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

Abstract

Compositions, methods, and systems for detecting MRSA, such as MRSA nasal colonization, are disclosed. In certain embodiments, the methods use phage-based signal amplification in the detection of bacteria and other microorganisms to detect MRSA. The methods for detecting MRSA can include preparing an assay comprising a selection agent and a mixture comprising 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 a positive detection of the indicator protein product indicates that MRSA is present in the sample.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 018,081, filed April 30, 2020, the entirety of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to compositions, methods, and systems for detecting Methicillin-resistant Staphylococcus Aureus (MRSA) using an infectious agent. BACKGROUND

[0004] There is a strong interest in detecting bacteria and other microorganisms that can lead to various forms of debilitating and lethal infections. Bacterial pathogens can cause a large number of illnesses in humans and domestic animals, as well as tremendous economic losses. Specifically, Methicillin-resistant Staphylococcus Aureus (MRSA) is an extremely important human pathogen that has the ability to cause lethal infections. MRSA is a leading cause of surgical site infections in hospitals, associated with longer patient hospital stays, higher readmission rates, decreased survival rates, and economic losses. Because of the severe clinical and financial burden on the healthcare industry, significant efforts have been made to understand and control the source of MRSA-related infections. Nasal carriage of MRSA has been found to be a major risk factor for subsequent disease, and most Staphylococcus aureus infections can be matched to an endogenous colonizing strain. Elimination of this risk factor by decolonization of MRSA nasal carriage has proven to be a successful strategy to reduce surgical site infections.

[0005] Traditional microbiological tests for detecting MRSA rely on non-selective and selective enrichment cultures followed by plating on selective media for further testing to confirm suspicious colonies from patient nasal swab specimens. Culture-based detection methods can involve the use of chromogenic and selective agar and often prove to have strong performance in terms of sensitivity and specificity. Despite often being significantly less expensive than some methods, one major drawback of culture-based methods is that results generally require 18-24 hours of incubation before detection.

[0006] A variety of rapid methods have been investigated and introduced into practice to reduce testing time. However, these methods also have disadvantages. For example, techniques involving immunoassays or genetic probes often require enrichment steps to achieve suitable sensitivity. Polymerase chain reaction (PCR) tests also include an amplification step, and thus can achieve very high sensitivity and selectivity. Detection of MRSA-specific DNA sequences using real-time PCR has proven to have excellent sensitivity and specificity, fast time to result, and overall clinical effectiveness. Although real-time PCR has produced promising results, the method also has disadvantages. First, new generations of real-time PCR must be constantly developed to match the ever-changing genetic landscape of MRSA resistance, as previous PCRs have resulted in some assays failing to detect new MRSA strains. Second, the high cost of real-time PCR relative to culture-based alternatives has raised questions about cost-effectiveness, particularly in areas where the prevalence of the disease is low.

[0007] Accordingly, there is a need for a faster, simpler, and sensitive method for detecting and identifying MRSA. SUMMARY

[0008] Embodiments of the present disclosure include compositions, methods, devices, systems, and kits for detecting MRSA nasal colonization. The present disclosure can be embodied in a variety of ways.

[0009] In some embodiments, the present disclosure provides a method of detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample. The method comprises: 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 to Staphylococcus aureus, and wherein the indicator gene encodes an indicator protein product; capturing the indicator protein product; and detecting a signal produced by the indicator protein product, wherein detection of the signal is used to determine the presence of MRSA in the sample.

[0010] In some embodiments, the present disclosure provides a method of detecting a microorganism in a sample, the method comprising: obtaining a sample; contacting the sample with a cocktail comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene and are specific to the microorganism, and wherein the indicator gene encodes 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 detection of the signal is used to determine the presence of the microorganism in the sample.

[0011] In some embodiments, the present disclosure utilizes a new recombinant bacteriophage for the detection of MRSA from nasal swab specimens. In some embodiments, the new recombinant bacteriophage is specific for S. aureus. The new diagnostic screen utilizes an assay comprising a recombinant bacteriophage that includes a luciferase reporter capable of recognizing S. aureus, while relying on antibiotic to limit the growth of non-MRSA strains. A wide variety of MRSA strains can be detected using the methods described herein.

[0012] In some embodiments, the present disclosure provides a method of detecting MRSA from a sample, comprising: (a) contacting the sample with a selective agent, (b) contacting the sample with a mixture comprising one or more infectious agents, wherein the infectious agent comprises an indicator gene and is specific for S. aureus, and wherein the indicator gene encodes an indicator protein product, and (c) detecting a signal produced by the indicator protein product, wherein detection of the signal is used to determine the concentration of MRSA in the sample. In some embodiments, the selective agent comprises an antibiotic (e.g., cefoxitin). In some embodiments, the sample is from a nasal swab.

[0013] In some embodiments, the infectious agent is a recombinant bacteriophage specific for S. aureus bacteria. In further embodiments, the indicator gene encodes an indicator protein product that generates an internal signal or a soluble enzyme that generates a signal upon reaction with a substrate.

[0014] In some embodiments, the present disclosure provides a method of detecting MRSA from a sample, comprising: contacting the sample with a selective agent, wherein the sample is from a nasal swab; contacting the sample with a mixture comprising one or more infectious agents, wherein the infectious agent comprises an indicator gene and is specific for S. aureus, and wherein the indicator gene encodes an indicator protein product, and detecting a signal produced by the indicator protein product, wherein detection of the signal is used to determine the presence of MRSA in the sample.

[0015] In some embodiments, the present disclosure provides kits and systems for detecting MRSA, comprising a nasal swab; and an assay comprising a recombinant bacteriophage specific for S. aureus and an antibiotic solution. In some embodiments, the present disclosure provides kits and systems for detecting microorganisms, comprising a nasal swab; an assay comprising a recombinant bacteriophage specific for S. aureus and optionally an antibiotic; and a surface for capturing an indicator protein product.

[0016] Certain embodiments of the present disclosure utilize methods and constructs described in U.S. Patent Publication No. 2015 / 0218616, the entire contents of which are incorporated herein by reference. DETAILED DESCRIPTION

[0017] Disclosed herein are compositions, methods, and systems that demonstrate surprising sensitivity in detecting various strains of methicillin-resistant Staphylococcus aureus (MRSA) in test samples (e.g., biological samples) in a shorter time frame than conventional methods. The compositions, methods, and systems disclosed herein can detect MRSA using genetically modified infectious bacteriophages in a shorter time frame than previously thought possible, with reduced culture time for enrichment, or in some embodiments, with minimal number of incubations (during which MRSA can potentially multiply) to detect MRSA. Surprisingly, assays using one or more recombinant bacteriophages in the presence of an antibiotic (e.g., cefoxitin) incubated with test samples generated very low colony forming unit (CFU) numbers to detect a variety of MRSA strains. Such low CFU concentrations were previously purported to be detected only after culture-based methods that required incubation for more than 24 hours. However, the assays described herein can facilitate the discovery, binding, and infection of small numbers of target cells. In some embodiments, the assays detect MRSA from nasal swab specimens in less than 10 hours, at a cost similar to longer time-based culture-based methods.

[0018] In some aspects, the bacteriophage-based MRSA assays described herein provide specific, sensitive, rapid, and low-cost detection of target bacteria and meet the growing diagnostic needs in multiple industries. Specifically, detecting MRSA nasal colonization and antibiotic susceptibility has a critical supporting role in preventing hospital-acquired infections and aiding in antibiotic stewardship. In some embodiments, the bacteriophage-based MRSA assays on nasal swab specimens utilize two luciferase reporter phages that are able to recognize genetically diverse S. aureus. In some embodiments, a beta-lactam antibiotic, cefoxitin, is included to distinguish between resistant (MRSA) and susceptible organisms. The bacteriophage-based MRSA assays surprisingly identify MRSA isolates unambiguously at low bacterial concentrations and produce appropriate negative results for non-MRSA S. aureus at higher concentrations of inoculum. Additionally, cross-reactivity of the phage mixture with other Staphylococcus and Bacillus species can be reduced under selective conditions. Thus, the bacteriophage-based MRSA assays described herein sensitively detect MRSA both in vitro and in human nasal matrices.

[0019] In some aspects, the disclosure provides recombinant bacteriophages comprising an indicator gene inserted into a late gene region of a bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified S. aureus-specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified bacteriophage genome derived from a bacteriophage that specifically recognizes S. aureus. In some embodiments, a mixture of bacteriophages comprises at least two different types of recombinant bacteriophages derived from a bacteriophage that specifically recognizes S. aureus. In some embodiments, an assay comprising a mixture of recombinant bacteriophages and a selection agent (e.g., an antibiotic) can distinguish MRSA in the presence of other types of bacteria, specifically, methicillin-sensitive S. aureus (MSSA).

