Indicator bacteriophages to select and monitor therapeutic efficacy and methods of use thereof

Abstract

Disclosed herein are methods and systems for detecting microorganisms in a sample and the use of such methods for selecting and monitoring therapies. The specificity of an indicator bacteriophage, such as a Staphylococcus specific bacteriophage, allows the detection of a particular microorganism, such as Staphylococcus, and the indicator signal can be amplified to optimize assay sensitivity.

Description

[0001] This application is a divisional of the parent application having application number 201980028152.3, filed on April 24, 2019, entitled “Select and Monitor Indicators of Phage Therapy Efficacy and Methods of Use Thereof.”

[0002] Cross Reference to Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 661,739, filed on April 24, 2018. The disclosures of U.S. Application Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, and 15 / 409,258, and U.S. Provisional Application 62 / 661,739 are hereby incorporated by reference in their entireties. TECHNICAL FIELD

[0004] The present invention relates to compositions, methods, systems, and kits for detecting microorganisms using infectious agents. BACKGROUND

[0005] There is strong interest in improving the speed and sensitivity of detection of bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples. Microbial pathogens can cause a large number of illnesses in humans and domestic animals, as well as tremendous economic losses. Moreover, given the outbreaks of life-threatening or fatal illnesses caused by consumption of food contaminated with certain microorganisms (e.g., Staphylococcus spp.), the detection of microorganisms is a high priority for the Food and Drug Administration (FDA) and the Centers for Disease Control (CDC).

[0006] Traditional microbiological tests for detecting bacteria rely on non-selective and selective enrichment cultures, followed by plating on selective media and further testing to confirm suspect colonies. Such procedures can take several days. A variety of rapid methods have been investigated and introduced into practice to reduce the time requirement. However, these methods have drawbacks. For example, techniques involving direct immunoassays or genetic probes often require an overnight enrichment step for adequate sensitivity. Polymerase chain reaction (PCR) tests also include an amplification step and, as a result, can have very high sensitivity and selectivity; however, the sample size that can be economically processed for PCR testing is limited. Using a diluted bacterial suspension, most small subsamples will have no cells and thus still require purification and / or lengthy enrichment steps.

[0007] The time required for traditional bio-enrichment is determined by the growth rate of the target bacterial population in the sample, the action of the sample matrix, and the required sensitivity. In practice, most high-sensitivity methods utilize overnight incubation, and the entire process takes about 24 hours. Due to the time required for culturing, these methods can take up to three days, depending on the organism to be identified and the source of the sample. This lag time is often inappropriate because contaminated food, water (or other products) can have already entered the livestock or humans. Furthermore, the increase in antibiotic resistant bacteria and considerations for bio-defense make rapid identification of bacterial pathogens in water, food, and clinical samples a high priority worldwide.

[0008] Accordingly, there is a need for faster, simpler, and more sensitive detection and identification of microorganisms, such as bacteria, viruses, and other potentially pathogenic microorganisms. SUMMARY

[0009] Embodiments of the present invention include compositions, methods, systems, and kits for detecting microorganisms. The present invention can be implemented in a variety of ways.

[0010] In some aspects, the present invention includes a recombinant bacteriophage comprising an indicator gene inserted into a late gene region of a bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Staphylococcus-specific bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Staphylococcus aureus-specific bacteriophage genome. In further embodiments, the Staphylococcus aureus is a methicillin-resistant Staphylococcus aureus (MRSA). For example, in certain embodiments, the recombinant bacteriophage is a genetically modified bacteriophage genome. In some embodiments, the bacteriophage used to make the recombinant bacteriophage specifically infects Staphylococcus. In one embodiment, the recombinant bacteriophage can distinguish Staphylococcus in the presence of over 100 other types of bacteria.

[0011] In some embodiments of the recombinant indicator bacteriophage, the indicator gene can be codon-optimized and can encode a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates a signal upon reaction with a substrate. Some recombinant bacteriophages further include an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region comprises a bacteriophage late gene promoter and a ribosomal entry site. In some embodiments, the indicator gene is a luciferase gene. The luciferase gene can be a naturally occurring gene, such as, for example, Oplophorus luciferase, firefly luciferase, Lucia luciferase, or sea pansy luciferase, or can be a genetically engineered gene, such as, for example, NanoLuc.

[0012] Also disclosed herein are methods for making recombinant indicator phages. Some embodiments include selecting a wild-type phage that specifically infects a target pathogenic bacteria; making a homologous recombination plasmid / vector comprising an indicator gene; transforming the homologous recombination plasmid / vector into the target pathogenic bacteria; infecting the transformed target pathogenic bacteria with the selected wild-type phage, thereby allowing homologous recombination between the plasmid / vector and the phage genome; and isolating specific clones of the recombinant phage. In some embodiments, the selected wild-type phage is a Staphylococcus-specific phage or a Staphylococcus aureus-specific phage. In some embodiments, the selected wild-type phage is T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or another naturally occurring phage having a genome that is 99, 98, 97, 96, 95, 94, 93, 92, 91 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, or 70% homologous to the phages disclosed above.

[0013] In some embodiments, making a homologous recombination plasmid / vector includes determining the native nucleotide sequence in the late region of the genome of the selected phage; annotating the genome and identifying the major capsid protein gene of the selected phage; designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence comprises a codon-optimized indicator gene; and incorporating the sequence designed for homologous recombination into a plasmid / vector. The step of designing a sequence can include inserting an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a phage late gene promoter and a ribosomal entry site. Thus, in some methods, the homologous recombination plasmid comprises an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a phage late gene promoter and a ribosomal entry site.

[0014] Some embodiments of the present application are compositions comprising recombinant indicator phages as described herein. For example, a composition can comprise one or more wild-type or genetically modified infectious agents (e.g., phages) and one or more indicator genes. In some embodiments, a composition can comprise a mixture of different indicator phages that can encode and express the same or different indicator proteins.

[0015] In some embodiments, the application includes a method for detecting a target microorganism in a sample, comprising the step of incubating the sample with a recombinant bacteriophage that infects the target microorganism, wherein the recombinant bacteriophage comprises an indicator gene inserted into a late gene region of the bacteriophage such that expression of the indicator gene during bacteriophage replication following host bacterial infection results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the target microorganism is present in the sample.

[0016] In some embodiments of the method for making a recombinant indicator bacteriophage, the wild-type bacteriophage is a Staphylococcus-specific bacteriophage and the target pathogenic bacteria is Staphylococcus. In some embodiments, isolating a particular clone of the recombinant bacteriophage includes a limiting dilution assay for isolating a clone that demonstrates expression of the indicator gene.

[0017] Other aspects of the application include a method for detecting bacteria (e.g., Staphylococcus) in a sample, comprising the steps of incubating the sample with a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Staphylococcus is present in the sample. In some embodiments, the application includes a method for detecting Staphylococcus aureus using a recombinant bacteriophage derived from Staphylococcus aureus. The sample can be a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

[0018] In some embodiments of the method for detecting bacteria, the sample is first incubated under conditions conducive to growth for an enrichment time of 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, or 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. In some embodiments, the total time to results is less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, or less than 6 hours. In some embodiments, the signal-to-background ratio of the detection of the indicator production is at least 2.0 or at least 2.5. In some embodiments, the method detects as few as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 of a particular bacteria in a standard size sample of the food safety industry.

[0019] Additional embodiments include systems and kits for detecting Staphylococcus, wherein the system or kit comprises a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage. In some embodiments, the system and kit can be used to detect S. aureus, wherein the system or kit comprises a recombinant bacteriophage derived from S. aureus. In some embodiments, the system and kit can be used to detect MRSA. Some embodiments further include a substrate for reacting with an indicator to detect a soluble protein product expressed by the recombinant bacteriophage. These systems or kits can include features described for the bacteriophages, compositions, and methods of the application. In other embodiments, the application includes a non-transitory computer readable medium for use with a method or system according to the application.

[0020] In another aspect, the application can include a method for selecting a treatment for a subject, comprising: (i) obtaining a biological sample from the subject; (ii) detecting a particular microbe or class of microbes in the biological sample using an indicator bacteriophage; and (iii) selecting a treatment based on the identification of the particular microbe detected in the biological sample.

[0021] In another aspect, the application can include a method for monitoring the efficacy of a treatment for a subject having a pathogenic medical condition, comprising: (i) obtaining a biological sample from the subject; (ii) detecting a particular microbe or class of microbes in the biological sample using an indicator bacteriophage; (iii) initiating a treatment for the subject; (iv) obtaining a second biological sample of the same type as the first biological sample from the subject; (v) detecting the particular microbe or class of microbes in the second biological sample using the indicator bacteriophage; and (vi) determining a decreasing, increasing, or stable level of the particular microbe or class of microbes in the subject based on the amounts detected in the first and second biological samples. BRIEF DESCRIPTION OF DRAWINGS

[0022] The application can be better understood by reference to the following non-limiting drawings.

[0023] Figure 1 Depicted are indicator bacteriophage constructs according to embodiments of the application, illustrating a genetic construct comprising a firefly luciferase gene and a T7 late promoter inserted into the late (class III) region of the bacteriophage. Also depicted are sequences comprising stop codons to prevent read-through and untranslated regions (UTRs) in all three reading frames.

[0024] Figure 2The genome of phage SEA1, a myovirus (related to T4 phage), is shown, which was obtained from the laboratory of Francisco Diez-Gonzalez and shares ~95% homology with the myovirus Salmonella phage S16. The gene 57A chaperone for tail fiber formation is located at the periphery of the late gene region, which consists of structural genes encoding virion proteins. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, as long as a late gene promoter and / or other similar control elements are used.

[0025] Figure 3 Two homologous recombination plasmid constructs carrying the luciferase gene of two different phages are shown, with ~500 bp of matching phage sequence upstream and downstream of the insertion site to facilitate homologous recombination. The luciferase is inserted into the pBAV1 k-T5-GFP plasmid backbone with an upstream untranslated region containing the phage late gene promoter and ribosome entry site. The luciferase is inserted into the pBAV1 k-T5-GFP plasmid backbone with an upstream untranslated region containing the phage late gene promoter and ribosome entry site. Recombinant phage plasmids are constructed to insert the luciferase into the late gene region, but kept at a distance from the major capsid protein (MCP) due to stability issues. The luciferase is inserted into the pBAV1 k-T5-GFP plasmid backbone with an upstream untranslated region containing the phage late gene promoter and ribosome entry site. Recombinant phage plasmids are constructed to insert the luciferase into the late gene region, but kept at a distance from the major capsid protein (MCP) due to stability issues.

[0026] Figure 4 A series of sequential infection and dilution steps are used to isolate recombinant phage from modifications of S. aureus phage, using plasmid constructs as shown in Figure 3 to identify recombinant phage expressing the indicator gene.

[0027] Figure 5 A "no-concentration assay" using modified phage to detect target bacteria is depicted according to embodiments of the application.

[0028] Figure 6 A "no-concentration assay" using modified phage to detect target bacteria is depicted according to embodiments of the application.

[0029] Figure 7 A "no-concentration assay" using modified phage to detect target bacteria is depicted according to embodiments of the application.

[0030] Figure 8A Hybrid Immuno-Phage (HIP) assay for detecting target bacteria using modified phage is depicted in accordance with embodiments of the application, wherein the microorganism is captured on the surface of an assay well using an antibody against the target microorganism prior to incubation with a recombinant infectious agent having an indicator gene. DETAILED DESCRIPTION

[0031] Disclosed herein are compositions, methods, and systems that demonstrate surprising sensitivity for detecting target microorganisms in test samples (e.g., biological samples, food samples, water samples, and clinical samples). Using genetically modified infectious agents, detection can be achieved in a shorter time frame than previously thought possible in assays performed without enrichment culture or, in some embodiments, with minimal incubation times in which microorganisms can potentially propagate. Also surprising is the success of using potentially high multiplicity of infection (MOI) or high concentration of plaque forming units (PFU) for incubation with test samples. Such high phage concentrations (PFU / mL) were previously thought to be detrimental in bacterial detection assays because they were said to cause "self lysis." However, high concentrations of phage facilitate the discovery, binding, and infection of low numbers of target cells.

[0032] Compositions, methods, systems, and kits of the present application can include infectious agents for detecting such microorganisms. In certain embodiments, the present application can include compositions comprising recombinant phage having an indicator gene inserted into the late gene region of the phage. In certain embodiments, expression of the indicator gene during phage replication following infection of the host bacteria results in production of a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene (i.e., class ΠΙ) region of the phage. The phage can be derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or a staphylococcus-specific phage, or another wild-type or engineered phage.

[0033] In some aspects, the present application includes methods for detecting a target microorganism. The methods can use an infectious agent for detecting the target microorganism. For example, in certain embodiments, the target microorganism is a bacterium and the infectious agent is a bacteriophage. Thus, in certain embodiments, the methods can include detecting a target microorganism in a sample by incubating the sample with a recombinant bacteriophage that infects the target microorganism. In certain embodiments, the recombinant bacteriophage comprises an indicator gene. In certain embodiments, the indicator gene can be inserted into a late gene region of the bacteriophage such that expression of the indicator gene during bacteriophage replication following infection of a host bacterium results in production of a soluble indicator protein product. The methods can include detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the target bacterium is present in the sample. In some embodiments, the indicator protein is soluble.

[0034] In certain embodiments, the present application can include systems. The systems can contain at least some of the compositions of the present application. In addition, the systems can include at least some of the components for performing the methods. In certain embodiments, the systems are formulated as kits. Thus, in certain embodiments, the present application can include systems for rapid detection of a target microorganism in a sample, comprising: a component for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent comprises an indicator moiety; and a component for detecting the indicator moiety. In yet other embodiments, the present application includes software for use with the methods or systems.

