Indicator bacteriophages for the selection of therapeutic agents and monitoring of effectiveness, and methods for using the same

Abstract

To provide more rapid, simple and sensitive detection and identification of microorganisms, such as bacteria and other potentially pathogenic microorganisms.SOLUTION: Disclosed herein are methods for preparing a recombinant indicator bacteriophage. Some embodiments include the steps for: selecting a wild-type bacteriophage that specifically infects target pathogenic bacteria; preparing a homologous recombination plasmid / vector comprising an indicator gene; transforming the homologous recombination plasmid / vector into target pathogenic bacteria; infecting the transformed target pathogenic bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid / vector and the bacteriophage genome; and isolating a particular clone of recombinant bacteriophage.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 661,739, filed on Apr. 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.

[0002] Field of the Invention The present invention relates to compositions, methods, systems, and kits for the detection of microorganisms using infectious agents.

Background Art

[0003] Background There is a strong interest in improving the speed and sensitivity for the detection of bacteria, viruses, and other microorganisms in biological samples, food samples, water samples, and clinical samples. Microbial pathogens can cause substantial pathological conditions and huge economic losses in humans and livestock animals. Also, considering the potential for life - threatening or fatal pandemics caused by the ingestion 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).

[0004] 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 suspicious colonies. Such procedures can take several days. Various rapid methods have been studied and introduced into practice to reduce the time requirements. However, these methods have drawbacks. For example, techniques involving direct immunoassays or gene probes generally require an overnight enrichment step to obtain appropriate sensitivity. Polymerase chain reaction (PCR) tests also involve an amplification step and thus have the ability to be both very sensitive and selective; however, economically, the sample size that can be subjected to PCR tests is limited. Using a dilute bacterial suspension, most small subsamples will have no cells and thus still require purification and / or a lengthy enrichment process.

[0005] The time required for traditional biological enrichment is defined by the growth rate of the target bacterial population in the above sample, by the effect of the above sample matrix, and by the required sensitivity. In practice, most high-sensitivity methods use an overnight incubation and overall take about 24 hours. Due to the time required for culturing, these methods can take up to 3 days, depending on the organism to be identified and the source of the above sample. This lag time is generally inappropriate because contaminated food, water (or other products) may already have entered livestock or humans. Furthermore, due to the increase in antibiotic-resistant bacteria and concerns about biodefense, the rapid identification of bacterial pathogens in water samples, food samples, and clinical samples has become a worldwide priority that cannot be lacking.

[0006] Therefore, there is a need for more rapid, simple, and sensitive detection and identification of microorganisms (e.g., bacteria and other potentially pathogenic microorganisms). Summary of the Invention Means for Solving the Problems

[0007] Abstract Embodiments of the present invention include compositions, methods, systems, and kits for the detection of microorganisms. The present invention can be embodied in various ways.

[0008] 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, Staphylococcus aureus is methicillin - resistant S. aureus (MRSA). For example, in certain embodiments, the recombinant bacteriophage is a genetically modified bacteriophage genome. In some embodiments, the bacteriophage used to prepare 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.

[0009] 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 endogenous 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, the untranslated region including a bacteriophage late gene promoter and a ribosome entry site. In some embodiments, the indicator gene is a luciferase gene. The luciferase gene can be a naturally occurring gene, such as the Oplophorus luciferase gene, the firefly luciferase gene, the Lucia luciferase gene, or the Renilla luciferase gene, or the luciferase gene can be a genetically engineered gene such as NanoLuc.

[0010] Also disclosed herein is a method for preparing a recombinant indicator bacteriophage. Some embodiments include selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, preparing a homologous recombination plasmid / vector containing an indicator gene, transforming the homologous recombination plasmid / vector into the target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby causing homologous recombination between the plasmid / vector and the bacteriophage genome, and isolating specific clones of the recombinant bacteriophage. In some embodiments, the selected wild-type bacteriophage is a Staphylococcus-specific bacteriophage or a Staphylococcus aureus-specific bacteriophage. In some embodiments, the selected wild-type bacteriophage is T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or another naturally occurring phage having a genome with at least 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% homology to the phages disclosed above.

[0011] In some embodiments, the step of preparing a homologous recombination plasmid / vector includes determining the native nucleotide sequence in the late region of the genome of the selected bacteriophage, annotating the genome, identifying the major capsid protein gene of the selected bacteriophage, designing a sequence for homologous recombination downstream of the major capsid protein gene, the sequence including a codon optimization indicator gene, and incorporating the sequence designed for homologous recombination into a plasmid / vector. The step of designing the sequence may further include inserting an untranslated region including a phage late gene promoter and a ribosome entry site upstream of the codon optimization indicator gene. Thus, in some methods, the homologous recombination plasmid includes an untranslated region including a bacteriophage late gene promoter and a ribosome entry site upstream of the codon optimization indicator gene.

[0012] Some embodiments of the invention are compositions comprising a recombinant indicator bacteriophage as described herein. For example, the composition may include one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the composition may include a cocktail of different indicator phages that can encode and express the same or different indicator proteins.

[0013] In some embodiments, the present invention includes a method for detecting a target microorganism in a sample, the method comprising 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 after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target microorganism in the sample.

[0014] In some embodiments of a method for preparing a recombinant indicator bacteriophage, the wild-type bacteriophage is a Staphylococcus-specific bacteriophage and the target pathogenic bacterium is Staphylococcus. In some embodiments, the step of isolating a particular clone of the recombinant bacteriophage comprises a limiting dilution assay to isolate a clone that exhibits expression of the indicator gene.

[0015] Another aspect of the present invention is a method for detecting bacteria such as Staphylococcus in a sample, the method comprising 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 a positive detection of the indicator protein product indicates the presence of Staphylococcus in the sample. In some embodiments, the present invention includes a method for detecting S. aureus using a recombinant bacteriophage derived from S. aureus. The sample can be a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

[0016] In some embodiments of the method for detecting bacteria, the sample is first incubated under conditions favorable for 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 result 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 generated by the step of detecting the indicator is at least 2.0 or at least 2.5. In some embodiments, the method detects a low number of specific bacteria, on the order of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100, in a standard-sized sample for the food safety industry.

[0017] Further embodiments include systems and kits for detecting Staphylococcus, wherein the system or kit includes a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage. In some embodiments, the systems and kits may be used to detect S. aureus, wherein the system or kit includes a recombinant bacteriophage derived from S. aureus. In some embodiments, the systems and kits may be used to detect MRSA. Some embodiments further include a substrate for reacting with an indicator for detecting a soluble protein product expressed by the recombinant bacteriophage. These systems or kits may include features described with respect to the bacteriophages, compositions, and methods of the present invention. In yet other embodiments, the present invention includes a non-transitory computer-readable medium for use with a method or system according to the present invention.

[0018] In another aspect, the present invention includes a method for selecting a treatment for a subject, the method comprising: (i) obtaining a biological sample from the subject; (ii) using an indicator phage to detect a particular microorganism or category of microorganisms in the biological sample; and (iii) selecting a treatment based on the identity of the particular microorganism detected in the biological sample.

[0019] In another aspect, the present invention is a method for monitoring the effectiveness of treatment for a subject having a pathogenic medical condition, the method comprising: (i) obtaining a biological sample from the subject; (ii) using an indicator phage to detect a specific microorganism or category of microorganisms in the biological sample; (iii) initiating treatment for the subject; (iv) obtaining a second biological sample of the same type as the first biological sample from the subject; (v) using an indicator phage to detect a specific microorganism or category of microorganisms in the second biological sample; and (vi) determining a decrease, increase, or steady level of the specific microorganism or category of microorganisms in the subject based on the amounts detected in the first and second biological samples.

[0020] The present invention may be better understood with reference to the following non-limiting drawings.

Brief Description of the Drawings

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[0029] **Detailed Description of the Invention** Compositions, methods, and systems are disclosed herein that exhibit surprising sensitivity for the detection of target microorganisms in test samples (e.g., biological samples, food samples, water samples, and clinical samples). Detection can be achieved in assays performed without culturing for enrichment or, in some embodiments, with minimal incubation times during which the microorganisms can potentially grow, using genetically modified infectious agents, in a time frame shorter than previously thought possible. Also surprising is the success of using potentially high multiplicity of infection (MOI), or high concentrations of plaque-forming units (PFU), to incubate with test samples. Such high phage concentrations (PFU / mL) were previously asserted to be detrimental in bacterial detection assays because they were claimed to cause "lysis from without". However, high concentrations of phage can facilitate the discovery, binding, and infection of a small number of target cells.

[0030] The compositions, methods, systems, and kits of the invention can include infectious agents for use in detecting such microorganisms. In certain embodiments, the invention can include compositions comprising recombinant bacteriophages having an indicator gene inserted into the late gene region of the bacteriophage. In certain embodiments, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in the production of a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene (i.e., class III) region of the bacteriophage. The bacteriophage can be derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or Staphylococcus-specific bacteriophage, or another wild-type or engineered bacteriophage.

[0031] In some aspects, the invention includes a method for detecting a target microorganism. The method may 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 method may include detecting a target bacterium in a sample by incubating the sample with a recombinant bacteriophage that infects the target bacterium. In certain embodiments, the recombinant bacteriophage includes an indicator gene. The indicator gene may be inserted in a late gene region of the bacteriophage in certain embodiments, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in production of an indicator protein product. The method may include detecting the indicator protein product, where a positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the indicator protein is soluble.

