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

Abstract

Disclosed are compositions, methods, and systems for detecting MRSA, e.g., nasal colonization by MRSA. In certain embodiments, the methods use bacteriophage-based amplification of signals in the detection of bacteria and other microorganisms to detect MRSA. A method for detecting MRSA can include preparing an assay containing a selection agent and a cocktail containing at least two different types of recombinant bacteriophage, 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-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 018,081, filed April 30, 2020, which is incorporated herein by reference in its entirety. Field

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

[0003] background There is a strong interest in detecting bacteria and other microorganisms that can cause various forms of debilitating and fatal infections. Bacterial pathogens can cause significant morbidity in humans and livestock, as well as significant economic losses. Specifically, methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen capable of causing fatal infections. MRSA is a leading cause of surgical site infections in hospitals, associated with prolonged hospital stays, increased readmission rates, reduced survival rates, and economic losses. Due to the significant clinical and financial burden on the healthcare industry, significant efforts have been made to understand and control the causes of MRSA-associated infections. Nasal carriage of MRSA has been found to be a major risk factor for subsequent disease, and the majority of S. aureus infections can be attributed to endogenous colonizing strains. Eliminating this risk factor through decolonization of MRSA nasal carriers has proven to be a successful strategy for reducing surgical site infections.

[0004] Traditional microbiological testing for detecting MRSA relies on nonselective and selective enrichment cultures, followed by plating on selective media and further testing to confirm suspect colonies from patient nasal swab specimens. Culture-based detection methods may involve the use of chromogenic and selective agar plates and often demonstrate strong performance in terms of sensitivity and specificity. While often significantly less expensive than some 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 researched and implemented. However, these methods also have drawbacks. For example, techniques involving immunoassays or gene probes generally require an enrichment step to achieve sufficient sensitivity. Polymerase chain reaction (PCR) tests also include an amplification step, thus enabling 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 produced promising results, this method also has drawbacks. First, several previous PCR assays for detecting novel MRSA strains have failed, so new generations of real-time PCR must be constantly developed to adapt to the changing genetic landscape of MRSA resistance. Second, compared with culture-based alternatives, the high cost of real-time PCR has led to uncertain cost-effectiveness, especially in areas with low community colonization rates.

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

[0007] A brief summary Embodiments of the present disclosure include compositions, methods, devices, systems and kits for detecting nasal colonization of MRSA. The present disclosure can be implemented in a variety of ways.

[0008] In some embodiments, the present disclosure provides a method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample, the method comprising obtaining a sample, adding a selection agent to the sample, contacting the sample with a cocktail comprising one or more infectious agents, the infectious agents comprising an indicator gene and 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, wherein the detection of the signal is used to determine the presence of MRSA in the sample.

[0009] In some embodiments, the present disclosure provides a method for detecting a microorganism in a sample, the method comprising obtaining a sample, contacting the sample with a cocktail comprising one or more infectious agents, the infectious agents comprising an indicator gene and specific for the 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 produced by the indicator protein product, wherein the detected signal is used to determine the presence of the microorganism in the sample.

[0010] In some embodiments, the present disclosure utilizes novel recombinant bacteriophages to detect MRSA from nasal swab specimens. In some embodiments, the novel recombinant bacteriophages are specific for Staphylococcus aureus. A novel diagnostic screen utilizes an assay that includes a recombinant bacteriophage containing a luciferase reporter that can recognize Staphylococcus aureus while relying on antibiotics to limit the growth of non-MRSA strains. The methods described herein can be used to detect a variety of MRSA strains.

[0011] In some embodiments, the present disclosure provides a method for detecting MRSA from a sample, the method comprising: (a) contacting the sample with a selection agent; (b) contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene and are specific for Staphylococcus aureus, and the indicator gene encodes an indicator protein product; and (c) detecting a signal produced by the indicator protein product, wherein the detection of the signal is used to determine the concentration of MRSA in the sample. In some embodiments, the selection agent 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 for Staphylococcus aureus bacteria. In further embodiments, the indicator gene encodes an indicator protein product that produces a unique signal or an enzyme that produces a signal upon reaction with a substrate.

[0013] In some embodiments, the disclosure provides a method for detecting MRSA from a sample, the method comprising contacting the sample with a selective agent, the sample being derived from a nasal swab; contacting the sample with a cocktail comprising one or more infectious agents, the infectious agents comprising an indicator gene and specific to Staphylococcus aureus, the indicator gene encoding 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 present disclosure provides kits and systems for detecting nasal swabs containing MRSA, and assays comprising a recombinant bacteriophage specific for Staphylococcus aureus and an antibiotic solution. In some embodiments, the present disclosure provides kits and systems for detecting nasal swabs containing a microorganism, assays comprising a recombinant bacteriophage specific for a microorganism and optionally an antibiotic, and surfaces for capturing an indicator protein product.

[0015] Certain embodiments of the present disclosure utilize methods and constructs described in U.S. Patent Application Publication No. 2015 / 0218616, which is incorporated herein by reference in its entirety. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description Disclosed herein are compositions, methods, and systems that demonstrate surprising sensitivity for detecting various strains of methicillin-resistant Staphylococcus aureus (MRSA) in test samples (e.g., biological samples) in shorter time frames than conventional methods. The compositions, methods, and systems disclosed herein can detect MRSA in shorter time frames than previously thought possible by using genetically modified, infectious bacteriophages that require reduced incubation times for enrichment, or, in some embodiments, minimize the incubation time during which MRSA can potentially grow. Surprisingly, assays using one or more recombinant bacteriophages in the presence of antibiotics (e.g., cefoxitin) for incubation with test samples detect various MRSA strains at concentrations that produce extremely low numbers of colony-forming units (CFUs). Such low CFU concentrations were previously believed to be detectable only after using culture-based methods requiring incubation times of more than 24 hours. However, the assays described herein can easily discover, bind, and infect low numbers of target cells. In some embodiments, the assay detects MRSA from nasal swab specimens in less than 10 hours, at a cost similar to longer culture-based methods.

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

[0018] In some aspects, the present disclosure provides a recombinant bacteriophage 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 cocktail of bacteriophages 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 selection agent (e.g., an antibiotic) can distinguish MRSA from the presence of other types of bacteria, particularly methicillin-sensitive Staphylococcus aureus (MSSA).

