incubation for more than 24 hours. However, the assays described herein can facilitate the discovery, binding, and infection of a small number of target cells. In some embodiments, this assay detects MRSA from nasal swab samples in less than 10 hours at a cost similar to methods based on longer cultures.

[0017] In several aspects, the bacteriophage-based MRSA assays described herein provide specific, highly sensitive, rapid, and low-cost detection of target bacteria, addressing the growing diagnostic needs in numerous industries. Specifically, detecting nasal colonization and antibiotic susceptibility of MRSA plays a crucial supporting role in preventing hospital-acquired infections and promoting appropriate antibiotic use. In some embodiments, the bacteriophage-based MRSA assay for nasal swab specimens utilizes two luciferase reporter phages capable of recognizing genetically diverse Staphylococcus aureus. In some embodiments, cefoxitin, a beta-lactam antibiotic, is included to distinguish between resistant (MRSA) and susceptible organisms. Surprisingly, the bacteriophage-based MRSA assays positively identified MRSA isolates at low bacterial concentrations, while non-MRSA Staphylococcus aureus yielded appropriate negative results in higher inoculum. Furthermore, cross-reactivity of the phage cocktail with other Staphylococcal and bacilli species can be mitigated under selective conditions. Therefore, the bacteriophage-based MRSA assays described herein detect MRSA with high sensitivity in both in vitro and human nasal matrix.

[0018] In some aspects, this disclosure provides recombinant bacteriophages comprising an indicator gene inserted into the late gene region of the bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Staphylococcus aureus-specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified bacteriophage genome derived from a bacteriophage that specifically recognizes Staphylococcus aureus. In some embodiments, the bacteriophage cocktail comprises at least two different types of recombinant bacteriophages derived from a bacteriophage that specifically recognizes Staphylococcus aureus. In some embodiments, an assay comprising a cocktail of recombinant bacteriophages and a selector (e.g., an antibiotic) can distinguish MRSA in the presence of other types of bacteria, particularly methicillin-sensitive Staphylococcus aureus (MSSA).

[0019] In some cases, a method for detecting MRSA may use an infectious agent for detecting Staphylococcus aureus. For example, in a particular embodiment, the microorganism of interest is MRSA, and the infectious agent is a bacteriophage that specifically infects Staphylococcus aureus. Therefore, in a particular embodiment, the method involves selecting one or more bacteriophages that specifically infect Staphylococcus aureus, preparing recombinant bacteriophages derived from Staphylococcus aureus bacteriophages, and preparing an assay comprising the recombinant bacteriophages and a selector (e.g., an antibiotic). The assay may include providing a sample from a nasal swab or similar source for analysis. In certain embodiments, the recombinant bacteriophage includes an indicator gene. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage such that the expression of the indicator gene during bacteriophage replication after infection with a host bacterium results in the production of an indicator protein product. The method may include detecting the indicator protein product, and positive detection of the indicator protein product indicates the presence of MRSA in the sample. In some embodiments, the indicator protein is soluble.

[0020] In some embodiments, compositions, methods, and systems can detect MRSA from diverse genetic backgrounds using assays comprising one or more recombinant bacteriophages and selectors, such as antibiotics. In some embodiments, the assay utilizes a selector, such as cefoxitin, to restrict the survival of susceptible bacteria while allowing the growth of MRSA. For example, the selector may kill or reduce the growth of all Staphylococcus aureus bacteria other than MRSA (e.g., MSSA). In this way, cefoxitin can identify diverse isolates of MRSA from competing organisms. As described herein, assays comprising cefoxitin result in high selectivity for MRSA and, importantly, do not interfere with the detection of MRSA strains. Furthermore, cefoxitin is effective in reducing false positives from several species of coagulase-negative staphylococci.

[0021] In some embodiments, the methods and systems described herein selectively detect low levels of MRSA from nasal swabs or similar samples. Each of the embodiments of the methods and systems of the present disclosure can be applied to the detection and quantification of a wide variety of MRSA strains. The methods and systems provide high detection sensitivity in a short time without requiring conventional biological enrichment and / or incubation that requires at least 24 hours. The method utilizes a novel bacteriophage-based MRSA diagnostic screening. The assay is a member of a new generation of luciferase phage reporter systems that utilize luciferase such as NANOLUC® to detect the target species with high sensitivity. The method has been found to be highly comprehensive and, in combination with cefoxitin selection, discriminates against most non-resistant strains. Furthermore, the screening was able to identify low burdens of MRSA in nasal samples with little or no interference.

[0022] In certain embodiments, the present disclosure may include a system. The system may contain at least some of the compositions of the present disclosure. Further, the system may include at least some of the components for performing the methods. In certain embodiments, the system is assembled as a kit. Thus, in some embodiments, a system for the rapid detection of MRSA from a nasal swab includes a component for incubating a sample with a recombinant infective agent specific for a microorganism of interest, wherein the recombinant infective agent includes an indicator moiety, a selection agent, and a component for detecting the indicator moiety. In other embodiments, the present disclosure includes software for use with the methods or systems.

[0023] Some embodiments of the disclosure described herein utilize the discovery that a single microorganism can recognize and bind to a specific infectious agent, such as a bacteriophage. After bacteriophage infection and replication, the success of the infection and the production of progeny phage can be detected via an indicator moiety expressed during bacteriophage replication. This principle enables the amplification of an indicator signal from one or a few cells based on the specific recognition of a microbial surface receptor. For example, even a single cell of bacteria can be exposed to multiple bacteriophages, and then, by enabling high-level expression of the encoded indicator gene product during bacteriophage amplification and replication, the indicator signal is amplified such that a single bacterium is detectable. Definitions

[0024] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature and these technologies used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are well known and commonly used in the art. Unless otherwise described, known methods and techniques are generally carried out according to conventional methods well known in the art and as described in various general and more specific references discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is well known and commonly used in the art. The following terms are to be understood to have the following meanings unless otherwise indicated.

[0025] As used herein, the terms "a," "an," and "the" may refer to one or more unless otherwise specified.

[0026] The use of the term “or” is used to mean “and / or” unless it is explicitly indicated that it refers only to the choices, or that the choices are mutually exclusive; however, this disclosure supports the definitions that refer only to the choices and “and / or.” As used herein, “another” may mean at least the second or more.

[0027] Throughout this application, the term "approximately" is used to indicate that the value includes inherent variations in the apparatus, the method used to determine the value, or variations present between samples.

[0028] The terms “solid support” or “support” refer to a structure that provides a substrate and / or surface upon which biomolecules can be bound. For example, a solid support may be an assay well (i.e., a microtiter plate or multiwell plate, etc.), or a solid support may be a position on a movable support such as a filter, array, or beads or membrane (e.g., a filter plate or lateral flow strip).

[0029] The term “binding agent” or “binding partner” refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction may be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction may be covalent.

[0030] The term "soluble binder" refers to a binder that is not bonded to a solid support (i.e., not bonded by covalent or non-covalent bonds).

[0031] The term "immobilized binding partner" refers to a binder that is bonded to a solid support (i.e., bonded covalently or noncovalently).

[0032] As used herein, “analyte” refers to the molecule, compound, or cell being measured. In certain embodiments, the analyte of interest may interact with the binder.

[0033] Where used herein, the term "analyte" may refer to the protein or peptide of interest. The analyte may be an agonist, antagonist, or regulator. Alternatively, the analyte may not have any biological effect. The analyte may include small molecules, sugars, oligosaccharides, lipids, peptides, peptide mimes, organic compounds, and the like.

[0034] The terms “detectable portion,” “detectable biomolecule,” “reporter,” “indicator,” or “indicator portion” refer to molecules that can be measured in a quantitative assay. For example, an indicator portion may include an enzyme that can be used to convert a substrate into a measurable product. An indicator portion may be an enzyme that catalyzes a reaction that produces bioluminescence (e.g., luciferase). Alternatively, an indicator portion may be a radioactive isotope that can be quantified. Alternatively, an indicator portion may be a fluorophore. Alternatively, other detectable molecules may be used.

[0035] As used herein, “bacteriophage” or “phage” includes one or more bacterial viruses. In this disclosure, the terms “bacteriophage” and “phage” include viruses such as mycobacteriophages (such as those against TB and para-TB), mycophages (such as those against fungi), mycoplasma phages, and any other terms referring to viruses that can invade living bacteria, fungi, mycoplasmas, protozoa, yeasts, and other microscopic living organisms and use them to replicate themselves. Here, “microscopic” means having a maximum dimension of one millimeter or less.