[0020] In some aspects, methods of detecting MRSA can use an infectious agent to detect S. aureus originally. For example, in certain embodiments, the microorganism of interest is MRSA and the infectious agent is a bacteriophage that specifically infects S. aureus. Thus, in certain embodiments, the method can comprise selecting one or more bacteriophages that specifically infect S. aureus bacteria, preparing recombinant bacteriophages derived from the S. aureus bacteriophage, preparing an assay comprising the recombinant bacteriophages and a selection agent (e.g., an antibiotic), 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 the indicator gene is expressed during bacteriophage replication after infecting the host bacteria, resulting in the production of an indicator protein product. The method can comprise detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates that MRSA is present in the sample. In some embodiments, the indicator protein is soluble.

[0021] In some embodiments, compositions, methods, and systems can detect MRSA from a wide variety of genetic backgrounds using an assay 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 viability of susceptible bacteria while allowing MRSA to grow. For example, the selection agent can kill or reduce the growth of all S. aureus bacteria (e.g., MSSA) except MRSA. In this way, cefoxitin is able to identify various isolates of MRSA from competing organisms. As described herein, assays comprising cefoxitin produce high selectivity for MRSA and importantly, do not interfere with detection of MRSA strains. Additionally, cefoxitin effectively reduces false positives from several species of coagulase-negative Staphylococcus.

[0022] 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 methods and systems of the present disclosure can be applied to the detection and quantification of a large number of MRSA strains. The methods and systems provide high detection sensitivity in a short time without the need for traditional biological enrichment and / or incubation, which takes at least 24 hours. The methods utilize a novel phage-based MRSA diagnostic screen. The assay utilizes luciferase such as a member of a new generation of luciferase-phage reporter gene systems that sensitively detects target species. The method proved to be inclusively high and, when combined with a cefoxitin selection, was able to discriminate most non-resistant strains. In addition, the screen was able to identify low load MRSA in nasal samples with little or no interference.

[0023] In certain embodiments, the present disclosure can include systems. The systems can comprise at least some of the compositions of the present disclosure. Additionally, the systems can include at least some of the components for performing the methods. In certain embodiments, the systems are configured as kits. Thus, in some embodiments, a system for rapid detection of MRSA from nasal swabs comprises: a component for incubating a sample with a recombinant infectious agent specific for a microorganism of interest, wherein the recombinant infectious agent comprises an indicator moiety; a selection agent; and a component for detecting the indicator moiety. In other embodiments, the present disclosure includes software for use with the methods or systems.

[0024] Some embodiments of the present disclosure described herein utilize the discovery that a single microorganism is able to recognize and bind a specific infectious agent, such as a bacteriophage. Upon phage infection and replication, the successful infection and production of progeny phage can be detected by an indicator moiety expressed during phage replication. This principle allows the amplification of the indicator signal from one or a few cells based on the specific recognition of a microorganism surface receptor. For example, by exposing even a single bacterial cell to multiple phages, followed by amplification of the phages and high level expression of the encoded indicator gene product during replication, the indicator signal is amplified such that a single bacterium can be detected.

[0025] Definitions

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures 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. Known methodologies and techniques are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are discussed throughout the present specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with the laboratory procedures and techniques described herein is intended to be interpreted in the same way as is customarily interpreted in the art. The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0027] As used herein, the terms "one," "a," and "the" can mean one or more, unless otherwise specifically indicated.

[0028] The use of the term "or" is used to mean "and / or", unless expressly indicated otherwise, although the disclosure supports a definition that is the "exclusive or" or "one or the other but not both." As used herein, "another" can mean at least a second or more.

[0029] Throughout this application, the term "about" is used to mean that a value includes the inherent variation of error for the device, method, or apparatus being employed to determine the value, or the variation that exists among the sample measurements obtained.

[0030] The term "solid phase support" or "support" means a structure that provides a substrate and / or surface upon which a biomolecule can be bound. For example, the solid phase support can be an assay well (i.e., such as a microtiter plate or multiwell plate), or the solid phase support can be a location on a filter, array, or movable support, such as a location on a bead or membrane (e.g., a filter plate or lateral flow strip).

[0031] As used herein, 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 noncovalent, for example, as a result of hydrogen bonding, Van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction can be covalent.

[0032] The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or noncovalently bound) with a solid phase support.

[0033] The term "immobilized binding partner" refers to a binding agent associated with (i.e., covalently or non-covalently bound to) a solid phase support.

[0034] As used herein, "analyte" refers to a molecule, compound, or cell that is measured. In certain embodiments, an analyte of interest can interact with a binding agent.

[0035] As described herein, the term "analyte" can refer to a protein or peptide of interest. The analyte can be an agonist, antagonist, or modulator. Alternatively, the analyte can have no biological effect. The analyte can include a small molecule, saccharide, oligosaccharide, lipid, peptide, peptidomimetic, organic compound, and the like.

[0036] The term "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator" or "indicating moiety" refers to a molecule that can be measured in a quantitative assay. For example, the indicating moiety can comprise an enzyme that can be used to convert a substrate to a product that can be measured. The indicating moiety can be an enzyme that catalyzes a reaction that generates bioluminescence (e.g., luciferase). Alternatively, the indicating moiety can be a radioisotope that can be quantified. Alternatively, the indicating moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0037] As used herein, "bacteriophage" or "phage" includes one or more of a variety of bacterial viruses. In the present disclosure, the terms "bacteriophage" and "phage" include viruses such as mycobacteriophages (such as for TB and co-TB), mycophages (such as for fungi), mycoplasmaphages, and any other term referring to viruses that can invade and utilize living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms to replicate themselves. Here, "microscopic" means a maximum dimension of 1 millimeter or less.

[0038] Bacteriophages are viruses that have evolved in nature to utilize bacteria as a means to replicate themselves. Phages do this by attaching themselves to a bacterium and injecting their DNA (or RNA) into the bacterium, and inducing it to replicate the phage hundreds or even thousands of times. This is called phage amplification.

[0039] As used herein, "late gene region" refers to a region in the viral genome that is transcribed late in the viral life cycle. The late gene region typically includes the most abundantly expressed genes (e.g., structural proteins that assemble into a bacteriophage particle). Late genes are synonymous with Class III genes and include genes with structural and assembly functions. For example, late genes (synonymous with Class III) are transcribed in bacteriophage T7, e.g., from 8 minutes post-infection until lysis, Class I (e.g., RNA polymerase) is early 4-8 minutes, Class II is 6-15 minutes, so there is overlap in timing of Class II and III. A late promoter is a promoter that is naturally positioned within this late gene region and is active.

[0040] As used herein, "culturing for enrichment" refers to traditional culturing, such as incubation in a medium that favors microbial propagation, and should not be confused with other possible uses of the word "enrichment," such as by removing a liquid component of a sample to concentrate microorganisms contained therein, or other forms of enrichment that do not include traditional promotion of microbial propagation. Culturing for enrichment that is performed for a very short period of time can be employed in some embodiments of the methods described herein, but is not required, and if used, is for a much shorter period of time than traditional culturing for enrichment.

[0041] As used herein "recombinant" refers to genetic (i.e., nucleic acid) modification, as is typically performed in a laboratory, to join together genetic material that would otherwise not be present together. This term is used interchangeably herein with the term "modified."

[0042] As used herein "RLU" refers to relative light units measured by a luminometer (e.g., 96) or similar light detecting instrument. For example, the reaction between luciferase and a suitable substrate (e.g., NANO- Glo®) is often reported in detected RLU.

[0043] As used herein "time to result" refers to the total amount of time from the initiation of sample incubation to the generation of a result. Time to result does not include any confirmatory testing time. Data collection can be completed at any time after the generation of a result.

[0044] Sample

[0045] Each embodiment of the methods and systems of the present disclosure can allow for rapid detection and quantification of MRSA in a sample. For example, the methods according to the present disclosure can be performed in a reduced time, with better results. Bacterial cells that can be detected by the present disclosure include, but are not limited to, various MRSA strains in vitro or from a nasal swab.

[0046] The sample can be liquid, solid, or semi-solid. The sample can be a swab of a surface. In some embodiments, the sample can be a nasal swab to detect nasal colonization of MRSA. In some embodiments, the sample can include a bodily substance, e.g., tissue fluid or nasal fluid. In some embodiments, the sample can be whole blood, plasma, serum, or a combination thereof.

[0047] In some embodiments, the sample can be used directly in the detection methods of the disclosure without preparation, concentration, or dilution. For example, a liquid sample, including but not limited to a nasal swab, can be assayed directly. The sample can be diluted or suspended in a solution, which can include, but is not limited to, a buffer solution or a bacterial culture medium. A sample that is solid or semi-solid can be suspended in a liquid by mincing, mixing, or soaking the solid in the liquid. The sample should be maintained within a pH range that promotes attachment of the bacteriophage to the host bacterial cell. Preferably, the sample is maintained at a temperature that sustains the viability of any pathogenic cells contained within the sample.