[0019] In some aspects, a method for detecting MRSA may use an infectious agent for detecting Staphylococcus aureus. For example, in certain embodiments, the microorganism of interest is MRSA, and the infectious agent is a bacteriophage that specifically infects Staphylococcus aureus. Thus, in certain embodiments, the method includes selecting one or more bacteriophages that specifically infect Staphylococcus aureus bacteria, preparing a recombinant bacteriophage derived from the Staphylococcus aureus bacteriophage, preparing an assay comprising the recombinant bacteriophage and a selection agent (e.g., an antibiotic), and and providing a sample from a nasal swab or similar source for analysis in the assay. In certain embodiments, the recombinant bacteriophage comprises an indicator gene. In certain embodiments, the indicator gene can be inserted into a late gene region of the bacteriophage such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in production of an indicator protein product. The method can include 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 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 a selection agent, e.g., an antibiotic. In some embodiments, the assay utilizes a selection agent, e.g., cefoxitin, to limit the survival of susceptible bacteria while allowing MRSA growth. For example, the selection agent can kill or reduce the growth of all Staphylococcus aureus bacteria other than MRSA (e.g., MSSA). In this way, cefoxitin can distinguish diverse isolates of MRSA from competing organisms. As described herein, assays comprising cefoxitin provide high selectivity for MRSA and, importantly, do not interfere with the detection of MRSA strains. Furthermore, cefoxitin is effective in reducing false positives from some 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 embodiment of the disclosed methods and systems 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 period, without the need for traditional biological enrichment and / or incubation, which requires at least 24 hours. The methods utilize a novel bacteriophage-based MRSA diagnostic screen. The assay is part of a new generation of luciferase phage reporter systems that utilize luciferases, such as NANOLUC®, for sensitive detection of target species. The method proved to be highly comprehensive and, when combined with cefoxitin selection, discriminated against the majority of non-resistant strains. Furthermore, the screen was able to identify low MRSA loads 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. Furthermore, the system may include at least some of the components for carrying out the method. In certain embodiments, the system is assembled as a kit. Thus, in some embodiments, a system for rapid detection of MRSA from nasal swabs includes components for incubating a sample with a recombinant infectious agent specific for a microorganism of interest, where the recombinant infectious 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 a method or system.

[0023] Some embodiments of the present disclosure described herein take advantage of the discovery that a single microorganism can recognize and bind to a specific infectious agent, such as a bacteriophage. After bacteriophage infection and replication, successful infection and production of progeny phage can be detected via an indicator moiety expressed during bacteriophage replication. This principle allows for the amplification of an indicator signal from one or a small number of cells based on specific recognition of a microbial surface receptor. For example, even a single bacterial cell can be exposed to multiple bacteriophages, and then by allowing high-level expression of the encoded indicator gene product during bacteriophage amplification and replication, the indicator signal is amplified so that a single bacterium can be detected. definition

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

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

[0026] Use of the term "or" is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0027] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists from sample to sample.

[0028] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface onto which a biomolecule can be bound. For example, a solid support can be an assay well (i.e., a microtiter plate or multiwell plate, etc.), or a solid support can be a filter, an array, or a location on a mobile support such as a bead 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 can be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction can be covalent.

[0030] The term "soluble binding agent" refers to a binding agent that is not bound (ie, not covalently or non-covalently attached) to a solid support.

[0031] The term "immobilized binding partner" refers to a binding agent that is attached (ie, covalently or non-covalently bound) to a solid support.

[0032] As used herein, "analyte" refers to the molecule, compound, or cell being measured. The analyte of interest may, in certain embodiments, interact with a binding agent.

[0033] As used herein, the term "analyte" may refer to a protein or peptide of interest. An analyte may be an agonist, antagonist, or modulator. Alternatively, an analyte may have no biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, etc.

[0034] The terms "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator" or "indicator moiety" refer to a molecule that can be measured in a quantitative assay. For example, an indicator moiety can include an enzyme that can be used to convert a substrate into a measurable product. An indicator moiety can be an enzyme (e.g., luciferase) that catalyzes a reaction that produces bioluminescence. Alternatively, an indicator moiety can be a radioisotope that can be quantified. Alternatively, an indicator moiety can be a fluorophore. Alternatively, other detectable molecules can 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 paraTB), mycophages (such as those against fungi), mycoplasma phages, and any other term that refers to a virus that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and use them to replicate itself. Here, "microscopic" means having a greatest dimension of 1 millimeter or less.

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

[0037] As used herein, "late gene region" refers to a region of a viral genome that is transcribed late in the viral life cycle. Late gene regions typically contain the most abundantly expressed genes (e.g., structural proteins that assemble into bacteriophage particles). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, in phage T7, late genes (synonymous with class III) are transcribed from, e.g., 8 minutes after infection until lysis; class I (e.g., RNA polymerase) is transcribed early, from 4 to 8 minutes, and class II is transcribed from 6 to 15 minutes, so there is an overlap in the timing of class II and class III. A late promoter is a promoter that is naturally located in and active in such a late gene region.

[0038] As used herein, "enrichment culture" refers to traditional culture, such as incubation in a medium that favors microbial growth, and should not be confused with other possible uses of the term "enrichment," such as enrichment by removing liquid components of a sample to concentrate the microorganisms contained therein, or other forms of enrichment that do not involve traditional promotion of microbial growth. In some embodiments of the methods described herein, very short enrichment cultures may be used, but are not required, and when very short enrichment cultures are used, they are much shorter than traditional enrichment cultures.

[0039] As used herein, "recombinant" refers to genetic (i.e., nucleic acid) modifications typically performed in a laboratory to bring together genetic material not found otherwise. The term is used interchangeably herein with the term "modified."

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

[0041] As used herein, "time to result" refers to the total amount of time from the start of sample incubation to the result being generated. The time to result does not include any confirmatory testing time. Data collection can occur any time after the result is generated. sample

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

[0043] The sample may be liquid, solid, or semi-solid. The sample may be a surface swab. In some embodiments, the sample may be a nasal swab to detect nasal colonization of MRSA. In some embodiments, the sample may include bodily 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, samples can be used directly in the detection methods of the present disclosure without preparation, concentration, or dilution. For example, liquid samples, including but not limited to nasal swabs, can be assayed directly. Samples can be diluted or suspended in solutions, including but not limited to buffer solutions or bacterial culture media. Solid or semi-solid samples can be suspended in liquid by chopping, mixing, or softening the solid in the liquid. The sample should be maintained within a pH range that promotes bacteriophage attachment to host bacterial cells. Preferably, the sample is maintained at a temperature that maintains 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 maintains the viability of any pathogen cells present in the sample. For example, during the stage in which bacteriophages are attaching to bacterial cells, it is preferable to maintain the sample at a temperature that promotes bacteriophage attachment. During the stage in which bacteriophages are replicating within infected bacterial cells or lysing such infected cells, it is preferable to maintain the sample at a temperature that promotes bacteriophage replication and host lysis. Such a temperature is 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 selection agent. To inhibit or promote microbial growth, a selection agent may be added to the assay, such as selective and non-selective antimicrobial agents that can inhibit or stop microbial growth, modulators (i.e., agents that can alter microbial growth but are not considered antimicrobial agents), or enrichment agents (e.g., substances that may be required for auxotrophic microorganisms, such as hemin, or substances that may be required by fastidious organisms), or other components that can promote microbial growth. In some embodiments, the selection agent is an antimicrobial agent, including, for example, cefoxitin.