[0035] Thus, some embodiments of the present application address the need for amplifying a detectable signal indicative of the presence of a bacterium by using a bacteriophage-based method. In certain embodiments, as few as a single bacterium is detected. The principles applied herein can be applied to detect a variety of microorganisms. Due to the numerous binding sites on the surface of a microorganism for an infectious agent, the ability to produce one hundred or more infectious agent progeny during infection, and the potential for high level expression of an encoded indicator moiety, the infectious agent or indicator moiety can be more easily detected than the microorganism itself. In this way, embodiments of the present application can achieve great signal amplification from even a single infected bacterium.

[0036] Various aspects of the present application take advantage of the high specificity of a binding agent (e.g., a binding component of an infectious agent) that can bind to a particular microorganism as a means of detecting and / or quantifying the particular microorganism in a sample. In some embodiments, the present application takes advantage of the high specificity of an infectious agent (e.g., a bacteriophage).

[0037] In some embodiments, detection is achieved by an indicator moiety associated with a binding agent specific for a target microorganism. For example, an infectious agent can comprise an indicator moiety, such as a gene encoding a soluble indicator. In some embodiments, the indicator can be encoded by the infectious agent (e.g., a bacteriophage), and the bacteriophage is designated an indicator bacteriophage.

[0038] Some embodiments of the application disclosed and described herein utilize the discovery that a single microorganism is capable of binding a specific recognition agent, such as a bacteriophage. Upon infection and replication of the bacteriophage, the progeny bacteriophage can be detected by an indicator moiety expressed during replication of the bacteriophage. This principle, based on the specific recognition of a microorganism surface receptor, allows amplification of the indicator signal from one or a few cells. For example, by exposing even a single bacterial cell to multiple bacteriophages, followed by allowing amplification of the bacteriophage and high level expression of the encoded indicator gene product during replication, the indicator signal amplification allows detection of a single bacterium.

[0039] Embodiments of the methods and systems of the application can be applied to detect and quantify a variety of microorganisms (e.g., bacteria, fungi, yeast) in a variety of environments, including but not limited to detecting pathogens from food samples, water samples, clinical samples, and commercial samples. In some embodiments, the presence of microorganisms in a clinical sample can be analyzed. The methods of the application can provide high detection sensitivity and specificity rapidly, and do not require traditional biological enrichment (e.g., enrichment culture), which is a surprising aspect as all available methods require culture. In some embodiments, detection is possible within a single replication cycle of the bacteriophage, which is unexpected.

[0040] Definitions

[0041] Unless otherwise defined herein, scientific and technical terms used in connection with the present application 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, unless otherwise indicated, generally in accordance with standard procedures, as discussed in various general and more specific references that are cited throughout this specification. Enzymatic reactions and purification techniques are performed according to manufacturer’s instructions, as commonly accomplished in the art or as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is that well-known and commonly used in the art.

[0042] The following terms are to be understood to have the following meanings unless otherwise indicated:

[0043] As used herein, the terms "a," "an," and "the" can refer to one or more, unless otherwise specifically indicated.

[0044] The use of the term "or" means "and / or" unless strictly indicated otherwise, even though "and / or" definitions are supported by the disclosure. As used herein, "another" can mean at least a second or more.

[0045] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the samples being tested.

[0046] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface upon which a biomolecule can be bound. For example, a solid support can be an assay well (i.e., e.g., a microtiter plate or multiwell plate), or a solid support can be a location on a filter, array, or mobility support (e.g., a bead or a membrane (e.g., a filter plate or a lateral flow strip)).

[0047] The term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) target molecule. The interaction can be noncovalent, e.g., as a result of a hydrogen bond, van der Waals interaction, or electrostatic or hydrophobic interaction, or it can be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or noncovalently bound) with a solid support.

[0048] "Analyte" as used herein refers to a molecule, compound, or cell that is measured. In certain embodiments, a target analyte can interact with a binding agent. The term "analyte" can refer to a target protein or peptide as described herein. An analyte can be an agonist, antagonist, or modulator. Alternatively, an analyte can have no biological effect. An analyte can include a small molecule, a sugar, an oligosaccharide, a lipid, a peptide, a peptidomimetic, an organic compound, etc.

[0049] The term "detectable moiety" or "detectable biomolecule" or "reporter molecule" or "indicator" or "indicating moiety" refers to a molecule that can be measured in a quantitative assay. For example, an indicating moiety can comprise an enzyme that can be used to convert a substrate to a measurable product. An indicating moiety can be an enzyme that catalyzes a reaction that generates a bioluminescent emission (e.g., luciferase). Alternatively, an indicating moiety can be a radioisotope that can be quantified. Alternatively, an indicating moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0050] 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 (e.g., mycobacteriophages (e.g., for TB and paraTB), mycophages (e.g., for fungi), mycoplasmophages) and any other term that refers to viruses that can invade and utilize living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms for their own replication. Here, "microscopic" means having a largest dimension of 1 millimeter or less. Phages are viruses that have evolved in nature to use bacteria as a means of replicating themselves. Bacteriophages do this by attaching themselves to a bacterium and injecting their DNA (or RNA) into the bacterium and inducing it to replicate the bacteriophage hundreds or even thousands of times. This is called phage amplification.

[0051] As used herein, "late gene region" refers to the region of the viral genome that is transcribed late in the viral life cycle. The late gene region typically includes the genes that are expressed in the greatest amount (e.g., structural proteins that are assembled into phage particles). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, late genes (synonymous with class III) are transcribed in phage T7, e.g., from 8 minutes post-infection until lysis, class I (e.g., RNA polymerase) is early from 4-8 minutes, and class II is from 6-15 minutes, so there is an overlap in time between II and III. A late promoter is a promoter that is naturally located in such a late gene region and is active therein.

[0052] As used herein, "enrichment culture" refers to traditional culturing, e.g., incubation in a medium that is conducive to the propagation of microorganisms, and is not to be confused with other possible uses of the word "enrichment" (e.g., enrichment by removing the liquid component of a sample to concentrate the microorganisms contained therein, or other forms of enrichment that do not include traditional promotion of microorganism propagation). In some embodiments of the methods described herein, very short periods of enrichment culture can be employed, but it is not necessary and if used it is much shorter in time compared to traditional enrichment culture.

[0053] As used herein, "recombinant" refers to genetic (i.e., nucleic acid) modifications typically made in the laboratory to bring together genetic material that would not otherwise be found together. The term is used interchangeably herein with the term "modified."

[0054] As used herein, "RLU" refers to relative light units measured by a luminometer (e.g., 96) or similar instrument that detects light. For example, detection of the reaction between luciferase and an appropriate substrate (e.g., and ) is typically reported as detected RLU.

[0055] "Time to result" as used herein refers to the total amount of time from the beginning of sample incubation to the generation of a result. Time to result does not include any confirmatory testing time. Data collection can be performed at any time after a result is generated.

[0056] Sample

[0057] Various embodiments of the methods and systems of the present application can allow for rapid detection and quantification of microorganisms in a sample. For example, methods according to the present application can be performed in a reduced time and with excellent results.

[0058] Microorganisms detected by the methods and systems of the present application include natural, commercial, medical, or veterinary pathogens. Such pathogens include gram-negative bacteria, gram-positive bacteria, and mycoplasma. Any microorganism for which an appropriate specific infectious agent has been identified can be detected by the methods of the present application. Those skilled in the art will appreciate that the application of the methods of the present application is not limited except by the availability of the necessary specific infectious agent / microorganism pair.

[0059] Bacterial cells detectable by the present application include, but are not limited to, bacterial cells that are food or water-borne pathogens. Bacterial cells detectable by the present application include, but are not limited to, all species of Salmonella, all strains of Escherichia coli, Cronobacter, Staphylococcus, all species of Listeria, including but not limited to L. monocytogenes, and all species of Campylobacter. Bacterial cells detectable by the present application include, but are not limited to, bacterial cells that are medically or veterinarily significant pathogens. Such pathogens include, but are not limited to, Bacillus spp., Bordetella pertussis, Campylobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens, Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Shigella sonnei, Staphylococcus aureus, and Streptococcus spp. In some embodiments, bacterial cells detectable by the present application include antibiotic resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)).

[0060] The sample can be an environmental or food sample or a water sample. Some embodiments can include a medical or veterinary sample. The sample can be a liquid, solid, or semi-solid. The sample can be a swab of a solid surface. The sample can include an environmental material such as a water sample, or a filter from an air sample, or an aerosol sample from a cyclone collector. The sample can be a vegetable, meat, fish, poultry, peanut butter, processed food, baby formula, powdered milk, tea, starch, egg, milk, cheese, or other dairy product. Medical or veterinary samples include, but are not limited to, blood, sputum, cerebral spinal fluid, and fecal samples, and different types of swabs.

[0061] In some embodiments, samples can be used directly in the detection methods of the present invention without preparation, concentration, or dilution. For example, liquid samples, including but not limited to milk and juice, can be directly measured. Samples can be diluted or suspended in a solution, which may include, but is not limited to, buffer solutions or bacterial culture media. Solid or semi-solid samples can be suspended in a liquid by chopping, mixing, or softening the solid. Samples should be maintained within a pH range that promotes phage attachment to host bacterial cells. Samples should also contain appropriate concentrations of divalent and monovalent cations, including but not limited to Na+. + Mg 2+ and Ca2 + Preferably, the sample is maintained at a temperature that preserves the viability of any pathogen cells contained within the sample.

[0062] Preferably, in the detection assay, the sample is maintained at a temperature that preserves the viability of any pathogen cells present in the sample. In the step where the phage attaches to bacterial cells, the sample is preferably maintained at a temperature conducive to phage attachment. In the step where the phage replicates or lyses infected cells within infected bacterial cells, the sample is preferably maintained at a temperature that promotes phage replication and host lysis. Such a temperature is at least about 25 degrees Celsius (C), more preferably not higher than about 45 degrees Celsius, and most preferably about 37 degrees Celsius. It is also preferable that the sample undergoes gentle mixing or shaking during phage attachment, replication, and cell lysis.

[0063] The assay can include a variety of suitable control samples. For example, a control sample that does not contain bacteriophages or a control sample that contains bacteriophages but does not contain bacteria can be used as a control for background signal levels.

[0064] Indicator bacteriophage

[0065] As described in more detail herein, the compositions, methods, systems, and kits of the present invention can include those for detecting infectious agents in pathogenic microorganisms. In some embodiments, the present invention includes recombinant indicator phages, wherein the phage genome is genetically modified to include an indicator gene or reporter gene. In some embodiments, the present invention can include compositions comprising recombinant phages having an indicator gene inserted into the genome of the phage.

[0066] The indicator phage can include a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during phage replication following infection of the host bacteria results in a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene region of the phage. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. The late gene region can be a class III gene region and can include the genes for the major capsid proteins.

[0067] Some embodiments include designing (and optionally preparing) a sequence for homologous recombination downstream of the major capsid protein gene. Other embodiments include designing (and optionally preparing) a sequence for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequence comprises a codon-optimized reporter gene preceded by an untranslated region. The untranslated region can comprise a phage late gene promoter and a ribosome entry site.

[0068] In some embodiments, the indicator phage is derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or a Staphylococcus-specific phage, or another phage having a genome that is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or a Staphylococcus-specific phage. In some embodiments, the indicator phage is derived from a phage that is highly specific for a particular pathogenic microorganism. Genetic modifications can avoid the deletion of wild-type genes and thus the modified phage can remain more similar to the wild-type infectious agent compared to many commercially available phages. Environmentally derived phages can be more specific to bacteria found in the environment and thus genetically different from commercially available phages.

[0069] Furthermore, phage genes that are considered non-essential can have unrecognized functions. For example, apparently non-essential genes can have important functions in elevating the burst size, such as fine cleavage, fitting, or trimming functions in assembly. Thus, deletion of the genes to insert the indicator can be detrimental. Most phages can package several percent more DNA than their native genome. With this in mind, smaller indicator genes can be a more suitable choice for modifying phages, especially phages with smaller genomes. OpLuc and Proteins are only about 20 kDa (encoding about 500-600 bp), while FLuc is about 62 kDa (encoding about 1,700 bp). For comparison, the genome of T7 is about 40 kbp, while the T4 genome is about 170 kbp, and the genome of a Staphylococcus-specific phage is about 157 kbp. Furthermore, the reporter gene should not be expressed endogenously by the bacteria (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be easy to detect in a timely manner. Promega's is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, the Oplophorus luciferase is used in combination with Promega's (furimazine) can provide a robust signal with low background.

[0070] In some bacteriophage embodiments, the indicator gene can be inserted into an untranslated region to avoid disruption of functional genes, leaving the wild-type phage genes intact, which can result in greater fitness when infecting non-laboratory bacterial strains. Additionally, inclusion of a stop codon in all three reading frames can help increase expression by reducing read-through (also known as leaky expression). This strategy can also eliminate the possibility of producing a fusion protein at low levels, which would appear as background signal that cannot be separated from the phage (e.g., luciferase).

[0071] The indicator gene can express a variety of biomolecules. An indicator gene is a gene that expresses a detectable product or produces 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 Renilla luciferase, or a modified luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as

[0072] Accordingly, in some embodiments, the present disclosure includes genetically modified bacteriophages that include a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. Use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is expressed not only at high levels like viral coat proteins, but also is not shut off like endogenous bacterial genes or even early viral genes.

[0073] ​In some embodiments, the late promoter is a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 promoter, or another phage promoter similar to that found in a selected wild-type phage (i.e., not genetically modified). The late gene region can be a class III gene region, and the phage can be derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus or Staphylococcus aureus specific phage, or another natural phage with a genome at least 70, 75, 80, 85, 90, or 95% homologous to T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus or Staphylococcus aureus specific phage.

[0074] Genetic modifications to the infectious agent can include insertion, deletion, or substitution of a small fragment of a nucleic acid, a substantial portion of a gene, or an entire gene. In some embodiments, the inserted or substituted nucleic acid comprises a non-native sequence. A non-native indicator gene can be inserted into the phage genome under the control of a phage promoter. In some embodiments, the non-native indicator gene is not part of a fusion protein. That is, in some embodiments, the genetic modification can be configured such that the indicator protein product does not comprise a polypeptide of the wild-type phage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present application includes a method for detecting a bacterium of interest comprising the step of incubating a test sample with such a recombinant phage.