[0036] Bacteriophages are viruses that have naturally evolved to use bacteria as a means of replicating themselves. A bacterium does this by attaching itself to a bacterium, injecting its DNA (or RNA) into the bacterium, and inducing the bacterium to replicate the bacterium hundreds or thousands of times. This is called phage amplification.

[0037] As used herein, “late gene region” refers to a region of the viral genome that is transcribed in the later stages of the viral life cycle. The late gene region typically contains the most abundantly expressed genes (e.g., structural proteins that assemble to form bacteriophage particles). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, late genes (synonymous with class III) are transcribed in phage T7, for example, from 8 minutes post-infection until lysis, while class I genes (e.g., RNA polymerase) are transcribed early, from 4–8 minutes, and class II genes are transcribed from 6–15 minutes, so there is an overlap in timing between II and III genes. A late promoter is a promoter that is naturally located within such a late gene region and is active within such a late gene region.

[0038] As used herein, “culturing for enrichment” refers to traditional culturing, such as incubation in a medium favorable to microbial growth, and should not be confused with other possible uses of the term “enrichment,” such as enrichment by removing the liquid component of a sample to concentrate the microorganisms contained therein, or other forms of enrichment that do not involve the traditional promotion of microbial growth. In some embodiments of the methods described herein, culturing for enrichment for very short periods may be used, but is not necessary, and when very short culturing for enrichment is used, it is for a much shorter period than conventional culturing for enrichment.

[0039] As used herein, “recombinant” refers to the modification of genes (i.e., nucleic acids) that are typically performed in a laboratory to combine genetic material that would not otherwise be found. This term is used herein interchangeably with the term “modified.”

[0040] As used herein, "RLU" refers to relative luminous units measured by a luminometer (e.g., GLOMAX® 96) or similar instrument that detects light. For example, the detection of a reaction between luciferase and a suitable substrate (e.g., NANOLUC® and NANO-GLO®) is often reported in the detected RLU.

[0041] As used herein, “time to result” refers to the total time from the start of sample incubation to the generated result. Time to result does not include any confirmation test time. Data collection can be performed at any time after the result has been generated. sample

[0042] Each embodiment of the methods and systems of this disclosure can enable rapid detection and quantification of MRSA in a sample. For example, the methods of this disclosure can be performed in a reduced time with excellent results. Bacterial cells detectable by this disclosure include, but are not limited to, various strains of MRSA derived in vitro or from nasal swabs.

[0043] The sample may be a liquid, solid, or semi-solid. The sample may be a surface swab. In some embodiments, the sample may be a nasal swab for detecting MRSA nasal colonization. In some embodiments, the sample may include body material, such as tissue or nasal fluid. In some embodiments, the sample may be whole blood, plasma, serum, or a combination thereof.

[0044] In some embodiments, the sample may be used directly in the detection method of the present disclosure without preparation, concentration, or dilution. For example, a liquid sample, including but not limited to a nasal swab, may be directly assayed. The sample may be diluted or suspended in a buffer solution or a bacterial culture medium, but not limited to these. A solid or semi-solid sample may be suspended in a liquid by finely chopping, mixing, or immersing the solid in the liquid to soften it. The sample should be maintained within a pH range that promotes the attachment of bacteriophages to host bacterial cells. Preferably, the sample is maintained at a temperature that preserves the viability of any pathogen cells contained within the sample.

[0045] In some embodiments of the detection assay, the sample is maintained at a temperature that preserves the viability of any pathogen cells present in the sample. For example, it is preferable to maintain the sample at a temperature that promotes bacteriophage attachment during the stage when bacteriophages are attaching to bacterial cells. It is also preferable to maintain the sample at a temperature that promotes bacteriophage replication and host lysis during the stage when bacteriophages are replicating within infected bacterial cells or lysing such infected cells. Such temperatures are at least about 25°C, more preferably about 45°C or less, and most preferably about 37°C.

[0046] In some embodiments, the assay may include a selective agent. To inhibit or promote microbial growth, the assay may include selective and non-selective antimicrobial agents capable of inhibiting or stopping microbial growth, modifiers (i.e., substances that can alter microbial growth but are not considered antimicrobial agents), or enrichers (e.g., substances that may be required by nutrient-dependent microorganisms such as hemin, or substances that may be required by preference organisms), or other components that may promote microbial growth. In some embodiments, the selective agent is, for example, an antimicrobial agent containing cefoxitin.

[0047] The assay may include a variety of suitable control samples. For example, control samples that do not contain bacteriophages or control samples that do not contain bacteria but contain bacteriophages may be assayed as controls for background signal levels. Bacteriophage

[0048] As described in more detail herein, the compositions, methods, systems, and kits of this disclosure may include infectious agents for use in the detection of MRSA. In certain embodiments, this disclosure provides recombinant indicator bacteriophages in which the bacteriophage genome is genetically modified to include an indicator gene or reporter gene. In some embodiments, the compositions may include recombinant bacteriophages having an indicator gene incorporated into the bacteriophage genome.

[0049] The compositions of this disclosure may comprise one or more genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may comprise a cocktail of different indicator phages capable of encoding and expressing the same or different indicator proteins. In some embodiments, the bacteriophage cocktail comprises at least two different types of recombinant bacteriophages derived from bacteriophages specific to Staphylococcus aureus.

[0050] Recombinant indicator bacteriophages may include a reporter gene or an indicator gene. In certain embodiments of the infectious agent, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage. Late genes generally encode structural proteins and are therefore expressed at higher levels than other phage genes. In some embodiments, the indicator bacteriophage is derived from a bacteriophage specific to Staphylococcus aureus.

[0051] Furthermore, phage genes that appear non-essential may possess unrecognized functions. For example, seemingly non-essential genes may have crucial functions in increasing production, such as subtle cutting, fitting, or trimming functions in aggregation. Therefore, deleting genes to insert indicators can be detrimental. Most phages can package DNA several percent larger than their natural genome. Considering this, smaller indicator genes may be a more appropriate choice for modifying bacteriophages, especially those with smaller genomes. OpLuc and NANOLUC® proteins are only about 20 kDa (coding at about 500-600 bp), while FLuc is about 62 kDa (coding at about 1,700 bp). In addition, reporter genes should not be endogenously expressed by bacteria (i.e., not part of the bacterial genome), should produce a high signal-to-background ratio, and should be readily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, NANOLUC® combined with Promega's NANO-GLO®, an imidazopyrazinon substrate (flimazine), can provide a robust signal with low background.

[0052] An indicator gene can express a variety of biomolecules. An indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. In alternative embodiments, as described in more detail herein, the luciferase is one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, sea urchin luciferase, or a modified luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene such as NANOLUC®.

[0053] Accordingly, in some embodiments, the present disclosure provides genetically modified bacteriophages that include a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-natural indicator gene is under the control of a late promoter. By using a viral late gene promoter, it is ensured that the reporter gene (e.g., luciferase) is not only expressed at high levels like a viral capsid protein, but also does not arrest like an endogenous bacterial gene or even an early viral gene.

[0054] Genetic modifications to infectious agents may include insertions, deletions, or substitutions of small fragments of nucleic acids, substantial portions of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acids contain non-natural sequences. Non-natural indicator genes may be inserted into the bacteriophage genome so as to be under the control of the bacteriophage promoter. Thus, in some embodiments, the non-natural indicator gene is not part of the fusion protein. In some embodiments, the indicator protein product is soluble. In some embodiments, the disclosure provides a method for detecting a bacterium of interest (e.g., Staphylococcus aureus), comprising the step of incubating a test sample with such recombinant bacteriophage.

[0055] In some embodiments, the expression of an indicator gene in progeny bacteriophages after infection with a host bacterium results in a free soluble protein product. In some embodiments, the non-native indicator gene is not contiguous with the gene encoding the structural phage protein and therefore does not produce a fusion protein. In some embodiments, the indicator or reporter ideally does not contain a bacteriophage structure; that is, the indicator or reporter is not bound to the phage structure. Therefore, the indicator or reporter gene is not fused with other genes in the recombinant phage genome. This can significantly increase the sensitivity of the assay (down to a single bacterium), simplify the assay, and, for some embodiments, allow the assay to be completed in two hours or less, unlike the several hours required due to the additional purification steps needed with constructs that produce detectable fusion proteins.