[0047] The assay may include a variety of appropriate control samples, for example, a control sample containing no bacteriophage or a control sample containing bacteriophage but no bacteria may be assayed as a control for background signal levels. Bacteriophage

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

[0049] The compositions of the present disclosure may include one or more genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may include a cocktail of different indicator phages that may encode and express the same or different indicator proteins. In some embodiments, the cocktail of bacteriophages includes at least two different types of recombinant bacteriophages derived from a bacteriophage specific for Staphylococcus aureus.

[0050] The recombinant indicator bacteriophage can include a reporter gene or indicator gene. In certain embodiments of the infectious agent, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product. In certain embodiments, the indicator gene can 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 for Staphylococcus aureus.

[0051] Furthermore, phage genes considered non-essential may have unrecognized functions. For example, seemingly non-essential genes may have important functions in increasing production, such as subtle cleavage, adaptation, or trimming functions during assembly. Therefore, deleting a gene to insert an indicator may be detrimental. Most phages can package DNA several percent larger than their native genome. With this in mind, a smaller indicator gene may be a more appropriate choice for modifying bacteriophages, especially those with smaller genomes. OpLuc and NANOLUC® proteins are only approximately 20 kDa (encoding approximately 500-600 bp), while FLuc is approximately 62 kDa (encoding approximately 1,700 bp). Furthermore, the reporter gene should not be endogenously expressed by the bacterium (i.e., not be part of the bacterial genome), should produce a high signal-to-background ratio, and should be easily 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 imidazopyrazinone substrate (furimazine), can provide a robust signal with low background.

[0052] The indicator gene may express a variety of biomolecules. The 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, the luciferase is one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase, as described in more detail herein. 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] Thus, in some embodiments, the present disclosure provides a genetically modified bacteriophage that includes a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels like the viral capsid protein, but also does not shut off like endogenous bacterial genes or even early viral genes.

[0054] Genetic modifications to infectious agents can include the insertion, deletion, or substitution of small fragments of nucleic acid, significant portions of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acid comprises a non-native sequence. The non-native indicator gene can be inserted into the bacteriophage genome so that it is under the control of a bacteriophage promoter. Thus, in some embodiments, the non-native indicator gene is not part of a fusion protein. In some embodiments, the indicator protein product is soluble. In some embodiments, the present disclosure provides a method for detecting a bacterium of interest (e.g., Staphylococcus aureus) comprising incubating a test sample with such a recombinant bacteriophage.

[0055] In some embodiments, expression of the indicator gene in the progeny bacteriophage following infection of the host bacterium results in a free, soluble protein product. In some embodiments, the non-native indicator gene is not contiguous with the genes encoding the structural phage proteins and therefore does not produce a fusion protein. In some embodiments, the indicator or reporter ideally does not comprise the bacteriophage structure; i.e., the indicator or reporter is not bound to the phage structure. Thus, the indicator or reporter gene is not fused to other genes in the recombinant phage genome. This can greatly increase the sensitivity of the assay (down to a single bacterium) and simplify the assay, allowing the assay to be completed in two hours or less for some embodiments, as opposed to the several hours required due to additional purification steps for 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 color change. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may function as indicator moieties. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0057] In some embodiments, preparation of recombinant bacteriophage stocks includes purification steps sufficient to remove substantially all residual indicator protein that may be associated with the bacteriophage prior to use in bacterial detection assays, and thus the resulting preparation of parent recombinant bacteriophage used to infect any target bacteria in a sample of interest is substantially free of indicator protein. Methods using infectious agents to detect MRSA

[0058] As described herein, in certain embodiments, the present disclosure provides methods of using infectious bacteriophages to detect MRSA or microorganisms. The methods of the present disclosure can be carried out in a variety of ways.

[0059] In some embodiments, the present disclosure provides a method for detecting a microorganism in a sample, the method comprising: obtaining a sample; contacting the sample with a cocktail comprising one or more infectious agents, the infectious agents comprising an indicator gene and specific for the 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 produced by the indicator protein product, wherein the detection of the signal is used to determine the presence of the microorganism in the sample.

[0060] In some embodiments, the present disclosure provides a method for detecting MRSA from a sample (e.g., from a nasal swab), comprising obtaining the sample, incubating the sample in an assay comprising a selection agent and one or more bacteriophages that infect Staphylococcus aureus, wherein the bacteriophage comprise an indicator gene such that expression of the indicator gene during bacteriophage replication following infection of the bacterium of interest results in 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 selection agent is an antibiotic, including cefoxitin.

[0061] In some embodiments, the method includes capturing the indicator protein product for detection. Capturing the indicator protein product on a surface improves 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 contacted with the surface. For example, the indicator protein product can adhere or bind to the surface during the capture step. In some embodiments, the surface can include, among others, a microtiter plate, latex particles, lateral flow strips, beads, magnetic particles, or a dipstick.

[0062] In some embodiments, the surface can contain immobilized binding partners. For example, to create an array for analyte recognition, one or more specific recognition elements can be immobilized on individual regions of the surface. The indicator protein product can be contacted with the surface containing the immobilized binding partners. In some embodiments, several different binding partners can be immobilized simultaneously on one surface. In some embodiments, the immobilized binding partner is an antibody or a fragment thereof.

[0063] In some embodiments, one or more different immobilized binding partners can be deposited (e.g., pipetted) onto a surface (e.g., a plate) to capture the indicator protein product. In some aspects, the surface can improve the accessibility and capture of the indicator protein product by orienting the immobilized binding partner. For example, an antibody can be deposited onto the plate and incubated for a period of time. In some embodiments, the antibody can be a rabbit antibody or a goat antibody. If necessary, the plate can be washed after incubation. The NANOLUC® antibody can then be deposited onto the coated plate. In some aspects, it is advantageous if the amount of indicator protein product deposited onto the surface with the immobilized binding partner is equal to or less than the amount of immobilized binding partner to form a monolayer on the surface as a solid support. For example, the immobilized binding partner can be an antibody that is bound to a layer on the surface of the solid support, making its specific binding epitope accessible.

[0064] In some embodiments, the disclosed methods may include various other steps to enhance sensitivity. The sensitivity of the 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 excess bacteriophage and / or luciferase or other indicator proteins that contaminate the bacteriophage preparation. Additionally, the captured microorganisms may be washed after incubation with the antibiotic and infectious agent and before the addition of the lysis buffer and substrate. These additional washing steps aid in the removal of excess parent phage and / or luciferase or other indicator proteins that contaminate the phage preparation. In some embodiments, the microorganisms may be captured, washed, and then infected with bacteriophage.