[0075] In some embodiments, expression of the indicator gene in progeny phage following infection of a host bacterium results in 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. Unlike systems that employ a fusion of a detection moiety to a capsid protein (i.e., a fusion protein), some embodiments of the present application express a soluble indicator or reporter molecule (e.g., soluble luciferase). In some embodiments, the indicator or reporter molecule is desirably free of phage structure. That is, the indicator or reporter molecule is not attached to phage structure. Thus, the indicator or reporter gene is not fused to other genes in the recombinant phage genome. For some embodiments, this can greatly increase the sensitivity of the assay (down to a single bacterium) and simplify the assay, allowing the assay to be completed in less than an hour, as opposed to hours required due to additional purification steps for constructs that produce a detectable fusion protein. Moreover, activity of a fusion protein can be lower than a soluble protein due to, for example, protein folding constraints that can alter the conformation of the active site of the enzyme or proximity to substrates.

[0076] Moreover, by definition, a fusion protein limits the number of moieties attached to a protein subunit in a phage. For example, using a commercially available system designed to be used as a fusion protein platform will result in about 415 copies of the fusion moiety, corresponding to about 415 copies of the gene 10B capsid protein in each T7 phage particle. Without this limitation, an infected bacterium can be expected to express more copies of a detection moiety (e.g., luciferase) than can fit on a phage. Additionally, large fusion proteins, such as capsid-luciferase fusion proteins, can inhibit assembly of phage particles, resulting in fewer phage progeny. Thus, a soluble, non-fusion indicator gene product can be preferred.

[0077] In some embodiments, the indicator phage encodes a reporter molecule, such as a detectable enzyme. The indicator gene product can produce light and / or can be detected by a color change. Various suitable enzymes are commercially available, such as, for example, alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can be used 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 produce a detectable signal can also be suitable indicator moieties.

[0078] In some embodiments, the use of soluble detection moieties eliminates the need to remove contaminating parental phage from lysates of infected sample cells. In the case of using a fusion protein system, any phage used to infect sample cells will have detection moieties attached and will not be distinguishable from progeny phage that also contain detection moieties. Since detection of sample bacteria relies on detection of newly created (de novo synthesized) detection moieties, use of a fusion construct requires additional steps to separate the newly created (progeny phage) moieties from the older (parental) moieties. This can be accomplished by multiple washes of infected cells before the phage life cycle is complete, by inactivating excess parental phage after infection by physical or chemical means, and / or by chemically modifying parental phage with a binding moiety (e.g. biotin) that can then be bound and separated (e.g. by streptavidin coated agarose gel beads). However, even with all these attempts, parental phage remain when high concentrations of parental phage are used to ensure infection of a small number of sample cells, producing background signal that can mask detection of signal from infected cell progeny phage.

[0079] In contrast, with the use of soluble detection moieties expressed in some embodiments of the application, there is no need to purify parental phage from the final lysate, since the parental phage do not have any detection moieties attached. Thus, any detection moieties present after infection must have been created de novo, indicating the presence of one or more bacteria that were infected. To take advantage of this benefit, production and preparation of parental phage can include purifying the phage from any free detection moieties produced during parental phage production in bacterial culture. Standard phage purification techniques can be employed to purify some embodiments of the phage according to the application, such as sucrose density gradient centrifugation, cesium chloride isopycnic centrifugation, HPLC, size exclusion chromatography, and dialysis or derivatization techniques (e.g., concentrators of the Amicon brand - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as part of the preparation of recombinant phage of the application to separate parental phage particles from contaminating luciferase protein produced when the phage are propagated in bacterial hosts. In this way, parental recombinant phage of the application are substantially free of any luciferase produced during production in bacteria. Removal of residual luciferase present in stocks of the phage can significantly reduce background signal when the recombinant phage are incubated with test samples.

[0080] In some embodiments of the modified bacteriophage, the late promoter (class III promoter, e.g., from T7, T4, or ViI) has high affinity for the RNA polymerase of the same bacteriophage that transcribes the genes for the structural proteins used to assemble into the bacteriophage particle. These proteins are the most abundant proteins produced by the bacteriophage, as each bacteriophage particle contains tens or hundreds of copies of these molecules. Use of a viral late promoter can ensure optimal high-level expression of the luciferase detection moiety. Use of a late viral promoter derived from, specific to, or active under the original wild-type bacteriophage from which the indicator bacteriophage is derived (e.g., a T4, T7, or ViI late promoter with a T4-, T7-, or ViI-based system) can further ensure optimal expression of the detection moiety. In some cases, use of standard bacterial (non-viral / non-bacteriophage) promoters can be disadvantageous for expression, as these promoters are typically down-regulated during bacteriophage infection (to allow the bacteriophage to preferentially utilize bacterial resources for bacteriophage protein production). Thus, in some embodiments, it is preferred to engineer the bacteriophage to encode and express the soluble (free) indicator moiety at a location in the genome that does not limit expression to the number of subunits of the bacteriophage structural component.

[0081] The compositions of the present application can comprise one or more wild-type or genetically modified infectious agents (e.g., bacteriophage) and one or more indicator genes. In some embodiments, the compositions can comprise a mixture of different indicator bacteriophage that can encode and express the same or different indicator proteins. In some embodiments, the mixture of bacteriophage comprises at least two different types of recombinant bacteriophage.

[0082] Methods of making indicator bacteriophage

[0083] Embodiments of the methods for making indicator bacteriophage begin with the selection of a wild-type bacteriophage for genetic modification. Some bacteriophage have a high degree of specificity for a target bacterium. This provides an opportunity for high-specificity detection.

[0084] Thus, the methods of the present application take advantage of the high specificity of the binding agent, which associates with the infectious agent, recognizes and binds to a specific target microorganism as a means to amplify the signal and thereby detect low levels of microorganisms (e.g., single microorganisms) present in a sample. For example, the infectious agent (e.g., bacteriophage) specifically recognizes a surface receptor of a particular microorganism and thus specifically infects these microorganisms. Thus, these infectious agents can be suitable binding agents for targeting the target microorganism.

[0085] Various infectious agents can be used. In alternative embodiments, phages, bacteriophages, mycobacteriophages (e.g., for TB and paraTB), mycophages (e.g., for fungi), mycoplasmophages, and any other viruses that can invade live bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms can be used to target the microorganism of interest. For example, in one embodiment, where the microorganism of interest is a bacterium, the infectious agent can include a bacteriophage. For example, bacteriophages of E. coli that have been well studied include T1, T2, T3, T4, T5, T7, and lambda; other E. coli bacteriophages available in the ATCC depository include, for example, phiX174, S13, Ox6, MS2, phiVl, fd, PR772, and ZIK1. As discussed herein, a bacteriophage can replicate inside a bacterium to generate hundreds of progeny bacteriophage. Detection of the product of an indicator gene inserted into the genome of the bacteriophage can be used as a measure of the amount of bacteria in a sample.

[0086] Some embodiments of the present application utilize the binding specificity and high level of gene expression capability of recombinant phages to rapidly and sensitively target and facilitate detection of the bacteria of interest. In some embodiments, a phage specific for Staphylococcus or Staphylococcus aureus is genetically modified to include a reporter gene. In some embodiments, the late gene region of the phage is genetically modified to include a reporter gene. In some embodiments, the reporter gene is located downstream of the major capsid genes. In other embodiments, the reporter gene is located upstream of the major capsid genes.

[0087] Some embodiments of a method for making a recombinant indicator phage include selecting a wild-type phage that specifically infects the target pathogenic bacteria; making a homologous recombination plasmid / vector containing an indicator gene; transforming the target pathogenic bacteria with the homologous recombination plasmid / vector; infecting the transformed target pathogenic bacteria with the selected wild-type phage, thereby allowing homologous recombination between the plasmid / vector and the phage genome; and isolating a specific clone of the recombinant phage.

[0088] Various methods for designing and making a homologous recombination plasmid are known. Various methods for transforming bacteria with a plasmid are known, including heat shock, F pilus-mediated bacterial conjugation, electroporation, and other methods. Various methods for isolating a specific clone after homologous recombination are also known. Some method embodiments described herein utilize specific strategies.

[0089] Accordingly, some embodiments of the method for making an indicator bacteriophage include the following steps: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; determining the native sequence in the late region of the genome of the selected bacteriophage; annotating the genome and identifying the major capsid protein gene of the selected bacteriophage; designing a sequence adjacent to the major capsid protein gene for homologous recombination, wherein the sequence comprises a codon-optimized reporter gene; incorporating the sequence designed for homologous recombination into a plasmid / vector; transforming the plasmid / vector into the target pathogenic bacterium; selecting the transformed bacteria; infecting the transformed bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination between the plasmid and the bacteriophage genome; determining the titer of the resulting recombinant bacteriophage lysate; and performing a limiting dilution assay to enrich and isolate the recombinant bacteriophage. Some embodiments include further repeating the limiting dilution and titering steps as needed after the first limiting dilution assay, as needed until a detectable fraction of the recombinant bacteriophage is represented in the mixture. For example, in some embodiments, the limiting dilution and titering steps can be repeated until at least 1 / 30 of the bacteriophage in the mixture is recombinant, prior to isolating a particular clone of the recombinant bacteriophage. In some embodiments, a 1:30 ratio of recombinant: wild-type is expected, with an average of 3.2 transduction units (TU) produced per 96 plaques (e.g., in a 96-well plate). The initial ratio of recombinant to wild-type bacteriophage can be determined by performing a limiting dilution assay based on TCID50 (tissue culture infective dose 50%), such as previously described in U.S. Patent Application No. 15 / 409,258. By the Poisson distribution, a 1:30 ratio yields a 96% chance of observing at least one TU somewhere in a 96-well.

[0090] Figure 1 A schematic depicting the genomic structure of a recombinant bacteriophage (indicator bacteriophage 415-Luc) of the present invention. For the Figure 1 embodiment depicted in FIG. 1, the detection moiety is encoded by a firefly luciferase gene 100 inserted within the late (class III) gene region 110, which is expressed late in the viral life cycle. Late genes are typically expressed at higher levels than other bacteriophage genes because they encode structural proteins. Accordingly, in the depicted embodiment of the recombinant bacteriophage, the indicator gene (i.e., firefly luciferase) is inserted into the late gene region, just after gene 10B (major capsid protein), and is a construct comprising the firefly luciferase gene 100. Figure 1 The construct depicted in FIG. 1 is designed to include a stop codon 120 in all three reading frames to ensure that luciferase is not incorporated into the gene 10B product. Also as Figure 1 depicted in FIG. 1, the construct is designed to include a stop codon 120 in all three reading frames to ensure that luciferase is not incorporated into the gene 10B product. Also as Figure 1The constructed organism described may contain a shared T7 late promoter 130 to drive the transcription and expression of the luciferase gene. The construct may also contain a complex untranslated region synthesized from several T7 UTR 140s. This construct ensures the production of soluble firefly luciferase, making expression not limited to the number of capsid proteins inherent in phage display systems.

[0091] As described herein, in some embodiments, infectious agents already isolated from the environment may be preferably used to generate the infectious agents of the present invention. In this way, infectious agents specific to naturally derived microorganisms can be generated.

[0092] For example, Figure 2 The genome of phage SEA1 is shown. SEA1 is a native phage with approximately 95% sequence homology to T4-associated myotail virus phage S16. SEA1 phage was obtained from Francisco Martinez's laboratory and its whole genome was sequenced using Illumina MiSeq with de novo sequence assembly. As discussed in the examples, the major capsid protein 220 and various other structural genes are composed of structural genes within the late gene region 210, which encode viral particle proteins. Gene 57A 230, encoding chaperone proteins to form long-tailed filaments, is located at the boundary of the late gene region. Because these viral particle proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided that a late gene promoter and / or other similar control elements are used.

[0093] Numerous known methods and commercial products are available for plasmid preparation. For example, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques can be combined to prepare plasmids. Synthetic plasmids are also commercially available (e.g., GeneWiz). Glusomes can also be utilized, or the CRISPR / CAS9 system can be used to selectively edit the phage genome. Some embodiments of methods for preparing recombinant indicator phages involve designing plasmids that can be readily recombined with the wild-type phage genome to generate a recombinant genome. When designing plasmids, some embodiments include adding codon-optimized reporter genes, such as luciferase genes. Some embodiments also include adding elements to the upstream untranslated region. For example, when designing plasmids for recombining with phage genomes specific to Staphylococcus or Staphylococcus aureus, the sequence encoding the C-terminus of the gp23 / major capsid protein can be combined with… An upstream untranslated region is added between the start codon of the reporter gene. The untranslated region can comprise a promoter, for example, a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 promoter. The untranslated region can also comprise a ribosomal entry / binding site (RBS), also known as a "Shine-Dalgarno sequence" with bacterial systems. One or both of these elements or other untranslated elements can be embedded within a short upstream untranslated region composed of random sequence containing approximately the same GC content as the rest of the phage genome. The random region should not contain an ATG sequence as it would serve as a start codon.

[0094] The compositions of the present application can comprise various infectious agents and / or indicator genes. For example, Figure 3 Two homologous recombination plasmid constructs carrying luciferase genes for two different phages are shown, with approximately 500 bp of matching phage sequence upstream and downstream of the insertion site to facilitate homologous recombination. The The luciferase is inserted into the pBAV1k-T5-GFP plasmid backbone with an upstream untranslated region containing a phage late gene promoter and ribosomal entry site. S. aureus phage recombination plasmids are constructed to insert into the late gene region, but kept at a distance from the major capsid protein (MCP) due to stability issues.

[0095] The major capsid protein fragment 416-915 is part of the structural gene encoding virion proteins. Because these virion proteins are expressed at high levels, any gene inserted into this region can be expected to have similar expression levels as long as a late gene promoter and / or other similar control elements are used.