[0056] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a change in color. Various suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may function as the indicator portion. In some embodiments, firefly luciferase is the indicator portion. In some embodiments, Oplophorus luciferase is the indicator portion. In some embodiments, NANOLUC® is the indicator portion. Other engineered luciferases or other enzymes that produce a detectable signal may also be suitable indicator portions.

[0057] In some embodiments, the preparation of recombinant bacteriophage stocks includes a purification step sufficient to remove substantially all residual indicator proteins that could associate with the bacteriophage before use in bacterial detection assays. Therefore, the resulting preparation of the parent recombinant bacteriophage used to infect any target bacteria in a sample of interest is substantially free of indicator proteins. Methods using infectious agents to detect MRSA

[0058] As described herein, in certain embodiments, the Disclosure provides a method for using an infectious bacteriophage to detect MRSA or a microorganism. The method of the Disclosure may be carried out in various forms.

[0059] In some embodiments, the present disclosure provides a method for detecting microorganisms in a sample. The method includes: acquiring a sample; contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene, which is specific to the microorganism, and the indicator gene encodes an indicator protein product; contacting the indicator protein product with a surface, wherein the surface comprises an immobilized binding partner for capturing the indicator protein product; and detecting a signal produced by the indicator protein product, the detection of which is used to determine the presence of the microorganism in the sample.

[0060] In some embodiments, the Disclosure provides a method for detecting MRSA from a sample (e.g., from a nasal swab), comprising the steps of: obtaining a sample; incubating the sample in an assay comprising a selector and one or more bacteriophages that infect Staphylococcus aureus, wherein the bacteriophages comprise an indicator gene such that the expression of an indicator gene during bacteriophage replication after infection with the bacterium of interest results in the production of a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of MRSA in the sample. In some embodiments, the selector is an antibiotic comprising cefoxitin.

[0061] In some embodiments, the method includes capturing an indicator protein product for detection. The step of capturing an indicator protein product on a surface improves the detection of microorganisms or various MRSA strains at concentrations that produce very few colony-forming units. To capture the indicator protein product on a surface, the indicator protein product can be brought into contact with the surface. For example, the indicator protein product may adhere to or bind to the surface during the capture step. In some embodiments, the surface may include, among other things, a microtiter plate, latex particles, a lateral flow strip, beads, magnetic particles, or a dipstick.

[0062] In some embodiments, the surface may include immobilized binding partners. For example, one or more specific recognition elements can be immobilized on separate regions of the surface to create an array for analyte recognition. An indicator protein product can be brought into contact with the surface containing the immobilized binding partners. In some embodiments, several different binding partners can be immobilized on a single surface simultaneously. In some embodiments, the immobilized binding partners are antibodies or fragments thereof.

[0063] In some embodiments, one or more different immobilized binding partners can be deposited on a surface (e.g., a plate) to capture an indicator protein product (e.g., by adding them with a pipette). In some aspects, the surface can improve the accessibility and capture of the indicator protein product by orienting the immobilized binding partners. For example, an antibody can be deposited on a plate and incubated for a certain period of time. In some embodiments, the antibody may be a rabbit antibody or a goat antibody. If necessary, the plate can be washed after incubation. Subsequently, NANOLUC® antibody can be deposited on the coated plate. In some aspects, it is advantageous if the amount of indicator protein product deposited on the surface with immobilized binding partners is equal to or less than the amount of immobilized binding partners required to form a monolayer on the surface as a solid support. For example, the immobilized binding partners may be antibodies that bind to a layer on the surface of the solid support, resulting in accessibility of their specific binding epitopes.

[0064] In some embodiments, the methods of the present disclosure may include various other steps to enhance sensitivity. The sensitivity of a method for detecting microorganisms or MRSA may be increased by one or more washing steps. For example, the method may include a step for washing the captured indicator protein product to remove luciferase or other indicator proteins that contaminate the bacteriophage and / or bacteriophage preparation. Furthermore, the captured microorganisms may be washed after incubation with antibiotics and infectious agents, and before the addition of lysis buffer and substrate. These additional washing steps help remove luciferase or other indicator proteins that contaminate the excess parent phage and / or phage preparation. In some embodiments, microorganisms may be captured, washed, and then infected with bacteriophages.

[0065] In some embodiments, the method involves adding a protein to an antibody to promote infection by a bacteriophage. Staphylococcus aureus binds to antibodies (e.g., IgG) in the blood, preventing bacteriophages from infecting these cells. In some embodiments, protein A is added to bind to antibodies in the blood, thereby preventing the antibodies from binding to Staphylococcus aureus. When Staphylococcus aureus cells divide in the presence of protein A, the antibodies are unable to bind to daughter cells, allowing bacteriophages to infect cells in the blood. In some embodiments, protein A is added to a phage cocktail. For example, protein A can be mixed with the phage cocktail before infection.

[0066] In certain embodiments, the assay may be performed to utilize a general concept that can be modified to suit different sample types or sizes and assay formats. Embodiments using recombinant bacteriophages (i.e., indicator bacteriophages) may use sizes of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 12, depending on the sample type, sample size, and assay format. Rapid detection of MRSA may be possible with total assay times of less than 5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, or 26.0 hours. For example, the time required may be somewhat shorter or longer depending on the bacteriophage strains and bacterial strains to be detected in the assay, the type and size of the sample being tested, the conditions required for the target's viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target bacterial contaminants. [Examples]

[0067] Examples The results shown in the following examples demonstrate the effectiveness of the compositions, methods, and systems described herein for detecting MRSA from nasal swab specimens with reduced time to results. These examples evaluated a novel bacteriophage-based assay used in the diagnostic screening methods and systems described herein. This assay is part of a new generation of luciferase-phage reporter systems that utilize NANOLUC® to detect target species. The method proved to be highly comprehensive and, when combined with cefoxitin selection, identifies most non-resistant strains. Furthermore, the method was able to identify low loadings of MRSA in nasal specimens with little or no evidence of problematic interference. Ultimately, the data indicate that this diagnostic screening may be a promising new tool for detecting MRSA colonization from nasal swab specimens. material and method bacterial strain

[0068] Bacterial strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), with the following exceptions: Salmonella enteritidis S492 was obtained from the University of Georgia Research Foundation, and Staphylococcus aureus RN4220 was obtained from the University of Iowa. Clinical strains of Staphylococcus aureus were procured in-house from the Clinical Microbiology Laboratory (Laboratory Corporation of America Holdings). MRSA isolates were obtained from anonymized human clinical specimens originating from three geographically distinct US locations (Burlington NC, Phoenix AZ, and Raritan NJ). MSSA isolates were similarly obtained from one location (Burlington, NC). The determination of whether a strain was MRSA or MSSA was confirmed by plating on the selective chromogenic agar medium MRSA Select II (Bio-Rad, Marnes-la-coquette, France). The strains were routinely propagated at 37°C in cerebral and cardiac exudate (BHI) broth (Becton Dickinson and Company, Sparks, MD, USA) while being shaken at 250 revolutions per minute (RPM). Preparation of bacteriophage sources and stocks

[0069] The assay includes two modified Staphylococcus aureus bacteriophages, MP115 and ISP. Staphylococcus aureus bacteriophages are members of the Myoviridae family, which includes large lytic Staphylococcus aureus bacteriophages. The MP115 bacteriophage was obtained from the Colorado School of Mines, and the ISP bacteriophage was obtained from Emory University.

[0070] Bacteriophage stocks were prepared as follows: For MP115, an overnight culture of RN4220 was diluted and grown to the exponential stage, then infected with an infection efficiency (MOI) of 0.01. The culture was monitored for a decrease in optical density (OD) to confirm viral growth. The bacteriophage solubilized solution was then clarified by centrifugation at 10,000 rpm for 10 minutes at 4°C. The clarified supernatant was again centrifuged at 4°C and 10,000 rpm for 2 hours. The pellet was resuspended overnight in 1×TMS (50 mM Tris-HCl, 10 mM MgCl2, and 300 mM NaCl). The bacteriophage preparations were then treated with 10 μg / mL DNase I and 5 μg / mL RNase. After treatment, the preparations were centrifuged at 5,000 rpm for 10 minutes at 4°C. The supernatant was removed and further purified by cesium chloride density gradient centrifugation (densities of 1.2, 1.3, 1.4, and 1.6) at 30,000 rpm at 20°C for 2 hours. The band containing phages was removed and the preparation was placed in a dialysis tube (Spectra / Por4, MWCO 12,000-14,000). Dialysis was performed for 1 hour in TMS containing 2.4 MNaCl, repeated in TMS containing 0.9 MNaCl, and again in TMS containing 0.3 MNaCl.