[0065] In some embodiments, the method includes adding a protein to antibodies to promote infection by bacteriophages. Staphylococcus aureus binds antibodies (e.g., IgG) in the blood, preventing bacteriophages from infecting these cells. In some embodiments, protein A is added to bind 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 cannot bind to daughter cells, allowing bacteriophages to infect the 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, assays can be performed to utilize a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments that use recombinant bacteriophages (i.e., indicator bacteriophages) can be performed at the following intervals depending on the sample type, sample size, and assay format: 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, 12.0, 12.5, 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, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 40.0 The assay may enable rapid detection of MRSA with a total assay time 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 assay time may be somewhat shorter or longer depending on the bacteriophage strain and bacterial strain to be detected in the assay, the type and size of the sample being tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target bacterial contaminants. [Example]

[0067] Example The results presented 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. The 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, discriminated against the majority of non-resistant strains. Furthermore, the method was able to identify low loads of MRSA in nasal samples with little or no evidence of problematic interference. Finally, the data indicate that this diagnostic screen may be a promising new tool for detecting MRSA colonization from nasal swab specimens. material and method Bacterial strains

[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 sourced in-house from a clinical microbiology laboratory (Laboratory Corporation of America Holdings). MRSA isolates were obtained from de-identified human clinical specimens from three geographically distinct US locations (Burlington, NC, Phoenix, AZ, and Raritan, NJ). MSSA isolates were similarly obtained from one location (Burlington, NC). MRSA or MSSA determinations were confirmed by plating on selective chromogenic agar, MRSA Select II (Bio-Rad, Marnes-la-coquette, France). Strains were routinely grown at 37°C in brain heart infusion (BHI) broth (Becton Dickinson and Company, Sparks, MD, USA) with shaking at 250 revolutions per minute (RPM). Bacteriophage source and stock preparation

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

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

[0071] For ISP, a similar procedure was used with the following exceptions: strain 12600 was used as the host, and exponential cultures were infected at an MOI of 0.05, followed by overnight pellet resuspension followed by a further centrifugation at 5,000 rpm for 10 min at 4°C before treatment with DNase and RNase. Stock titers were determined by standard methods using plaque counts performed on host strains grown in semi-solid agar. Engineering luciferase reporter phage

[0072] The target bacteriophage was transformed with a homologous recombination donor construct designed with a host-specific promoter and codon-optimized NANOLUC® placed between two 500-bp flanking sequences with homology corresponding to the suspected late gene region in the ISP. This construct was inserted into the PstI site of pBAV1KT5gfp (accession HQ191434). The host-specific promoter was modeled after previous studies. Cloning and codon optimization of NANOLUC® were performed by Genewiz (South Plainfield, NJ, USA). Because the regions of homology share 99.9% identity, this donor construct was utilized for both ISP and MP115 engineering.

[0073] Electroporation-competent S. aureus was generated from RN4220. To accomplish this, an overnight culture of RN4220 was diluted and grown to mid-logarithmic phase in tryptone soya broth (TSB) (Oxiod, Hampshire, United Kingdom). The bacteria were then chilled 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 as aliquots for storage at -80°C. 100 ng of donor construct plasmid DNA was then added to the thawed aliquot 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 plated on TSB agar containing 50 μg / mL kanamycin (Sigma, St. Louis, MO, USA). Transformants were isolated and confirmed by expression of NANOLUC®. Colonies were grown in TSB with kanamycin for 3 hours before being tested. A mixture of 10 μL of culture, 50 μL of NanoGlo buffer, 15 μL of Renilla lysis buffer, and 1 μL of NanoGlo substrate (Promega, Madison, WI, USA) was prepared and analyzed using a GloMax Navigator (Promega, Madison, WI, USA).

[0074] NANOLUC®-positive cultures of transformed RN4220 were grown to early logarithmic phase and infected with either MP115 or ISP at an MOI of 0.1 and incubated at 37°C for 3 hours with shaking at 225 rpm. The phage lysate was centrifuged to remove cell debris, filtered through a 0.45 μM Whatman Puradisc filter (GE Health, Pittsburgh, PA, USA), and finally buffer-exchanged into TMS using a 100K MWCO protein concentrator (Pierce). Limiting dilution enrichment was then performed to increase the frequency of recombinants 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 for 2 hours at 37°C. After infection, 50 μL of NanoGlo buffer, 15 μL of Renilla lysis buffer, and 1 μL of NanoGlo substrate were added to each well, which was then evaluated using a GloMax Navigator. Positive wells with high signals were filtered, diluted, and used to infect the next passage. This was repeated until three consecutive passages yielded 100% positive and considered pure plaques. In vitro phage detection assays - sensitivity, inclusiveness and MSSA exclusivity

[0075] Overnight cultures were diluted with brain heart infusion (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 number of colony-forming units (CFU) per well (e.g., 10, 1,000, or 1,000 CFU). Two additional wells containing 135 μL of BHI broth alone were used to determine the medium background. One well for each sample served as a control well and received 15 μL of BHI broth. The other well served as a selective well and received 15 μL of BHI broth containing 22 μg / mL cefoxitin (Alfa Aesar, Ward Hill, MA, USA). The selective well had a final cefoxitin concentration of 2.2 μg / mL. Where indicated, the actual CFU for each sample was confirmed by plate counting on BHI agar. The 96-well strip plates were sealed with cover film (Thermo Fisher Scientific, Rochester, NY, USA) and incubated at 37°C for 4 h to facilitate enrichment and selection. Phage cocktails were prepared in lysogeny broth (LB) (Gibco, Grand Island, NY, USA) at 1.6 × 10 per mL each. 8 The plates contained both engineered phages in plaque-forming units (PFU). 10 μL of the phage cocktail was added to each well, mixed by pipetting, and then re-covered with film. To promote phage infection and luciferase production in the presence of MRSA, plates were incubated at 37°C for 4 hours. 65 μL of detection solution, consisting of 50 μL of NanoGlo Buffer, 15 μL of Renilla lysis buffer, and 1 μL of NanoGlo Substrate, was added to each well and mixed by pipetting. Samples were read using a GloMax Navigator with a 3-minute wait time and 1-second integration. Results were assessed at a cutoff of 600 relative light units (RLU), approximately three times the background observed with medium alone. In vitro phage detection assays - non-Staphylococcus aureus exclusivity and bacterial interference

[0076] Overnight cultures of competitor 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). An additional four wells, each containing only 125 μL of BHI broth, were utilized to determine the medium background and baseline signal for MRSA (BAA-1720). Two wells from each sample were assigned to the exclusivity test, while the other two wells were used to evaluate 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 and received an additional 15 μL of BHI broth, while the other served as the selective well and received 15 μL of BHI broth containing 22 μg / mL cefoxitin. 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-collected 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 the second nostril. Specimens were stored overnight at 4°C before processing. 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 bona fide MRSA colonization, a reference method using both direct plating and enriched culture was used. For direct plating, 135 μL of the nasal eluate used in screening was plated on MRSA Select II agar. For enriched culture, one swab was placed in 3 mL of TSB containing 6.5% NaCl (Fisher Scientific, Geel, Belgium) and grown overnight at 37°C with shaking at 250 rpm. The next day, the culture was streaked onto MRSA Select II agar. In both cases, the manufacturer's instructions were followed to identify the presence or absence of MRSA colonization. If either method (direct plating or enriched culture) produced a positive result on selective agar, the swab was considered MRSA positive.