[0048] In some embodiments of the detection assay, the sample is maintained at a temperature that sustains the viability of any pathogenic cells present in the sample. For example, during the step in which the bacteriophage attaches to the bacterial cell, the sample is preferably maintained at a temperature that promotes attachment of the bacteriophage. During the step in which the bacteriophage replicates within the infected bacterial cell or lyses this infected cell, the sample is preferably maintained at a temperature that promotes replication of the bacteriophage and lysis of the host. This temperature is at least about 25 degrees Celsius (°C), more preferably no more than about 45 °C, and most preferably about 37 °C.

[0049] In some embodiments, the assay can include a selection agent. Selection agents can be added to the assay to inhibit or promote the growth of microorganisms, such as selective and non-selective antimicrobial agents that can inhibit or prevent the growth of microorganisms, modulators (i.e., agents that can alter the growth of microorganisms but are not considered antimicrobial agents), or enrichment agents (e.g., substances necessary for auxotrophic microorganisms, such as hematin chloride, or substances necessary for oligotrophic organisms) or other components that can promote the growth of microorganisms. In some embodiments, the selection agent is an antimicrobial agent comprising, e.g., cefoxitin.

[0050] The assay can include various appropriate control samples. For example, a control sample containing no bacteriophage or a control sample containing bacteriophage but no bacteria can be assayed to determine the background signal level.

[0051] Bacteriophage

[0052] As described in greater detail herein, the compositions, methods, systems, and kits of the present disclosure can comprise an infectious agent for 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 compositions can comprise a recombinant bacteriophage having an indicator gene incorporated into the genome of the bacteriophage.

[0053] The compositions of the present disclosure can comprise one or more genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions can include a mixture of different indicator phages, which can encode and express the same or different indicator proteins. In some embodiments, the mixture of bacteriophages comprises at least two different types of recombinant bacteriophages derived from bacteriophages specific to Staphylococcus aureus.

[0054] The recombinant indicator bacteriophage can include a reporter gene or an indicator gene. In certain embodiments of the infectious agent, expression of the indicator gene during bacteriophage replication following infection of a host bacterium produces a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into a late gene region of the bacteriophage. Late genes are expressed at generally higher levels than other phage genes because they encode structural proteins. In some embodiments, the indicator bacteriophage is derived from a bacteriophage specific to Staphylococcus aureus.

[0055] Furthermore, bacteriophage genes considered non-essential can have unrecognized functions. For example, apparently non-essential genes can have important functions in increasing the burst size, such as fine cleavage, assembly, or trimming functions in assembly. Thus, deleting a gene to insert an indicator can be detrimental. Most bacteriophages can package several percent more DNA than their native genome. Given this consideration, smaller indicator genes can be a more suitable choice for modifying bacteriophages, particularly bacteriophages having smaller genomes. OpLuc and Proteins are only about 20 kDa (coding sequence is about 500-600 bp), whereas FLuc is about 62 kDa (coding sequence is about 1,700 bp). Furthermore, the reporter gene should not be endogenously expressed by the bacteria (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily detectable in a timely manner. Promega’s is a modified Oplophorus gracilirostris (abyssal shrimp) luciferase. In some embodiments, Promega’s The combination of a furimazine substrate and a luciferase enzyme can provide robust signals with low background.

[0056] An indicator gene can express a wide variety of biomolecules. An indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase. Various types of luciferases can be used. In alternative embodiments, and as described in greater detail herein, the luciferase is one of an Oplophorus luciferase, a firefly luciferase, a Lucia luciferase, a sea pansy luciferase, or an engineered luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as

[0057] Accordingly, in some embodiments, the present disclosure provides genetically modified bacteriophages comprising a non-bacteriophage indicator gene in a 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 expressed at high levels not only like viral coat proteins, but also does not shut down like endogenous bacterial genes or even early viral genes.

[0058] Genetic modifications to the infectious agent can include insertion, deletion, or substitution of a small piece of nucleic acid, a large portion of a gene, or an entire gene. 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 such that it is under the control of a bacteriophage promoter. Accordingly, 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 methods of detecting a bacterium of interest (e.g., Staphylococcus aureus) comprising the step of incubating a test sample with such a recombinant bacteriophage.

[0059] In some embodiments, expression of the indicator gene in progeny bacteriophage following infection of a host bacterium produces a free, soluble protein product. In some embodiments, the non-native indicator gene is not contiguous with a gene encoding a structural phage protein, and thus does not produce a fusion protein. In some embodiments, the indicator or reporter desirably does not contain a bacteriophage structure. That is, the indicator or reporter is not attached to a phage structure. As such, in the recombinant phage genome, the gene for the indicator or reporter is not fused with other genes. This can greatly increase the sensitivity of the assay (down to a single bacterium), and simplify the assay, allowing for some embodiments to be completed in 2 hours or less, as opposed to several hours required due to the requirement for additional purification steps with constructs that produce detectable fusion proteins.

[0060] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product can generate light and / or can be detected by a color change. Various suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can act as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal can also be suitable indicator moieties.

[0061] In some embodiments, the preparation of the recombinant bacteriophage stock includes a purification step to remove substantially all residual indicator protein that can be associated with the bacteriophage prior to use in a bacterial detection assay. The resulting preparation of parent recombinant bacteriophage is substantially free of indicator protein for use in infecting any target bacteria in a sample of interest.

[0062] Methods of using infectious bacteriophage to detect MRSA

[0063] As described herein, in certain embodiments, the present disclosure provides methods of using infectious bacteriophage to detect MRSA or microorganisms. The methods of the present disclosure can be implemented in a variety of ways.

[0064] In some embodiments, the disclosure provides a method of detecting a microorganism in a sample. The method comprises: obtaining a sample; contacting the sample with a mixture comprising one or more infectious agents, wherein the infectious agents comprise an indicator gene and are specific for a microorganism, and wherein the indicator gene encodes 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 detection of the signal is used to determine the presence of the microorganism in the sample.

[0065] In some embodiments, the disclosure provides a method of detecting MRSA from a sample (e.g., from a nasal swab), comprising the steps of: obtaining a sample, incubating the sample in an assay comprising a selection agent and one or more bacteriophages that infect Staphylococcus aureus, wherein the bacteriophages comprise an indicator gene, such that expression of the indicator gene during bacteriophage replication following infection of the bacteria 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 that MRSA is present in the sample. In some embodiments, the selection agent is an antibiotic comprising cefoxitin.

[0066] In some embodiments, the method comprises capturing the indicator protein product for detection. The step of capturing the indicator protein product on a surface improves detection of microorganisms that produce concentrations of colony forming units that are very low, or of multiple strains of MRSA. The indicator protein product can be contacted with a surface to capture the indicator protein product on the surface. For example, the indicator protein product can attach or bind to the surface during the step of capturing. In some embodiments, the surface can comprise a microtiter plate, latex particles, a lateral flow strip, a bead, a magnetic particle, an impregnated test strip, and the like.

[0067] In some embodiments, the surface can comprise an immobilized binding partner. For example, one or more specific recognition elements can be immobilized in discrete regions of a surface to generate an array for analyte recognition. The indicator protein product can be contacted with the surface comprising an immobilized binding partner. In some embodiments, several different binding partners can be immobilized on one surface at the same time. In some embodiments, the immobilized binding partner is an antibody or fragment thereof.

[0068] In some embodiments, one or more different immobilized binding partners can be deposited (e.g., pipetted) onto a surface (e.g., a plate) for capture of the indicator protein product. In some aspects, the surface can increase accessibility and capture the indicator protein product by directional immobilized binding partners. For example, antibodies can be deposited on a plate and incubated for a period of time. In some embodiments, the antibodies can be rabbit or goat antibodies. Optionally, the plate can be washed after incubation. Subsequently, Antibodies can be deposited on a coated plate. In some aspects, it is advantageous if the amount of indicator protein product to be deposited on the surface is equal to or less than the amount of immobilized binding partner used to form a monolayer on the surface as a solid support. For example, the immobilized binding partner can be antibodies that bind to a layer on the surface of the solid support, creating accessibility to their specific binding epitopes.

[0069] In some embodiments, the methods of the disclosure can include various other steps to increase sensitivity. The sensitivity of the method of detecting microorganisms or MRSA can be increased by one or more washing steps. For example, the method can include a step of washing the captured indicator protein product to remove excess bacteriophage and / or luciferase or other indicator proteins contaminating the bacteriophage preparation. Additionally, the captured microorganisms can be washed after incubation with antibiotics and infectious agents, then lysing buffer and substrate are added. These additional washing steps aid in the removal of excess parental bacteriophage and / or luciferase or other indicator proteins contaminating the bacteriophage preparation. In some embodiments, the microorganisms can be captured, washed, and then infected with bacteriophage.

[0070] In some embodiments, the method includes adding a protein to the antibodies to facilitate infection by the bacteriophage. Antibodies (e.g., IgG) in the blood bind to S. aureus preventing the bacteriophage from infecting the cells. In some embodiments, protein A is added to bind to the antibodies in the blood, thereby preventing the antibodies from binding to S. aureus. When S. aureus is lysed in the presence of protein A, the antibodies cannot bind to the daughter cells, allowing the bacteriophage to infect the cells in the blood. In some embodiments, protein A is added to the bacteriophage mixture. For example, protein A can be mixed with the bacteriophage mixture prior to infection.