[0071] For ISP, the same procedure was used, except as follows: strain 12600 was used as the host, and exponential cultures were infected with a MOI of 0.05. After overnight pellet resuspension, further centrifugation was performed at 5,000 rpm for 10 minutes at 4°C before treatment with DNase and RNase. Stock titers were determined by a standard method using plaque counting performed on host strains grown in semi-solid agar. Luciferase reporter phage manipulation

[0072] Target bacteriophages were transformed with a homologous recombination donor construct designed using a host-specific promoter and codon-optimized NANOLUC®, positioned between two 500 bp adjacent sequences having homology to the suspected late gene region in ISP. This construct was inserted into the PstI site of pBAV1KT5gfp (accession HQ191434). The host-specific promoter was modeled after previous studies. NANOLUC® was cloned and codon-optimized by Genewiz (South Plainfield, NJ, USA). Because the homology region shares 99.9% identity, this donor construct was used for both ISP and MP115 manipulation.

[0073] Electroporation-ready Staphylococcus aureus was prepared from RN4220. To achieve this, overnight cultures of RN4220 were diluted and grown in tryptone Sawyer broth (TSB) (Oxiod, Hampshire, United Kingdom) until metaphase logarithmic. The bacteria were then cooled on ice for 1 hour, centrifuged at 4,000 g for 10 minutes at 4°C, and washed three times with ice-cold sterile deionized water. After washing, the final pellet was suspended in ice-cold 10% glycerol and prepared in fixed volumes for storage at -80°C. Then, 100 ng of donor construct plasmid DNA was added to the thawed fixed volumes and incubated at room temperature for 30 minutes before electroporation. Electroporation was performed using a MicroPulser Plus (1.8 kV voltage, 1 pulse, 2.5 msec time constant) with a 0.2 cm cuvette (Bio-Rad, Marnes-la-coquette, France). Cells were harvested in B2 medium (10 g / L peptone, 25 g / L yeast extract, 25 g / L NaCl, 1 g / L K2HPO4, pH 7.5) and spread on TSB agar containing 50 μg / mL kanamycin (Sigma, St. Louis, MO, USA). Transformants were isolated and confirmed by NANOLUC® expression. Colonies were grown in kanamycin-containing TSB for 3 hours before testing. A mixture of 10 μL of culture, 50 μL of NanoGlo buffer, 15 μL of sea urchin lysis buffer, and 1 μL of NanoGlo substrate (Promega, Madison, WI, USA) was prepared and analyzed using GloMax Navigator (Promega, Madison, WI, USA).

[0074] Transformed RN4220 NANOLUC®-positive cultures were grown to the early logarithmic phase, infected with either MP115 or ISP at a MOI of 0.1, and incubated at 37°C for 3 hours with shaking at 225 rpm. The phage solubilizer was centrifuged to remove cell debris, filtered through a 0.45 μM Whatman Puradisc filter (GE Health, Pittsburgh, PA, USA), and finally buffered during TMS using a 100K MWCO protein concentrator (Pierce). Limiting dilution enrichment was then performed to increase recombinant frequency before isolation by plaque screening on semi-solid agar. Individual plaques were isolated using a sterile pipette tip and mixed with 100 μL of TMS buffer. 10 μL of this suspension was used to infect 100 μL of strain 12600 in TSB at 37°C for 2 hours. After infection, 50 μL of NanoGlo buffer, 15 μL of sea urchin lysis buffer, and 1 μL of NanoGlo substrate were added to each well and evaluated with GloMax Navigator. Positive wells with high signal were filtered, diluted, and used to infect the next passage. This was repeated until three consecutive passages were 100% positive and produced plaques that were considered pure. In vitro phage detection assay - Sensitivity, inclusion, and MSSA exclusivity

[0075] The cultures were diluted overnight with brain and heart extract (BHI) broth, and 135 μL of the BHI-diluted culture was transferred to two wells of a 96-well strip plate (Griener Bio-One GmbH, Frickenhausen, Germany) to obtain the desired colony-forming units (CFU) per well (e.g., 10, 1000, or 1000 CFU). Two additional wells containing only 135 μL of BHI broth were used to determine the medium background. One well for each sample served as a control well, to which 15 μL of BHI broth was added. The other well served as a selective well, to which 15 μL of BHI broth containing 22 μg / mL of cefoxitine (Alfa Aesar, Ward Hill, MA, USA) was added. The selective well had a final concentration of cefoxitine of 2.2 μg / mL. Where indicated, the actual CFU for each sample was confirmed by plate counting on BHI agar. 96-well strip plates were sealed with cover film (Thermo Fisher Scientific, Rochester, NY, USA) and incubated at 37°C for 4 hours to facilitate enrichment and selection. Phage cocktails were prepared in lysogenic broth (LB) (Gibco, Grand Island, NY, USA) at a concentration of 1.6 × 10⁶ per 1 mL. 8 Both manipulated phages were contained in plaque-forming units (PFUs). 10 μL of the phage cocktail was added to each well, mixed by pipetting, and then covered again with film. The plates were incubated at 37°C for 4 hours to promote phage infection and luciferase production in the presence of MRSA. 65 μL of detection solution consisting of 50 μL of NanoGlo Buffer, 15 μL of sea urchin lysis buffer, and 1 μL of NanoGlo substrate was added to each well and mixed by pipetting. Samples were read using GloMax Navigator with a 3-minute waiting time and 1-second integration. Results were evaluated at a cutoff of 600 relative light units (RLU), which is approximately three times the background observed in the medium alone. In vitro phage detection assay - non-Staphylococcus aureus exclusivity and bacterial interference

[0076] Overnight cultures of competing organisms were diluted with BHI broth, and 125 μL of the diluted culture was transferred to four wells of a 96-well strip plate to obtain the desired CFU per well (e.g., 10, 1000, or 1000 CFU). Four additional wells, each containing only 125 μL of BHI broth, were used to determine the medium background and baseline signals of MRSA (BAA-1720). Two wells from each sample were assigned to an exclusivity test, and the other two wells were used to assess bacterial interference. For exclusivity, 10 μL of BHI broth was added to both wells, while 10 μL of BHI broth containing MRSA was added to the bacterial interference well. For each condition, one well served as the control well, to which an additional 15 μL of BHI broth was added, and the other served as the selective well, to which 15 μL of BHI broth containing 22 μg / mL of cefoxitin was added. Enrichment, phage infection, and CFU were then determined as described above. Nasal swab phage detection - endogenous samples, MRSA spikes, and autoluminescence

[0077] BBL CultureSwab Liquid Stuart Double swab (Becton Dickinson and Company, Sparks, MD, USA) was used in the experiments described herein. Rayon nasal swabs were self-recovered from volunteers who were instructed to insert the swab into one nostril, rotate it at least five times, and repeat with the same swab in a second nostril. Before processing, the samples were stored overnight at 4°C. To evaluate endogenous nasal samples, one swab was eluted by vortexing in 1 mL of BHI broth for 15 seconds. 135 μL of this nasal eluate was added to two wells of a 96-well strip plate. These wells were evaluated in the same manner as the 135 μL diluted cultures described above.

[0078] To identify genuine MRSA colony formation, a reference method using both direct plating and enriched culture was employed. For direct plating, 135 μL of nasal eluate used in screening was plated onto MRSA Select II agar. For enriched culture, one swab was placed in 3 mL of TSB (Fisher Scientific, Geel, Belgium) containing 6.5% NaCl and grown overnight at 37°C with shaking at 250 rpm. The culture was streaked onto MRSA Select II agar the following day. In both cases, the manufacturer's instructions were followed to determine the presence or absence of MRSA colony formation. A swab was considered MRSA-positive if either method (direct plating or enriched culture) yielded a positive result on selective agar.