[0079] The ability to detect MRSA in the nasal matrix was evaluated by adding diluted MRSA cultures to the 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 samples were evaluated, with eight samples allocated for each MRSA strain tested (BAA-1707, BAA-1717, BAA-1720, BAA-1763, and BAA-1766). As a control, 10 μL of each MRSA strain was also added to 125 μL of BHI broth. After addition, two wells were evaluated in the same manner as for the 135 μL diluted cultures described above.

[0080] The autoluminescence of each nasal sample was assessed by mixing each sample with a detection solution (phage cocktail) that did not contain a luciferase source. To accomplish this, 135 μL of each nasal eluate was combined with 25 μL of BHI broth in a 96-well strip plate. 65 μL of detection solution was then added to each well and mixed by pipetting. The plate was read in a luminometer. Example 1. Sensitivity and inclusion studies

[0081] The methods and systems described herein are capable of identifying MRSA strains from diverse genetic backgrounds (Table 1). Comprehensive strains of MRSA were obtained from academic sources, as shown in Table 1. For the majority of strains, detection of various MRSA strains could be achieved at or below 100 CFU. This limit of detection and analytical sensitivity are similar to previously described PCR-based screening.

[0082] The bacteriophage-based MRSA screening involved a 4-hour enrichment period, a 2-hour infection period, and subsequent detection of emitted light in a luminometer. Two wells of a 96-well strip plate were run for each sample: one control well and one selective well. The selective wells were used for MRSA determination and contained the MRSA selective agent cefoxitin, while the control wells contained bacterial culture medium only and were primarily used to measure 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 bacteriophage encoding recombinant luciferase. Luciferase production, indicating successful viral infection, was measured by detecting emitted light in a luminometer after the addition of substrate. Seventeen diverse MRSA strains were evaluated using this method with a starting target of 10, 100, or 1,000 colony-forming units (CFU) in triplicate wells (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 cutoff of 600 RLU, which is approximately three times the background observed in culture medium alone.

[0083] Positive results were obtained in 51 of 51 wells (100%) tested at both 100 and 1,000 CFU per well in the control conditions. At 10 CFU / well, 48 of 51 wells (94.1%) were positive. Three unique strains of MRSA were positive in only two of three wells at 10 CFU. These results highlight the ability of the phage cocktail to recognize diverse MRSA isolates. When cefoxitin was included for MRSA determination, positive signals were still detectable in 51 of 51 wells (100%) at 1,000 CFU / well and in 48 of 51 wells (94.1%) at 100 CFU / well. The inability to detect BAA-42, also known as HDE288, at 100 CFU under selection was not entirely unexpected. This strain belongs to an "early clone" of MRSA with low-level and heterogeneous methicillin resistance. As shown in Table 1, 44 of 51 selective wells (86.3%) remained positive with only 10 CFU. The limit of detection was determined for each strain based on the lowest CFU required for 100% detection in both control and selective wells. Thirteen of the 17 MRSA strains tested could be reliably detected with 10 CFU per well, while three required 100 CFU per well. BAA-42 was the only strain requiring >100 CFU per well for consistent positive detection with MRSA selection. As shown in Table 1, the MRSA assay demonstrates 100% coverage with 17 MRSA strains tested at 100 CFU. The MRSA assay also demonstrates selectivity for 48 of the 51 MRSA strains tested. Overall, these results demonstrate the ability of this screen 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-susceptible Staphylococcus aureus (MSSA) strains. Table 2 shows five well-characterized strains of MSSA evaluated using the methods described herein at 100, 1,000, and 10,000 CFU in triplicate wells, providing CFU and RLU values ​​determined from plate counts. MRSA control wells contained no cefoxitin, while MRSA-selective wells contained cefoxitin. As expected, MSSA strains were positive in 100% of control wells at CFU levels of 100, 1,000, and 10,000. The inclusion of cefoxitin in selective wells resulted in a significant reduction in positive results. In the MRSA-selective wells containing cefoxitin, 0 of 15 (0%) selective wells were positive at 100 CFU, while only 1 of 15 (6.7%) selective wells were positive at 1,000 CFU and 10,000 CFU. These results support the ability of the MRSA assay to distinguish most MSSA strains. [Table 2] [Table S2]

[0085] Exclusivity of MRSA screening was evaluated in vitro against a panel of 40 strains encompassing 21 unique genera and 32 different species beyond MSSA, as shown in Table 3. CFU and RLU values ​​(determined from plate counts) are shown in Table S3. CFU for each exclusivity strain was greater than 1,500 CFU per well (median CFU of 15,950). When specificity was assessed, Table 3 shows that 6 of 40 strains (15%) were positive in control wells. Positive signals in this condition were the result of cross-reactivity of the phage cocktail and were observed with Staphylococcus and Bacillus species. Many S. aureus phages are polyvalent and have been demonstrated to lyse both coagulase-positive and coagulase-negative Staphylococcus species. Adsorption of staphylococcal phages by Bacillus species has been previously reported and may be related to the similarity of their cell wall teichoic acids (WTAs). Despite this cross-reactivity, the selective conditions would have yielded 0 of 40 positive strains and no false positives for MRSA. These results demonstrate the specificity of the phage cocktail used in the experiments described herein and the exclusivity of the overall assay.

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

[0087] Example 3. Screening performance among circulating S. 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, NC; Phoenix, AZ; and Raritan, NJ). MSSA isolates were similarly obtained from a single location (Burlington, NC). Identification of MRSA or MSSA was confirmed by plating on selective chromogenic agar. A total of 390 clinical MRSA strains were isolated from unique specimens and evaluated in the MRSA screen. RLU and CFU values ​​for each strain are provided (Table S4).

[0089] Table 4 shows that the median MRSA load tested was 47 CFU per well. As shown in Table 4, 388 of 390 clinical MRSA strains (99.5%) were detected positive in the control wells. Under cefoxitin selection, 381 of 390 clinical MRSA strains (97.7%) were positive and identified as MRSA by screening. Clinical MSSA strains were tested for exclusion at higher loads, i.e., either 10 or 100 times the MRSA level (500 and 5,000 CFU, respectively). 122 of 123 clinical MSSA strains (99.2%) were detected positive in the control conditions for either inoculum. However, in the selective wells, the positive signal from 500 CFU dropped to 8 of 123 MSSA strains (6.5%). At approximately 5,000 CFU per well, this false-positive rate increased to 21 of 123 strains (17.1%). This suggests that while most MSSA strains are negative, some may overwhelm selection at high loads, resulting in false positives. Importantly, of the 513 clinical S. aureus isolates tested, 510 (99.4%) were positive in the control condition. This continues to support the idea that the phage cocktail utilized in the described method and system confers broad-host-range coverage. Overall, these results demonstrate the ability of this screen 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 with human nasal swabs.

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

[0092] To perform screening using these specimens, swabs were eluted in bacterial culture medium and added to wells containing (selective) or without (control) cefoxitin. A positive result in the selective condition was considered a positive MRSA result. The control condition was not required or utilized for MRSA determination but was included to demonstrate the effectiveness of the selection. Positive results were expected in most control wells due to the high nasal colonization rate of Staphylococcus species and previously described cross-reactivity with the phage cocktail. As expected, 36 of 40 (90%) samples were positive in the control wells. RLU values ​​for the endogenous samples are provided (Table S5).