[0032] In certain embodiments, the invention may include a system. The system may include at least some of the compositions of the invention. The system may also include at least some of the components for performing the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the invention may include a system for rapid detection of a target microorganism in a sample, the system including a component for incubating the sample with an infectious agent specific for the target microorganism, where the infectious agent includes an indicator moiety; and a component for detecting the indicator moiety. In yet other embodiments, the invention includes software for use with the method or system.

[0033] Accordingly, some embodiments of the present invention solve the need by using a bacteriophage-based method for amplifying a detectable signal indicative of the presence of bacteria. In certain embodiments, as few as about one bacterium is detected. The principles applied herein can be applied to the detection of various microorganisms. Since there are many binding sites for infectious agents on the surface of microorganisms, the ability to generate 100 or more progeny agents during infection, and the potential for high-level expression of the encoded indicator moiety, the infectious agent or indicator moiety can be more readily detectable than the microorganism itself. In this way, embodiments of the present invention can achieve a very large signal amplification from even a single infected cell.

[0034] Aspects of the present invention utilize the high specificity of binding factors that can bind to specific microorganisms, such as binding components of infectious agents, as a means of detecting and / or quantifying specific microorganisms in a sample. In some embodiments, the present invention utilizes the high specificity of an infectious agent (e.g., a bacteriophage).

[0035] In some embodiments, detection is achieved through an indicator moiety associated with a binding factor specific for the target microorganism. For example, the infectious agent can include an indicator moiety (e.g., 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 referred to as an indicator phage.

[0036] Some embodiments of the invention disclosed and described herein utilize the discovery that a single microorganism can bind a specific recognition factor (e.g., a phage). After infection and replication of the phage, progeny phage can be detected via an indicator moiety expressed during phage replication. This principle enables amplification of an indicator signal from one or a few cells based on specific recognition of a microbial surface receptor. For example, by exposing as few as a single cell of bacteria to multiple phage and then enabling high level expression of the encoded indicator gene product during amplification and replication of the phage, the indicator signal is amplified such that the single bacterium is detectable.

[0037] Embodiments of the methods and systems of the invention can be applied to the detection and quantification of various microorganisms (e.g., bacteria, fungi, yeast) in a variety of environments including, but not limited to, the detection of pathogens from food samples, water samples, clinical samples and commercial samples. In some embodiments, clinical samples can be analyzed for the presence of microorganisms. The methods of the invention provide high detection sensitivity and specificity quickly and without the need for traditional biological enrichment (e.g., culturing for enrichment). This is a surprising aspect as all available methods require culturing. In some embodiments, detection is possible within a single replication cycle of a bacteriophage, which is unexpected.

[0038] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention should have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, the singular terms should include the plural, and the plural terms should include the singular. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein, as well as these techniques, are well known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods well known in the art and as described in various general and more specific references discussed throughout this specification, unless otherwise indicated. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is well known and commonly used in the art.

[0039] The following terms should be understood to have the following meanings, unless otherwise indicated:

[0040] As used herein, the terms "a", "an", and "the" can refer to one or more, unless otherwise specifically noted.

[0041] The use of the term "or" is used to mean "and / or" unless it is clearly indicated that only the alternatives are being referred to or the alternatives are mutually exclusive, although the disclosure supports definitions that refer only to the alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0042] Throughout this application, the term "about" is used to indicate that a value includes the variability of the inherent error of the device, method used to determine the value, or the variability that exists between samples.

[0043] The term "solid support" or "support" means a structure that provides a substrate and / or surface to which a biomolecule can bind. For example, the solid support can be an assay well (i.e., for example, a microtiter plate or multiwell plate), or the solid support can be a position on a filter, an array, or a mobile support (e.g., beads) or a membrane (e.g., a filter plate or lateral flow strip).

[0044] The term "binding agent" refers to a molecule that can bind specifically and selectively to a second (i.e., different) target molecule. The interaction can be non-covalent as a result of, for example, hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction can be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or non-covalently) with a solid support.

[0045] As used herein, "analyte" refers to the molecule, compound or cell being measured. The analyte of interest can interact with a binding agent in certain embodiments. As described herein, the term "analyte" can refer to the protein or peptide of interest. The analyte can be an agonist, antagonist, or modulator. Alternatively, the analyte may not have a biological effect. Examples of analytes can include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, and organic compounds.

[0046] The terms "detectable moiety", "detectable biomolecule", "reporter", "indicator", or "indicator moiety" refer to a molecule that can be measured in a quantitative assay. For example, the indicator moiety can include an enzyme that can be used to convert a substrate into a measurable product. The indicator moiety can be an enzyme that catalyzes a reaction that results in the emission of bioluminescence (e.g., luciferase). Alternatively, the indicator moiety can be a radioisotope that can be quantified. Alternatively, the indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0047] As used herein, "bacteriophage" or "phage" includes one or more of a plurality of bacterial viruses. In the present disclosure, the terms "bacteriophage" and "phage" refer to viruses that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic-level living organisms, and that use the above organisms to replicate the virus itself, including mycobacteriophages (e.g., those related to TB and para-TB), mycophages (e.g., those related to fungi), mycoplasma phages, and any other terms, such as viruses. Here, "microscopic-level" means that the maximum dimension is 1 millimeter or less. Bacteriophages are viruses that have evolved to use bacteria in nature as a means of replicating them. Phages do this by attaching the phage itself to a bacterium, injecting its DNA (or RNA) into the bacterium, and inducing the bacterium to replicate the phage hundreds or even thousands of times. This is also referred to as phage amplification.

[0048] As used herein, the term "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 most abundantly expressed genes (e.g., the structural proteins that assemble into the bacteriophage particles). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, in phage T7, the late genes (synonymous with class III) are transcribed at times overlapping those of classes II and III, where class I (e.g., RNA polymerase) is transcribed early, at 4 - 8 minutes after infection, class II at 6 - 15 minutes, and the late genes are transcribed from 8 minutes after infection until lysis. A late promoter is a promoter that is naturally located and active in such a late gene region.

[0049] As used herein, "culturing for enrichment" refers to traditional culturing (e.g., incubation in a medium conducive to microbial growth), and should not be confused with other possible uses of the term "enrichment" (e.g., enrichment by removing the liquid components of a sample and concentrating the microorganisms contained therein), or other forms of enrichment that do not include the traditional promotion of microbial growth. Culturing for enrichment over a very short period can be used in some embodiments of the methods described herein, but if used at all, it is not essential and is much shorter than traditional culturing for enrichment.

[0050] As used herein, "recombinant" refers to genetic (i.e., nucleic acid) modification, typically done in a laboratory, to bring together genetic material not found together in other ways. This term is used interchangeably herein with the term "modified".

[0051] As used herein, "RLU" refers to the relative light units measured by a luminometer (e.g., GLOMAX® 96) or similar device that detects light. For example, detection of the reaction product between luciferase and a suitable substrate (e.g., NANOLUC® and NANO-GLO®) is often reported as the RLU detected.

[0052] As used herein, "time to result" refers to the total amount of time from the start of incubation of a sample to the generation of a result. The time to result does not include any confirmation test time. Data collection can be performed at any time after the result has been generated.

[0053] Sample Each of the embodiments of the methods and systems of the present invention can enable rapid detection and quantification of microorganisms in a sample. For example, the methods according to the present invention can be performed in a shortened period with excellent results.

[0054] Microorganisms detected by the methods and systems of the present invention include pathogens that are of natural, commercial, medical or veterinary concern. Such pathogens include Gram-negative bacteria, Gram-positive bacteria, and mycoplasma. Any microorganism for which a specific infectious agent has been identified can be detected by the methods of the present invention. Those skilled in the art will recognize that there are no limitations to the application of the methods of the present invention other than the availability of the specific infectious agent / microorganism pairs that are essential.

[0055] Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are pathogens transmitted in food or water. Bacterial cells detectable by the present invention include, but are not limited to, the following: all species of Salmonella, Escherichia coli, Cronobacter, all strains of Staphylococcus, all species of Listeria (including but not limited to L. monocytogenes), and all species of Campylobacter. Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are medically or veterinarily important pathogens. Such pathogens include, but are not limited to, the following: 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 invention include antibiotic-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)).

[0056] The sample can be an environmental sample, a food sample, or a water sample. Some embodiments can include medical samples or veterinary samples. The sample can be liquid, solid, or semi-solid. The sample can be a swab of a solid surface. Examples of samples include environmental materials (e.g., water samples), or filters derived from air samples or aerosol samples from a cyclone collector. The sample can be a sample of vegetables, meat, fish, poultry, peanut butter, processed foods, powdered infant formula, powdered milk, tea, starch, eggs, milk, cheese, or other dairy products. Examples of medical samples or veterinary samples include, but are not limited to, blood samples, sputum samples, cerebrospinal fluid samples, and fecal samples and various types of swabs. In some embodiments, the sample can be used directly in the detection method of the present invention without preparation, concentration, or dilution. For example, liquid samples (including, but not limited to, milk and juice) can be assayed directly. The sample can be diluted or suspended in a solution including, but not limited to, a buffered solution or a bacterial culture medium. A sample that is solid or semi-solid can be suspended in a liquid by finely chopping, mixing, or macerating the solid in the liquid. The sample should be maintained within a pH range that promotes bacteriophage attachment to host bacterial cells. The sample should also contain appropriate concentrations of divalent and monovalent cations (Na + Mg 2+ Ca2 + including, but not limited to). Preferably, the sample is maintained at a temperature that maintains the viability of any pathogen cells contained within the sample. Preferably throughout the detection assay, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. During the step in which the bacteriophage attaches to the bacterial cell, it is preferred to maintain the sample at a temperature that promotes bacteriophage attachment. During the step in which the bacteriophage replicates within the infected bacterial cell or lyses such infected cells, it is preferred to maintain the sample at a temperature that promotes bacteriophage replication and lysis of the host. Such temperature is at least about 25 degrees Celsius (°C), more preferably about 45 °C or less, and most preferably about 37 °C. It is also preferred that the sample be subjected to gentle mixing or agitation during bacteriophage attachment, replication, and cell lysis.