[0079] The detection ability of MRSA in the nasal cavity matrix was evaluated by adding diluted MRSA cultures to nasal eluate. For this purpose, 125 μL of nasal eluate was added to two wells of a 96-well strip plate for each sample. 10 μL of diluted MRSA culture was added to both wells. Forty unique nasal cavity samples were evaluated, with eight samples assigned to each MRSA strain tested (BAA-1707, BAA-1717, BAA-1720, BAA-1763, BAA-1766). As a control, 10 μL of each MRSA strain was also added to 125 μL of BHI broth. After addition, the two wells were evaluated in the same manner as with the 135 μL of diluted culture described above.

[0080] The autoluminescence of each nasal cavity sample was evaluated by mixing each sample with a detection solution (phage cocktail) that did not contain a luciferase source. To achieve this, 135 μL of each nasal cavity eluate was combined with 25 μL of BHI broth in a 96-well strip plate. Then, 65 μL of the detection solution was added to each well and mixed by pipetting. The plate was read using a luminometer. Example 1. Study on sensitivity and inclusion

[0081] The methods and systems described herein can identify MRSA strains from diverse genetic backgrounds (Table 1). As shown in Table 1, the MRSA-containing strains were obtained from academic sources. For the majority of strains, detection of various MRSA strains could be achieved with 100 CFU or less. The limits of detection and analytical sensitivity are similar to those of PCR-based screenings described previously.

[0082] Bacteriophage-based MRSA screening involved a 4-hour enrichment period, a 2-hour infection period, and subsequent detection of emitted light using a luminometer. Two wells of a 96-well strip plate were used for each sample, consisting of one control well and one selective well. The selective well was used for MRSA determination and contained cefoxitin, an MRSA selector, while the control well contained only bacterial culture medium and primarily measured phage performance during assay development. Cefoxitin has been shown to be an excellent choice for phenotypic identification of MRSA in disk diffusion and agar dilution assays. Samples were enriched in these wells for 4 hours to facilitate the recovery, growth, and selection of resistant bacteria. This was followed by a 2-hour infection period with a bacteriophage encoding recombinant luciferase. Luciferase production, indicating successful viral infection, was measured by detecting emitted light using a luminometer after substrate addition. Seventeen diverse MRSA strains were evaluated using this method in triplicate wells with starting targets of 10, 100, or 1,000 colony-forming units (CFUs) (Table 1). CFU (determined from plate counts) and relative light units (RLU) values ​​are shown in Table S1. Positive results were determined based on a 600 RLU cutoff, which is approximately three times the background observed in culture medium alone.

[0083] Under control conditions, positive results were obtained in all 51 wells (100%) tested at both 100 CFU / well and 1,000 CFU / well. At 10 CFU / well, 48 wells (94.1%) were positive. Three specific strains of MRSA were positive in only two of the three wells at 10 CFU / well. These results highlight the ability of the phage cocktail to recognize diverse MRSA isolates. When cefoxitin was included for MRSA detection, positive signals could still be detected in all 51 wells (100%) at 1,000 CFU / well and in all 48 wells (94.1%) at 100 CFU / well. The failure to detect BAA-42, also known as HDE288, at 100 CFU / well was not entirely unexpected. This strain belongs to an "early clone" of MRSA with low levels and heterogeneous methicillin resistance. As shown in Table 1, 44 out of 51 selective wells (86.3%) remained positive with as little as 10 CFU. Detection limits were determined for each strain based on the minimum CFU required for 100% detection in both control and selective wells. Of the 17 MRSA strains tested, 13 could be reliably detected with 10 CFU per well, while three required 100 CFU per well. BAA-42 was the only strain requiring more than 100 CFU per well for consistent positive detection by MRSA selection. As shown in Table 1, the MRSA assay demonstrates 100% inclusion for the 17 MRSA strains tested with 100 CFU. The MRSA assay also demonstrates selectivity for 48 out of 51 MRSA strains tested. Overall, these results demonstrate the ability of this screening to detect the presence of genetically diverse MRSA strains at low bacterial loads. [Table 1] [Table S1-1] [Table S1-2] Example 2. Exclusivity and specificity of in vitro MRSA screening

[0084] In addition to highly sensitive MRSA detection, a successful MRSA assay must also demonstrate the ability to exclude the majority of methicillin-sensitive Staphylococcus aureus (MSSA) strains. Table 2 shows five well-characterized strains of MSSA evaluated using the method described herein at 100, 1,000, and 10,000 CFU levels in a set of three wells, providing CFU values ​​determined from plate counts and RLU values. The MRSA control wells did not contain cefoxitin, while the MRSA selective wells contained cefoxitin. As expected, MSSA strains were positive in 100% of the control wells at 100, 1,000, and 10,000 CFU levels. The inclusion of cefoxitin in the selective wells resulted in a significant reduction in positive results. In the MRSA selective wells containing cefoxitin, 0 out of 15 selective wells (0%) were positive at 100 CFU, while only 1 out of 15 selective wells (6.7%) were positive at 1,000 CFU and 10,000 CFU. These results support the ability of the MRSA assay to identify most MSSA strains. [Table 2] [Table S2]

[0085] As shown in Table 3, the exclusivity of MRSA screening was evaluated in vitro against a panel of 40 strains encompassing 21 specific genera and 32 distinct species, beyond MSSA. CFU and RLU values ​​(determined from plate counts) are shown in Table S3. The CFU for each exclusive strain was over 1,500 CFU per well (median CFU of 15,950). Assessing specificity, Table 3 shows that 6 of the 40 strains (15%) were positive in the control well. The positive signal under this condition was a result of phage cocktail cross-reactivity, observed in both Staphylococcus and Bacillus species. Many Staphylococcus aureus phages are polyvalent and have been demonstrated to lyse both coagulase-positive and coagulase-negative Staphylococcus species. Adsorption of Staphylococcus phages by Bacillus species has been previously reported and may be related to the similarity of their cell wall teichoic acid (WTA). Despite this cross-reactivity, under selective conditions, zero out of 40 strains were positive, which would not have resulted in false positives for MRSA. These results demonstrate the specificity of the phage cocktail used in the experiments described herein and the exclusivity of the entire assay.

[0086] The ability of the MRSA screening to detect small numbers of MRSA in the presence of excessive competitive bacterial loads was evaluated. For this purpose, approximately 50 CFU of MRSA was combined with each strain from the exclusivity panel in at least 20-fold excess (Table 3). The CFU and RLU values ​​(determined from plate counts) are shown in Table S3. Surprisingly, 39 out of 40 wells (97.5%) and 40 out of 40 wells (100%) were positive under control and selective conditions, respectively, in the presence of competing species. Streptococcus pneumoniae inhibited detection under control conditions when tested in 100-fold excess. This is not surprising, given that antagonism between these species is known both in vitro and in vivo. Importantly, this effect was lost in the presence of cefoxitin (MRSA selective condition) and therefore would not result in false negatives for MRSA. This data demonstrates the ability of this screening to detect low levels of MRSA in environments containing excessive competing organisms. [Table 3-1] [Table 3-2] [Table S3-1] [Table S3-2]

[0087] Example 3. Screening performance among circulating Staphylococcus aureus clinical isolates in vitro.

[0088] MRSA isolates from human clinical specimens were obtained in-house from three geographically distinct clinical microbiology laboratories in the United States (Burlington, North Carolina; Phoenix, Arizona; and Raritan, New Jersey). MSSA isolates were similarly obtained from one location (Burlington, North Carolina). Identification of MRSA or MSSA was confirmed by plating on selective chromogenic agar. A total of 390 clinical MRSA strains were isolated from characteristic specimens and evaluated by MRSA screening. RLU and CFU values ​​for each strain are provided (Table S4).

[0089] Table 4 shows that the median MRSA loading per well tested was 47 CFU. As shown in Table 4, 388 out of 390 clinical MRSA strains (99.5%) were detected positively in the control well. Under cefoxitin selection, 381 out of 390 clinical MRSA strains (97.7%) were positive and identified as MRSA by screening. Clinical MSSA strains were tested for exclusion at higher loadings, i.e., 10-fold or 100-fold MRSA levels (500 CFU and 5,000 CFU, respectively). 122 out of 123 clinical MSSA strains (99.2%) were detected positively under the control conditions of either inoculum. However, in the selective wells, the positive signal from 500 CFU decreased to 8 out of 123 MSSA strains (6.5%). At approximately 5,000 CFU per well, this false-positive rate increased to 21 out of 123 strains (17.1%). This suggests that while most MSSA strains are negative, some may overwhelm selection with high loadings, potentially leading to false positives. Importantly, of the 513 clinical Staphylococcus aureus isolates tested, 510 (99.4%) were positive under control conditions. This continues to support the idea that the phage cocktail utilized in the described method and system provides broad host-range applicability. Overall, these results demonstrate the ability of this screening to successfully recognize and detect the majority of clinical MRSA strains while excluding most clinical MSSA strains. [Table 4] [Table S4-1] [Table S4-2] [Table S4-3] [Table S4-4] [Table S4-5] [Table S4-6] [Table S4-7] [Table S4-8] [Table S4-9] [Table S4-10] [Table S4-11] [Table S4-12] [Table S4-13] [Table S4-14] [Table S4-15]

[0090] Example 4. Specificity and screening performance using human nasal swabs.