[0071] In certain embodiments, assays can be performed to utilize general concepts that can be modified to accommodate different sample types or sizes and assay formats. Embodiments employing recombinant bacteriophage (i.e., indicator bacteriophage) can allow for rapid detection of MRSA with total assay times of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 12.5, 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, depending on the sample type, sample size, and assay format. For example, the amount of time required can be somewhat shorter or longer, depending on the bacteriophage strain to be detected in the assay and the bacterial strain, the type and size of the sample to be tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target bacterial contaminants.

[0072] Examples

[0073] The results described in the following examples demonstrate the effectiveness of the compositions, methods, and systems described herein in detecting MRSA from nasal swab specimens in a reduced time to result. The examples evaluate a new bacteriophage-based assay for use in the diagnostic screening methods and systems described herein. The assay employs a new generation luciferase-bacteriophage reporter system member that detects the target species. The method proved to be inclusive and was able to discriminate most non-resistant strains when combined with a cefoxitin selection. In addition, the method was able to identify low load MRSA in nasal samples with little or no evidence of problematic interference. Finally, the data show that this diagnostic screen can be a promising new tool for detecting MRSA colonization from nasal swab specimens. The results described in the following examples demonstrate the effectiveness of the compositions, methods, and systems described herein in detecting MRSA from nasal swab specimens in a reduced time to result. The examples evaluate a new bacteriophage-based assay for use in the diagnostic screening methods and systems described herein. The assay employs a new generation luciferase-bacteriophage reporter system member that detects the target species. The method proved to be inclusive and was able to discriminate most non-resistant strains when combined with a cefoxitin selection. In addition, the method was able to identify low load MRSA in nasal samples with little or no evidence of problematic interference. Finally, the data show that this diagnostic screen can be a promising new tool for detecting MRSA colonization from nasal swab specimens.

[0074] Materials and Methods

[0075] Bacterial Strains

[0076] Bacterial strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) except where noted below. 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 S. aureus were obtained from the internal Laboratory Corporation of America Holdings. MRSA isolates from de-identified human clinical specimens originated from three geographically distinct US locations (Burlington NC, Phoenix AZ, and Raritan NJ). MSSA isolates were obtained in a similar fashion from one location (Burlington, NC). Determination of MRSA or MSSA was 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) and shaken at 250 revolutions per minute (RPM).

[0077] Bacteriophage Source and Stock Preparation

[0078] Assays included 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 the Colorado School of Mines, and ISP bacteriophage was obtained from Emory University.

[0079] 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. Loss of optical density (OD) of the culture was monitored as confirmation of viral propagation. Bacteriophage lysate was then clarified by centrifugation at 10,000 rpm for 10 minutes at 4°C. The clarified supernatant was again centrifuged at 10,000 rpm for 2 hours at 4°C. The pellet was resuspended in lx TMS (50 mM Tris-HCL, 10 mM MgCl2, and 300 mM NaCl) overnight. The bacteriophage preparation was then treated with 10 pg / mL DNase I and 5 pg / 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 of 1.2, 1.3, 1.4, and 1.6) at 30,000 rpm for 2 hours at 20°C. The band containing the phage was removed and the preparation was placed in a dialysis tube (Spectra / Por 4, 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 repeated again in TMS containing 0.3 M NaCl.

[0080] For ISP, a similar procedure was used with the following differences: strain 12600 was used as the host, the exponential phase culture was infected at a MOI of 0.05, an additional centrifugation at 5,000 rpm for 10 minutes at 4°C was performed after the pellet was resuspended overnight, and then treated with DNase and RNase. Stock titer was determined by standard methods using plaque counts on host strains grown in semi-solid agar.

[0081] Engineering of Luciferase Reporter Bacteriophage

[0082] Target bacteriophages were transformed with a homologous recombination donor construct that utilized a host-specific promoter and a codon-optimized design placed between two 500 bp flanking sequences with homology to the putative late gene region in ISP. This construct was inserted into the Pstl site of pBAV1 KT5gfp (Accession HQ191434). The host-specific promoter was constructed after previous studies. Cloning and codon optimization of the gfp gene was performed by Genewiz (South Plainfield, NJ, USA). This donor construct was used for engineering of both ISP and MP115, as the homology regions shared 99% identity.

[0083] Electroporation-competent S. aureus was made from RN4220. To achieve this, an overnight culture of RN4220 was diluted and grown to mid-log phase in tryptic soy broth (TSB) (Oxiod, Hampshire, United Kingdom). The bacteria were then chilled on ice for 1 h, centrifuged at 4,000 g for 10 min at 4°C, and washed three times with ice-cold sterile deionized water. After washing, the final pellet was suspended in ice-cold 10% glycerol and prepared in aliquots for storage at -80°C. Then, 100 ng of donor construct plasmid DNA was added to a thawed aliquot and incubated for 30 min at room temperature 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 recovered in B2 medium (10 g / L proteose peptone, 25 g / L yeast extract, 25 g / L NaCl, 1 g / L K2HPO4, pH 7.5) and plated on TSB agar containing 50 pg / mL kanamycin (Sigma, St. Louis, MO, USA). Transformants were isolated and confirmed by expression of the Before testing, colonies were grown for 3 h in TSB containing kanamycin. A mixture of 10 pL culture, 50 pL NanoGlo buffer, 15 pL Renilla lysis buffer, and 1 pL NanoGlo substrate (Promega, Madison, WI, USA) was prepared and analyzed using a GloMax Navigator (Promega, Madison, WI, USA).

[0084] Characterization of transformed RN4220 Positive cultures were grown to the early logarithmic growth phase and infected with MP115 or ISP at an MOI of 0.1 and incubated at 37°C for 3 hours with shaking at 225 rpm. Phage lysates were centrifuged to remove cell debris, filtered through a 0.45 μM Whatman Puradisc filter (GE Health, Pittsburgh, PA, USA), and finally exchanged for TMS using a 100K MWCO protein concentrator (Pierce) buffer. Limiting dilutions were then performed to enrich and increase the frequency of recombinants, followed by plaque screening on semi-solid agar. Individual plaques were isolated using sterile pipette tips 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 Renshin lysis buffer, and 1 μL of NanoGlo substrate were added to each well, and the results were evaluated on GloMax Navigator. High-signal positive cells are filtered, diluted, and used to infect the next generation. This process is repeated until three consecutive generations produce 100% positive plaques that are considered pure.

[0085] In Vitro Bacteriophage Detection Assay - Sensitivity, Inclusivity, and MSSA Exclusion

[0086] Overnight cultures were diluted in 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 colony-forming units (CFU) per well (e.g., 10, 1000, or 1000 CFU). The culture background was determined using two additional wells consisting solely of 135 μL of BHI broth. One well for each sample served as a control well and received 15 μL of BHI broth. The other wells served as selection wells and received 15 μL of BHI broth containing 22 μg / mL cefoxitin (Alfa Aesar, Ward Hill, MA, USA). The selection wells contained a final concentration of 2.2 μg / mL cefoxitin. When indicated, the actual CFU for each sample was confirmed by plaque counting on the BHI agar. 96-well strip plates were sealed with a covering membrane (Thermo Fisher Scientific, Rochester, NY, USA) and incubated at 37°C for 4 hours to promote enrichment and selection. Phage mixtures containing 1.6 x 10⁻⁶ cells each were prepared in lysozyme broth (LB) (Gibco, Grand Island, NY, USA). 8Two engineered phage in plaque forming units (PFU) per mL. 10 μL of phage mixture was added to each well and mixed by pipette, then covered again with film. Plates were incubated at 37°C for 4 hours to promote phage infection in the presence of MRSA and luciferase production. 65 μL of detection solution consisting of 50 μL NanoGlo buffer, 15 μL Renilla lysis buffer, and 1 μL NanoGlo substrate was added to each well and mixed by pipette. Samples were read using a GloMax Navigator with a 3 minute wait time and 1 second integration. Results were evaluated with a cutoff of 600 relative light units (RLU), which is approximately 3 times the background observed with media alone.

[0087] In Vitro Bacteriophage Detection Assay - Non-Staphylococcus aureus Exclusion and Bacterial Interference

[0088] Overnight cultures of competing organisms were diluted in BHI broth and 125 μL of the diluted culture was transferred to 4 wells of a 96-well strip plate to obtain the desired CFU per well (e.g., 10, 1000, or 1000 CFU). An additional 4 wells consisting of 125 μL of BHI broth alone were used to determine the background of the media and the baseline signal of MRSA (BAA-1720). 2 wells of each sample were assigned to exclusivity testing while the other two wells were used to assess bacterial interference. For exclusivity, 10 μL of BHI broth was added to the two wells while 10 μL of BHI broth containing MRSA was added to the bacterial interference wells. For each condition, one well served as a control well and received an additional 15 μL of BHI broth while the other wells served as selection wells and received 15 μL of BHI broth containing 22 μg / mL cefoxitin. Enrichment, phage infection, and CFU determination were then performed as previously described.