[0057] The assay may include various suitable control samples. For example, a control sample without bacteriophage or a control sample containing bacteriophage without bacteria can be assayed as a control for background signal levels.

[0058] Indicator bacteriophage As described in more detail herein, the compositions, methods, systems, and kits of the present invention may include infectious agents for use in the detection of pathogenic microorganisms. In certain embodiments, the present invention includes recombinant indicator bacteriophages, wherein the bacteriophage genome is genetically modified to include an indicator or reporter gene. In some embodiments, the present invention may include a composition comprising a recombinant bacteriophage having an indicator gene incorporated into the genome of the bacteriophage.

[0059] Recombinant indicator bacteriophages may contain 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 bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage. Since late genes encode structural proteins, they are generally expressed at higher levels than other phage genes. The late gene region may be a class III gene region and may contain a gene for the major capsid protein.

[0060] Some embodiments include designing (and optionally preparing) sequences for homologous recombination downstream of the major capsid protein gene. Other embodiments include designing (and optionally preparing) sequences for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequence contains a codon-optimized reporter gene following an untranslated region. The untranslated region may include a phage late gene promoter and a ribosome entry site.

[0061] In some embodiments, the indicator bacteriophage is derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 or a Staphylococcus-specific bacteriophage, or another bacteriophage having a genome with 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% homology to T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 or a Staphylococcus-specific bacteriophage. In some embodiments, the indicator phage is derived from a bacteriophage that is highly specific for a particular pathogenic microorganism. Genetic modifications can avoid deletions of wild-type genes, and thus, the modified phage can remain more similar to wild-type infectious agents than many commercially available phages. Bacteriophages derived from the environment can be more specific for bacteria found in the environment and thus can be genetically distinct from commercially available phages.

[0062] Furthermore, phage genes that are considered non-essential may have unrecognized functions. For example, a gene that appears non-essential may have an important function in increasing burst size, such as a subtle cleavage, fitting, or trimming function in assembly. Therefore, deleting a gene and inserting an indicator can be harmful. Most phages can package DNA that is several percent larger than their native genome. In this consideration, smaller indicator genes may be a more appropriate choice for modifying bacteriophages (especially bacteriophages with smaller genomes). The OpLuc and NANOLUC® proteins are only about 20 kDa (encoding approximately 500 - 600 bp), while FLuc is about 62 kDa (encoding approximately 1,700 bp). For comparison, the T7 genome is about 40 kbp, while the T4 genome is about 170 kbp, and the genome of a Staphylococcus-specific bacteriophage is about 157 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacterium (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be readily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, Promega's NANO-GLO® NANOLUC® in combination with an imidazopyrazinone substrate (furimazine) can provide a robust signal with low background.

[0063] In some indicator phage embodiments, the indicator gene can be inserted into the untranslated region to avoid disruption of functional genes while leaving the wild-type phage gene intact, which can result in greater fitness when infecting non-laboratory strain bacteria. Further, including stop codons 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 that a fusion protein that appears as a background signal (e.g., luciferase) that cannot be separated from the phage is produced at low levels.

[0064] Indicator genes can express various biomolecules. The indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferase can be used. In alternative embodiments, and as described in more detail herein, the luciferase is one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene such as NANOLUC®.

[0065] Accordingly, in some embodiments, the invention includes a genetically modified bacteriophage comprising 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. Using a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like a viral capsid protein, but also not shut off like an endogenous bacterial gene or even an early viral gene.

[0066] In some embodiments, the late promoter is the T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 promoter, or another phage promoter similar to the promoter found in a selected wild-type phage, i.e., without genetic modification. The late gene region can be the class III gene region, and the bacteriophage can be derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, a Staphylococcus or S. aureus-specific bacteriophage, or another natural bacteriophage having a genome with at least 70, 75, 80, 85, 90 or 95% homology to T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, a Staphylococcus or S. aureus-specific bacteriophage.

[0067] Genetic modifications to infectious agents can include the insertion, deletion, or substitution of small fragments of nucleic acid, substantial portions of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acid includes a non-natural sequence. The non-natural indicator gene can be inserted into the bacteriophage genome such that it is under the control of a bacteriophage promoter. In some embodiments, the non-natural 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 include the polypeptide of the wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the invention includes a method for detecting a bacterium of interest, the method including the step of incubating a test sample with such a recombinant bacteriophage.

[0068] In some embodiments, expression of an indicator gene in progeny bacteriophages after infection of a host bacterium results in a free soluble protein product. In some embodiments, the non-native indicator gene is not linked to a gene encoding a phage structural protein, so no fusion protein is produced. Unlike systems that use fusions of detection moieties to capsid proteins (i.e., fusion proteins), some embodiments of the present invention express a soluble indicator or reporter (e.g., soluble luciferase). In some embodiments, the indicator or reporter ideally does not include a bacteriophage structure. That is, the indicator or reporter is not attached to the phage structure. Thus, the gene for the indicator or reporter is not fused to other genes within the recombinant phage genome. This can greatly increase the sensitivity of the assay (down to a single bacterium), simplify the assay, and, in contrast to the hours required for additional purification steps required for constructs that produce detectable fusion proteins, allow the assay to be completed in less than one hour for some embodiments. Further, fusion proteins may be less active than soluble proteins due to protein folding constraints that can alter, for example, the conformation of an enzyme active site or access to a substrate.

[0069] Furthermore, by definition, the fusion protein limits the number of portions attached to the protein subunits in the bacteriophage. For example, using a commercially available system designed to act as a platform for the fusion protein, approximately 415 copies of the fusion portion, corresponding to approximately 415 copies of the gene 10B capsid protein, occur in each T7 bacteriophage particle. Without this constraint, the infected bacteria could be predicted to express more copies of the detection portion (e.g., luciferase) that can fit in the bacteriophage. Furthermore, a large fusion protein (e.g., a capsid-luciferase fusion) can inhibit the assembly of the bacteriophage particles and thus result in fewer bacteriophage progeny. Therefore, a soluble non-fusion indicator gene product may be preferred.

[0070] In some embodiments, the indicator phage encodes a reporter such as a detectable enzyme. The indicator gene product can produce light and / or be detectable by a color change. Various suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes can act as the indicator portion. In some embodiments, firefly luciferase is the indicator portion. In some embodiments, Oplophorus luciferase is the indicator portion. In some embodiments, NANOLUC® is the indicator portion. Luciferases made by other engineering techniques or other enzymes that generate a detectable signal can also be suitable indicator portions.

[0071] In some embodiments, the use of a soluble detection moiety obviates the need to remove contaminating parental phage from the lysate of the infected sample cells. When using a fusion protein system, any bacteriophage used to infect the sample cells has an attached detection moiety and cannot be distinguished from the daughter bacteriophage that also contains the detection moiety. Since the detection of sample bacteria relies on the detection of newly created (de novo synthesized) detection moieties, the use of fusion constructs requires additional steps to separate the old (parental) moieties from the newly created (daughter bacteriophage) moieties. This can be accomplished by washing the infected cells multiple times prior to completion of the bacteriophage life cycle, inactivating excess parental phage after infection by physical or chemical means, and / or chemically modifying the parental bacteriophage with a binding moiety (e.g., biotin), which can then be bound and separated (e.g., by streptavidin-coated sepharose beads). However, even when all of these attempts are used during removal, parental phage can remain when high concentrations of parental phage are used to ensure infection of a small number of sample cells, creating a background signal that can obscure the detection of signals from the progeny phage of the infected cells.

[0072] In contrast, when using the soluble detection moieties expressed in some embodiments of the present invention, purification of the parental phage from the final lysate is not required. This is because the parental phage does not have any attached detection moieties. Thus, any detection moieties present after infection must be newly created, indicating the presence of one or more infected bacteria. To take advantage of this benefit, generation and preparation of the parental phage can include purification of the phage from any free detection moieties generated during generation of the parental bacteriophage in a bacterial culture. Standard bacteriophage purification techniques can be used to purify some embodiments of the phages according to the present invention (e.g., sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography, and dialysis or derivative techniques (e.g., Amicon brand concentrators - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as part of the preparation of the recombinant phages of the present invention to separate the parental phage particles from contaminating luciferase proteins generated during growth of the phage in the bacterial host. In this way, the parental recombinant bacteriophages of the present invention are substantially free of any luciferase generated during production in the bacteria. Removal of the remaining luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with test samples.

[0073] In some embodiments of the modified bacteriophage, the late promoter (class III promoter, e.g., from T7, T4 or ViI) has a high affinity for the RNA polymerase of the same bacteriophage that transcribes the gene of the structural protein that assembles into the bacteriophage particle. These proteins are the most abundant proteins made by the phage as each bacteriophage particle contains dozens or hundreds of copies of these molecules. The use of viral late promoters can, optimally, ensure a high level of expression of the luciferase detection moiety. The use of a late viral promoter from which the indicator phage is derived, specific to or active under this of the original wild-type bacteriophage can further ensure optimal expression of the detection moiety. The use of standard bacterial (non-viral / non-bacteriophage) promoters can, in some cases, be detrimental to expression because these promoters are often down-regulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production). Thus, in some embodiments, the phage is preferably engineered to encode and express the soluble (free) indicator moiety using an arrangement within the genome that does not limit expression to the number of subunits of the phage structural components.