[0091] Anterior nasal cavity specimens were self-collected from 40 adult volunteers using rayon swabs. Previous studies have confirmed the effectiveness of self-collection for detecting MRSA colonization. Before processing, specimens were stored overnight at 4°C to mimic possible sample transport conditions. Reference methods using both direct plating and enriched cultures were used to identify genuine MRSA colonization. All 40 human nasal cavity specimens were negative by both reference methods and determined to lack MRSA colonization (Table 5). The absence of detection in 40 individuals is not surprising, as the estimated rate of MRSA colonization in healthy adults is less than 2%.

[0092] To screen these samples, swabs were eluted into bacterial culture medium and added to wells containing (selective) or not containing (control) cefoxitin. Positive results under the selective condition were considered to be positive MRSA results. The control condition was included to demonstrate the effectiveness of selection, although it was not required or utilized for MRSA determination. Due to the high nasal colonization rate of Staphylococcus species and previously described cross-reactivity with phage cocktails, positive results were expected in most control wells. As expected, 36 out of 40 (90%) samples were positive in the control wells. RLU values ​​for endogenous samples are provided (Table S5).

[0093] Of the 40 samples, 36 (90.0%) were negative for MRSA detection, consistent with the reference method. False positives were identified in four samples, with median RLU signals less than 5 times the signal cutoff. All nasal samples were negative when directly tested with luciferase substrates, indicating that nonspecific autoluminescence was not a significant source of false positives (Table S5). The exact mechanism behind the false positive signals in these samples remains unclear, but it may be related to methicillin-resistant coagulase-negative staphylococci. Furthermore, several MSSA strains have been previously observed to produce false positive results at high bacterial loads (Table 4). Overall, this method successfully screened out the majority (90%) of MRSA-negative samples. [Table 5] [Table S5-1] [Table S5-2]

[0094] To determine whether this method can successfully detect MRSA in the nasal matrix, five well-characterized MRSA strains were added to the eluates from the aforementioned 40 non-colonizing nasal swabs. RLU and CFU values ​​for each sample are provided (Table S5). The median MRSA loading was 87 CFU per well. 40 out of 40 (100%) MRSA-added samples were positive under both control and selective conditions (Table 5). The absence of invalid samples suggests the absence of assay inhibitors in these individuals. The successful detection of five distinctive MRSA strains when added at low loadings in these samples supports the effectiveness of bacteriophage-based screening in the nasal matrix.

[0095] As illustrated in the examples, this disclosure provides a culture-based approach to an MRSA luciferase phage reporter assay that achieves highly sensitive and rapid detection of MRSA from nasal swabs. As shown in Table 1, diagnostic screening using the MRSA luciferase phage reporter assay was able to identify MRSA strains from diverse genetic backgrounds in approximately 6 hours. For most MRSA strains, successful detection required the presence of only 10–100 CFU per well, equivalent to approximately 75–750 CFU per nasal swab. This detection limitation is similar to that of previously described PCR-based screenings. The median MRSA load recovered from carrier nasal swabs was found to exceed 10,000 CFU. Furthermore, individuals with high nasal colonization loads are more likely to harbor MRSA in multiple body sites and be vectors of transmission. Therefore, the sensitivity of this assay appears to be well-suited to addressing the expected load from clinical nasal specimens, regardless of whether the goal is to eliminate MRSA carriers or limit patient-to-patient transmission.

[0096] In several respects, the performance of luciferase reporter phage assays is highly dependent on the selection of bacteriophages. The MRSA diagnostic screening in this example utilized NanoLuc-expressing recombinants of two phages, ISP and MP115, members of the large lytic Staphylococcus bacteriophage bacteriophage family of the Myoviridae family. These phages bind to the host surface primarily via the highly conserved WTA, resulting in broad host range capabilities. WTA-deficient mutants are thought to be resistant to all or at least most Staphylococcus phages. While resistant WTA-deficient mutants are hypothetically possible, previous studies have shown that WTA is necessary for both nasal colonization and methicillin resistance. Generally, the loss of WTA also results in an overall reduction in in vivo adaptation costs and pathogenicity. Therefore, it is reasonable to expect all current and future MRSA strains involved in nasal colonization to possess the receptor targeted by this screening. Furthermore, this conclusion is further supported by the data in Table 4, which shows positive phage signals detected in 99.5% of the clinically tested MRSA isolates.

[0097] As shown in the results in Table 4, of 513 clinical Staphylococcus aureus strains, two MRSA isolates (BNC159 and PHX079) and one MSSA isolate (MSSA090) failed to produce a positive signal under control conditions. One of these isolates (PHX079) appeared to have growth defects in culture (data not shown). Poor growth during enrichment may have contributed to the inability to reliably detect this MRSA strain. The inability to detect BNC159 and MSSA090 may be related to phage resistance through restriction modification systems or capsule production. Restriction modification systems target and eliminate foreign DNA, often identified through the presence or absence of DNA methylation at specific motifs. Staphylococcal phages have evolved under the pressure of these pathways, and there is evidence that some phages completely lack specific sequences targeted by these systems. Nevertheless, the diversity of restriction modification systems across Staphylococcus aureus is extensive and may contribute to the resistance observed in these isolates. Separately, capsule production is associated with phage resistance in Staphylococcus aureus via shielding of surface receptors. While some common strains of Staphylococcus aureus do not produce capsule polysaccharides, this mechanism may promote the rare (<1%) resistance observed.

[0098] Furthermore, Table 4 also shows that the combination of the MRSA luciferase phage reporter assay and a selector (e.g., an antibiotic) limited the viability and proliferation of non-MRSA and did not interfere with MRSA detection. For example, the MRSA luciferase phage reporter assay utilized cefoxitin to limit the viability and proliferation of non-MRSA. The results in Table 4 demonstrate the effectiveness of this selection, as only 6.5% of clinical MSSA strains were positive when tested at approximately 500 CFU per well. Surprisingly, cefoxitin did not interfere with MRSA detection, as 97.7% of clinical MRSA strains remained positive in the selective wells at approximately 50 CFU per well. Furthermore, Table 3 shows that this selector was also beneficial in limiting false positives from several Bacillus species and coagulase-negative staphylococci, while simultaneously preventing interference from Streptococcus pneumoniae. Cefoxitin has demonstrated as an excellent choice for MRSA selection and can identify a diverse range of isolates. Despite the high detection rate of clinical MRSA, some strains yielded false-negative results in the presence of cefoxitine. Since the clinical MRSA strains were evaluated at particularly low loadings in some cases, these strains are likely to exhibit low levels of resistance or hetero-resistance. Such strains may exhibit detection limits exceeding 100 CFU per well, similar to those found for BAA-42 (Table 1).

[0099] Regarding performance with nasal swabs, Table 5 shows that 90.0% of MRSA-negative samples yielded negative test results under selection, consistent with the reference method. Therefore, false positives were detected in 10% of the nasal elutes. These false positives may stem from three origins. Firstly, autoluminescence may occur, but this was ruled out in these samples by demonstrating the need for luciferase addition, as shown in Table S5. Secondly, high loadings of certain MSSA strains may result in false positives (Table 4). Finally, some cross-reactive species of coagulase-negative staphylococci can become methicillin-resistant via the same resistance mechanism as MRSA. These species may contribute to the weak false MRSA positivity observed in four samples.

[0100] The methods and systems for detecting MRSA described herein are unique in that they assess sample validity by requiring the viability of the endogenous nasal microbiota. To replicate the endogenous nasal microbiota, one of five MRSA strains was added to the nasal eluate (Table 5). As shown in Table 5, positive detection of low MRSA loadings in the nasal matrix was achieved in 100% of the added samples. Importantly, this indicates that successful bacteriophage infection and luciferase production can occur in the nasal matrix. Furthermore, this reveals that the previously observed negative control wells in 10% of endogenous samples were not the result of assay inhibitors. Overall, these results strongly suggest that if MRSA colonization is present, it will be detected in nasal specimens.