[0089] Nasal Swab Bacteriophage Detection - Endogenous Samples, MRSA Spiking, and Autofluorescence

[0090] BBL CultureSwab Liquid Stuart Double swabs (Becton Dickinson and Company, Sparks, MD, USA) were used in the experiments described herein. Synthetic fiber nasal swabs were self-collected by volunteers who were instructed to insert the swab into one nostril, rotate at least 5 times, and repeat with the same swab in the second nostril. Samples were stored at 4°C overnight prior to 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 2 wells of a 96-well strip plate. These wells were evaluated in the same manner as the 135 μL diluted cultures described above.

[0091] Reference methods using direct plating and enrichment culture were employed to identify true MRSA colonization. For direct plating, 135 μL of the nasal eluate used in the screening was plated on MRSA Select II agar. For the enrichment culture method, a 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 on MRSA Select II agar. In both cases, the manufacturer's instructions were followed to identify the presence or absence of MRSA colonization. A swab was considered MRSA positive if either method (direct plating or enrichment culture) produced a positive result on the selective agar.

[0092] The ability to detect MRSA in the nasal matrix was evaluated by spiking a dilute culture of MRSA into the nasal eluate. To do this, 125 μL of the nasal eluate was added to 2 wells of a 96-well strip plate for each sample. The two wells received 10 μL of the dilute MRSA culture. Forty independent nasal samples were evaluated, with 8 samples assigned to each MRSA strain tested (BAA-1707, BAA-1717, BAA-1720, BAA-1763, BAA-1766). As a control, 10 μL of each MRSA strain was also spiked into 125 μL of BHI broth. After spiking, the two wells were evaluated in the same manner as the 135 μL dilute culture described above.

[0093] The spontaneous luminescence of each nasal sample was evaluated by mixing each sample with a detection solution that did not have a source of luciferase (phage mix). To do this, 135 μL of each nasal eluate was combined with 25 μL of BHI broth in a 96-well strip plate. 65 μL of the detection solution was then added to each well and mixed by pipetting. The plate was read on a luminometer.

[0094] Example 1. Selectivity and inclusivity studies

[0095] The methods and systems described herein were able to identify MRSA strains from a wide variety of genetic backgrounds (Table 1). As shown in Table 1, the inclusivity strains of MRSA were obtained from academic sources. For the vast majority of strains, detection of the various MRSA strains could be achieved at 100 CFU or less. This limit of detection and analytical sensitivity is similar to previously described PCR-based screens.

[0096] MRSA selection based on bacteriophage includes a 4 hour enrichment, a 2 hour infection, and subsequent detection of emitted light on a luminometer. Two wells of a 96 well strip plate are assayed for each sample, the two wells consisting of one control well and one selection well. The selection well is used for MRSA determination and contains the MRSA selective agent cefoxitin, while the control well contains only bacterial media and primarily calibrates phage performance during the assay color development. Cefoxitin was shown to be the best choice for phenotypic identification of MRSA in paper disk diffusion and agar dilution assays. The samples are enriched in these wells for 4 hours, which promotes recovery, growth, and selection of resistant bacteria. Thereafter, a 2 hour infection period with a recombinant bacteriophage encoding luciferase is performed. Luciferase production is measured by detecting emitted light with a luminometer after addition of substrate, which production indicates a successful viral infection. This method was used to evaluate 17 different MRSA strains at a starting target of 10, 100, or 1,000 colony forming units (CFU) in triplicate wells (Table 1). Values for CFU (determined from plating) and relative light units (RLU) are provided in Table S1. A positive result is determined based on a cutoff value of 600 RLU. This cutoff is approximately 3 times the background observed with media alone.

[0097] Fifty-one of 51 (100%) wells tested were positive at 100 CFU and 1,000 CFU per well under control conditions. Forty-eight of 51 (94.1%) were positive at 10 CFU / well. At 10 CFU, only two of three unique strains of MRSA were positive in three wells. These results highlight the ability of the phage cocktail to recognize a variety of MRSA isolates. When cefoxitin was included for the MRSA assay, 51 of 51 (100%) wells were still detectable for a positive signal at 1,000 CFU / well and 48 of 51 (94.1%) were still detectable for a positive signal at 100 CFU / well. At 100 CFU, BAA-42 (also known as HDE288) was not detectable after selection was not entirely unexpected. This strain belongs to an "old clone" of MRSA associated with low levels and heterogeneous methicillin resistance. As shown in Table 1, with only 10 CFU, 44 of 51 (86.3%) of the selection wells were still positive. Based on the lowest CFU detected in both control and selection wells, a limit of detection was determined for each strain. Of the 17 MRSA strains tested, 13 were reliably detectable at 10 CFU / well, while three required 100 CFU / well. BAA-42 was the only strain that required greater than 100 CFU / well to obtain a consistent positive detection with the MRSA selection. As shown in Table 1, the MRSA assay demonstrated 100% inclusivity for the 17 MRSA strains tested at 100 CFU. The MRSA assay also demonstrated selectivity for 48 of the 51 MRSA strains tested. In summary, these results demonstrate the ability of this screen to detect the presence of genetically diverse MRSA strains at low bacterial loads.

[0098] Table 1

[0099]

[0100] 1 Strain ID corresponds to American Type Culture Collection (ATCC) catalog number.

[0101] 2 SCCmec type and pulse field gel electrophoresis (PFGE) available from (ATCC).

[0102] 3 Positive cells were defined based on a signal cutoff of 600 relative light units (RLU).

[0103] 4 Limit of detection (LoD) defined as the lowest colony forming units (CFU) that exhibited 100% positive results in both control and selection wells.

[0104] Table S1

[0105]

[0106]

[0107] 1 CFU was determined by plating samples with 100 CFU of target in duplicate and calculating from dilutions of samples with 10 and 1000 CFU of target.

[0108] 2 BHI broth was used instead of bacterial culture to show assay background.

[0109] Example 2. Exclusivity and specificity of in vitro MRSA screening

[0110] In addition to sensitive MRSA detection, a successful MRSA assay must also demonstrate the ability to exclude most methicillin-susceptible Staphylococcus aureus (MSSA) strains. Table 2 shows five well-characterized strains of MRSA evaluated using the method described herein at 100, 1,000, and 10,000 CFU in triplicate wells and provides CFU values determined from plating counts and RLU values. MRSA control wells did not include cefoxitin, and MRSA selection wells included cefoxitin. As expected, at the 100, 1,000, and 10,000 CFU levels, the MSSA strains were positive in 100% of the control wells. The inclusion of cefoxitin in the selection wells resulted in a significant decrease in positive results. In the MRSA selection wells including cefoxitin, 0 of 15 (0%) were positive at 100 CFU, and only 1 of 15 (6.7%) were positive at 1,000 and 10,000 CFU. These results support the ability of the MRSA assay to distinguish most MSSA strains.

[0111] Table 2

[0112]

[0113] 1 Strain ID corresponds to American Type Culture Collection (ATCC) catalog number.

[0114] 2 Positive cells were defined based on a signal cutoff of 600 RLU.

[0115] Table S2

[0116]

[0117] 1CFU were determined by plating samples with 100 CFU per well (in duplicate) and calculating from dilutions of samples with 10 and 1000 CFU of target.

[0118] 2 BHI broth was used instead of bacterial culture to identify assay background.

[0119] As shown in Table 3, the exclusivity of the MRSA screen was evaluated in vitro against a panel of 40 strains encompassing 21 unique genera and 32 different species, with the exception of MSSA. Values for CFU (determined by plating) and RLU are provided in Table S3. CFU for each exclusive strain were greater than 1,500 CFU per well (median CFU was 15,950). When assessing specificity, Table 3 shows that 6 of the 40 strains (15%) were positive in the control well. Positive signals under these conditions are a result of cross-reactivity of the phage cocktail, observed against Staphylococcus and Bacillus species. Many Staphylococcus phages have been demonstrated to be polyvalent, lysing both coagulase-positive and coagulase-negative Staphylococcus species. Bacillus species have been previously reported to adsorb Staphylococcus phages and this can be related to similarities with their cell wall teichoic acids (WTA). Despite this cross-reactivity, there were 0 positives out of 40 strains under selection conditions and would not result in false positives for MRSA. These results demonstrate the specificity of the phage cocktail used in the experiments described herein and the exclusivity of the overall assay.

[0120] The ability of the MRSA screen to detect low numbers of MRSA in the presence of excess competitor loads was evaluated. To do this, approximately 50 CFU of MRSA were combined with at least a 20-fold excess of each strain from the exclusivity panel (Table 3). Values for CFU (determined by plating) and RLU are provided in Table S3. Surprisingly, in the presence of the competitor species, 39 out of 40 wells (97.5%) and 40 out of 40 wells (100%) were positive under control and selection conditions, respectively. S. pneumoniae inhibited detection under control conditions when tested at a 100-fold excess. This is not surprising given the known antagonism between these species in vitro and in vivo. Crucially, this effect disappeared in the presence of cefoxitin (MRSA selection condition) 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 competing organisms.