[0096] In some embodiments, the indicator phage according to the present application comprises a Staphylococcus-specific phage genetically engineered to comprise a reporter gene, for example a luciferase gene. For example, the indicator phage can be a Staphylococcus-specific phage, wherein the genome comprises the sequence of the gene. The recombinant Staphylococcus-specific NanoLuc phage genome can further comprise a promoter, for example, a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus-specific, ViI, or another late promoter. In some embodiments, the Staphylococcus-specific phage is a Staphylococcus aureus-specific phage.

[0097] Thus, in embodiments of the recombinant phage generated by recombination, the indicator gene (i.e. ) insertion into the late gene region, immediately downstream of the gene encoding the major coat protein, and thus creating a recombinant phage genome comprising the luciferase gene. The construct can additionally comprise the consensus promoter of T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, staphylococcus specific phage, ViI or another late promoter or another suitable promoter to drive transcription and expression of the luciferase gene. The construct can also comprise a composite untranslated region synthesized from several UTRs. This construct ensures the production of soluble luciferase, so that expression is not limited to the amount of coat protein inherent in the phage display system.

[0098] Figure 4 depicts isolation of recombinant phage from a mixture of wild type and recombinant phage resulting from homologous recombination.

[0099] In a first step 402, S. aureus bacteria transformed with a homologous recombination plasmid are infected with S. aureus phage K to produce progeny phage 434 with a mixture of parent and recombinant phage in a ratio of approximately 186 wild type to 1 recombinant phage. The resulting mixture of recombinant phage is diluted 404 into 96-well plates 406 to result in an average of 5 recombinant transduction units (TU) (9.3 PFU / well) per plate. The 96-well plates are assayed for luciferase activity to identify wells 436 containing recombinant phage as compared to wells 440 containing wild type phage. Bacteria 438 are added 408; for example, each well can contain approximately 50 μL of turbid S. aureus culture. This allows the phage to replicate and produce luciferase 442. After incubation at 37°C for 2 hours as shown at 410, the wells can be screened for the presence of luciferase 442. Any positive well can have been inoculated with a single recombinant phage, and at this stage the mixture can contain a ratio of approximately 9.3 wild type phage: 1 recombinant, enriched from the initial 186: 1 ratio. In one embodiment, one of five wells is found to contain soluble luciferase by the luciferase assay, containing phage in a ratio of approximately 2.4: 1 recombinant to total. If necessary (i.e., if the ratio of recombinant to total is less than 1:30), progeny from this enrichment can undergo additional one or more limiting dilution assays 414 to increase the ratio and determine the actual concentration of recombinant phage transduction units. For example, if the ratio is 1:384 recombinant to PFU, approximately 5 recombinant TU and 1920 contaminating total phage (5 x 384 = 1920), each 96-well plate 416 can be aliquoted 414 from the previous positive well, resulting in a second dilution assay plate inoculated with approximately 20 mostly wild type phage per well (1920 PFU / 96 well = 20 PFU / well) 420. Any positive luciferase well can have been inoculated with a single recombinant and 19 wild type phage. These wells can be analyzed for the presence of luciferase 442.

[0100] After addition of bacteria and incubation (e.g., 2 hours at 37°C) 418, soluble luciferase and phage are present in approximately a 20: 1 ratio of total to recombinants 420. This ratio can be verified by TU50 titration for recombinants and plaque assay for total PFU. Finally, a plaque assay 422 can be performed to screen for recombinants expressing luciferase 446. A small number of individual (e.g., n = 48) plaques can be individually picked and screened in a third multi-well plate 426 for luciferase activity 436. In one embodiment, this method should ensure that enough plaques are screened such that, based on the known ratio of recombinants to total phage, about 3 recombinants are in the mixture of plaques to be screened. One plaque can be removed from the plate into each well of a 96-well plate 424 and a luciferase assay 426 performed to determine which wells contain phage exhibiting luciferase activity 442. Wells 428 showing luciferase activity represent pure recombinant phage 434, while wells 430 showing no luciferase activity represent pure wild-type phage 432.

[0101] Individual plaques can then be suspended in buffer (e.g., 100 μΐ TMS) or media and an aliquot (e.g., about 5 μΐ) added to a well containing a turbid S. aureus culture and assayed after incubation (e.g., about 45 minutes to 1 hour at 37°C). Positive wells are expected to contain pure recombinant phage culture. Certain embodiments can include additional rounds of plaque purification.

[0102] Thus, as Figure 4 As illustrated, recombinant phage generated by homologous recombination of a plasmid designed to recombine with wild-type phage genomes can be isolated from a mixture containing only 0.005% of the total phage genomes. After isolation, large-scale production can be performed to obtain a high-titer recombinant indicator phage stock suitable for use in S. aureus detection assays. Furthermore, cesium chloride isopycnic gradient centrifugation can be used to separate contaminating luciferin from phage particles to reduce background.

[0103] Methods of detecting microorganisms using an infectious agent

[0104] As described herein, in certain embodiments, the present application can include methods of detecting microorganisms using an infectious agent. The methods of the present application can be practiced in a variety of ways.

[0105] In one embodiment, the present application can include a method for detecting a target bacterium in a sample, comprising the steps of: incubating a bacteriophage that infects the target bacterium with the sample, wherein the bacteriophage comprises an indicator gene such that expression of the indicator gene during replication of the bacteriophage following infection of the target bacterium 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 the target bacterium is present in the sample.

[0106] In another embodiment, the present application can include a method for detecting a target antibiotic resistant bacterium in a sample, comprising the steps of: (i) incubating the sample with at least one antibiotic to allow enrichment of bacteria that are resistant to the antibiotic, (ii) incubating the enriched sample with a bacteriophage that infects the target bacterium, wherein the bacteriophage comprises an indicator gene such that expression of the indicator gene during replication of the bacteriophage following infection of the target bacterium results in production of a soluble indicator protein product; and (iii) detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the target antibiotic resistant bacterium is present in the sample.

[0107] In certain embodiments, the assay can be performed to utilize the general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments utilizing the recombinant bacteriophage of the present application (i.e., indicator bacteriophage) can allow for rapid detection of a specific bacterial strain with a total assay time of less than 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, or 20.0 hours, depending on the sample type, sample size, and assay format. For example, the amount of time required can be slightly less or more depending on the strain of bacteriophage and the strain of bacteria to be detected in the assay, the type and size of 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.

[0108] Figure 5A strategy using an indicator phage that produces soluble luciferase is shown according to embodiments of the application. In this method, a phage (e.g., T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 phage) can be engineered to express soluble luciferase during phage replication. Expression of luciferase is driven by a viral capsid promoter (e.g., phage T7 or T4 late promoter), resulting in high expression. The parental phage will not contain luciferase, so the luciferase detected in the assay must come from replication of progeny phage during infection of the bacterial cell. Thus, there is typically no need to separate the parental phage from the progeny phage.

[0109] In these experiments, at least a portion of the sample 500 containing bacteria 502 to be quantified is placed into a spin column filter and centrifuged to remove the LB broth, and a suitable multiplicity of phage 504 that has been genetically engineered to express soluble luciferase 503 is added. The infected cells can be incubated for a time sufficient for progeny phage replication and cell lysis to occur (e.g., 30-90 minutes at 37°C). The parental phage 504 and progeny phage 516 plus free luciferase 503 in the lysate can then be collected (e.g., by centrifugation), and the level of luciferase in the filtrate is quantified using a luminometer 518. Alternatively, a high-throughput method can be utilized, in which the bacterial sample is applied to a 96-well filter plate, and after all of the above-listed operations are performed, luciferase can be assayed directly in the original 96-well filter plate, without a final centrifugation step.

[0110] Figure 6 A filter plate assay using modified phage according to embodiments of the application to detect target bacteria is depicted. Briefly, a sample 616 including target bacteria 618 can be added to a well 602 of a multi-well filter plate 604 and spun 606 to concentrate the sample by removing liquid from the sample. Genetically modified phage 620 are added to the well and incubated with additional media added for a time 608 sufficient to adsorb, subsequently infect the target bacteria and proceed through the phage life cycle 610 (e.g., ~45 minutes). Finally, a luciferase substrate is added and reacts with any luciferase 624 present. The emission produced is measured in a luminometer 614 that detects luciferase activity 626.

[0111] In certain embodiments, the assay can be performed without concentrating the bacteria on or near a capture surface. Figure 7An "unconcentrated assay" for detecting target bacteria using modified phage according to embodiments of the application is illustrated by way of example. An aliquot of indicator phage 714 is dispensed into individual wells 702 of a multi-well plate 704, and then an aliquot of test sample containing bacteria 712 is added and incubated 706 (e.g., for 45 minutes at 37°C) for a period of time sufficient for the phage to replicate and generate soluble indicator 716 (e.g., luciferase). The plate wells 708 containing soluble indicator and phage can then be assayed 710 to measure indicator activity (e.g., luciferase assay) on a plate 718. In this embodiment, the test sample is not concentrated (e.g., by centrifugation), but is simply incubated directly with the indicator phage for a period of time and subsequently assayed for luciferase activity.

[0112] In some embodiments, a sample can be enriched prior to testing by incubation under conditions that promote growth. In such embodiments, the enrichment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours or more, depending on the sample type and size.

[0113] In some embodiments, the indicator phage comprises a detectable indicator moiety, and infection of a single pathogen cell (e.g., a bacterium) can be detected by an amplified signal generated via the indicator moiety. Thus, the method can comprise detecting the indicator moiety produced during phage replication, wherein detection of the indicator indicates that the target bacteria is present in the sample.

[0114] In one embodiment, the application can comprise a method for detecting a target bacteria in a sample, comprising the steps of: incubating the sample with a recombinant phage that infects the target bacteria, wherein the recombinant phage comprises an indicator gene inserted into a late gene region of the phage, such that expression of the indicator gene during phage replication following host bacterial infection 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 the target bacteria is present in the sample. In some embodiments, the amount of indicator moiety detected corresponds to the amount of target bacteria present in the sample.

[0115] As described in greater detail herein, the methods and systems of the present application can utilize a range of concentrations of the parental indicator phage to infect bacteria present in a sample. In some embodiments, the indicator phage is added to the sample at a concentration sufficient to rapidly find, bind to, and infect target bacteria (e.g., single cells) present in the sample at very low numbers. In some embodiments, the phage concentration can be sufficient to find, bind to, and infect the target bacteria in less than one hour. In other embodiments, these events can occur in less than two hours or less than three hours after the indicator phage is added to the sample. For example, in certain embodiments, the phage concentration used for the incubation step is greater than 1 x 1010PFU / mL, greater than 1 x 1011PFU / mL, or greater than 1 x 1012PFU / mL. 5 PFU / mL, greater than 1 x 1011PFU / mL, or greater than 1 x 1012PFU / mL. 6 PFU / mL, greater than 1 x 1011PFU / mL, or greater than 1 x 1012PFU / mL. 7 PFU / mL.

[0116] In certain embodiments, the recombinant infectious agent can be purified to be free of any residual indicator protein that can have been generated in the production of the infectious agent stock. Thus, in certain embodiments, the recombinant phage can be purified using a cesium chloride density gradient, or the like, prior to incubation with the sample. When the infectious agent is a phage, this purification can have the additional benefit of removing phage that do not have DNA (i.e., empty phage or "ghosts").

[0117] In some embodiments of the methods of the present application, the microorganism can be detected without the need for any separation or purification of the microorganism from the sample. For example, in certain embodiments, a sample containing one or several microorganisms of interest can be applied directly to an assay vessel, e.g., a spin column, a microtiter well, or a filter, and the assay is performed in the assay vessel. Various embodiments of such assays are disclosed herein.

[0118] An aliquot of the test sample can be dispensed directly into a well of a multi-well plate, the indicator phage added, and after a period of time sufficient for infection, a lysis buffer can be added along with an indicator moiety substrate (e.g., luciferase substrate for a luciferase indicator) and the detection of the indicator signal assayed. Some embodiments of this method can be performed on a filter plate. Some embodiments of this method can be performed with or without concentration of the sample prior to infection with the indicator phage.

[0119] For example, in many embodiments, a multi-well plate is used to perform the assay. The choice of plate (or any other vessel in which the detection can be performed) can affect the detection steps. For example, some plates can include a colored or white background, which can affect the detection of light emission. In general, white plates have higher sensitivity, but also produce higher background signals. Plates of other colors can produce lower background signals, but also have slightly lower sensitivity. Additionally, one cause of background signal is light leakage from one well to an adjacent well. There are plates that have white wells, but the rest of the plate is black. This allows for high signal inside the well, but prevents light leakage from well to well, and thus can reduce background. Thus, the choice of plate or other assay vessel can affect the sensitivity and background signal of the assay.

[0120] The methods of the application can include various other steps to increase sensitivity. For example, as discussed in more detail herein, the methods can include a step of washing the captured and infected bacteria after the addition of the bacteriophage, but before the incubation, to remove excess parental bacteriophage and / or reporter protein of luciferase or other contaminating bacteriophage preparations.

[0121] In some embodiments, the detection of the microorganism of interest can be accomplished without the need for culturing the sample as a means of increasing the population of the microorganism. For example, in certain embodiments, the total time required for detection is less than 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, 1.0 hour, 45 minutes, or less than 30 minutes. Minimizing the time to result is critical for food and environmental testing of pathogens.

[0122] In contrast to assays known in the art, the methods of the present application can detect a single microorganism. Thus, in certain embodiments, the methods can detect < 10 microbial cells present in a sample (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 microbes). For example, in certain embodiments, the recombinant bacteriophage is highly specific for Staphylococcus. In some embodiments, the Staphylococcus species is Staphylococcus aureus. In further embodiments, the Staphylococcus aureus can be methicillin resistant (MRSA). In one embodiment, the recombinant bacteriophage can distinguish Staphylococcus in the presence of over 100 other types of bacteria. In other embodiments, the recombinant bacteriophage can distinguish Staphylococcus aureus in the presence of over 100 other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a particular type in a sample. In certain embodiments, the recombinant bacteriophage detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 of a particular bacterium in a sample.