[0074] The compositions of the invention 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 composition can comprise a cocktail of different indicator phages that can encode and express the same or different indicator proteins. In some embodiments, the bacteriophage cocktail comprises at least two different types of recombinant bacteriophage.

[0075] Method for preparing an indicator bacteriophage Embodiments of a method for producing an indicator bacteriophage begin with the selection of a wild-type bacteriophage for genetic modification. Some bacteriophages are highly specific for target bacteria. This provides an opportunity for highly specific detection.

[0076] Thus, the method of the present invention utilizes the high specificity of a binding factor associated with an infectious agent that recognizes and binds to a particular microorganism of interest as a means of amplifying a signal and thereby detecting low levels of microorganisms (e.g., a single microorganism) present in a sample. For example, an infectious agent (e.g., a bacteriophage) specifically recognizes surface receptors of a particular microorganism and thus specifically infects those microorganisms. Thus, these infectious agents can be suitable binding factors for targeting the microorganism of interest.

[0077] A variety of infectious agents can be used. In alternative embodiments, bacteriophages, phages, mycobacteriophages (e.g., with respect to TB and para-TB), mycophages (e.g., with respect to fungi), mycoplasma phages, and any other virus that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic-level organisms can be used to target the microorganism of interest. For example, in one embodiment, when the microorganism of interest is a bacterium, the infectious agent can include a bacteriophage. For example, well-studied phages of E. coli include T1, T2, T3, T4, T5, T7, and lambda; other E. coli phages available in the ATCC collection include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1. As discussed herein, bacteriophages can replicate inside the bacterium to produce hundreds of progeny phages. Detection of the product of an indicator gene inserted into the bacteriophage genome can be used as a measure of the bacteria in the sample.

[0078] Some embodiments of the present invention utilize the binding specificity and high-level gene expression ability of recombinant bacteriophages for rapid and sensitive targeting to facilitate the detection of target bacteria by infection. In some embodiments, Staphylococcus or S. aureus-specific bacteriophages are genetically modified to contain a reporter gene. In some embodiments, the late gene region of the bacteriophage is genetically modified to contain a reporter gene. In some embodiments, the reporter gene is placed downstream of the major capsid gene. In other embodiments, the reporter gene is placed upstream of the major capsid gene.

[0079] Some embodiments of a method for preparing a recombinant indicator bacteriophage include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, preparing a homologous recombination plasmid / vector containing an indicator gene, transforming the homologous recombination plasmid / vector into the target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby causing homologous recombination between the plasmid / vector and the bacteriophage genome, and isolating specific clones of the recombinant bacteriophage.

[0080] Various methods for designing and preparing homologous recombination plasmids are known. Various methods for transforming bacteria with plasmids are known, including heat shock, F-pilus-mediated bacterial conjugation, electroporation, and other methods. Various methods for isolating specific clones after homologous recombination are also known. Some method embodiments described herein utilize specific strategies.

[0081] Accordingly, some embodiments of a method for preparing an indicator bacteriophage include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, determining a 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 for homologous recombination adjacent to the major capsid protein gene, the sequence including 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 for the transformed bacterium, infecting the transformed bacterium with the selected wild-type bacteriophage, thereby causing 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 further include repeating the limiting dilution step and the titer step as necessary until the recombinant bacteriophage represents a detectable proportion of the mixture after a first limiting dilution assay. For example, in some embodiments, the limiting dilution step and the titer step may be repeated until at least 1 / 30 of the bacteriophages in the mixture are recombinant before isolating a particular clone of the recombinant bacteriophage. A 1:30 recombinant:wild-type ratio is predicted to give an average of 3.2 transduction units (TUs) per 96 plaques (e.g., in a 96-well plate) in some embodiments. The initial ratio of recombinant phage to wild-type phage can be determined by performing a limiting dilution assay based on the TCID50 (50% tissue culture infective dose) as previously described in U.S. Application No. 15 / 409,258. By the Poisson distribution, a 1:30 ratio results in a 96% probability of observing at least 1 TU in any of the 96 wells.

[0082] Figure 1 shows a schematic diagram of the genomic structure of the recombinant bacteriophage of the present invention, the indicator phage T7SELECT® 415-Luc. For the embodiment shown in Figure 1, the detection portion is encoded by a firefly luciferase gene 100 inserted within a late (class III) gene region 110 that is expressed late in the viral life cycle. Since late genes encode structural proteins, they are generally expressed at higher levels than other phage genes. Thus, in the embodiment of the recombinant phage shown in Figure 1, the indicator gene (i.e., firefly luciferase) is inserted into the late gene region immediately following gene 10B (major capsid protein) and is a construct that includes the firefly luciferase gene 100. The construct shown in Figure 1 was designed to include stop codons 120 in all three reading frames to ensure that luciferase is not incorporated into the gene 10B product. Also, as shown in Figure 1, the construct may include a consensus T7 late promoter 130 that drives the transcription and expression of the luciferase gene. The construct may also include a composite untranslated region synthesized from several T7 UTRs 140. This construct ensures that soluble firefly luciferase is produced so that expression is not limited to the number of capsid proteins specific to the phage display system.

[0083] As noted herein, in certain embodiments, it may be preferable to utilize an infectious agent isolated from an environment suitable for the production of the infectious agent of the present invention. In this way, infectious agents specific for naturally occurring microorganisms can be produced.

[0084] For example, FIG. 2 shows the genome of bacteriophage SEA1, a natural phage having approximately 95% sequence homology with T4-related myovirus bacteriophage S16. The SEA1 bacteriophage was obtained from the lab of Francisco Martinez and whole-genome sequencing was performed using Illumina MiSeq and de novo sequence assembly. As discussed in the examples, the major capsid protein 220 and various other structural genes are within the late gene region 210 consisting of structural genes encoding virion proteins. Gene 57A 230, which encodes a chaperone for long tail fiber formation, is at the boundary of the late gene region. Since these virion proteins are expressed at very high levels, any gene inserted into this region can be predicted to have similar expression levels as long as the late gene promoter and / or other similar regulatory elements are used.

[0085] There are numerous known methods and products for preparing plasmids. For example, for preparing plasmids, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques can be used in combination. Synthetic plasmids can also be ordered commercially (e.g., GeneWiz). Cosmids can be used to selectively edit bacteriophage genomes, or the CRISPR / CAS9 system can also be used. Some embodiments of methods for preparing recombinant indicator bacteriophages involve designing a plasmid that can be readily recombined with a wild-type bacteriophage genome to generate a recombinant genome. In plasmid design, some embodiments involve the addition of a codon-optimized reporter gene, such as the luciferase gene. Some embodiments further involve the addition of elements to the upstream untranslated region. For example, in the design of a plasmid for recombination with a Staphylococcus or S. aureus-specific bacteriophage genome, an upstream untranslated region can be added between the sequence encoding the C-terminus of gp23 / major capsid protein and the start codon of the NANOLUC® reporter gene. The untranslated region can include a promoter, such as the T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, or SAPJV1 promoter. The untranslated region can also include a ribosome entry / binding site (RBS), also known as the "Shine Dalgarno sequence" in bacterial systems. Any one or both of these elements, or other untranslated elements, can be embedded within a short upstream untranslated region made up of a random sequence having approximately the same GC content as the rest of the phage genome. The random region should not contain an ATG sequence, as it would act as a start codon.

[0086] The composition of the present invention may contain various infectious agents and / or indicator genes. For example, FIG. 3 shows two homologous recombination plasmid constructs having the luciferase gene for two different phages having matching phage sequences of approximately 500 bp upstream and downstream of the insertion site to promote homologous recombination. NANOLUC® luciferase is inserted into the pBAV1k-T5-GFP plasmid backbone having an upstream untranslated region containing a phage late gene promoter and a ribosome entry site. The S. aureus phage recombination plasmid was constructed to insert NANOLUC® within the late gene region, but at a certain distance from the major capsid protein (MCP) due to stability issues.

[0087] The major capsid protein fragment 416-915 is part of the structural gene encoding the virion protein. Since these virion proteins are expressed at very high levels, any gene inserted into this region can be predicted to have a similar expression level as long as the late gene promoter and / or other similar regulatory elements are used.

[0088] In some embodiments, the indicator phage according to the present invention comprises a Staphylococcus - specific bacteriophage that is genetically engineered to contain a reporter gene such as a luciferase gene. For example, the indicator phage may be a Staphylococcus - specific bacteriophage whose genome contains the sequence of the NANOLUC® gene. The recombinant Staphylococcus - specific NanoLuc bacteriophage genome may further contain a promoter such as 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 bacteriophage is a Staphylococcus aureus - specific bacteriophage.

[0089] Thus, in embodiments of the recombinant phage produced as a result of recombination, the indicator gene (i.e., NANOLUC®) is inserted into the late gene region immediately downstream of the gene encoding the major capsid protein, thus giving rise to a recombinant bacteriophage genome containing the NANOLUC® gene. The construct may further comprise a consensus promoter of T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus-specific bacteriophage, ViI, or another late promoter, or another suitable promoter driving the transcription and expression of the luciferase gene. The construct may also include a composite untranslated region synthesized from several UTRs. This construct ensures that soluble luciferase is produced so that expression is not limited to the number of capsid proteins inherent in the phage display system.

[0090] Figure 4 shows the isolation of recombinant phage from a mixture of wild-type bacteriophage and recombinant bacteriophage resulting from homologous recombination.