[0121] Table 3

[0122]

[0123]

[0124] 1 Strain ID for all strains corresponds to ATCC catalog number, except for Salmonella enterica S492 strain.

[0125] 2 Positive cells were defined based on a signal cutoff of 600 RLU.

[0126] 3 For exclusivity, each competitor strain was evaluated individually at greater than 1,500 CFU / well individually.

[0127] 4 For bacterial interference, MRSA (BAA-1720) was added at approximately 50 CFU / well, while the indicated competitor was added in excess (at least 20-fold).

[0128] Table S3

[0129]

[0130]

[0131] 1 CFU was determined using dilution samples for competitors or direct plating (in duplicate) for MRSA.

[0132] 2 BHI broth was used instead of bacterial culture identification assays to determine background.

[0133] 3 For exclusivity, each competitor strain was evaluated individually at the indicated CFU / well individually.

[0134] 4 For bacterial interference, MRSA (BAA-1720) was added at the indicated load combined with the indicated competitor CFU / well.

[0135] Example 3. In vitro screening performance among circulating Staphylococcus aureus clinical isolates.

[0136] MRSA isolates from human clinical specimens were obtained from three geographically distinct clinical microbiology laboratories internally (Burlington NC, Phoenix AZ, and Raritan NJ). MSSA isolates were obtained in a similar fashion from one site (Burlington, NC). MRSA or MSSA identification was confirmed by plating on selective chromogenic agar. A total of 390 clinical MRSA strains were isolated from independent specimens and evaluated using the MRSA screen. RLU and CFU values are provided for each strain (Table S4).

[0137] Table 4 shows the median load of MRSA tested was 47 CFU / well. As shown in Table 4, 388 of 390 (99.5%) of the clinical MRSA strains were detected positive in the control wells. After cefoxitin selection, 381 of 390 (97.7%) of the clinical MRSA strains were positive and identified as MRSA by this screen. At higher loads, 10- or 100-fold MRSA levels (500 CFU and 5,000 CFU, respectively), the exclusion of clinical MSSA strains was tested. In the control condition for each inoculum, 122 of 123 (99.2%) of the clinical MSSA strains were detected positive. However, in the selection wells, the positive signal from 500 CFU dropped to 8 positive of 123 MSSA strains (6.5%). At approximately 5,000 CFU / well, this false positive rate increased to 21 positive of 123 strains (17.1%). This suggests that although most MSSA strains are negative, some can withstand selection at high loads and result 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 notion that the phage cocktail utilized in the described method and system produces a broad host range coverage. In summary, these results show the ability of this screen to successfully identify and detect most clinical MRSA strains while excluding most clinical MSSA strains.

[0138] Table 4

[0139]

[0140] 1 A positive cell was defined based on a signal cutoff of 600 RLU.

[0141] 2 The median CFU tested for clinical MRSA strains was 47 CFU / well. The load of each strain can be found in the Appendix.

[0142] 3 The median CFU / well tested for clinical MSSA strains was 850 CFU at "500" and 8,500 CFU at "5,000".

[0143] Table S4

[0144] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0145]

[0146] Table S4

[0147] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0148]

[0149] Table S4

[0150] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0151]

[0152] Table S4

[0153] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0154]

[0155] Table S4

[0156] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0157]

[0158] Table S4

[0159] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0160]

[0161] Table S4

[0162] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0163]

[0164] Table S4

[0165] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0166]

[0167] Table S4

[0168] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0169]

[0170] Table S4

[0171] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0172]

[0173] Table S4

[0174] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0175]

[0176] Table S4

[0177] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0178]

[0179] Table S4

[0180] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0181]

[0182] Table S4

[0183] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0184]

[0185] Table S4

[0186] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0187]

[0188] Table S4

[0189] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0190]

[0191] Table S4

[0192] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0193]

[0194] Table S4

[0195] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0196]

[0197] Table S4

[0198] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0199]

[0200] Table S4

[0201] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0202]

[0203] Table S4

[0204] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0205]

[0206] Table S4

[0207] CFU and RLU for MRSA screening with clinical S. aureus (Table 4)

[0208]

[0209] Example 4. Specificity and screening performance with human nasal swabs.

[0210] Anterior nasal specimens were self-collected by 40 adult human volunteers using a synthetic fiber swab. Previous studies have demonstrated the efficacy of self-collection for detecting MRSA colonization. Prior to processing, specimens were stored overnight at 4°C to best mimic possible sample shipping conditions. Both reference methods using direct plating and enrichment culture were employed to identify true MRSA colonization. By both reference methods, all 40 human nasal specimens were negative and were determined to lack MRSA colonization (Table 5). The lack of detection among the 40 individuals was not surprising as the MRSA colonization rate is estimated to be less than 2% in healthy adults.

[0211] To screen using these specimens, swabs were eluted into bacterial broth and added to wells containing (selection) or not containing (control) cefoxitin. Positive results in the selection condition were considered positive MRSA results. The control condition is not required or used for the MRSA assay, but was included to demonstrate the effectiveness of the selection. Due to the high nasal colonization rate of S. aureus species and the cross-reactivity described earlier for the phage cocktail, positive results were expected in most control wells. As expected, 36 of 40 samples (90%) were positive in the control wells. RLU values for endogenous samples are provided (Table S5).

[0212] For MRSA detection, 36 of 40 specimens (90.0%) were negative, consistent with the reference method. Four samples were identified as false positives, with median RLU signals less than 5-fold the signal cutoff. All nasal samples were negative when tested directly with luciferase substrate, indicating that non-specific auto luminescence was not the primary source of false positives (Table S5). The exact mechanism behind the false positive signals in these samples remains unknown, but can potentially be linked to methicillin-resistant coagulase-negative staphylococci. In addition, some MSSA strains were previously observed to produce false positive results at high bacterial loads (Table 4). In summary, the majority (90%) of MRSA-negative samples can be successfully screened out by this method.

[0213] Table 5. Screening performance of non-colonized nasal swabs

[0214]

[0215] 1 Positive wells were defined based on a signal cutoff of 600 RLU.

[0216] 2 Nasal swabs were eluted in bacterial broth and assayed directly.

[0217] 3 One of five MRSA strains at -100 CFU / well was spiked into the nasal eluate prior to testing.

[0218] 4 A combination of direct plating and enrichment culture was used as the reference method using MRSA Select II agar.

[0219] Table S5

[0220]

[0221]

[0222] 1 Nasal swabs were eluted in BHI and assayed directly.

[0223] 2 Nasal eluates were spiked with the indicated MRSA strain at the CFU / well indicated.

[0224] 3Nasal eluates were combined with luciferase substrate and buffer in the absence of luciferase reporter phage. The signal in these wells was considered to be autofluorescence, possibly the result of non-specific activation of the substrate or pre-existing luminescence in the sample.

[0225] 4 CFU were determined directly by plating (in duplicate).

[0226] To determine if this method could successfully detect MRSA in nasal matrices, 5 well-characterized MRSA strains were spiked into eluates from 40 non-colonized nasal swabs described previously. RLU and CFU values are provided for each sample (Table S5). The median load of MRSA spiking was 87 CFU / well. Under control and selection conditions, 40 of 40 (100%) of the MRSA inoculated samples were positive (Table 5). The lack of any failed samples indicates the lack of assay inhibitors in these individuals. The successful detection of 5 independent MRSA strains when spiked into these samples at low loads supports the efficacy of the bacteriophage-based screen in nasal matrices.

[0227] As shown in the Examples, the present disclosure provides a MRSA luciferase phage reporter assay that enables sensitive and rapid detection of MRSA from nasal swabs in a culture-based manner. As shown in Table 1, diagnostic screening with the MRSA luciferase phage reporter assay was able to identify MRSA strains from a variety of genetic backgrounds in approximately 6 hours. For the vast majority of MRSA strains, successful detection required the presence of only 10-100 CFU / well, roughly equivalent to 75-750 CFU / nasal swab. This limit of detection is similar to previously described PCR-based screens. The median load of MRSA recovered from the nasal swabs of carriers was found to be greater than 10,000 CFU. In addition, individuals with high loads of nasal colonization are more likely to carry MRSA on multiple body sites and are carriers of transmission. Thus the sensitivity of this assay appears well suited to address the expected load problem from clinical nasal specimens, whether the goal is to eradicate MRSA colonizers or limit patient-to-patient spread.