[0123] As described herein, in certain embodiments, the present application can include methods of using recombinant bacteriophage to detect resistance of a microorganism to an antibiotic or in another way to detect efficacy of an antibiotic on a microorganism. In another embodiment, the present application includes methods for selecting an antibiotic for treatment of an infection. Additionally, the methods can include methods for detecting antibiotic resistant bacteria in a sample. The methods of the present application can be embodied in a variety of ways.

[0124] The methods can include contacting a sample comprising a microorganism with an antibiotic and an infectious agent as described above. In some embodiments, the disclosure provides a method of determining an effective dose of an antibiotic in killing or inhibiting growth of a microorganism, comprising: (a) incubating each of one or more antibiotic solutions separately with one or more samples comprising a microorganism, wherein the concentration of the one or more antibiotic solutions is different and defines a range, (b) incubating the microorganism in the one or more samples with an infectious agent comprising an indicator gene, and wherein the infectious agent is specific to the microorganism of interest: and (c) detecting an indicator protein product produced by the infectious agent in the one or more samples, wherein detection of the indicator protein product in one or more of the plurality of samples indicates that the concentration of the one or more antibiotic solutions used to treat the one or more samples is ineffective, and lack of detection of the indicator protein indicates that the antibiotic is effective, thereby determining the effective dose of the antibiotic.

[0125] In some embodiments, the antibiotic and the infectious agent are added to the sample simultaneously, such that the sample is contacted with both the antibiotic and the infectious agent. In other embodiments, the antibiotic and the infectious agent are added sequentially, e.g., the sample is contacted with the antibiotic before the sample is contacted with the infectious agent. In certain embodiments, the method can include incubating the sample with the antibiotic for a period of time before the sample is contacted with the infectious agent. The incubation time can vary depending on the nature of the antibiotic and the microorganism, e.g., based on the doubling time of the microorganism. In some embodiments, the incubation time is less than 24 hours, less than 18 hours, less than 12 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. The incubation time of the microorganism with the infectious agent can also vary depending on the life cycle of the particular infectious agent, in some cases, the incubation time is less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. Microorganisms that are resistant to the antibiotic will survive and can multiply, and the infectious agent specific to the microorganism will replicate; conversely, microorganisms that are sensitive to the antibiotic will be killed and thus the infectious agent will not replicate. The infectious agent according to the method comprises an indicator moiety in an amount corresponding to the amount of microorganism present in the sample that has been treated with the antibiotic. Thus, positive detection of the indicator moiety indicates that the microorganism is resistant to the antibiotic.

[0126] In some embodiments, the method can be used to determine whether an antibiotic-resistant microorganism is present in a clinical sample. For example, the method can be used to determine whether a patient is infected with Staphylococcus aureus that is resistant or susceptible to a particular antibiotic. A clinical sample obtained from the patient can then be incubated with an antibiotic specific to Staphylococcus aureus. The sample can then be incubated with a recombinant bacteriophage specific to Staphylococcus aureus for a period of time. In samples with Staphylococcus aureus that is resistant to the antibiotic, detection of the indicator protein produced by the recombinant bacteriophage will be positive. In samples with Staphylococcus aureus that is susceptible to the antibiotic, detection of the indicator protein will be negative. In some embodiments, the method for detecting antibiotic resistance can be used to select an effective therapeutic agent to which the pathogenic bacteria is susceptible.

[0127] In certain embodiments, the total time required for detection is less than 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, or less than 1.0 hour. The total time required for detection will depend on the target bacteria, the type of bacteriophage, and the antibiotic being tested.

[0128] Optionally, the method further comprises lysing the microorganism prior to detecting the indicator moiety. Any solution capable of lysing the microorganism can be used. In some cases, the lysis buffer can contain a non-ionic detergent, a chelating agent, an enzyme, or a proprietary combination of various salts and reagents. Lysis buffers are also commercially available from Promega, Sigma-Aldrich, or Thermo-Fisher. Experiments have shown that in some embodiments, infected unlysed cells can be detected after the addition of luciferase substrate. It is hypothesized that luciferase can exit the cell and / or luciferase substrate can enter the cell without complete cell lysis. Thus, for embodiments utilizing a spin filter system, where only luciferase released into the lysate (and not luciferase still inside intact bacteria) is analyzed in the luminometer, lysis is required for detection. However, as described below, for embodiments utilizing a filter plate or 96-well plate and phage infected samples in solution or suspension, where both intact and lysed cells can be directly measured in the luminometer, lysis can not be necessary for detection. Thus, in some embodiments, the method of detecting antibiotic resistance does not involve lysing the microorganism.

[0129] A surprising aspect of embodiments of the assay is that the step of incubating the microorganism in the sample with the infectious agent only requires a sufficient amount of time for a single life cycle of the infectious agent (e.g., phage). It was previously thought that the amplification capability of phage required more time, such that the phage would replicate several cycles. According to some embodiments of the present application, a single replication of the indicator phage can be sufficient to facilitate sensitive and rapid detection. Another surprising aspect of embodiments of the assay is that a high concentration of phage (i.e., high MOI) used to infect the test sample has successfully enabled detection of very small amounts of antibiotic resistant target microorganism that have been treated with an antibiotic. Factors including the amount of phage lysis can influence the number of phage life cycles and thus the time required for detection. Phage with a large amount of lysis (approximately 100 PFU) can only require one cycle for detection, while phage with a smaller amount of lysis (e.g., 10 PFU) can require multiple phage cycles for detection. In some embodiments, the incubation of the phage with the test sample only requires a sufficient amount of time for a single phage life cycle. In other embodiments, the incubation of the phage with the test sample is for an amount of time greater than a single life cycle. The concentration of phage for the incubation step will vary depending on the type of phage used. In some embodiments, the concentration of phage for this incubation step is greater than 1.0 x 10 5 greater than 1.0 x 10 6 greater than 1.0 x 10 7 or greater than 1.0 x 10 8PFU / mL. The success of using such a high concentration of phage is surprising because such a large amount of phage has been associated with "autolysis" which immediately kills the target cell and thereby prevents the generation of a useful signal from the early phage assay. The purification of the phage stock described herein can help alleviate this problem (e.g., purification by CsCl2 density gradient ultracentrifugation) because in addition to removing any contaminating luciferase associated with the phage, this purification can also remove ghost particles (particles with missing DNA). Such ghost particles can lyse the bacterial cell by "autolysis", prematurely killing the cell and thereby preventing the generation of an indicator signal. Electron microscopy has demonstrated that crude recombinant phage lysates (i.e., prior to CsCl2 purification) can have more than 50% ghosts. These ghost particles can cause premature death of the microbe by the action of many phage particles that puncture the cell membrane. Thus, ghost particles can cause the problem noted previously where high PFU concentrations were reported to be detrimental.

[0130] Any indicator moiety as described in the present disclosure can be used to detect the viability of a microbe after antibiotic treatment, and thereby detect antibiotic resistance. In some embodiments, the indicator moiety associated with the infectious agent can be detected during or after replication of the infectious agent. For example, as described above, in some cases the indicator moiety can be a protein that emits an intrinsic signal, such as a fluorescent protein (e.g., green fluorescent protein or others). The indicator can generate light and / or can be detectable by a color change. In some embodiments, a luminometer can be used to detect the indicator (e.g., luciferase). However, other machines or devices can also be used. For example, a spectrophotometer, a CCD camera, or a CMOS camera can detect color changes and other light emissions.

[0131] In some embodiments, it can be desirable to optimize sensitivity by exposing the sample to the antibiotic for 5 minutes or more and detecting at various time points. For example, aliquots of the primary sample treated with the antibiotic can be taken at different time intervals (e.g., at 5 minutes, 10 minutes, or 15 minutes). Then, the samples from the different time intervals are infected with the phage and the indicator moiety is measured after addition of the substrate.

[0132] In some embodiments, detection of the signal is used to determine antibiotic resistance. In some embodiments, the signal produced by the sample is compared to an experimentally determined value. In other embodiments, the experimentally determined value is the signal produced by a control sample. In some embodiments, a control without microorganisms is used to determine a background threshold. In some embodiments, the experimentally determined value is a background threshold calculated from the average background signal plus 1-3 standard deviations of the average background signal or more. In some embodiments, the background threshold can be calculated from the average background signal plus 2 standard deviations of the average background signal. In other embodiments, the background threshold can be calculated from the average background signal multiplied by a certain factor (e.g., 2 or 3). Detection of a sample signal greater than the background threshold indicates the presence of one or more antibiotic resistant microorganisms in the sample. For example, the average background signal can be 250 RLU. The threshold background value can be calculated by multiplying the average background signal (e.g., 250) by 3 to calculate a value of 750 RLU. A sample of bacteria with a signal value greater than 750 RLU is determined to be positive for containing antibiotic resistant bacteria.

[0133] Alternatively, the experimentally determined value is the signal produced by a control sample. Assays can include various suitable control samples. For example, a sample containing no infectious agent specific for the microorganism or a sample containing the infectious agent but no microorganism can be assayed as a control for background signal levels. In some cases, a sample containing microorganisms that have not been treated with an antibiotic is assayed as a control for use of the infectious agent to determine antibiotic resistance.

[0134] In some embodiments, the sample signal is compared to a control signal to determine whether antibiotic resistant microorganisms are present in the sample. An unchanged signal detection compared to a control sample that was contacted with the infectious agent but not the antibiotic indicates that the microorganism is resistant to the antibiotic, and a reduced detection of a fraction compared to a control sample that was contacted with the infectious agent but not the antibiotic indicates that the microorganism is susceptible to the antibiotic. Unchanged detection refers to a detection signal from a sample that has been treated with the antibiotic and the infectious agent that is at least 80%, at least 90%, or at least 95% of the signal from a control sample that has not been treated with the antibiotic. Reduced detection refers to a detection signal from a sample that has been treated with the antibiotic and the infectious agent that is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or at least 30% of the signal from a control sample that has not been treated with the antibiotic.

[0135] Optionally, the sample comprising the microorganism of interest is an uncultured sample. Optionally, the infectious agent is a bacteriophage and comprises an indicator gene inserted into a late gene region of the bacteriophage such that expression of the indicator gene results in a soluble indicator protein product during bacteriophage replication following infection of the host bacteria. The features of each composition used in this method can also be used in a method of detecting antibiotic resistance of a microorganism of interest, as described above.

[0136] Also provided herein are methods of determining an effective dose of an antibiotic for killing a microorganism. In some embodiments, the antibiotic is effective to kill a Staphylococcus species. For example, the antibiotic can be cefoxitin, which is effective against most methicillin-sensitive Staphylococcus aureus (MSSA). Typically, one or more antibiotic solutions are prepared having different concentrations, such that the different concentrations of the solutions define a range. In some cases, the concentration ratio range of the lowest concentrated antibiotic solution to the highest concentrated antibiotic solution is 1 :2 to 1 :50, e.g., 1 :5 to 1 :30, or 1 :10 to 1 :20. In some cases, the lowest concentration of the one or more antibiotic solutions is at least 1 pg / mL, e.g., at least 2 pg / mL, at least 5 pg / mL, at least 10 pg / mL, at least 20 pg / mL, at least 40 pg / mL, at least 80 pg / mL, or at least 100 pg / mL. Each of the one or more antibiotic solutions is incubated with one aliquot of a sample comprising the target microorganism. In some cases, an infectious agent comprising an indicator moiety specific for the microorganism is added concurrently with the antibiotic solution. In some cases, the aliquot of the sample is incubated with the antibiotic solution for a period of time, and then the infectious agent is added. The indicator moiety can be detected, and a positive detection indicates that the antibiotic solution is not effective and a negative detection indicates that the antibiotic solution is effective, and the concentration of the antibiotic solution is the effective dose. Thus, in some embodiments, a method of determining an effective dose of an antibiotic in killing a target microorganism comprises separately incubating each of one or more antibiotic solutions with the target microorganism in a sample, wherein the concentrations of the one or more antibiotic solutions are different and define a range; incubating the microorganism in the one or more samples with an infectious agent comprising an indicator moiety; detecting the indicator moiety of the infectious agent in the one or more samples, wherein a positive detection of the indicator moiety in one or more of the one or more samples indicates that the concentration of the antibiotic solution used to treat the one or more samples is not effective, and a lack of detection of the indicator protein indicates that the antibiotic is effective, thereby determining the effective dose of the antibiotic. In some embodiments, two or more antibiotic solutions are tested, and the concentration ratio range of the lowest concentrated solution to the highest concentrated solution in the one or more antibiotic solutions is 1 :2 to 1 :50, e.g., 1 :5 to 1 :30, or 1 :10 to 1 :20. In some cases, the lowest concentration of the one or more antibiotic solutions is at least 1 pg / mL, e.g., at least 2 pg / mL, at least 5 pg / mL, at least 10 pg / mL, at least 20 pg / mL, at least 40 pg / mL, at least 80 pg / mL, or at least 100 pg / mL.

[0137] In some embodiments, the present application includes methods for detecting antibiotic resistant microorganisms in the presence of antibiotic sensitive microorganisms. In certain cases, detection of antibiotic resistant bacteria can be used to prevent the spread of infection in a medical facility. In some embodiments, patients in a medical facility environment can be monitored for colonization by antibiotic resistant bacteria. Preventative measures can then be taken to prevent the spread of antibiotic resistant bacteria.

[0138] In some embodiments of the methods for detecting antibiotic resistant microorganisms, the sample can comprise both antibiotic resistant bacteria and bacteria sensitive to the antibiotic. For example, the sample can comprise both MRSA and MSSA. In some embodiments, MRSA can be detected in the presence of MSSA without the need to isolate MRSA from the sample. In the presence of the antibiotic, MSSA does not generate a signal above a threshold value, but MRSA present in the sample is able to produce a signal above the threshold value. Thus, if both are present in the sample, a signal above the threshold value indicates the presence of an antibiotic resistant strain (e.g., MRSA).