[0101] The bacteriophage-based MRSA assay described herein is a member of a new generation of luciferase reporter phage systems that utilize NanoLuc for highly sensitive detection of the target species. The method proved highly comprehensive and, when combined with cefoxitin selection, identifies most non-resistant strains. Furthermore, the screening was able to identify low loadings of MRSA in nasal samples without evidence of problematic interference. Additionally, MRSA detection performed within 6 hours allows for immediately usable results to be obtained in a single work shift. Ultimately, these data demonstrate that the bacteriophage-based MRSA assay described herein may be a promising new tool for detecting MRSA colonization from nasal swabs.

[0102] Example 5. Direct coating of NanoLuc onto medium and high protein binding plates.

[0103] Staphylococcus aureus (ATCC12600) in trypsin-soybean broth (TSB) at a logarithmic phase (0.41 OD). 600The cultures were grown to the desired load by diluting the cultures with TSB and confirming this by plating them onto TSB agar to obtain colony-forming units (CFUs). 37.5 μL of TSB or human blood in 96-well strips (high binding; Grenier Bio-One, reference no. 762074) were directly added to 12.5 μL of each dilution. Where indicated, some strips contained conjugated anti-NanoLuc antibody (purified mouse monoclonal IgG clone #965808; catalog no. MAB10026) for capture. Human blood was collected from a single donor using heparin sodium as an anticoagulant. For blood samples, 100 μL of polyanethole sulfone was added. TSB containing sodium phosphate (SPS) was added to obtain a 25% human blood matrix. The final concentration of SPS in the well (150 μL volume) was 0.05%. For the TSB sample, 100 μL of TSB was added to achieve the same 150 μL volume. The test strips were then sealed with cover film and incubated at 37°C for 30 minutes. After this short enrichment period, 20 μL of phage work stock was added to the wells containing the TSB matrix. The phage work stock was 8 × 10⁶. 7Both MP115.NL and SAPJV1.NL were included in plaque-forming units / mL. To enable infection in wells containing blood matrix, 0.5 mg of recombinant Staphylococcus protein A (pro-356, Prospec, Ness-Ziona, Israel) per well was included in 20 μL of phage working stock, as indicated. The assay strips were again sealed with cover film and incubated at 37°C for 3 hours. After infection, these strips were washed three times with 300 μL of PBS-T (10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20, pH 7.4). Washing was performed using an automated plate washer (AccuWash, Thermo Fisher Scientific, Waltham, MA, USA). 100 μL of NanoGlo buffer (Promega, Madison, WI, USA) containing 1 μL of NanoGlo substrate (Promega, Madison, WI, USA) was added to each well. After a 3-minute waiting period, the signal output of each sample as relative light units (RLU) was determined using GloMax Navigator (Promega, Madison, WI, USA). The signal against background (S / B) was calculated by dividing the RLU from each sample by the RLU observed in the medium control against its test matrix. [Table 6]

[0104] In these examples, the anti-NanoLuc antibody is the immobilized binding partner. Table 6 shows a significant increase in signal detection when the indicator protein is captured by the immobilized binding partner. For example, in samples with low or high loadings of Staphylococcus aureus, the RLU is significantly higher when the indicator protein is captured by the anti-NanoLuc antibody than when the sample is not captured using the control strip. Surprisingly, if Staphylococcus aureus has bound IgG, infection by Staphylococcus aureus cannot occur. The addition of protein A allows infection by Staphylococcus aureus. Red blood cells and other serum proteins do not interfere with the capture of the expressed indicator protein. Furthermore, signal quenching seen in the control by red blood cells is eliminated, and the signal is maintained or increased above the background. Thus, the indicator protein can be detected using whole blood samples with minimal interference from other components (e.g., proteins) in the sample. Conventionally, serum or plasma is isolated from blood for reliable detection of the indicator protein product. Advantageously, the examples demonstrate that the detection method can be performed on whole blood samples collected directly from patients by using this capture step.

[0105] Example 6. Antibiotic susceptibility testing in human blood

[0106] Methicillin-resistant Staphylococcus aureus (MRSA) strains (ATCC BAA-1720, CDC AR0480) and methicillin-sensitive Staphylococcus aureus (MSSA) strain (ATCC 12600) were sterilized in trypsin-soybean broth (TSB) during a logarithmic period (OD in the range of 0.16 to 0.4). 600The cultures were grown to the desired load. The cultures were diluted with TSB to obtain the desired load, which was confirmed by plating onto TSB agar to obtain colony-forming units (CFUs). 50 μL of each dilution was added to test strips. Where indicated, some strips contained anti-NanoLuc antibody (purified mouse monoclonal IgG clone #965808; catalog number MAB10026) bound to a medium-binding plate (Grenier Bio-One, Strips Plate 12xF8, PS, F-Bottom, White, Lumitrac, Med Binding, reference no. 762075) or a high-binding plate (Grenier Bio-One, reference no. 762074) for capture. Either 85 μL of TSB or human blood diluted with TSB and sodium polyanethole sulfonate (SPS) was added. Human blood was collected from a single donor using heparin sodium as an anticoagulant. Next, 15 μL of either TSB or 22 μg / mL cefoxitin (FOX) in TSB was added to each well. The final concentrations of each component in the wells (150 μL volume) were 25% human blood, 0.0375% SPS, and 2.2 μg / mL FOX. The test strips were then sealed with cover film and incubated at 37°C for 2 hours. After this selective enrichment, 20 μL of phage work stock was added to the wells containing the TSB matrix. The phage work stock was 8 × 10⁶ 7 MP115.NL and 6.9 × 10⁻¹⁰ plaque formation units / mL (pfu / mL) 8The assay strips contained SAPJV1.NL at pfu / mL. For wells containing blood matrix, 0.5 mg of recombinant Staphylococcus aureus protein A (pro-356, Prospec, Ness-Ziona, Israel) was added per well in 20 μL of phage work stock. The assay strips were resealed with cover film and incubated at 37°C for 3 hours. After infection, the anti-NanoLuc capture and control strips were washed three times with 300 μL of PBS-T (10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20, pH 7.4). Washing was performed using an automated plate washer (AccuWash, Thermo Fisher Scientific, Waltham, MA, USA). 100 μL of NanoGlo buffer (Promega, Madison, WI, USA) containing 1 μL of NanoGlo substrate (Promega, Madison, WI, USA) was added to each well. The "no washing + no capture" strips were not washed; instead, 65 μL of a master mix containing 50 μL of NanoGlo buffer, 15 μL of TSB, and 1 μL of NanoGlo substrate was added. Strips blocked with 5% BSA (bovine serum albumin, Sigma Life Science product number A9647) were washed. The BSA-blocked strips were blocked with BSA for nonspecific binding sites. After a 3-minute waiting period, the signal output of each sample as relative light units (RLU) was determined using GloMax Navigator (Promega, Madison, WI, USA). The signal against background (S / B) was calculated by dividing the RLU from each sample by the RLU observed in the medium control against its test matrix. [Table 7]

[0107] In Table 7, the “no capture + no washing” example demonstrated the generation of a full signal and the reduction of the signal by cefoxitin when the assay was performed in culture medium only (TSB). When performed in the presence of blood, the signal is quenched. When a capture strip is used, there is a significant increase in the signal due to the removal of the quenching performed by the blood. A strip blocked with 5% BSA (bovine serum albumin, Sigma Life Science product number A9647) is used to demonstrate nonspecific binding. Here again, the example demonstrates a significant increase in signal detection when the indicator protein is captured by an immobilized binding partner for whole blood samples. Furthermore, signal detection was significantly improved by the capture step for whole blood samples containing antibiotics. Surprisingly, the indicator protein can be detected using whole blood samples with minimal interference from other components in the sample.

[0108] Example 7. Titration of a plate coated with NanoLuc.