[0093] Thirty-six of 40 (90.0%) specimens were negative for MRSA detection, consistent with the reference method. Four false-positives were identified, with a median RLU signal less than five times the signal cutoff. All nasal samples were negative when tested directly with luciferase substrate, 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 may be related to methicillin-resistant coagulase-negative staphylococci. Furthermore, some MSSA strains have previously been observed to produce false-positive results at high bacterial loads (Table 4). Overall, the majority (90%) of MRSA-negative samples could be successfully screened out by this method. [Table 5] [Table S5-1] [Table S5-2]

[0094] To determine whether this method could successfully detect MRSA in the nasal matrix, five well-characterized MRSA strains were spiked into the eluate from 40 noncolonized nasal swabs described previously. RLU and CFU values ​​for each sample are provided (Table S5). The median MRSA spike load was 87 CFU per well. 40 of 40 (100%) MRSA-spiked samples were positive in both the control and selective conditions (Table 5). The lack of invalid samples suggests the absence of assay inhibitors in these individuals. The successful detection of five unique MRSA strains when spiked at low loads in these samples supports the effectiveness of bacteriophage-based screening in the nasal matrix.

[0095] As shown in the Examples, the present disclosure provides a culture-based approach using an MRSA luciferase phage reporter assay to achieve 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 the majority of MRSA strains, successful detection required the presence of only 10–100 CFU per well, equivalent to approximately 75–750 CFU per nasal swab. This limit of detection is similar to previously described PCR-based screening. The median MRSA load recovered from nasal swabs of colonized individuals was found to exceed 10,000 CFU. Furthermore, individuals with high nasal colonization loads are more likely to harbor MRSA at multiple body sites and be vectors of transmission. Thus, the sensitivity of this assay appears well suited to addressing the expected burden from clinical nasal specimens, whether the goal is to eliminate MRSA carriage or limit patient-to-patient transmission.

[0096] In some respects, the performance of luciferase reporter phage assays depends heavily on the choice of bacteriophage. This MRSA diagnostic screen in the examples utilized NanoLuc-expressing recombinants of two phages, ISP and MP115, which are members of the Myoviridae family of large, lytic staphylococcal bacteriophages. These phages primarily bind to host surfaces via a highly conserved WTA, conferring broad-host-range capabilities. Mutants lacking WTA are likely resistant to all, or at least most, staphylococcal phages. While resistant WTA-deficient mutants are hypothetical, previous studies have demonstrated that WTA is required for both nasal colonization and methicillin resistance. In general, loss of WTA also results in a fitness cost and an overall reduction in virulence in vivo. Therefore, it is reasonable to expect that all current and future MRSA strains involved in nasal carriage will possess the receptor targeted by this screen. Furthermore, this conclusion is further supported by the data in Table 4, which show that positive phage signals were detected for 99.5% of the clinical MRSA isolates tested.

[0097] As shown in the results in Table 4, among the 513 clinical strains of S. aureus, two MRSA isolates (BNC159 and PHX079) and one MSSA isolate (MSSA090) failed to generate a positive signal under control conditions. One of these isolates (PHX079) appeared to have a growth defect in culture (data not shown). Poor growth during the enrichment period 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 evolve under the pressure of these pathways, and there is evidence that some phages completely lack specific sequences targeted by these systems. Despite this, the diversity of restriction-modification systems across S. aureus is extensive and may contribute to the resistance seen in these isolates. Separately, capsule production has been associated with phage resistance in S. aureus through shielding of surface receptors. Although some common strains of S. aureus do not produce capsular polysaccharides, this mechanism may facilitate the rare (<1%) resistance observed.

[0098] Furthermore, Table 4 also shows that the combination of the MRSA luciferase phage reporter assay with a selective agent (e.g., antibiotic) limited the viability and growth of non-MRSA strains without interfering with MRSA detection. For example, the MRSA luciferase phage reporter assay utilized cefoxitin to limit the viability and growth of non-MRSA strains. The results in Table 4 demonstrate the effectiveness of this selection, as only 6.5% of clinical MRSA 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 selective wells at approximately 50 CFU per well. Furthermore, Table 3 shows that this selective agent was also beneficial in limiting false positives from some species of Bacillus and coagulase-negative Staphylococci while also preventing interference from Streptococcus pneumoniae. Cefoxitin has been demonstrated as an excellent choice for MRSA selection, allowing for the identification of a wide variety of isolates. Despite the high detection rate of clinical MRSA, some strains produced false-negative results in the presence of cefoxitin. Clinical MRSA strains were evaluated at particularly low loads in some instances, making these strains likely to express low-level resistance or heteroresistance. Such strains may exhibit detection limits above 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 nasal eluates. These false positives could stem from three sources. First, autoluminescence may occur, but was ruled out in these samples by demonstrating the need for luciferase addition, as shown in Table S5. Second, the high load of certain MSSA strains can 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 could potentially contribute to the weak false MRSA positives observed in four samples.

[0100] The method and system for detecting MRSA described herein is unique in that it requires viability of endogenous nasal flora to assess sample adequacy. To replicate endogenous nasal flora, nasal eluates were spiked with one of five MRSA strains (Table 5). As shown in Table 5, positive detection of low MRSA loads in the nasal matrix was achieved in 100% of spiked samples. Importantly, this demonstrates that successful bacteriophage infection and luciferase production can occur in the nasal matrix. Furthermore, this demonstrates that the negative control wells previously observed in 10% of endogenous samples were not the result of assay inhibitors. Overall, these results strongly suggest that MRSA colonization, if present, 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 utilizes NanoLuc for highly sensitive detection of target species. The method proved to be highly comprehensive and, when combined with cefoxitin selection, discriminated against the majority of non-resistant strains. Furthermore, the screen was able to identify low MRSA loads in nasal samples without evidence of problematic interference. Furthermore, MRSA detection was performed within 6 hours, allowing ready-to-use results to be obtained in a single work shift. Finally, the present data indicate 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 (ATCC 12600) was cultured in tryptic soy broth (TSB) at logarithmic phase (OD of 0.41). 600) cultures were diluted with TSB to obtain the desired loading, which was confirmed by plating on TSB agar to obtain colony-forming units (CFU). 12.5 μL of each dilution was added directly to 37.5 μL of TSB or human blood in 96-well strips (high binding; Grenier Bio-One, ref. no. 762074). 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 sodium heparin as the anticoagulant. Blood samples were collected using 100 μL of polyanethole sulfone TSB containing sodium phosphate (SPS) was added to obtain a 25% human blood matrix. The final concentration of SPS in the wells (150 μL volume) was 0.05%. For TSB samples, 100 μL of TSB was added to achieve the same 150 μL volume. The test strips were then sealed with a cover film and incubated at 37°C for 30 minutes. After this brief enrichment, 20 μL of phage working stock was added to the wells containing the TSB matrix. The phage working stock contained 8 x 10 7The strips contained plaque-forming units / mL of both MP115.NL and SAPJV1.NL. To allow infection in wells containing blood matrix, 0.5 mg of recombinant Staphylococcus aureus protein A (pro-356, Prospec, Ness-Ziona, Israel) was included per well 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 h. After infection, the 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 a GloMax Navigator (Promega, Madison, WI, USA). Signal to background (S / B) was calculated by dividing the RLU from each sample from the RLU observed in the medium control for that test matrix. [Table 6]