[0139] Accordingly, aspects of the present application provide methods for detecting microorganisms in a test sample by indicator moieties. In some embodiments, when the microorganism of interest is a bacterium, the indicator moiety can be associated with an infectious agent, such as an indicator bacteriophage. The indicator moiety can react with a substrate to emit a detectable signal or can emit an intrinsic signal (e.g., a fluorescent protein). In some embodiments, the detection sensitivity can reveal the presence of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the microorganism of interest in the test sample. In some embodiments, even a single cell of the microorganism of interest can produce a detectable signal. In some embodiments, the bacteriophage is a T4-like or a ViI-like bacteriophage. In some embodiments, the recombinant bacteriophage is derived from a Staphylococcus specific bacteriophage. In certain embodiments, the recombinant Staphylococcus specific bacteriophage has a high degree of specificity for Staphylococcus.

[0140] In some embodiments, the indicator moiety encoded by the infectious agent can be detected during or after replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicator moieties are known in the art, and many are commercially available. In some embodiments, the indicator phage comprises an enzyme for use as an indicator moiety. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator can emit light and / or can be detectable by a color change. Various suitable enzymes are commercially available, for example, alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can be used as indicator moieties. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, other engineered luciferases or other enzymes that generate detectable signals can also be suitable indicator moieties.

[0141] Thus, in some embodiments, the recombinant phage of the method, system, or kit is prepared from a wild-type Staphylococcus-specific phage. In some embodiments, the indicator gene encodes a protein that emits an intrinsic signal, for example, a fluorescent protein (e.g., green fluorescent protein or others). The indicator can emit light and / or can be detectable by a color change. In some embodiments, the indicator gene encodes an enzyme that interacts with a substrate to generate a signal (e.g., luciferase). In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is one of Oplophorus luciferase, firefly luciferase, Renilla luciferase, external Gaussia luciferase, Lucia luciferase, or an engineered luciferase, for example, Rluc8.6-535 or Orange Nano-lantern.

[0142] Detecting the indicator can include detecting emission of light. In some embodiments, a luminometer can be used to detect the reaction of the indicator (e.g., luciferase) with a substrate. Detection of RLU can be accomplished with a luminometer, or other machines or devices can also be used. For example, a spectrophotometer, a CCD camera, or a CMOS camera can detect color changes and other light emissions. Absolute RLU is important for detection, but for reliably detecting single cells or small numbers of cells, the signal-to-background ratio also needs to be high (e.g., >2.0, >2.5, or >3.0).

[0143] In some embodiments, the indicator phage is genetically engineered to comprise a gene for an enzyme (e.g., luciferase) that is produced only after infection of bacteria to which the phage specifically recognizes and infects. In some embodiments, the indicator moiety is expressed at a late stage in the viral life cycle. In some embodiments, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits the copy number of the indicator, as described herein.

[0144] Thus, in some embodiments utilizing indicator phage, the present application includes a method for detecting a microorganism of interest comprising the steps of: capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phage; allowing time for infection and replication to occur to produce progeny phage and express a soluble indicator moiety; and detecting the progeny phage, or preferably the indicator, wherein detection of the indicator demonstrates that the bacterium is present in the sample.

[0145] For example, in some embodiments, the test sample bacteria can be captured by binding to the surface of a plate, or by filtering the sample through a phage filter (e.g., a 0.45 μιη pore size spin filter or a plate filter). In one embodiment, the infectious agent (e.g., indicator phage) is added directly to the captured sample on the filter in a minimal volume. In one embodiment, the microorganisms captured on the filter or plate surface are then washed one or more times to remove excess unbound infectious agent. In one embodiment, media (e.g., Luria-Bertani Broth, also referred to herein as LB, or Tryptic Soy Broth or Tryptone Soy Broth, also referred to herein as TSB) can be added for a further incubation time to allow for replication of the bacterial cells and phage and high level expression of the gene encoding the indicator moiety. However, a surprising aspect of some embodiments of the test assay is that the incubation step with the indicator phage only requires sufficient time for a single phage life cycle. It was previously thought that the amplification capacity of phage required more time so that the phage could replicate several cycles. According to some embodiments of the present application, a single replication cycle of the indicator phage is sufficient to facilitate sensitive and rapid detection.

[0146] In some embodiments, an aliquot of a test sample comprising bacteria can be applied to a spin column, and after infection with the recombinant phage and optional washing to remove any excess phage, the amount of soluble indicator detected will be proportional to the amount of phage produced by the infected bacteria.

[0147] The soluble indicator (e.g., luciferase) released into the surrounding liquid upon bacterial lysis can then be measured and quantified. In one embodiment, the solution is spun through a filter and the filtrate collected in a new vessel (e.g., in a luminometer) after addition of a substrate for the indicator enzyme (e.g., luciferase substrate) for the assay. Alternatively, the indicator signal can be measured directly on the filter.

[0148] In various embodiments, the purified parental indicator phage does not include the detectable indicator itself, as the parental phage can be purified prior to use for incubation with the test sample. Expression of late (class III) genes occurs late in the viral life cycle. In some embodiments of the application, the parental phage can be purified to exclude any indicator protein (e.g., luciferase) that can be present. In some embodiments, expression of the indicator gene during phage replication following infection of the host bacteria results in soluble indicator protein product. Thus, in many embodiments, it is not necessary to separate the parental phage from the progeny phage prior to the detection step. In one embodiment, the microorganism is a bacterium and the indicator phage is a bacteriophage. In one embodiment, the indicator moiety is a soluble luciferase, which is released upon lysis of the host microorganism.

[0149] Thus, in alternative embodiments, the indicator substrate (e.g., luciferase substrate) can be incubated with the portion of the sample that remains on the filter or bound to the surface of the plate. Thus, in some embodiments, the solid support is a 96-well filter plate (or a conventional 96-well plate) and the substrate reaction is detected by placing the plate directly in a luminometer.

[0150] For example, in one embodiment, the application can include a method for detecting MRSA, comprising the steps of: infecting cells captured on a 96-well filter plate with a plurality of parental indicator phage capable of expressing luciferase upon infection; washing away excess phage; adding LB broth with antibiotic and allowing time for phage replication and lysis of the specific S. aureus target (e.g., 30-90 minutes); and detecting the indicator luciferase by adding luciferase substrate and measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity indicates that MRSA is present in the sample.

[0151] In some embodiments, lysis of the bacteria can occur before, during, or after the detection step. Experiments have shown that in some embodiments, infected unlysed cells can be detected after addition of luciferase substrate. It is hypothesized that luciferase can exit the cell and / or luciferase substrate can enter the cell without complete cell lysis. Thus, for embodiments utilizing a spin filter system, where only luciferase released into the lysate (and not luciferase still inside intact bacteria) is analyzed in the luminometer, lysis is required for detection. However, for embodiments utilizing a filter plate or 96-well plate and samples in solution or suspension, where both intact and lysed cells are directly assayed in the luminometer, lysis is not necessary for detection.

[0152] In some embodiments, the reaction of the indicator moiety (e.g., luciferase) with the substrate can continue for 30 minutes or more, and taking readings at various time points can be desirable to optimize sensitivity. For example, in embodiments using a 96-well filter plate as a solid support and luciferase as the indicator, luminometer readings can be taken at the initial and at 10- or 15-minute intervals until the reaction is complete.

[0153] Surprisingly, the use of high concentrations of bacteriophage for infecting test samples has successfully enabled the detection of very small amounts of target microorganisms in a very short time frame. In some embodiments, the incubation of bacteriophage with the test sample only requires a period of time sufficient for a single bacteriophage life cycle. In some embodiments, the concentration of bacteriophage used for this incubation step is greater than 7 x 10 6 , 8 x 10 6 , 9 x 10 6 , 1.0 x 10 7 , 1.1 x 10 7 , 1.2 x 10 7 , 1.3 x 10 7 , 1.4 x 10 7 , 1.5 x 10 7 , 1.6 x 10 7 , 1.7 x 10 7 , 1.8 x 10 7 , 1.9 x 10 7 , 2.0 x 10 7 , 3.0 x 10 7 , 4.0 x 10 7 , 5.0 x 10 7 , 6.0 x 10 7 , 7.0 x 10 7 , 8.0 x 10 7 , 9.0 x 10 7 , or 1.0 x 108 PFU / mL.

[0154] The success of using such high concentrations of phage is surprising because large amounts of phage have previously been associated with "autolysis" which kills the target cell and thereby prevents the generation of a useful signal from the early phage assay. The removal of the phage stocks prepared as described herein can help alleviate this problem (e.g., removal by CsCl2 gradient ultracentrifugation) because in addition to removing any contaminating luciferase associated with the phage, this removal can also remove ghost particles (particles with missing DNA). Ghost particles can lyse the bacterial cell through "autolysis", prematurely killing the cell and thereby preventing the generation of an indicator signal. Electron microscopy has demonstrated that crude phage lysates (i.e., prior to CsCl2 removal) can have more than 50% ghosts. These ghost particles can cause premature death of the microbe through the action of many phage particles puncturing the cell membrane. Thus, ghost particles can cause the problem previously reported where high PFU concentrations were detrimental. In addition, very clean phage preparations allow the assay to be performed without a wash step, which makes it possible to perform the assay without an initial concentration step. Some embodiments do include an initial concentration step, and in some embodiments, this concentration step makes the enrichment incubation time shorter.

[0155] Some embodiments of the test method can further include a confirmatory assay. Various assays for confirming initial results are known in the art, often at a later time point. For example, the sample can be cultured (e.g., as described in Example 4 The assay), the presence of microbial DNA can be confirmed using PCR, or other confirmatory assays can be used to confirm the initial results.

[0156] In certain embodiments, in addition to detection with an infectious agent, the methods of the application combine the use of a binding agent (e.g., an antibody) to purify and / or concentrate the target microbe from the sample. For example, in certain embodiments, the application includes a method for detecting a target microbe in a sample, comprising the steps of: capturing the microbe from the sample on a prior support using a capture antibody specific for the target microbe; incubating the sample with a recombinant phage that infects the target microbe, wherein the recombinant phage comprises an indicator gene inserted into a late gene region of the phage, such that expression of the indicator gene during phage replication after infection of the host bacterium results in a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the target microbe is present in the sample.

[0157] For example, Figure 8A hybrid immuno-pbage (HIP) assay for detecting target bacteria using modified phage is depicted in accordance with embodiments of the application. A sample is first applied to microtiter plate wells coated with bacteria-specific antibodies 802. The plate is then centrifuged to promote binding of bacteria to the capture antibodies 804. After sufficient time to allow for complete capture of bacteria, a solution containing bacteria-specific - a solution of phage is added to each sample 806. Incubation with the phage results in binding and attachment of single or multiple phage to the captured bacteria 808. Finally, the samples are incubated to promote phage replication and luciferase expression, which results in cell lysis and release of soluble luciferase 810.

[0158] Recent advances in synthetic biology have increased interest in phage-based therapeutic approaches to treating pathogenic diseases (see, e.g., Phage Therapy in the Era of Synthetic Biology, Cold Spring Harb Perspect Biol 2016; 8: a023879; and U.S. Patent No. 9,597,407 and U.S. Patent Application Nos. 20170266306 and 20160331804, the contents of which are incorporated by reference herein as if fully detailed herein. Designing and engineering phage to detect the presence of a particular microorganism (e.g., a particular type of bacteria) in a medical sample from a patient can enhance the usefulness of such therapeutic phage.

[0159] In some embodiments, indicator phage can be used to test for the presence of a particular pathogen (e.g., a particular genus or species of bacteria) in an initial patient sample. In some embodiments, indicator phage can be used to detect a particular pathogen in a clinical sample. In this way, indicator phage can be used in a similar manner to a companion diagnostic in order to assess the potential efficacy of a particular therapy (e.g., a particular antibiotic, other drug, or therapeutic phage), e.g., in the context of a given patient infection or other pathogenic medical condition. In some embodiments, as previously described, diagnostic indicator phage can be prepared by making genetic modifications to naturally occurring phage.

[0160] In some embodiments, indicator phages prepared by synthetic techniques can be used for non-clinical purposes. For example, indicator phages can be used as food safety diagnostics to identify the presence of specific bacteria in food. In other embodiments, diagnostic indicator phages can be prepared by synthetic techniques. For example, synthetic phage genomes can be designed and constructed for transformation and propagation of the corresponding phage in various types of bacteria. In some cases, synthetic biology techniques can be used to generate indicator phages using the target bacteria of the indicator phage. In other cases, more convenient bacteria can be used to generate the indicator phage. For example, synthetic genomes for indicator phages targeting Listeria can be generated in Listeria or a more convenient species (e.g., Lactobacillus).

[0161] In some embodiments, synthetic phages are designed to optimize desired traits for pathogen detection assays. In some embodiments, previous analyses of bioinformatics and genetic modifications are employed to optimize desired traits. For example, in some embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to specific species of bacteria. In other embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to entire genera or specific groups of species within a genus of bacteria. In this way, phages can be optimized to detect a broader or narrower group of pathogens. In some embodiments, synthetic phages can be designed to improve expression of reporter genes. Additionally and / or alternatively, in some cases, synthetic phages can be designed to increase the burst size of the phage to improve detection.

[0162] In some embodiments, the stability of the phage can be optimized to improve shelf life. For example, enzybiotic solubility can be increased in order to increase subsequent phage stability. Additionally and / or alternatively, the thermal stability of the phage can be optimized. Heat-resistant phages better retain functional activity during storage, increasing shelf life. Thus, in some embodiments, thermal stability and / or pH tolerance can be optimized.

[0163] Some species of bacteria establish biofilm walls to protect themselves from the immune system. These biofilms can make it difficult to effectively target the bacteria. A number of enzymes have been identified that are capable of breaking down bacterial biofilms (e.g., glycoside hydrolases PelAh and PslGh). In some embodiments, phages can be modified to encode soluble or fusion virion proteins to allow incorporation of enzymes to break down biofilms.