[0091] In the first step 402, S. aureus bacteria transformed with a homologous recombination plasmid are infected with S. aureus bacteriophage K, resulting in progeny phages with a mixture of parental phages and recombinant phages having an approximate ratio of 186 wild type:1 recombinant phage 434. The resulting recombinant phage mix is diluted 404 into a 96-well plate 406 to give an average of 5 recombinant transduction units (TU) / plate (9.3 PFU / well). The 96-well plate is assayed for luciferase activity to identify wells 436 containing recombinant phages as compared to wells 440 containing wild-type bacteriophage. Bacteria 438 are added 408; for example, each well may contain approximately 50 μL of a turbid S. aureus culture. This allows the phages to replicate and produce the luciferase enzyme 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 wells may have been seeded with a single recombinant phage, and at this stage, the mixture may contain an enrichment exceeding the original 186:1 ratio, an approximate ratio of 9.3 wild-type phages:1 recombinant type. In one embodiment, one of five wells was found to contain soluble luciferase via a luciferase assay and contained phages at an approximate ratio of 2.4 total:1 recombinant type. If necessary (i.e., if the recombinant type:total ratio is lower than 1:30), progeny from this enriched culture 412 can be subjected to further limiting dilution assay(s) 414 to increase the ratio and determine the actual concentration of recombinant phage transduction units. For example, if the ratio is 1:384 recombinant type:PFU, approximately 5 recombinant TUs per 96-well plate 416 may be aliquoted from a previous positive well with 1920 (5×384 = 1920) contaminating total phages 414, resulting in an approximate seeding of 20 (1920 PFU / 96 wells = 20 PFU / well) of mainly wild-type phages per well of a second dilution assay plate 420. Any positive luciferase wells may have been seeded with a single recombinant type along with 19 wild-type phages.These wells can be analyzed for the presence of luciferase 442.

[0092] After addition of bacteria and incubation (e.g., 2 hours at 37°C) 418, soluble luciferase and phage are present in an approximate ratio of 20 total:1 recombinant 420. This ratio may be verified by TU50 titration for the recombinant and plaque assay for total PFU. Finally, the plaque assay can be performed to screen for recombinants expressing luciferase 446. A few individual plaques (e.g., n = 48) can be individually picked and screened for luciferase activity 436 in a third multi-well plate 426. In one embodiment, this approach should ensure that sufficient plaques are screened such that, based on the known ratio of recombinant to total phage, approximately three recombinants are in the mix of plaques being screened. One plaque is removed from the plate into each well of a 96-well plate 424, and a luciferase assay can be performed to determine which wells contain phage showing luciferase activity 442 426. Wells showing luciferase activity 428 represent pure recombinant phage 434, while wells without luciferase activity 430 represent pure wild-type phage 432.

[0093] The individual plaques can then be suspended in buffer (e.g., 100 μL TMS) or medium, and an aliquot (e.g., approximately 5 μL) can be added to wells containing a turbid S. aureus culture and assayed after incubation (e.g., about 45 minutes to 1 hour at 37°C). Positive wells are predicted to contain a pure culture of recombinant phage. Certain embodiments may include additional rounds of plaque purification.

[0094] Thus, as illustrated in FIG. 4, recombinant phages generated by homologous recombination of plasmids designed for recombination in the wild-type phage genome can be isolated from a mixture containing only 0.005% of the entire phage genome. After isolation, large-scale production can be carried out to obtain a high-titer recombinant indicator phage stock suitable for use in the S. aureus detection assay. Furthermore, cesium chloride isopycnic density gradient centrifugation can be used to separate phage particles from contaminating luciferase protein to reduce background.

[0095] Method of using an infectious agent to detect a microorganism As noted herein, in certain embodiments, the present invention may include methods for using infectious particles for detecting microorganisms. The methods of the present invention may be embodied in various ways.

[0096] In one embodiment, the present invention is a method for detecting a target bacterium in a sample, comprising the step of incubating the sample with a bacteriophage that infects the target bacterium, wherein the bacteriophage contains an indicator gene, such that expression of the indicator gene during bacteriophage replication after infection of the target bacterium results in a soluble indicator protein product, and the step of detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target bacterium in the sample.

[0097] In another embodiment, the present invention is a method for detecting a target antibiotic-resistant bacterium in a sample, comprising: (i) incubating the sample with at least one antibiotic to enrich bacteria that are resistant to the antibiotic; (ii) incubating the enriched sample with a bacteriophage that infects the target bacterium, wherein the bacteriophage contains an indicator gene, such that expression of the indicator gene during bacteriophage replication after infection of the target bacterium results in the production of a soluble indicator protein product; and (iii) detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target antibiotic-resistant bacterium in the sample.

[0098] In certain embodiments, the assay can be performed using general concepts that can be modified to accommodate different sample types or sizes and assay formats. Embodiments using the recombinant bacteriophages of the present invention (i.e., indicator bacteriophages) can enable rapid detection of specific bacterial strains in a total assay time of less than 1.5 hours, 2.0 hours, 2.5 hours, 3.0 hours, 3.5 hours, 4.0 hours, 4.5 hours, 5.0 hours, 5.5 hours, 6.0 hours, 6.5 hours, 7.0 hours, 7.5 hours, 8.0 hours, 8.5 hours, 9.0 hours, 9.5 hours, 10.0 hours, 10.5 hours, 11.0 hours, 11.5 hours, 12 hours, 12.5 hours, 13.0 hours, 13.5 hours, 14.0 hours, 14.5 hours, 15.0 hours, 15.5 hours, 16.0 hours, 16.5 hours, 17.0 hours, 17.5 hours, 18.0 hours, 18.5 hours, 19.0 hours, 19.5 hours or 20.0 hours, depending on the sample type, sample size and assay format. For example, the amount of time required can vary slightly shorter or longer depending on the strain of bacteriophage and the strain of bacteria detected in the assay, the type and size of the sample being tested, the conditions required for the viability of the target, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target contaminating bacteria.

[0099] Figure 5 shows a strategy using an indicator phage that produces soluble luciferase according to an embodiment of the present invention. 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. The expression of luciferase is driven by a viral capsid promoter (e.g., bacteriophage T7 or T4 late promoter), resulting in high expression. Since the parental phage does not contain luciferase, the luciferase detected in the assay must have originated from the replication of progeny phages during the infection of bacterial cells. Thus, generally, there is no need to separate the parental phage from the progeny phages.

[0100] In these experiments, at least a portion of sample 500 containing quantified bacterium 502 is placed in a spin column filter and centrifuged to remove the LB broth, and an appropriate multiplicity of phages 504 that have 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). Then, in addition to parental phages 504 and progeny phages 516 in the lysate, free luciferase 503 can be collected, for example, by centrifugation, and the level of luciferase in the filtrate can be quantified using a luminometer 518. Alternatively, a high-throughput method may be employed, in which the bacterial sample is applied to a 96-well filter plate, and after performing all of the operations listed above, luciferase can be directly assayed in the original 96-well filter plate without performing the final centrifugation step.

[0101] Figure 6 shows a filter plate assay for detecting a target bacterium using a modified bacteriophage according to an embodiment of the present invention. Briefly, a sample 616 containing the target bacterium 618 can be added to a well 602 of a multi-well filter plate 604 and concentrated by spinning 606 to remove liquid from the sample. A genetically modified phage 620 is added to the well and incubated for a sufficient time for adsorption 608 with additional medium, followed by infecting the target bacterium and allowing the phage life cycle to proceed 610 (e.g., about 45 minutes). Finally, a luciferase substrate is added and reacted with any luciferase 624 present. The resulting luminescence is measured with a luminometer 614 that detects luciferase activity 626.

[0102] In certain embodiments, the assay can be performed without concentrating bacteria on or near the capture surface. Figure 7 illustrates a "No Concentration Assay" for detecting a target bacterium using a modified bacteriophage according to an embodiment of the present invention. An aliquot of an indicator phage 714 is dispensed into individual wells 702 of a multi-well plate 704, and then an aliquot of a test sample containing bacteria 712 is added and incubated 706 for a sufficient period (e.g., 45 minutes at 37°C) for the phage to replicate and produce a soluble indicator 716 (e.g., luciferase). The plate wells 708 containing the soluble indicator and phage can then be assayed 710 (e.g., a luciferase assay) to measure the indicator activity 718 in the plate. 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 then assayed for luciferase activity.

[0103] In some embodiments, the sample can be enriched prior to testing by incubation under conditions that promote growth. In such embodiments, the enrichment period can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours, or longer, depending on the sample type and size.

[0104] In some embodiments, the indicator bacteriophage comprises a detectable indicator moiety, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by an amplified signal generated via the indicator moiety. Accordingly, the method can include detecting an indicator moiety generated during phage replication, where detection of the indicator indicates the presence of the bacterium of interest in the sample.

[0105] In one embodiment, the present invention is a method for detecting a bacterium of interest in a sample, the method comprising incubating the sample with a recombinant bacteriophage that infects the bacterium of interest, 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 after infection of the host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the bacterium of interest in the sample. In some embodiments, the amount of the detected indicator moiety corresponds to the amount of the bacterium of interest present in the sample.

[0106] As described in more detail herein, the methods and systems of the present invention can utilize a series of concentrations of parental indicator bacteriophages to infect bacteria present in a sample. In some embodiments, the indicator bacteriophages are added to the sample at a concentration sufficient to rapidly find, bind to, and infect target bacteria that are present in very low numbers in the sample, such as single cells. In some embodiments, the phage concentration can be sufficient to find, bind to, and infect target bacteria in less than 1 hour. In other embodiments, these events can occur in less than 2 hours, or less than 3 hours, after the addition of the indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubating step is higher than 1×10 5 PFU / mL, higher than 1×10 6 PFU / mL, or higher than 1×10 7 PFU / mL.