[0109] A 1.5 mg / mL purified NANOLUC stock solution was diluted to 1 ng / mL in PBS. Serial 10-fold dilutions in PBS were prepared from 1 ng / mL to 0.001 pg / mL. Rabbit anti-mouse IgG (Abcam, catalog number 46540) or goat anti-mouse IgG (Abcam, catalog number 6708) was diluted to 10 μg / mL in PBS and pipetted into 100 μL / well. The plates were incubated at 2–8°C for 18–20 hours, then washed three times with 300 μL of PBS / well / wash. Mouse anti-NanoLuc antibody (purified mouse monoclonal IgG, clone #965808, R&D Systems, catalog number MAB10026) was diluted to 1 μg / mL in PBS and pipetted into 100 μL / well of plates coated with rabbit or goat anti-mouse IgG. The assay strips included 5% BSA-blocked strips for nonspecific binding determination and uncoated strips for Nanoluc activity measurement. The assay strips were sealed with cover film and incubated at 37°C for 3 hours. The antibody-coated strips were washed three times with 300 μL / well of PBS-T (10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20, pH 7.4). Washing was performed using an automated plate washer (AccuWash, Thermo Fisher Scientific, Waltham, MA, USA). 100 μL of NanoGlo buffer (Promega, Madison, WI, USA) containing 1 μL of NanoGlo substrate (Promega, Madison, WI, USA) was added to each well. After a 3-minute waiting period, the signal output of each sample as relative light units (RLU) was determined using a GloMax Navigator (Promega, Madison, WI, USA). The signal-to-background ratio (S / B) was calculated by dividing the RLU from each sample by the RLU observed in the PBS control for that test. [Table 8] [Table 9]

[0110] Tables 8 and 9 demonstrate that plates coated with rabbit anti-mouse IgG or goat anti-mouse IgG gave improved orientation of mouse anti-nanoluc luciferase to enhance the capture / binding surface. Indeed, plates coated with rabbit anti-mouse IgG or goat anti-mouse IgG were more readily available for binding the indicator protein product. The plate coating showed improved signal detection, which may be due to the orientation of mouse anti-nanoluc luciferase and the availability of the binding site for the indicator protein. Example

[0111] Example 1: A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample, comprising: obtaining a sample; adding a selectant to the sample; and contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene that is specific to Staphylococcus aureus, the indicator gene encoding an indicator protein product, capturing the indicator protein product, and detecting a signal produced by the indicator protein product, the detection of which is used to determine the presence of MRSA in the sample.

[0112] Example 2 is an exemplary preceding or succeeding method in which the selective agent includes an antibiotic.

[0113] Example 3 is an exemplary preceding or succeeding method in which the antibiotic comprises cefoxitin.

[0114] Example 4 is an exemplary preceding or succeeding method in which the sample originates from a nasal swab.

[0115] Example 5 is any preceding or succeeding exemplary method in which the method detects 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacterium in a sample.

[0116] Example 6 is an exemplary preceding or succeeding method in which the cocktail comprises at least two different types of recombinant bacteriophages, at least one of which is derived from ISP, MP115, or a combination thereof.

[0117] Example 7 is one of the preceding or following exemplary methods, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates a unique signal or a soluble enzyme that generates a signal upon reaction with a substrate.

[0118] Example 8 is one of the preceding or following exemplary methods, further comprising an untranslated region upstream of a codon-optimized indicator gene, wherein the untranslated region comprises a bacteriophage late gene promoter.

[0119] Example 9 is an exemplary method, either preceding or succeeding, in which the capture step includes bringing the indicator protein product into contact with a surface.

[0120] Example 10 is an exemplary preceding or succeeding method in which the surface is a microtiter plate, latex particles, a lateral flow strip, beads, magnetic particles, or a dipstick.

[0121] Example 11 is an exemplary preceding or succeeding method, further comprising depositing an immobilized binding partner onto the surface before capturing the indicator protein product.

[0122] Example 12 is an exemplary preceding or succeeding method in which the immobilized binding partner is an antibody or a fragment thereof.

[0123] Example 13 is an exemplary preceding or succeeding method, further comprising cleaning the surface containing the immobilized bonding partner.

[0124] Example 14 is an exemplary method, either preceding or succeeding, further comprising washing the surface after capturing the indicator protein product.

[0125] Example 15 is an exemplary preceding or succeeding method in which the signal-to-background ratio generated by detecting the indicator protein product is at least 2.0 or at least 2.5.

[0126] Example 16 is an exemplary preceding or succeeding method in which the sample is first incubated under conditions favorable to growth for an enrichment period of 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or less than 2 hours.

[0127] Example 17 is an exemplary preceding or succeeding method comprising: obtaining a sample; contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprising an indicator gene and being specific to a microorganism, the indicator gene encoding an indicator protein product; contacting the indicator protein product with a surface comprising an immobilized binding partner for capturing the indicator protein product; and detecting a signal generated by the indicator protein product, the detection of which is used to determine the presence of the microorganism in the sample.

[0128] Example 18: A kit for detecting methicillin-resistant Staphylococcus aureus, comprising a nasal swab, an assay comprising a recombinant bacteriophage and antibiotic specific to Staphylococcus aureus, and a surface for capturing an indicator protein product.

[0129] Example 19 is a kit of either a preceding or succeeding example, which includes a bonding partner on which the surface is immobilized.

[0130] Example 20 is an exemplary preceding or succeeding kit in which the antibiotic comprises cefoxitin.

[0131] This disclosure is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art are included within this disclosure as defined by the claims. The present invention provides, for example, the following items: (Item 1) A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample, Obtaining a sample, Adding a selective agent to the aforementioned sample, The method involves contacting the sample with a cocktail containing one or more infectious agents, wherein the infectious agent includes an indicator gene that is specific to Staphylococcus aureus, and the indicator gene encodes an indicator protein product. To capture the aforementioned indicator protein product, The detection of a signal generated by the aforementioned indicator protein product is used to determine the presence of MRSA in the sample. Methods that include... (Item 2) The method according to item 1, wherein the selected agent includes an antibiotic. (Item 3) The method according to item 2, wherein the antibiotic comprises cefoxitin. (Item 4) The method described in item 1, wherein the sample is derived from a nasal swab. (Item 5) The method according to item 1, wherein the method detects 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacterium in the sample. (Item 6) The method according to item 1, wherein the cocktail comprises at least two different types of recombinant bacteriophages, at least one of which is derived from ISP, MP115, or a combination thereof. (Item 7) The method according to item 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates a unique signal or a soluble enzyme that generates a signal upon reaction with a substrate. (Item 8) The method according to item 1, further comprising an untranslated region upstream of a codon-optimized indicator gene, wherein the untranslated region comprises a bacteriophage late gene promoter. (Item 9) The method according to item 1, wherein the capturing step includes bringing the indicator protein product into contact with the surface. (Item 10) The method according to item 9, wherein the surface is a microtiter plate, latex particles, a lateral flow strip, beads, magnetic particles, or a dipstick. (Item 11) The method according to item 9, further comprising depositing an immobilized binding partner onto the surface before capturing the indicator protein product. (Item 12) The method according to item 11, wherein the immobilized binding partner is an antibody or a fragment thereof. (Item 13) The method according to item 11, further comprising cleaning the surface containing the immobilized bonding partner. (Item 14) The method according to item 13, further comprising washing the surface after capturing the indicator protein product. (Item 15) The method according to item 1, wherein the signal-to-background ratio generated by detecting the indicator protein product is at least 2.0 or at least 2.5. (Item 16) The method according to item 1, wherein the sample is first incubated under conditions favorable to growth for an enrichment period of 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or less than 2 hours. (Item 17) A method for detecting microorganisms in a sample, Obtaining a sample, The method involves contacting the sample with a cocktail containing one or more infectious agents, wherein the infectious agents include an indicator gene, are specific to the microorganism, and the indicator gene encodes an indicator protein product. The method involves bringing the indicator protein product into contact with a surface, wherein the surface is equipped with an immobilized binding partner for capturing the indicator protein product. The detection of a signal generated by the aforementioned indicator protein product is used to determine the presence of the microorganism in the sample. Methods that include... (Item 18) A kit for detecting methicillin-resistant Staphylococcus aureus, Nasal swab and, Assays including recombinant bacteriophages and antibiotics specific to Staphylococcus aureus, A surface for capturing indicator protein products, A kit that includes this. (Item 19) The kit according to item 18, wherein the surface comprises an immobilized bonding partner. (Item 20) The kit described in item 18, comprising the aforementioned antibiotic cefoxitin.

Claims

[Claim 1] The invention described in the specification.

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