[0164] In some embodiments, the genetically modified or synthetically derived bacteriophage comprises a detectable indicator. In some embodiments, the indicator is luciferase. In some embodiments, the bacteriophage genome comprises an indicator gene (e.g., a luciferase gene or another gene that encodes a detectable indicator).

[0165] In another aspect, the application can include a method for selecting a treatment for a subject comprising: (i) obtaining a biological sample from the subject; (ii) detecting a particular microbe or class of microbes in the biological sample using an indicator bacteriophage; and (iii) selecting a treatment based on the identification of the particular microbe detected in the biological sample.

[0166] In some embodiments, whether synthetically prepared or not, the indicator bacteriophage can be used to detect pathogens in patient samples after the initiation of a certain type of treatment. In some embodiments, the treatment can be a bacteriophage-based therapeutic. In other embodiments, the treatment can be an antibiotic (e.g., a traditional antibiotic such as penicillin or cyclosporine). In other embodiments, the treatment can be another type of drug or therapy. In this way, the indicator bacteriophage can be used to monitor the progress or efficacy of any type of treatment or therapy. In some embodiments, the indicator bacteriophage can be used to detect and monitor the pathogen content of patient samples taken hours or days after the initiation of treatment. In some embodiments, the indicator bacteriophage can be used to monitor samples in the case of a chronic infection, and can be days, weeks, months, or years after the initiation of treatment.

[0167] Systems and kits of the application

[0168] In some embodiments, the application includes systems (e.g., automated systems or kits) that include components for performing the methods disclosed herein. In some embodiments, the indicator bacteriophage is included in a system or kit according to the application. The methods described herein can also utilize such indicator bacteriophage systems or kits. Some embodiments described herein are particularly suitable for automation or kits in view of the small amount of reagents and materials needed to perform the methods. In certain embodiments, each component of the kit can comprise a self-contained unit that can be transferred from a first location to a second location.

[0169] In some embodiments, the present application includes a system or kit for rapid detection of a target microorganism in a sample. In certain embodiments, the system or kit includes a component that incubates a sample with an infectious agent specific for the target microorganism and a component for detecting an indicator moiety, wherein the infectious agent comprises an indicator moiety. In some embodiments of both the system and the kit of the present application, the infectious agent is a recombinant bacteriophage that infects the target bacteria, and the recombinant bacteriophage comprises an indicator gene inserted into a late gene region of the bacteriophage as the indicator moiety, such that expression of the indicator gene during bacteriophage replication after infection of the host bacteria results in a soluble indicator protein product. Some systems further include a component for capturing the target microorganism on a solid support.

[0170] In other embodiments, the present application includes a method, system or kit for rapid detection of a target microorganism in a sample, which includes a component of an infectious agent specific for the target microorganism and a component for detecting an indicator moiety, wherein the infectious agent comprises an indicator moiety. In some embodiments, the bacteriophage is a T4-like, ViI, ViI-like, staphylococcus-specific bacteriophage. In one embodiment, the recombinant bacteriophage is derived from a staphylococcus-specific bacteriophage. In certain embodiments, the recombinant bacteriophage is highly specific for a particular bacteria. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Staphylococcus. In one embodiment, the recombinant bacteriophage can distinguish Cronobacter in the presence of over 100 other types of bacteria. In another embodiment, the recombinant bacteriophage can distinguish Staphylococcus in the presence of over 100 other types of bacteria. In certain embodiments, the system or kit detects a single bacteria of a particular type in a sample. In certain embodiments, the system or kit detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 of a particular bacteria in a sample.

[0171] In certain embodiments, the system and / or kit can further include a component for washing the captured microorganism sample. Additionally or alternatively, the system and / or kit can further include a component for determining the amount of indicator moiety, wherein the amount of indicator moiety detected corresponds to the amount of microorganism in the sample. For example, in certain embodiments, the system or kit can include a luminometer or other device for measuring luciferase activity.

[0172] In some systems and / or kits, the same component can be used for multiple steps. In some systems and / or kits, the steps are automated or controlled by a user via computer input and / or wherein a liquid handling robot performs at least one step.

[0173] Thus, in certain embodiments, the present application can include a system or kit for rapid detection of a target microorganism in a sample, comprising: a component for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent comprises an indicator moiety; a component for capturing the microorganism from the sample on a solid support; a component for washing the captured microorganism sample to remove unbound infectious agent; and a component for detecting the indicator moiety. In some embodiments, the same component can be used for the steps of capturing and / or incubating and / or washing (e.g., a filter component). Some embodiments additionally include a component for determining the amount of target microorganism in the sample, wherein the amount of indicator moiety detected corresponds to the amount of microorganism in the sample. Such a system can include various embodiments and subembodiments similar to those described above for the method for rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage. In a computerized system, the system can be fully automated, semi-automated, or directed by a user through a computer (or some combination thereof).

[0174] In some embodiments, the system can include a component for separating the target microorganism from other components in the sample.

[0175] In one embodiment, the present application includes a system or kit comprising components for detecting a target microorganism, comprising: a component for separating at least one microorganism from other components in a sample; a component for infecting the at least one microorganism with a plurality of parental infectious agents; a component for lysing the at least one infected microorganism to release progeny infectious agents present in the microorganism; and a component for detecting the progeny infectious agents, or, with greater sensitivity, a soluble protein encoded and expressed by the infectious agents, wherein detection of the infectious agents or the soluble protein product of the infectious agents indicates that the microorganism is present in the sample. The infectious agents can comprise Staphylococcus-specific NANOLUC bacteriophages.

[0176] The system or kit can include various components for detecting the progeny infectious agents. For example, in one embodiment, the progeny infectious agents (e.g., bacteriophages) can include an indicator moiety. In one embodiment, the indicator moiety in the progeny infectious agents (e.g., bacteriophages) can be a detectable moiety expressed during replication, e.g., a soluble luciferase protein.

[0177] In other embodiments, the present application can include a kit for rapid detection of a target microorganism in a sample, the system comprising: a component for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent comprises an indicator moiety; a component for capturing the microorganism from the sample on a solid support; a component for washing the captured microorganism sample to remove unbound infectious agent; and a component for detecting the indicator moiety. In some embodiments, the same component can be used for the steps of capturing and / or incubating and / or washing. Some embodiments additionally include a component for determining the amount of target microorganism in the sample, wherein the amount of indicator moiety detected corresponds to the amount of microorganism in the sample. Such kits can include various embodiments and subembodiments similar to those described above for the methods for rapid detection of microorganisms. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

[0178] In some embodiments, the kit can include a component for separating the target microorganism from other components in the sample.

[0179] The systems and kits of the present application include various components. The term "component" as used herein is broadly defined and includes any suitable device or collection of devices suitable for carrying out the methods described. The components need not be integrally connected or arranged relative to each other in any particular way. The present application includes any suitable arrangement of the components relative to each other. For example, the components need not be in the same space. But in some embodiments, the components are connected to each other in a complete unit. In some embodiments, the same component can perform multiple functions.

[0180] Computer systems and computer readable media

[0181] The systems described in the technology of the present application, or any component thereof, can be embodied in the form of a computer system. Typical examples of computer systems include general purpose computers, programmed microprocessors, micro-controllers, peripheral integrated circuit elements, and other devices or elements of equipment that are capable of carrying out the steps of the methods of the present technology by means of a collective interaction of their functional parts.

[0182] The computer system can include a computer, input devices, a display unit and / or the Internet. The computer can further include a microprocessor. The microprocessor can be connected to a communication bus. The computer can also include a memory. The memory can include a random access memory (RAM) and a read-only memory (ROM). The computer system can further include a storage device. The storage device can be a hard disk drive or a removable storage drive, such as a floppy disk drive, a optical disk drive, etc. The storage device can also be other similar means for loading computer programs or other instructions to the computer system. The computer system can also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet through the I / O interface. The communication unit allows data to be transferred to and from other databases and the Internet. The communication unit can include a modem, an Ethernet card or any similar device capable of connecting the computer system to databases and networks, such as LAN, MAN, WAN and the Internet. The computer system is thus advantageously adapted to access the system through the I / O interface from a user through the input devices.

[0183] The computing device will typically include an operating system that provides executable program instructions for the general administration and operation of that computing device, and typically will include a computer-readable storage medium (e.g., a hard disk, random access memory, read only memory, etc.) on which instructions stored, that, when executed by the processor of the server allow the computing device to perform its intended functions. Suitable implementations for the operating system and general functionality of a computing device are known or commercially available and are readily implemented by persons having ordinary skill in the art, particularly in light of the disclosure herein.

[0184] The computer system executes a set of instructions that are stored in one or more storage elements, in order to process input data. The storage elements can also be used for storing temporary variable or other intermediate information during execution of instructions by the computer system. The storage element can be in the form of a database maintained by a server, a physical storage element (e.g., a disk drive, a memory, etc.), or any other suitable storage elements.

[0185] The environment can include various data stores such as above discussed and other memory and storage media as appropriate. These can be of the same type or different types, and stored via any access method appropriate to the computer technology. By way of example, memory can be of a fixed or removable type such as, for example, read-only memory (ROM), random access memory (RAM), programmable ROM (PROM), erasable PROM (EPROM), electrically erasable PROM (EEPROM), magnetic disk drives, magnetic tape drives, optical disk drives, flash memory, or other similar memory. The data stores can also include other persistent like computer-readable storage media, and / or can include random access memory (RAM), dynamic RAM (DRAM), another volatile memory storage technology, cache, and / or others. The drawings show a block diagram of a computing device that can be used in accordance with the embodiments. The computing device includes a processor; e.g., a central processing unit (CPU), a graphics processing unit (GPU), or both; memory; e.g., RAM, ROM, flash, etc.; and numerous other elements and functionalities typical of today's computing devices, such as one or more processing units, multi-format card readers, storage, user input (e.g., keyboards, displays, touch screens, etc.), and so on. The processor executes instructions, codes, computer programs, scripts, or others, that it accesses from the memory. While the processor can be hard coded to perform specific functions, the processor can also execute software that enables the computer to be programmable. That is, whereas specific implementations can delete from the processor the ability to execute specific functions, the processor is not ill-equipped to perform other functions.

[0186] Such devices also can include a computer-readable storage media reader; a communications device (e.g., a modem, a network card (wireless or wired), an infrared communication device, etc.); and working memory as described above. The computer- readable storage media reader can be connected with a computer-readable storage medium, or the like, such as a network or data storage.

[0187] For purposes of this disclosure, a computer-readable medium can include any suitable medium employed to provide computing device access to the instructions, data structures, program modules or other data. A computer-readable medium can also include, by way of example, a floppy diskette, a DVD, a CD, a Blu-Ray disc, an optical disc, a hard disk, a magnetic tape, a memory chip, a RAM, a ROM, a FLASH memory, a PROM, an EPROM, an EEPROM or other memory technology, a carrier wave used to transmit a computer program or other data, or any other medium from which a computer can read instructions or data. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will appreciate other ways and / or methods to implement the various embodiments.

[0188] A computer-readable medium can include, but is not limited to, an electronic, optical, magnetic, or other storage device capable of providing a processor with computer-readable instructions. Other examples include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, SRAM, DRAM, content addressable memory ("CAM"), DDR, flash (e.g., NAND flash or NOR flash), ASIC, configured processor, optical storage, magnetic tape, or other storage device, or any other medium from which a computer can read instructions. In one embodiment, a computing device can include a single type of computer-readable medium, such as, for example, random access memory (RAM). In other embodiments, a computing device can include two or more types of computer-readable media, such as, for example, random access memory (RAM), a disk drive, and a cache.

[0189] As discussed above, embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions can include processor-specific instructions generated by a compiler and / or an interpreter from code written in any suitable computer-programming language including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, a computing device includes a single processor. In other embodiments, a device includes two or more processors. Such processors can include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and state machines. Such processors can further include programmable electronic devices such as PLCs, programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electronically programmable read-only memories (EPROMs or EEPROMs), or other similar devices.

[0190] A computing device includes a network interface. In some embodiments, a network interface is configured to communicate over wired or wireless communication links. For example, a network interface can allow communication over a network using Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, a network interface can allow communication over a network using, for example, a CDMA, GSM, UMTS, or other cellular communication network. In some embodiments, a network interface can allow point-to-point connection with another device, for example, through a Universal Serial Bus (USB), 1394 FireWire, serial, or parallel, or similar interface. Some embodiments of suitable computing devices can include two or more network interfaces for communication over one or more networks. In some embodiments, a computing device can include a data store in addition to or in place of a network interface.

[0191] Some embodiments of suitable computing devices can include or communicate with a variety of external or internal devices such as a mouse, a CD-ROM, DVD, a keyboard, a display, a speaker, one or more microphones, or any other input or output devices. For example, a computing device can communicate with various user interface devices and displays. A display can use any suitable technology including, but not limited to, LCD, LED, CRT, etc.

[0192] A set of instructions for execution by a computer system can include various commands that instruct a processing machine to perform specific tasks such as the steps that comprise the methods of the inventive technology. The set of instructions can be in the form of a software program. Additionally, the software can be in the form of a collection of separate programs, a program module with a larger program or a portion of a program module as in the technology herein. The software can also include modular programming in the form of object-oriented programming. Processing of input data by the processing machine can be in response to user commands, in response to results of previous processing, or in response to a request made by another processing machine.

[0193] While the application has been disclosed with reference to certain implementations, it is apparent that modifications and changes can be made without departing from the scope and spirit of the application as defined in the following claims. It is therefore intended that the application not be limited to the described implementations, but that the full scope of equivalents be contemplated by the language of the following claims. Example

[0194] The results described in the following examples demonstrate the detection of small numbers of cells, even single bacteria, in a reduced time to result.