[0107] In certain embodiments, the recombinant infectious agent can be purified so as not to contain any residual indicator proteins that may be generated during the production of the infectious agent stock. Thus, in certain embodiments, the recombinant bacteriophage can be purified using cesium chloride isopycnic density gradient centrifugation prior to incubation with the sample. When the infectious agent is a bacteriophage, this purification can have the additional advantage of removing bacteriophages that do not have DNA (i.e., empty phages or "ghosts").

[0108] In some embodiments of the methods of the present invention, the microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or several target microorganisms 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 that assay vessel. Various embodiments of such assays are disclosed herein.

[0109] Test sample aliquots can be directly dispensed into the wells of a multi-well plate, an indicator phage can be added, and after a sufficient period for infection, a lysis buffer can be added, as well as a substrate for the indicator moiety (e.g., a luciferase substrate for a luciferase indicator), and assayed for detection of the indicator signal. 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.

[0110] For example, in many embodiments, a multi-well plate is used to perform the above assay. The choice of plate (or any other container on which the detection step can be performed) can affect the above detection step. For example, some plates can include a colored or white background that can affect the detection of light emission. Generally, white plates have higher sensitivity but also produce a higher background signal. Other colors of plates can produce a lower background signal but can have slightly lower sensitivity. Additionally, one reason for the background signal is light leakage from one well to another adjacent well. There are some plates with white wells, while the rest of the plates are black. This allows for a high signal inside the wells but prevents light leakage from well to well and thus can reduce the background. Thus, the choice of plate or other assay container can affect the sensitivity and background signal for the above assay.

[0111] The method of the present invention may include various other steps for increasing sensitivity. For example, as discussed in more detail herein, the method may include a step of washing the captured and infected bacteria after adding the bacteriophage but before incubating to remove excess parent bacteriophage and / or luciferase or other reporter proteins contaminating the bacteriophage preparation.

[0112] In some embodiments, detection of the target microorganism can be accomplished without the need to culture the sample as a method 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 in food and environmental testing for pathogens.

[0113] In contrast to assays known in the art, the methods of the invention can detect individual microorganisms. Thus, in certain embodiments, the method can detect ≤10 cells of a microorganism present in a sample (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 microorganisms). For example, in certain embodiments, the recombinant bacteriophage is highly specific for Staphylococcus. In some embodiments, the Staphylococcus species is S. aureus. In further embodiments, S. 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 yet 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 specific type of single bacterium in a sample. In certain embodiments, the recombinant bacteriophage detects specific bacteria in amounts as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 in a sample.

[0114] As noted herein, in certain embodiments, the invention can include methods of using recombinant bacteriophage to detect the resistance of a microorganism to an antibiotic or, in another method described, to detect the effectiveness of an antibiotic against a microorganism. In another embodiment, the invention includes methods for selecting an antibiotic for the treatment of an infectious disease. Further, the method can include methods for detecting antibiotic-resistant bacteria in a sample. The methods of the invention can be embodied in various ways.

[0115] The method may include contacting a sample containing a microorganism with an antibiotic and an infectious agent as described above. In some embodiments, the present disclosure is a method for determining an effective dose of an antibiotic in killing a microorganism or inhibiting the growth of a microorganism, comprising: (a) separately incubating each of one or more antibiotic solutions with one or more samples containing a microorganism, wherein the concentrations of the one or more antibiotic solutions are different and define a range; (b) incubating the microorganism in the one or more samples with an infectious agent containing an indicator gene, 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 antibiotic solution used to treat one or more of the plurality of samples is not effective, and the absence of detection of the indicator protein indicates that the antibiotic is effective, thereby determining an effective dose of the antibiotic.

[0116] 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 and then 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 and then contacting the sample 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, and 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 resistant to the antibiotic can survive and proliferate, and the infectious agent specific to the microorganism is replicated; conversely, microorganisms sensitive to the antibiotic die and thus the infectious agent is not replicated. The infectious agent by this method includes an indicator moiety, the amount of which corresponds to the amount of microorganisms present in the sample treated with the antibiotic. Thus, a positive detection of the indicator moiety indicates that the microorganism is resistant to the antibiotic.

[0117] 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 sensitive to a particular antibiotic. A clinical sample obtained from the patient can then be incubated with an antibiotic specific for S. aureus. The sample can then be incubated for a period of time with a recombinant phage specific for S. aureus. In a sample having S. aureus resistant to the antibiotic, detection of the indicator protein produced by the recombinant phage will be positive. In a sample having S. aureus sensitive to the antibiotic, detection of the indicator protein will be negative. In some embodiments, the method for detection of antibiotic resistance can be used to select an effective therapeutic agent to which the pathogenic bacterium is sensitive.

[0118] 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 varies depending on the bacterium of interest, the type of phage, and the antibiotic being tested.

[0119] Optionally, the method further comprises lysing the microorganism prior to detecting the indicator portion. Any solution capable of lysing the microorganism may be used. In some cases, the lysis buffer may contain a non-ionic detergent, a chelating agent, an enzyme, or a unique combination of various salts and factors. Lysis buffers are also commercially available from Promega, Sigma-Aldrich, or Thermo-Fisher. Experiments suggest that infected, unlysed cells may be detectable upon addition of the luciferase substrate in some embodiments. Perhaps luciferase can exit the cell and / or the luciferase substrate can enter the cell without complete cell lysis. Thus, for embodiments utilizing a spin filter system, lysis is required for detection if only the luciferase released into the lysate (and not the luciferase still within intact bacteria) is analyzed in the luminometer. However, as described below, lysis may not be required for detection in embodiments utilizing a filter plate or 96-well plate with phage-infected samples in solution or suspension that can be directly assayed in the luminometer for both intact and lysed cells. Thus, in some embodiments, the method for detecting antibiotic resistance does not involve lysis of the microorganism.

[0120] A surprising aspect of the assay embodiments is that the step of incubating the microorganisms in the sample with the infectious agent only requires a time sufficient for a single life cycle of the infectious agent, such as a phage. The amplification power of using phages was previously thought to require more time for the phages to replicate over several cycles. A single replication of the indicator phage can be sufficient to facilitate sensitive and rapid detection according to some embodiments of the present invention. Another surprising aspect of the assay embodiments is that a high concentration of phages (i.e., high MOI) utilized to infect the test sample has successfully detected a very small number of antibiotic-resistant target microorganisms treated with antibiotics. Several factors, including the burst size of the phage, can affect the number of phage life cycles and thus the amount of time required for detection. Phages with a large burst size (approximately 100 PFU) may only require 1 cycle for detection, while phages with a smaller burst size (e.g., 10 PFU) may require multiple phage cycles for detection. In some embodiments, the incubation of the phage with the test sample only requires a time sufficient for a single phage life cycle. In other embodiments, the incubation of the phage with the test sample is an incubation over a time period longer than a single life cycle. The phage concentration in the incubation step varies depending on the type of phage used. In some embodiments, the phage concentration in this incubation step is higher than 1.0×10 5 PFU / mL, higher than 1.0×10 6 PFU / mL, higher than 1.0×10 7 PFU / mL, or higher than 1.0×10 8It is higher than PFU / mL. Such success at high concentrations of phage is surprising because many of the phages have been previously associated with "non-infectious lysis", which killed the target cells immediately, thereby preventing the generation of useful signals from earlier phage assays. The purification of the phage stock described herein (e.g., by cesium chloride isopycnic density gradient ultracentrifugation) may help to mitigate this problem because in addition to removing any contaminating luciferase associated with the phage, this purification can also remove ghost particles (particles that have lost their DNA). Ghost particles can lyse bacterial cells via "non-infectious lysis", killing the cells prematurely and thereby preventing the generation of an indicator signal. Electron microscopy clearly shows that crude recombinant phage lysates (i.e., before cesium chloride purification) can have more than 50% ghosts. These ghost particles can contribute to the premature death of the microorganism through the action of many phage particles that puncture the cell membrane. Thus, ghost particles may have been a factor in the previous problem where high PFU concentrations were reported to be detrimental.

[0121] Any of the indicator moieties described in this disclosure can be used to detect the viability of a microorganism after antibiotic treatment and thereby detect antibiotic resistance. In some embodiments, an indicator moiety associated with an infectious agent can be detectable 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 endogenous signal, such as a fluorescent protein (e.g., green fluorescent protein, etc.). The indicator can generate light and / or be detectable by a change in color. In some embodiments, a luminometer can be used to detect the indicator (e.g., luciferase). However, other machines or devices may also be used. For example, a spectrophotometer, a CCD camera, or a CMOS camera can detect color changes and other light emissions.

[0122] In some embodiments, the exposure of the sample to the antibiotic can last for 5 minutes or longer, and detection at various time points can be desirable to optimize sensitivity. For example, aliquots of the primary sample treated with the antibiotic can be taken at different time intervals (e.g., 5 minutes, 10 minutes, or 15 minutes). Samples from the various time intervals can then be infected with phage, and the indicator moiety can be measured after the addition of the substrate.

[0123] In some embodiments, the detection of the signal is used to determine antibiotic resistance. In some embodiments, the signal generated by the sample is compared to an experimentally determined value. In further embodiments, the experimentally determined value is the signal generated by a control sample. In some embodiments, the background threshold is determined using a control that does not contain microorganisms. In some embodiments, the experimentally determined value is a background threshold calculated from the mean background signal + 1 to 3 times or greater than the standard deviation of the mean background signal. In some embodiments, the background threshold can be calculated from the mean background signal + 2 times the standard deviation of the mean background signal. In other embodiments, the background threshold can be calculated from several multiples (e.g., 2 or 3) of the mean background signal. Detection of a sample signal that exceeds the background threshold indicates the presence of one or more antibiotic-resistant microorganisms in the sample. For example, the mean background signal can be 250 RLU. The threshold background value can be calculated by multiplying the mean background signal (e.g., 250) by 3 to calculate a value of 750 RLU. A sample with bacteria having a signal value exceeding 750 RLU is determined to be positive for the presence of antibiotic-resistant bacteria.