[0104] In these examples, 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 S. aureus loading, 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, when S. aureus has bound IgG, S. aureus infection cannot occur. The addition of Protein A allows S. aureus to become infected. Erythrocytes and other serum proteins do not interfere with the capture of the expressed indicator protein. Furthermore, quenching of the signal seen in the control by erythrocytes is eliminated, and the signal is maintained or increased above background. Therefore, the indicator protein can be detected using a whole blood sample with minimal interference from other components (e.g., proteins) in the sample. Traditionally, serum or plasma is isolated from blood to reliably detect the indicator protein product. Advantageously, the examples demonstrate that the present detection method can be performed on whole blood samples taken 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-susceptible Staphylococcus aureus (MSSA) strains (ATCC 12600) were cultured in tryptic soy broth (TSB) at logarithmic phase (OD ranging from 0.16 to 0.4). 600The cultures were grown to a concentration of 0.01%. The cultures were diluted with TSB to obtain the desired loading amount, which was confirmed by plating on TSB agar to obtain colony-forming units (CFUs). 50 μL of each dilution was added to a test strip. Where indicated, some strips contained anti-NanoLuc antibody (purified mouse monoclonal IgG clone #965808; catalog number MAB10026) coupled to a medium-binding plate (Grenier Bio-One, Strips Plate 12xF8, PS, F-Bottom, White, Lumitrac, Med Binding, reference number 762075) or a high-binding plate (Grenier Bio-One, reference number 762074) for capture. Either 85 μL of TSB or human blood diluted with TSB and sodium polyanethol sulfonate (SPS) was added. Human blood was collected from a single donor using sodium heparin as the anticoagulant. Each well then received 15 μL of either TSB or 22 μg / mL cefoxitin (FOX) in TSB. The final concentrations of each component in the well (150 μL volume) were 25% human blood, 0.0375% SPS, and 2.2 μg / mL FOX. The test strips were then sealed with a cover film and incubated at 37°C for 2 hours. After this selective enrichment, 20 μL of phage working stock was added to the wells containing the TSB matrix. The phage working stock contained 8 x 10 7 plaque-forming units / mL (pfu / mL) of MP115.NL and 6.9 x 10 8The wells contained 20 pfu / mL of SAPJV1.NL. For wells containing blood matrix, 0.5 mg of recombinant Staphylococcus aureus protein A (pro-356, Prospec, Ness-Ziona, Israel) was included per well in 20 μL of phage working stock. The assay strips were again sealed with cover film and incubated at 37°C for 3 h. 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. "No Wash + No Capture" strips were not washed; instead, 65 μL of master mix containing 50 μL of NanoGlo buffer, 15 μL of TSB, and 1 μL of NanoGlo substrate was added. 5% BSA-blocked strips (bovine serum albumin, Sigma Life Science Product No. A9647) were washed. BSA-blocked strips were blocked with BSA to remove nonspecific binding sites. 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). Signal to background (S / B) was calculated by dividing the RLU from each sample from the RLU observed in the media control for that test matrix. [Table 7]

[0107] In Table 7, the "No Capture + No Wash" example demonstrates the generation of a total signal and the reduction of signal with cefoxitin when the assay is performed in medium alone (TSB). When performed in the presence of blood, the signal is quenched. When capture strips are used, there is a significant increase in signal due to the elimination of quenching caused by blood. Strips blocked with 5% BSA (bovine serum albumin, Sigma Life Science Product No. A9647) are used to demonstrate nonspecific binding. Again, the example demonstrates a significant increase in signal detection when the indicator protein is captured by the 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 NanoLuc-coated plates.

[0109] A 1.5 mg / mL stock solution of purified NanoLuc was diluted to 1 ng / mL in PBS. Serial 10-fold dilutions in PBS were made from 1 ng / mL to 0.001 pg / mL. Rabbit anti-mouse IgG (Abcam, catalog no. 46540) or goat anti-mouse IgG (Abcam, catalog no. 6708) was diluted to 10 μg / mL in PBS and pipetted at 100 μL / well. The plate was 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 no. MAB10026) was diluted to 1 μg / mL in PBS and pipetted at 100 μL / well onto the rabbit or goat anti-mouse IgG-coated plate. 5% BSA-blocked strips were included for determining nonspecific binding, and uncoated strips were included for measuring Nanoluc activity. Assay strips were sealed with cover film and incubated at 37°C for 3 hours. 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). Signal to background (S / B) was calculated by dividing the RLU from each sample from the RLU observed in the PBS control for that test. [Table 8] [Table 9]

[0110] Tables 8 and 9 demonstrate that coating plates with rabbit anti-mouse IgG or goat anti-mouse IgG provided improved orientation of the mouse anti-nanoluc luciferase to improve the capture / binding surface. Indeed, plates coated with rabbit anti-mouse IgG or goat anti-mouse IgG were more available to bind the indicator protein product. Plate coating showed improved signal detection, which may be due to the orientation of the mouse anti-nanoluc luciferase and the availability of binding sites 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 selective agent to the sample; contacting the sample with a cocktail comprising one or more infectious agents, the infectious agents comprising an indicator gene and 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, wherein the detection of the signal is used to determine the presence of MRSA in the sample.

[0112] Example 2 is any of the preceding or following exemplary methods, wherein the selection agent comprises an antibiotic.

[0113] Example 3 is any preceding or following exemplary method wherein the antibiotic comprises cefoxitin.

[0114] Example 4 is either the preceding or following exemplary method, wherein the sample is derived from a nasal swab.

[0115] Example 5 is an exemplary method either preceding or following the method, wherein the method detects 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacterium in a sample.

[0116] Example 6 is any preceding or subsequent exemplary method, wherein the cocktail comprises at least two different types of recombinant bacteriophage, and at least one of the recombinant bacteriophages is derived from ISP, MP115, or a combination thereof.

[0117] Example 7 is an exemplary method, either preceding or following, in which the indicator gene is codon-optimized and encodes a soluble protein product that produces a unique signal or a soluble enzyme that produces a signal upon reaction with a substrate.

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

[0119] Example 9 is any preceding or subsequent exemplary method, wherein the capturing step comprises contacting the indicator protein product with a surface.

[0120] Example 10 is any preceding or following exemplary method, wherein the surface is a microtiter plate, latex particles, lateral flow strips, beads, magnetic particles, or a dipstick.