[0228] In some aspects, the performance of the luciferase reporter phage assay is highly dependent on the selection of the bacteriophage. This MRSA diagnostic screen in the Examples utilizes a recombinant of two phages (ISP and MP115) expressing NanoLuc, which are members of the Myoviridae family of large lytic S. aureus bacteriophages. These phages bind to the host surface primarily through a highly conserved WTA, which creates the ability for broad host range. Mutants lacking WTA are thought to be resistant to all, or at least most, staphylococcal phages. Although resistant WTA-deficient mutants are postulated to be possible, previous studies have revealed that WTA is essential for both nasal colonization and methicillin resistance. Typically, loss of WTA also results in health costs in vivo as well as overall reduction in virulence. Therefore, it is a reasonable expectation that all existing and future MRSA strains that participate in nasal carriage have the receptor targeted by this screen. Furthermore, this conclusion is further supported by the data in Table 4, which shows 99.5% of the tested clinical MRSA isolates detected positive phage signal.

[0229] As shown in the results in Table 4, of the 513 S. aureus clinical strains, two MRSA isolates (BNC159 and PHX 079) and one MSSA isolate (MSSA 090) were unable to produce a positive signal under control conditions. One of these isolates (PHX 079) appeared to have a growth defect in culture (data not shown). Poor growth during the enrichment period can have facilitated the inability to reliably detect this MRSA strain. The inability to detect BNC 159 and MSSA 090 can be related to phage resistance or capsule production through restriction-modification systems. Restriction-modification systems target and eliminate foreign DNA, often identified by the presence or absence of DNA methylation at specific motifs. There is evidence that staphylococcal phages have evolved under pressure at these pathways, and several phages completely lack specific sequences targeted by these systems. Nonetheless, the diversity of restriction-modification systems among S. aureus is extensive, and can have contributed to the observed resistance in these isolates. Respectively, capsule production has been linked to phage resistance in S. aureus through masking surface receptors. Although several common lineages of S. aureus do not produce capsular polysaccharide, this mechanism can contribute to the observed rare (<1%) resistance.

[0230] In addition, Table 4 also shows that the combination of the MRSA luciferase phage reporter assay and a selection agent (e.g., antibiotic) limits the viability and growth of non-MRSA and does not interfere with MRSA detection. For example, the MRSA luciferase phage reporter assay utilizes cefoxitin to limit the viability and growth of non-MRSA. The results in Table 4 demonstrate the efficacy of this selection because only 6.5% of the clinical MSSA strains were positive when tested at approximately 500 CFU / well. Surprisingly, cefoxitin does not interfere with MRSA detection because 97.7% of the clinical MRSA strains remained positive in the selection well at approximately 50 CFU / well. In addition, Table 3 shows that this selection agent also benefits from limiting false positives from Bacillus as well as several species of coagulase-negative Staphylococcus while also preventing interference from Streptococcus pneumoniae. Cefoxitin has proven to be a preferred selection for MRSA, capable of identifying a variety of isolates. Despite the high rate of detection of clinical MRSA, some strains produced false negative results in the presence of cefoxitin. Since the clinical MRSA strains were evaluated at particularly low loads in some embodiments, it appears plausible that these strains express low levels of resistance or heterogenous resistance. Such strains can exist above the detection limit of 100 CFU / well, similar to that found for BAA-42 (Table 1).

[0231] With respect to performance using nasal swabs, Table 5 provides that 90.0% of the MRSA-negative samples yielded negative test results after selection, consistent with the reference method. Thus false positives were detected in 10% of the nasal eluates. These false positives can originate from three sources. First, spontaneous light can occur, but was ruled out by demonstrating the need to add luciferase in these samples, as provided in Table S5. Second, high loads of certain MSSA strains can result in false positives (Table 4). Finally, some of the coagulase-negative Staphylococcus species can become methicillin-resistant through the same resistance mechanism as MRSA. These species can potentially contribute to the weak false MRSA positives observed in 4 samples.

[0232] The methods and systems described herein for detecting MRSA require the viability of the endogenous nasal flora, which is unique in evaluating the viability of a sample. To replicate the endogenous nasal flora, one of the five MRSA strains was spiked into the nasal eluate (Table 5). As shown in Table 5, positive detection of low MRSA loads in the nasal matrix was achieved in 100% of the spiked samples. Importantly, this indicates that successful bacteriophage infection and luciferase production can occur in the nasal matrix. Furthermore, this reveals that the negative control wells previously seen in 10% of the endogenous samples were not the result of assay inhibitors. In summary, the results strongly suggest that MRSA persister, when present, will be detected in nasal specimens.

[0233] The bacteriophage-based MRSA assay described herein is a member of a new generation of luciferase reporter phage system that utilizes NanoLuc to sensitively detect target species. The method demonstrated high inclusivity and was able to discriminate most non-drug resistant strains when combined with cefoxitin selection. In addition, the screen was able to identify low load MRSA in nasal samples with no evidence of problematic interference. Additionally, as MRSA detection was completed within 6 hours, actionable results were available within a single shift. Finally, data showed that the bacteriophage-based MRSA assay described herein can be a promising new tool for detecting MRSA colonization from nasal swabs.

[0234] Example 5. Direct plating of NanoLuc on culture media and high protein binding plates.

[0235] Staphylococcus aureus (ATCC 12600) was grown in tryptic soy broth (TSB) to log phase (OD 600 The culture was diluted in TSB to obtain the desired load, which was confirmed by colony forming units (CFU) by plating on TSB agar. 12.5 μΐ^of each dilution was added directly to 37.5 μΐ^of TSB or human blood in 96-well strip plates (high binding; Grenier Bio-One, Ref# 762074). When indicated, some strips contained bound anti-NanoLuc antibody (purified mouse monoclonal IgG clone #965808; Cat# MAB10026) for capture. Human blood was collected from a single donor using sodium heparin as anticoagulant. For blood samples, 100 μΐ^of TSB containing sodium poly-m- aminostilbene sulfonate (SPS) was added to achieve a 25% human blood matrix. The final concentration of SPS in the wells (150 μΐ^volume) was 0.05%. For TSB samples, 100 μΐ^of TSB was added to achieve the same 150 μΐ^volume. The test strips were then sealed with a film lid and incubated at 37°C for 30 minutes. After this short enrichment, 20 μΐ^of phage working stock was added to the wells containing the TSB matrix. The phage working stock contained 8 x 10 7MP115.NL and SAPJV1.NL plaque forming units / mL. To allow infection in wells containing blood matrix, 0.5 mg recombinant staphylococcal protein A (pro-356, Prospec, Ness-Ziona, Israel) per well was included in 20 μL phage working stock as indicated. Assay strips were again sealed with a cover film and incubated at 37 °C for 3 hours. After infection, these strips were washed 3 times with 300 μL 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 NanoGlo buffer (Promega, Madison, WI, USA) containing 1 μL NanoGlo substrate (Promega, Madison, WI, USA) was added to each well. After a 3 minute wait period, each sample was measured for signal output as relative light units (RLU) using a GloMax Navigator (Promega, Madison, WI, USA). The RLU from each sample was divided by the RLU observed in the media control for that test matrix to calculate the signal-to-background ratio (S / B).

[0236]

[0237] In these examples, the anti-NanoLuc antibody is the immobilized binding partner. Table 6 demonstrates a large increase in signal detection when the indicator protein is captured by the immobilized binding partner. For example, in samples containing low or high loadings of S. aureus, the RLU when the indicator protein is captured by the anti-NanoLuc antibody is significantly higher than when the sample is not captured using a control strip. Surprisingly, infection of S. aureus can not occur if S. aureus has already bound IgG. The addition of protein A allows S. aureus to be infected. Red blood cells and other serum proteins do not interfere with the capture of the expressed indicator protein. In addition, the quenching of the signal by red blood cells as seen in the control is eliminated and the signal-to-background ratio is maintained or increased. Thus, the indicator protein can be detected using a whole blood sample with minimal interference from other components (e.g., proteins) in the sample. Conventionally, serum or plasma is separated from blood to reliably detect indicator protein products. Advantageously, the examples demonstrate that the detection method can be accomplished on a whole blood sample directly taken from a patient by using this capture step.

[0238] Example 6. Antibiotic susceptibility testing in human blood.

[0239] Methicillin-resistant S. aureus (MRSA) strains (ATCC BAA-1720, CDC AR0480) and methicillin-susceptible S. aureus (MSSA) strain (ATCC 12600) were grown in tryptic soy broth (TSB) to log phase (OD 600 ranging from 0.16-0.4). Cultures were diluted in TSB to obtain the desired load, which was confirmed by plating for colony forming units (CFU) on TSB agar. 50 μL of each dilution was added to the test strips. When indicated, some strips contained bound anti-NanoLuc antibody (purified mouse monoclonal IgG clone #965808; Cat #MAB10026) for capture of bound on the media binding plate (Grenier Bio-One, Strip Plate 12x F8, PS, F-bottom, white, Lumitrac, Med Binding, Ref# 762075) or high binding plate (Grenier Bio-One, Ref# 762074). 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 anticoagulant. Each well then received 15 μL 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 1010 7 MP115.NL and 6.9 x 1010 8SAPJV1.NL at 109pfu / mL. For wells containing blood matrix, 0.5 mg of recombinant staphylococcal protein A (pro-356, Prospec, Ness-Ziona, Israel) per well was included in the 20 pL phage working stock. Assay strips were again sealed with a cover film and incubated at 37 °C for 3 hours. Following infection, anti-NanoLuc capture and control strips were washed 3 times with 300 pL 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 pL of NanoGlo buffer (Promega, Madison, WI, USA) containing 1 pL of NanoGlo substrate (Promega, Madison, WI, USA) was added to each well. The “no wash + no capture” strip was not washed and instead received 65 pL of master mix containing 50 pL of NanoGlo buffer, 15 pL of TSB, and 1 pL of NanoGlo substrate. The 5% BSA (bovine serum albumin, Sigma Life Science Product #A9647) blocked strip. The BSA blocked strip was used to block non-specific binding sites with BSA. After a 3 minute wait period, each sample was measured for signal output as relative light units (RLU) using a GloMax Navigator (Promega, Madison, WI, USA). The RLU from each sample was divided by the RLU observed in the media control for that test matrix to calculate the signal-to-background ratio (S / B).