[0124] Alternatively, the experimentally determined value is the signal generated by the control sample. The assay can include various appropriate control samples. For example, a sample that does not contain an infectious agent specific to the microorganism, or a sample that contains an infectious agent but does not contain the microorganism, can be assayed as a control for the background signal level. In some cases, a sample containing microorganisms that have not been treated with an antibiotic is assayed as a control for determining antibiotic resistance using an infectious agent.

[0125] In some embodiments, the sample signal is compared to the control signal to determine whether an antibiotic-resistant microorganism is present in the sample. No change in the detection of the signal compared to a control sample that has been contacted with the infectious agent but not with the antibiotic indicates that the microorganism is resistant to the antibiotic, and a decrease in the detection of the indicator moiety compared to a control sample that has been contacted with the infectious agent but not with the antibiotic indicates that the microorganism is sensitive to the antibiotic. No change in detection means that the signal detected from the sample treated with the antibiotic and the infectious agent is at least 80%, at least 90%, or at least 95% of the signal from the control sample that has not been treated with the antibiotic. A decrease in detection means that the signal detected from the sample treated with the antibiotic and the infectious agent is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or at least 30% of the signal from the control sample that has not been treated with the antibiotic.

[0126] Optionally, the sample containing the microorganism of interest is an uncultured sample. Optionally, the infectious agent is a phage that contains an indicator gene inserted into the 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. As described above, each feature of the composition used in the method can also be utilized in a method for detecting the antibiotic resistance of a microorganism of interest.

[0127] Methods for determining the effective dose of an antibiotic for killing microorganisms are also provided herein. In some embodiments, the antibiotic is effective for killing Staphylococcus species. For example, the antibiotic can be cefoxitin, which is effective against most methicillin-sensitive S. aureus (MSSA). Typically, one or more antibiotic solutions having different concentrations are prepared such that different concentration ranges of the solutions are defined. In some cases, the concentration ratio of the least concentrated antibiotic solution to the most concentrated antibiotic solution is in the range of 1:2 to 1:50, such as 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 μg / mL, such as at least 2 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 20 μg / mL, at least 40 μg / mL, at least 80 μg / mL, or at least 100 μg / mL. Each of the one or more antibiotic solutions is incubated with one aliquot of a sample containing the microorganism of interest. In some cases, an infectious agent that is specific for the microorganism and contains an indicator moiety is added simultaneously with the antibiotic solution. In some cases, the aliquot of the sample is incubated with the antibiotic solution for a certain period of time prior to the addition of the infectious agent. The indicator moiety can be detected, and a positive detection indicates that the antibiotic solution is not effective, while a negative detection indicates that the antibiotic solution is effective and that the concentration of the antibiotic solution is the effective dose.Thus, in some embodiments, a method for determining an effective dose of an antibiotic in killing a target microorganism comprises the steps of 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 microorganisms 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 one or more of the one or more samples is ineffective, and the absence 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 of the lowest and highest concentration solutions in the one or more antibiotic solutions ranges from 1:2 to 1:50, such as 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 μg / mL, such as at least 2 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 20 μg / mL, at least 40 μg / mL, at least 80 μg / mL, or at least 100 μg / mL.

[0128] In some embodiments, the present invention includes a method for detecting an antibiotic-resistant microorganism in the presence of an antibiotic-sensitive microorganism. In certain cases, the detection of antibiotic-resistant bacteria can be used to prevent the spread of infection in a medical setting. In some embodiments, patients in a medical setting can be monitored for colonization with antibiotic-resistant bacteria. Preventive measures can then be implemented to prevent the spread of antibiotic-resistant bacteria.

[0129] In some embodiments of the method for detecting antibiotic-resistant microorganisms, the sample can contain both antibiotic-resistant bacteria and antibiotic-sensitive bacteria. For example, the sample can contain 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 an antibiotic, MSSA does not generate a signal exceeding the threshold, while MRSA present in the sample can generate a signal exceeding the threshold. Thus, when both are present in the sample, a signal exceeding the threshold indicates the presence of an antibiotic-resistant strain (e.g., MRSA).

[0130] Accordingly, aspects of the present invention provide a method for detecting microorganisms in a test sample via an indicator moiety. In some embodiments, when the microorganism of interest is a bacterium, the indicator moiety can be associated with an infectious agent (e.g., an indicator bacteriophage). The indicator moiety can react with a substrate to emit a detectable signal or can emit an endogenous 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, as few as 1 cell of the microorganism of interest can generate a detectable signal. In some embodiments, the bacteriophage is a T4-like or ViI-like bacteriophage. In some embodiments, the recombinant bacteriophage is derived from a Staphylococcus-specific bacteriophage. In certain embodiments, the recombinant Staphylococcus-specific bacteriophage is highly specific for Staphylococcus.

[0131] In some embodiments, the indicator portion encoded by the infectious agent may be detectable during or after replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicator portions are known in the art and many are commercially available. In some embodiments, the indicator phage comprises an enzyme, which serves as the indicator portion. 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 may be detectable by emitting light and / or by a change in color. A variety of suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes may serve as the indicator portion. In some embodiments, firefly luciferase is the indicator portion. In some embodiments, Oplophorus luciferase is the indicator portion. In some embodiments, NANOLUC® is the indicator portion. Luciferases made by other engineering techniques or other enzymes that generate a detectable signal may also be suitable indicator portions.

[0132] Thus, in some embodiments, the recombinant bacteriophage of the method, system or kit is prepared from a wild-type Staphylococcus-specific bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an endogenous signal, such as a fluorescent protein (e.g., green fluorescent protein, etc.). The indicator can emit light and / or be detectable by a change in color. In some embodiments, the indicator gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to generate a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is Oplophorus luciferase, firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase or a luciferase made by engineering techniques, such as NANOLUC®, Rluc8.6-535 or orange nano-lantern.

[0133] The step of detecting the indicator may include the step of detecting the emission of light. In some embodiments, a luminometer can be used to detect the reaction of an indicator (e.g., luciferase) with a substrate. Detection of RLU can be achieved 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 a change in color and other light emissions. Although absolute RLU is important for detection, a high signal-to-background ratio (e.g., >2.0, >2.5 or >3.0) is also required to ensure the detection of a single cell or a small number of cells.

[0134] In some embodiments, the indicator phage is genetically engineered to contain a gene for an enzyme (e.g., luciferase) that is produced only upon infection of bacteria that the phage specifically recognizes and infects. In some embodiments, the indicator moiety is expressed late in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits its copy number.

[0135] Accordingly, in some embodiments utilizing an indicator phage, the invention encompasses a method for detecting a target microorganism, the method comprising the steps of capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phages; allowing time for infection and replication to produce progeny phages and express a soluble indicator moiety; and detecting the progeny phages, preferably the indicator, wherein detection of the indicator comprises indicating the presence of the bacterium in the sample.

[0136] For example, in some embodiments, test sample bacteria can be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a spin filter or plate filter with a 0.45 μm pore size). In one embodiment, the infectious agent (e.g., an indicator phage) is added in a minimal volume directly to the sample captured on the filter. In one embodiment, the microorganisms captured on the filter or plate surface are then washed one or more times to remove any unbound excess infectious agent. In one embodiment, a medium (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 over a further incubation time to allow replication of bacterial cells and phages and high - level expression of the gene encoding the indicator moiety. However, a surprising aspect of some embodiments of the test assay is that only the incubation step with the indicator phage needs to be long enough for a single phage life cycle. The amplifying power of using bacteriophages was previously thought to require more time for the phages to replicate over several cycles. A single replication cycle of the indicator phage can be sufficient to facilitate sensitive and rapid detection according to some embodiments of the present invention.

[0137] In some embodiments, an aliquot of a test sample containing bacteria can be applied to a spin column, and after infection with a recombinant bacteriophage and optional washing to remove any excess bacteriophage, the amount of soluble indicator detected is proportional to the amount of bacteriophage produced by the infected bacteria.

[0138] The soluble indicator (e.g., luciferase) released into the surrounding liquid upon lysis of the bacterium can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter and the filtrate is collected in a new container for assay (e.g., in a luminometer), and then a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

[0139] In various embodiments, the purified parent indicator phage does not contain the detectable indicator itself. This is because the parent phage can be purified before it is used for incubation with the test sample. Expression of late (class III) genes occurs late in the viral life cycle. In some embodiments of the present invention, the parent phage can be purified to eliminate any existing indicator protein (e.g., luciferase). In some embodiments, expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. Thus, in many embodiments, it is not essential to separate the parent 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 soluble luciferase, which is released upon lysis of the host microorganism.

[0140] Thus, in an alternative embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with a portion of the sample that remains on the filter or remains bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a normal 96-well plate) and the substrate reaction can be detected by placing the plate directly into the luminometer.

[0141] For example, in one embodiment, the present invention is a method for detecting MRSA, comprising the steps of infecting cells captured on a 96-well filter plate with a plurality of parental indicator phages capable of expressing luciferase upon infection, washing away excess phage, adding LB broth containing an antibiotic to allow a time (e.g., 30-90 minutes) for the phage to replicate and lyse a specific Staphylococcus aureus target, and adding a luciferase substrate and detecting the indicator luciferase by directly measuring the luciferase activity in the 96-well plate, wherein detection of the luciferase activity includes indicating the presence of MRSA in the sample.