[0195] Example 1. Bacterial detection using S. aureus specific phage NanoLuc indicator phage after incubation of sample in media

[0196] Samples of methicillin-resistant S. aureus (MRSA) were rendered susceptible by the addition of 135 μΐ of a sample containing S. aureus to a rich media. The rich media contained a sub-inhibitory antibiotic (cefoxitin) to allow for specific enrichment and induction of MRSA. The sample was incubated in the rich media for 1-2 hours. After rendering the sample susceptible, additional cefoxitin was added to the media and the sample was incubated for an additional 2 hours. S. aureus specific phage NanoLuc indicator phage was added to the sample and incubated for 2 hours. Lysis buffer and Reagents. After 5 minutes of incubation, bioluminescence measurements were performed using a Victor® 96 instrument. A signal / background ratio >750 RLU indicated positive detection of S. aureus. A signal / background ratio <750 RLU indicated that the sample was negative for S. aureus.

[0197] Table 1.1 Detection of Staphylococcus aureus by phage mediation

[0198]

[0199]

[0200] Table 1.2 Detection of Staphylococcus aureus by phage mediation

[0201]

[0202]

[0203] Table 1.3 Detection of Staphylococcus aureus by phage mediation

[0204]

[0205]

[0206] Table 1.4 Detection of Staphylococcus aureus by phage mediation

[0207]

[0208] Table 1.5 Detection of Staphylococcus aureus by phage mediation

[0209]

[0210]

[0211] Example 2. After incubating the sample in media, use Staphylococcus specific phage Indicator phage diagnose and monitor Staphylococcus infection

[0212] A clinical sample is obtained from a patient. The clinical sample is incubated with Staphylococcus specific phage Indicator phage for 2 hours. After incubation, lysis buffer and reagents are added and incubated for 5 minutes. Then, the bioluminescence is measured using a Victor® 96 instrument to determine the presence of Staphylococcus in the clinical sample. A signal / background ratio > 750 RLU indicates a positive detection of Staphylococcus. A signal / background ratio < 750 RLU indicates the sample is negative for Staphylococcus.

[0213] The patient with the sample positive for Staphylococcus is treated with a therapeutic phage specific for Staphylococcus. A second clinical sample is obtained from the patient 72 hours after treatment with the therapeutic phage specific for Staphylococcus. The clinical sample is then incubated with Staphylococcus specific phage Indicator phage for 2 hours. After incubation, lysis buffer and reagents are added and incubated for 5 minutes. Then, the ​96Instrument measures bioluminescence to monitor changes in the presence of Staphylococcus in clinical samples post-treatment. A signal / background ratio >750 RLU indicates a positive detection of Staphylococcus. A signal / background ratio <750 RLU indicates the sample is negative for Staphylococcus. If the sample is positive for Staphylococcus, treatment with therapeutic phage specific for Staphylococcus is continued and the Staphylococcus specific phage Indicator phage monitor treatment efficacy.

[0214] Exemplary embodiments:

[0215] 1. A recombinant phage comprising an indicator gene inserted into a late gene region of a phage genome.

[0216] 2. The recombinant phage of embodiment 1, wherein the recombinant phage specifically infects Staphylococcus.

[0217] 3. The recombinant phage of embodiment 2, wherein the Staphylococcus is Staphylococcus aureus.

[0218] 4. The recombinant phage of embodiment 3, wherein the Staphylococcus aureus is methicillin resistant.

[0219] 5. The recombinant phage of embodiment 1, wherein the indicator gene is codon optimized and encodes a soluble protein product that produces an intrinsic signal or a soluble enzyme that produces a signal upon reaction with a substrate.

[0220] 6. The recombinant phage of embodiment 5, further comprising an untranslated region upstream of the codon optimized indicator gene, wherein the untranslated region comprises a phage late gene promoter and a ribosomal entry site.

[0221] 7. A cocktail composition comprising at least two different types of recombinant phage, wherein at least one of the recombinant phage comprises an indicator gene according to embodiment 1.

[0222] 8. A method of making a recombinant indicator phage, comprising:

[0223] selecting a wild-type phage that specifically infects a target pathogenic bacteria;

[0224] making a homologous recombination plasmid / vector comprising an indicator gene;

[0225] transforming the homologous recombination plasmid / vector into the target pathogenic bacteria;

[0226] infecting the transformed target pathogenic bacteria with a selected wild-type bacteriophage, thereby allowing homologous recombination between the plasmid / vector and the bacteriophage genome; and

[0227] isolating specific clones of the recombinant bacteriophage.

[0228] 9. The method of embodiment 8, wherein preparing the homologously recombined plasmid / vector comprises:

[0229] determining the native nucleotide sequence in the late region of the genome of the selected bacteriophage;

[0230] annotating the genome and identifying the major capsid protein gene of the selected bacteriophage;

[0231] designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence comprises a codon-optimized indicator gene; and

[0232] incorporating the sequence designed for homologous recombination into a plasmid / vector.

[0233] 10. The method of embodiment 9, wherein designing a sequence further comprises inserting an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late gene promoter and a ribosomal entry site.

[0234] 11. The method of embodiment 8, wherein the homologously recombined plasmid comprises an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late gene promoter and a ribosomal entry site.

[0235] 12. The method of embodiment 8, wherein the wild-type bacteriophage is a Staphylococcus- specific bacteriophage and the target pathogenic bacteria is Staphylococcus.

[0236] 13. The method of embodiment 12, wherein the Staphylococcus is Staphylococcus aureus.

[0237] 14. The method of embodiment 13, wherein the Staphylococcus aureus is methicillin-resistant (MRSA).

[0238] 15. The method of embodiment 8, wherein isolating specific clones of the recombinant bacteriophage comprises a limiting dilution assay for isolating clones that demonstrate expression of the indicator gene.

[0239] 16. A method for detecting Staphylococcus in a sample, comprising:

[0240] incubating the sample with a recombinant bacteriophage derived from a Staphylococcus- specific bacteriophage, the recombinant bacteriophage comprising an indicator gene inserted into a late gene region of the bacteriophage genome; and

[0241] detecting an indicator protein product produced by the recombinant bacteriophage, wherein a positive detection of the indicator protein product indicates that Staphylococcus is present in the sample.

[0242] 17. The method of embodiment 16, wherein the Staphylococcus is Staphylococcus aureus.

[0243] 18. The method of embodiment 17, wherein the Staphylococcus aureus is methicillin resistant.

[0244] 19. The method of embodiment 16, wherein the sample is a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

[0245] 20. The method of embodiment 16, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacterium in a standard size sample of the food safety industry.

[0246] 21. The method of embodiment 19, wherein the food sample comprises meat, fish, vegetables, eggs, or baby formula.

[0247] 22. The method of embodiment 16, wherein the sample is incubated with a cocktail composition comprising at least two different types of recombinant bacteriophage, wherein at least one of the recombinant bacteriophage comprises an indicator gene according to embodiment 16.

[0248] 23. The method of embodiment 16, wherein the sample is first incubated under conditions conducive to growth for an enrichment time of 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less.

[0249] 24. The method of embodiment 16, wherein the total time to results is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, or less than 6 hours.

[0250] 25. The method of embodiment 16, wherein the signal-to-background ratio generated by detecting the indicator is at least 2.0 or at least 2.5.

[0251] 26. A kit for detecting Staphylococcus comprising a recombinant bacteriophage derived from a Staphylococcus specific bacteriophage.

[0252] 27. The kit of embodiment 26, wherein the Staphylococcus is Staphylococcus aureus and the Staphylococcus specific bacteriophage is a Staphylococcus aureus specific bacteriophage.

[0253] 28. The kit of embodiment 27, wherein the S. aureus is methicillin-resistant and the S. aureus-specific bacteriophage is a methicillin-resistant S. aureus-specific bacteriophage.

[0254] 29. The kit of embodiment 26, further comprising a substrate for reaction with an indicator to detect a soluble protein product expressed by the recombinant bacteriophage.

[0255] 30. A system for detecting Staphylococcus comprising a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage.

[0256] 31. The system of embodiment 30, wherein the Staphylococcus is S. aureus.

[0257] 32. The system of embodiment 31, wherein the S. aureus is methicillin-resistant.

[0258] 33. A method for selecting a treatment for a subject, comprising:

[0259] (i) obtaining a biological sample from the subject;

[0260] (ii) detecting a particular microbe or class of microbes in the biological sample using an indicator bacteriophage; and

[0261] (iii) selecting a treatment based on the identification of the particular microbe detected in the biological sample.

[0262] 34. The method of embodiment 33, wherein the indicator bacteriophage is a synthetically prepared bacteriophage.

[0263] 35. The method of embodiment 33, wherein the indicator bacteriophage is a genetically modified naturally occurring bacteriophage.

[0264] 36. A method for monitoring the efficacy of a treatment for a subject having a pathogenic medical condition, comprising:

[0265] (i) obtaining a biological sample from the subject;

[0266] (ii) detecting a particular microbe or class of microbes in the biological sample using an indicator bacteriophage;

[0267] (iii) initiating a treatment for the subject;

[0268] (iv) obtaining a second biological sample of the same type as the first biological sample from the subject;

[0269] (v) detecting the specific microbe or class of microbes in the second biological sample using the indicator bacteriophage; and

[0270] (vi) determining a decreasing, increasing, or stable level of the specific microbe or class of microbes in the subject based on the amounts detected in the first and second biological samples.

Claims

1. Use of an indicator phage in the manufacture of a reagent for a method of selecting a treatment for a subject, the method comprising: (i) obtaining a biological sample from the subject; (ii) detecting a particular microorganism or class of microorganisms in the biological sample using an indicator phage, wherein the indicator phage infects methicillin-resistant Staphylococcus aureus (MRSA), and the detecting comprises the steps of: (a) incubating the sample with at least one antibiotic to allow enrichment of bacteria resistant to the antibiotic; (b) incubating the enriched sample with a phage that infects the target bacteria, wherein the phage comprises an indicator gene such that expression of the indicator gene during phage replication following infection of the target bacteria results in production of a soluble indicator protein product; and (c) detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the target antibiotic-resistant bacteria are present in the sample; and (iii) selecting a treatment based on the identification of the particular microorganism detected in the biological sample.

2. The use of claim 1, wherein the indicator phage is a synthetically manufactured phage.

3. The use of claim 1, wherein the indicator phage is a naturally occurring phage that is genetically modified.

4. Use of an indicator phage in the manufacture of a reagent for a method of monitoring the efficacy of a treatment of a subject having a pathogenic medical condition, the method comprising: (i) obtaining a biological sample from the subject; (ii) detecting a particular microorganism or class of microorganisms in the biological sample using an indicator phage, wherein the indicator phage infects methicillin-resistant Staphylococcus aureus (MRSA), and the detecting comprises the steps of: (a) incubating the sample with at least one antibiotic to allow enrichment of bacteria resistant to the antibiotic; (b) incubating the enriched sample with a phage that infects the target bacteria, wherein the phage comprises an indicator gene such that expression of the indicator gene during phage replication following infection of the target bacteria results in production of a soluble indicator protein product; and (c) detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the target antibiotic-resistant bacteria are present in the sample; (iii) initiating a treatment for the subject; (iv) obtaining a second biological sample of the same type as the first biological sample from the subject; (v) detecting the particular microorganism or class of microorganisms in the second biological sample using the indicator phage; and (vi) determining a level of reduction, increase, or stability of the particular microorganism or class of microorganisms in the subject based on the amount detected in the first and second biological samples.

5. The use of claim 4, wherein the indicator phage is a synthetically manufactured phage.

6. The use of claim 4, wherein the indicator phage is a naturally occurring phage that is genetically modified.

7. A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in a sample for non-diagnostic purposes, the method comprising: (i) incubating the sample with at least one antibiotic to allow enrichment of bacteria resistant to the antibiotic; (ii) incubating the enriched sample with a recombinant bacteriophage that infects the target bacteria, wherein the bacteriophage comprises an indicator gene such that expression of the indicator gene during replication of the bacteriophage following infection of the target bacteria results in production of a soluble indicator protein product; and (iii) detecting the indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that the target antibiotic resistant bacteria are present in the sample.

8. The method of claim 7, wherein the sample is a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

9. The method of claim 7, wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacteria in a sample of standard size for the food safety industry.

10. The method of claim 7, wherein the food sample comprises meat, fish, vegetables, eggs, or baby formula.

11. The method of claim 7, wherein the sample is incubated with a cocktail composition comprising at least two different types of recombinant bacteriophage, wherein at least one of the recombinant bacteriophage comprises the indicator gene of claim 7.

12. The method of claim 7, wherein the incubation with at least one antibiotic to allow enrichment is for a time period of 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less.

13. The method of claim 7, wherein the total time to results is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, or less than 6 hours.

14. The method of claim 7, wherein the signal-to-background ratio generated by detecting the indicator is at least 2.0 or at least 2.

5.

15. The method of claim 1, wherein the enriched sample is incubated with bacteriophage at a concentration of greater than 1.0 X 10 5 PFU / mL, greater than 1.0 X 10 6 PFU / mL, greater than 1.0 X 10 7 PFU / mL, or greater than 1.0 X 10 8 PFU / mL.

16. The method of claim 4, wherein the enriched sample is incubated with a concentration of bacteriophage greater than 1.0 X 10 5 PFU / mL, greater than 1.0 X 10 6 PFU / mL, greater than 1.0 X 10 7 PFU / mL, or greater than 1.0 X 10 8 PFU / mL.

17. The method of claim 7, wherein the enriched sample is incubated with phage at a concentration of greater than 1.0 X 10 5 PFU / mL, greater than 1.0 X 10 6 PFU / mL, greater than 1.0 X 10 7 PFU / mL, or greater than 1.0 X 10 8 PFU / mL.

18. The method of claim 1, wherein the enriched sample is incubated with the bacteriophage that infects the target bacteria for less than 4 hours, less than 3 hours, or less than 2 hours.

19. The method of claim 4, wherein the enriched sample is incubated with the bacteriophage that infects the target bacteria for less than 4 hours, less than 3 hours, or less than 2 hours.

20. The method of claim 7, wherein the enriched sample is incubated with the bacteriophage that infects the target bacteria for less than 4 hours, less than 3 hours, or less than 2 hours.

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