[0240]

[0241] In Table 7, the “no capture + no wash” example demonstrates the total signal generated when the assay is completed in media (TSB) only and the signal drop caused by cefoxitin. When completed in the presence of blood, the signal is quenched. When a capture strip is used, the signal is greatly increased due to the elimination of quenching caused by blood. The 5% BSA blocked strip (bovine serum albumin, Sigma Life Science Product #A9647) was used to show non-specific binding. The example again demonstrates that for whole blood samples, signal detection is greatly increased when the indicator protein is captured by an immobilized binding partner. Additionally, the capture step significantly improves signal detection 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.

[0242] Example 7. Titration of NanoLuc coated plates.

[0243] 1.5 mg / mL stock of purified NANOLUC diluted to 1 ng / mL in PBS. Serial 10-fold dilutions from 1 ng / mL to 0.001 pg / mL were prepared in PBS. Rabbit anti-mouse IgG (Abeam, Cat# 46540) or goat anti-mouse IgG (Abeam, Cat# 6708) were diluted to 10 pg / mL in PBS and pipetted to 100 pL / well. Plates were incubated at 2-8 °C for 18-20 hours and then washed 3 times using 300 pL PBS / well / wash. Mouse anti-NanoLuc antibody (purified mouse monoclonal IgG, clone #965808, R&D Systems, Cat# MAB10026) was diluted to 1 pg / mL in PBS and pipetted to 100 pL / well to plates coated with rabbit or goat anti-mouse IgG. A 5% BSA blocking strip was included to determine non-specific binding, and an uncoated strip was used to measure Nanoluc activity. Assay strips were sealed with lids and incubated at 37 °C for 3 hours. Antibody-coated strips were washed 3 times with 300 pL / well 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 pL of NanoGlo buffer (Promega, Madison, WI, USA) containing 1 pL of NanoGlo substrate (Promega, Madison, WI, USA) was added to each well. After a 3-minute wait period, each sample was assayed for signal output as relative light units (RLU) using a GloMax Navigator (Promega, Madison, WI, USA). The RLU from each sample was divided by the RLU observed in the PBS control used for the test to calculate the signal-to-background ratio (S / B).

[0244]

[0245]

[0246]

[0247] Tables 8 and 9 demonstrate that plates coated with rabbit anti-mouse IgG or goat anti-mouse IgG provide improved orientation for mouse anti-nanoluc luciferase, enhancing the capture / binding surface. Indeed, coating plates with rabbit anti-mouse IgG or goat anti-mouse IgG offers greater accessibility to the binding indicator protein product. The improved signal detection observed with plate coating is likely due to the orientation of mouse anti-nanoluc luciferase and the accessibility of the binding site to the indicator protein.

[0248] Implementation Plan

[0249] Implementation Scheme 1: A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample, the method comprising: obtaining a sample; adding a selector to the sample; contacting the sample with a mixture containing one or more infectious agents, wherein the infectious agents contain an indicator gene and are specific to Staphylococcus aureus, and wherein the indicator gene encodes an indicator protein product; capturing the indicator protein product; and detecting a signal generated by the indicator protein product, wherein the detection of the signal is used to determine the presence of MRSA in the sample.

[0250] Implementation scheme 2 is the method described in any of the foregoing or subsequent implementation schemes, wherein the selector includes an antibiotic.

[0251] Implementation scheme 3 is the method described in any of the foregoing or subsequent implementation schemes, wherein the antibiotic includes cefoxitin.

[0252] Implementation scheme 4 is the method described in any of the foregoing or subsequent implementation schemes, wherein the sample is derived from a nasal swab.

[0253] Implementation scheme 5 is any of the foregoing or subsequent implementation schemes of the method, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacterium in a sample.

[0254] Implementation 6 is the method described in any of the foregoing or subsequent implementations, wherein the mixture comprises at least two different types of recombinant bacterial phages, and at least one of the recombinant bacterial phages is derived from ISP, MP115, or a combination thereof.

[0255] Implementation 7 is the method described in any of the foregoing or subsequent implementations, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an internal signal or a soluble enzyme that generates a signal upon reaction with a substrate.

[0256] Implementation 8 is the method described in any of the foregoing or subsequent implementations, further comprising a codon-optimized untranslated region upstream of an indicator gene, wherein the untranslated region comprises a bacterial bacteriophage late gene promoter.

[0257] Embodiment 9 is the method of any preceding or subsequent embodiment, wherein the capturing step comprises contacting the indicator protein product with a surface.

[0258] Embodiment 10 is the method of any preceding or subsequent embodiment, wherein the surface is a microtiter plate, latex particle, lateral flow strip, bead, magnetic particle, or dipstick.

[0259] Embodiment 11 is the method of any preceding or subsequent embodiment, further comprising depositing an immobilized binding partner on the surface prior to capturing the indicator protein product.

[0260] Embodiment 12 is the method of any preceding or subsequent embodiment, wherein the immobilized binding partner is an antibody or fragment thereof.

[0261] Embodiment 13 is the method of any preceding or subsequent embodiment, further comprising washing the surface comprising the immobilized binding partner.

[0262] Embodiment 14 is the method of any preceding or subsequent embodiment, further comprising washing the surface after capturing the indicator protein product.

[0263] Embodiment 15 is the method of any preceding or subsequent embodiment, wherein the signal-to-background ratio produced by detecting the indicator protein product is at least 2.0 or at least 2.5.

[0264] Embodiment 16 is the method of any preceding or subsequent embodiment, wherein the sample is first incubated under conditions conducive to 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.

[0265] Embodiment 17 is the method of any preceding or subsequent embodiment, the method comprising: obtaining a sample; contacting the sample with a mixture comprising one or more infectious agents, wherein the infectious agent comprises an indicator gene and is specific for a microorganism, and wherein the indicator gene encodes 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 detection of the signal is used to determine the presence of the microorganism in the sample.

[0266] Embodiment 18: A kit for detecting methicillin-resistant Staphylococcus aureus comprising: a nasal swab; an assay comprising a recombinant bacteriophage specific for Staphylococcus aureus and an antibiotic; and a surface for capturing an indicator protein product.

[0267] Embodiment 19 is the kit of any preceding or subsequent embodiment, wherein the surface comprises an immobilized binding partner.

[0268] Embodiment 20 is the kit of any preceding or subsequent embodiment, wherein the antibiotic comprises cefoxitin.

[0269] The present disclosure is not limited to the exact details shown and described, variations apparent to those skilled in the art being included within the scope of the present disclosure as defined by the claims.

Claims

1. A method of detecting Methicillin-resistant Staphylococcus Aureus (MRSA) in a sample for non-diagnostic purposes, the method comprising: obtaining a sample; adding a selection agent to the sample, wherein the selection agent comprises an antibiotic; contacting the sample with a mixture comprising one or more recombinant bacteriophage, wherein at least one of the recombinant bacteriophage specifically recognizes S. aureus and comprises a genetically modified S. aureus-specific bacteriophage genome, wherein the genetically modified S. aureus-specific bacteriophage genome comprises an indicator gene, and wherein the indicator gene encodes an indicator protein product; capturing the indicator protein product with a substrate comprising an immobilized binding partner for capturing the indicator protein product; washing the substrate comprising the captured indicator protein product; and detecting a signal produced by the captured indicator protein product, wherein detection of the signal is used to determine the presence of MRSA in the sample.

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

3. The method of claim 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an internal signal or a soluble enzyme that generates 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, wherein the untranslated region comprises a bacteriophage late gene promoter.

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

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 an impregnated test strip.

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 fragment thereof.

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

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

11. 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. The method of claim 1, wherein the sample is first incubated under conditions conducive to 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, comprising: ​ an assay comprising one or more recombinant bacteriophages and an antibiotic, wherein at least one of the recombinant bacteriophages specifically recognizes S. aureus and comprises a genetically modified S. aureus- specifically recognizing phage genome, wherein the genetically modified S. aureus- specifically recognizing phage genome comprises an indicator gene, and wherein the indicator gene encodes an indicator protein product; and a surface comprising an immobilized binding partner for capturing the indicator protein product.

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

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