[0121] Example 11 is any of the preceding or subsequent exemplary methods, further comprising depositing an immobilized binding partner on the surface prior to capturing the indicator protein product.

[0122] Example 12 is any of the preceding or following exemplary methods, wherein the immobilized binding partner is an antibody or fragment thereof.

[0123] Example 13 is any of the preceding or subsequent exemplary methods, further comprising washing the surface containing the immobilized binding partners.

[0124] Example 14 is any of the preceding or subsequent exemplary methods above, further comprising washing the surface after capturing the indicator protein product.

[0125] Example 15 is any preceding or subsequent exemplary method, wherein the signal to background ratio produced by detecting said indicator protein product is at least 2.0 or at least 2.5.

[0126] Example 16 is an exemplary method, either preceding or following, in which the sample is first incubated under conditions favoring growth for an enrichment period of less than 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 2 hours.

[0127] Example 17 is any of the preceding or subsequent exemplary methods, including obtaining a sample, contacting the sample with a cocktail containing one or more infectious agents, the infectious agents containing an indicator gene and specific to a microorganism, the indicator gene encoding an indicator protein product, contacting the indicator protein product with a surface, the surface comprising an immobilized binding partner for capturing the 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 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 an antibiotic specific for Staphylococcus aureus, and a surface for capturing an indicator protein product.

[0129] Example 19 is any of the preceding or following exemplary kits, wherein the surface comprises an immobilized binding partner.

[0130] Example 20 is any of the preceding or subsequent exemplary kits, wherein the antibiotic comprises cefoxitin.

[0131] The present disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the present disclosure as defined by the claims. The present invention provides, for example, the following items. (Item 1) 1. A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample, comprising: obtaining a sample; adding a selection agent to the sample; contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene and are specific for Staphylococcus aureus, and the indicator gene encodes an indicator protein product; capturing the indicator protein product; 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; A method comprising: (Item 2) 2. The method of claim 1, wherein the selection agent comprises an antibiotic. (Item 3) 3. The method of claim 2, wherein the antibiotic comprises cefoxitin. (Item 4) 2. The method of claim 1, wherein the sample is derived from a nasal swab. (Item 5) 2. The method of claim 1, wherein the method detects 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacterium in a sample. (Item 6) 2. The method of claim 1, wherein the cocktail comprises at least two different types of recombinant bacteriophages, and at least one of the recombinant bacteriophages is derived from ISP, MP115, or a combination thereof. (Item 7) 2. The method of claim 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that produces a unique signal or a soluble enzyme that produces a signal upon reaction with a substrate. (Item 8) 2. The method of claim 1, further comprising an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region comprises a bacteriophage late gene promoter. (Item 9) 2. The method of claim 1, wherein the capturing step comprises contacting the indicator protein product with a surface. (Item 10) 10. The method of claim 9, wherein the surface is a microtiter plate, latex particles, lateral flow strips, beads, magnetic particles, or a dipstick. (Item 11) 10. The method of claim 9, further comprising depositing an immobilized binding partner on the surface prior to capturing the indicator protein product. (Item 12) 12. The method of claim 11, wherein the immobilized binding partner is an antibody or a fragment thereof. (Item 13) 12. The method of claim 11, further comprising washing the surface containing the immobilized binding partner. (Item 14) 14. The method of claim 13, further comprising washing the surface after capturing the indicator protein product. (Item 15) 2. The method of claim 1, wherein the signal-to-background ratio produced by detecting the indicator protein product is at least 2.0 or at least 2.5. (Item 16) 2. The method of claim 1, wherein the sample is initially incubated under conditions favoring growth for an enrichment period of less than 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 2 hours. (Item 17) 1. A method for detecting a microorganism in a sample, comprising: obtaining a sample; contacting the sample with a cocktail comprising one or more infectious agents, the infectious agents comprising an indicator gene and specific for a microorganism, the indicator gene encoding an indicator protein product; contacting the indicator protein product with a surface, the surface comprising an immobilized binding partner for capturing the indicator protein product; detecting a signal produced by the indicator protein product, wherein the detection of the signal is used to determine the presence of the microorganism in the sample; and A method comprising: (Item 18) A kit for detecting methicillin-resistant Staphylococcus aureus, comprising: Nasal swabs and an assay comprising a recombinant bacteriophage and an antibiotic specific for Staphylococcus aureus; a surface for capturing an indicator protein product; Includes a kit. (Item 19) 19. The kit of claim 18, wherein the surface comprises an immobilized binding partner. (Item 20) 19. The kit of item 18, wherein the antibiotic comprises cefoxitin.

Claims

1. A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a blood sample, comprising: adding a selection agent to the blood sample, the selection agent comprising an antibiotic; contacting the blood sample with a cocktail comprising one or more recombinant bacteriophages, at least one of which is specific for Staphylococcus aureus and comprises a genetically modified Staphylococcus aureus-specific bacteriophage genome comprising an indicator gene, the indicator gene encoding an indicator protein product; capturing the indicator protein product on a substrate comprising an immobilized binding partner for capturing the indicator protein product; washing the substrate containing the captured indicator protein product; detecting a signal produced by the captured indicator protein product, wherein the detection of the signal is used to determine the presence of MRSA in the blood sample; A method comprising:

2. 10. The method of claim 1, wherein the antibiotic comprises cefoxitin.

3. 2. The method of claim 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that produces a unique signal or a soluble enzyme that produces a signal upon reaction with a substrate.

4. The method of claim 1, further comprising an untranslated region upstream of the codon-optimized indicator gene, said untranslated region comprising a bacteriophage late gene promoter.

5. 10. The method of claim 1, wherein the capturing step comprises contacting the indicator protein product with a surface.

6. 6. The method of claim 5, wherein the surface is a microtiter plate, a latex particle, a lateral flow strip, a bead, a magnetic particle, or a dipstick.

7. The method of claim 5 , further comprising depositing an immobilized binding partner on the surface prior to capturing the indicator protein product.

8. The method of claim 7, wherein the immobilized binding partner is an antibody or a fragment thereof.

9. The method of claim 7, further comprising washing the surface containing the immobilized binding partner.

10. 10. The method of claim 9, further comprising washing the surface after capturing the indicator protein product.

11. 2. The method of claim 1, wherein the signal to background ratio produced by detecting the indicator protein product is at least 2.0 or at least 2.

5.

12. 2. The method of claim 1, wherein the blood sample is initially incubated under conditions that favor growth for an enrichment period of less than 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 2 hours.

13. A kit for detecting methicillin-resistant Staphylococcus aureus in a blood sample, comprising: an assay comprising one or more recombinant bacteriophages and an antibiotic, wherein at least one of the recombinant bacteriophages is specific for Staphylococcus aureus and comprises a genetically modified Staphylococcus aureus-specific bacteriophage genome comprising an indicator gene, the indicator gene encoding an indicator protein product; a surface comprising an immobilized binding partner for capturing said indicator protein product; Includes a kit.

14. 14. The kit of claim 13, wherein the antibiotic comprises cefoxitin.

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