[0142] In some embodiments, lysis of the bacteria can occur before, during, or after the detection step. Experiments suggest that infected but unlysed cells can be detectable in some embodiments upon addition of the luciferase substrate. Perhaps luciferase can exit the cell and / or the luciferase substrate can enter the cell without complete cell lysis. Thus, for embodiments utilizing a spin filter system, lysis is required if only the luciferase released into the lysate (and no luciferase is further present in intact bacteria) is analyzed in the luminometer. However, for embodiments utilizing a filter plate or 96-well plate with a sample of solution or suspension, lysis is not essential for detection if the original plate filled with intact and lysed cells is assayed directly in the luminometer.

[0143] In some embodiments, the reaction between the indicator moiety (e.g., luciferase) and the substrate can continue over 30 minutes or longer, and detection at various time points can be desirable to optimize sensitivity. For example, in embodiments using a 96-well filter plate as the solid support and luciferase as the indicator, luminometer readings can be taken initially and at 10 or 15 minute intervals until the reaction is complete.

[0144] Surprisingly, high concentrations of phage utilized to infect test samples successfully achieved detection of very few target microorganisms in a very short time frame. Incubating the phage with the test sample in some embodiments requires only a time sufficient for a single phage life cycle. In some embodiments, the concentration of bacteriophage for this incubating step is 7×10 6 、8×10 6 、9×10 6 、1.0×10 7 、1.1×10 7 、1.2×10 7 、1.3×10 7 、1.4×10 7 、1.5×10 7 、1.6×10 7 、1.7×10 7 、1.8×10 7 、1.9×10 7 、2.0×10 7 、3.0×10 7 、4.0×10 7 、5.0×10 7 、6.0×10 7 、7.0×10 7 、8.0×10 7 、9.0×10 7 、or higher than 1.0×10 8 PFU / mL.

[0145] Such success of phages at such high concentrations is surprising because many of the phages were previously associated with "non-infectious lysis" which killed the target cells, thereby preventing the generation of useful signals from earlier phage assays. The clean-up of the prepared phage stocks described herein is thought to help mitigate this problem (e.g., purification by cesium chloride isopycnic density gradient ultracentrifugation). This is because in addition to removing any contaminating luciferase associated with the phage, this purification can also remove ghost particles (particles that have lost their DNA). The ghost particles can lyse bacterial cells via "non-infectious lysis", killing the cells prematurely and thereby preventing the generation of an indicator signal. Electron microscopy clearly shows that crude phage lysates (i.e., before cesium chloride purification) can have more than 50% ghosts. These ghost particles can contribute to the premature death of the microorganisms through the action of many phage particles that puncture the cell membrane. Thus, ghost particles may have been a contributing factor to the previous problem where high PFU concentrations were reported to be detrimental. Furthermore, a very clean phage preparation allows the assay to be performed without a washing step, which in turn allows the assay to be performed without an initial concentration step. Some embodiments include an initial concentration step, and in some embodiments, this concentration step allows for a shorter enrichment incubation time.

[0146] Some embodiments of the test method may further include a confirmation assay. To confirm initial results at a later time point, various assays are known in the art. For example, a sample can be cultured (e.g., the CHROMAGAR™ / DYNABEADS™ assay described in Example 4), PCR can be utilized to confirm the presence of microbial DNA, or other confirmation assays can be used to confirm the initial results.

[0147] In certain embodiments, the methods of the invention combine, in addition to detection of infectious agents, the use of binding factors (e.g., antibodies) to purify and / or concentrate the microorganism of interest from a sample. For example, in certain embodiments, the invention provides a method for detecting a microorganism of interest in a sample, the method comprising capturing microorganisms from the sample on a support onto the capture antibody specific for the microorganism of interest, incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the microorganism of interest in the sample.

[0148] For example, FIG. 8 illustrates a Hybrid Immuno-Phage (HIP) assay for detecting a bacterium of interest using a modified bacteriophage according to an embodiment of the invention. A sample is first applied 802 to a microtiter plate that is well-coated with a bacterium-specific antibody. The plate is then centrifuged 804 to facilitate binding of the bacteria to the capture antibody. After sufficient time has elapsed to allow complete bacterium capture, a solution containing the bacterium-specific NANOLUC® phage is added 806 to each sample. Incubation with the phage results 808 in binding and attachment of the single or multiple phages to the captured bacteria. Finally, the samples are incubated 810 to promote phage replication and luciferase expression, leading to cell lysis and release of soluble luciferase.

[0149] Recent advances in synthetic biology have increased interest in bacteriophage-based therapeutic approaches for treating pathogenic diseases (see, e.g., Phage Therapy in the Era of Synthetic Biology, Cold Spring Harb Perspect Biol 2016; 8:a023879; as well as U.S. Patent No. 9,597,407, and U.S. Patent Application Nos. 20170266306 and 20160331804, the contents of which are incorporated herein by reference as if fully set forth herein. Bacteriophages designed and engineered to detect pathogens in a medical sample from a patient for the presence of a particular microorganism, such as a particular type of bacterium, can enhance the usefulness of such therapeutic phages.

[0150] In some embodiments, an indicator phage can be utilized to test an initial sample from a patient for the presence of a particular pathogen, e.g., a bacterium of a particular genus or species. In some embodiments, an indicator phage can be used to detect a particular pathogen in a clinical sample. In this way, an indicator phage can be used in a manner similar to a companion diagnostic to evaluate the potential effectiveness of a particular treatment, such as a particular antibiotic, other drug, or therapeutic phage, e.g., in the context of an infectious or other pathogenic medical condition of a given patient. In some embodiments, a diagnostic indicator phage can be prepared by genetic modification of a naturally occurring bacteriophage, as described above.

[0151] In some embodiments, indicator phages prepared through synthetic techniques can be used for non-clinical applications. For example, the indicator phage can be used as a food safety diagnosis to identify the presence of specific bacteria in food. In other embodiments, diagnostic indicator phages can be prepared through synthetic techniques. For example, synthetic phage genomes can be designed and constructed for the transformation and propagation of the corresponding phages in various types of bacteria. In some cases, synthetic biology techniques can be used to generate indicator phages using indicator phage target bacteria. In other cases, more convenient bacteria can be used to generate indicator phages. For example, a synthetic genome for an indicator phage targeting Listeria can be generated in Listeria, or a more convenient species (e.g., Lactobacillus).

[0152] In some embodiments, synthetic phages are designed to optimize desired traits for use in pathogen detection assays. In some embodiments, prior analysis of bioinformatics and genetic modification is used to optimize the 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 an entire genus of bacteria, or a specific group of species within a genus. 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 the 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.

[0153] In some embodiments, the stability of the phage can be optimized to improve the shelf life. For example, the enzybiotic solubility may be increased to enhance subsequent phage stability. Additionally and / or alternatively, the thermal stability of the phage may be optimized. Thermostable phages better preserve their functional activity during storage, thereby improving the shelf life. Thus, in some embodiments, the thermal stability and / or pH tolerance can be optimized.

[0154] Some species of bacteria construct biofilm walls to protect themselves from attack by the immune system. These biofilms can make it difficult to effectively target the bacteria. Some enzymes (e.g., glycoside hydrolases PelAh and PslGh) that can degrade bacterial biofilms have been identified. In some embodiments, the phage can be modified to encode either a soluble or a fusion virion protein such that the biofilm is degraded by the incorporation of the enzyme.

[0155] In some embodiments, the genetically modified or synthetically derived phage contains a detectable indicator. In some embodiments, the indicator is luciferase. In some embodiments, the phage genome contains an indicator gene (e.g., a luciferase gene or another gene encoding a detectable indicator).

[0156] In another aspect, the invention can include a method for selecting a treatment for a subject, the method including: (i) obtaining a biological sample from the subject; (ii) using an indicator phage to detect a specific microorganism or category of microorganisms in the biological sample; and (iii) selecting a treatment based on the identity of the specific microorganism detected in the biological sample.

[0157] In some embodiments, the indicator phage can be used to detect pathogens in a patient sample and subsequently initiate several types of treatment, whether prepared synthetically or not. In some embodiments, the treatment can be a phage-based therapeutic. In other embodiments, the treatment can be an antibiotic (e.g., a conventional antibiotic such as penicillin or cyclosporine). In other embodiments, the treatment can be another type of drug or therapy. In this way, the indicator phage can be used to monitor the progress or effectiveness of any type of treatment or therapy. In some embodiments, the indicator phage can be used to detect and monitor the pathogen content of a patient sample taken hours or days after the start of treatment. In some embodiments, the indicator phage can be used to monitor samples in the context of chronic infections, which can be days, weeks, months, or years after the start of treatment.

[0158] The systems and kits of the present invention In some embodiments, the present invention includes a system (e.g., an automated system or kit) that includes components for performing the methods disclosed herein. In some embodiments, the systems or kits according to the present invention include an indicator phage. The methods described herein can also utilize such indicator phage systems or kits. Some of the embodiments described herein are particularly suitable for automation or kits, considering the minimal amounts of reagents and materials required to perform the above methods. In certain embodiments, each of the components of the above kit can include a self-sufficient unit that is deliverable from a first site to a second site.

[0159] In some embodiments, the present invention includes a system or kit for rapid detection of a target microorganism in a sample. The system or kit, in certain embodiments, comprises components for incubating the sample with an infectious agent specific to the target microorganism, where the infectious agent may comprise a component including an indicator portion, and a component for detecting the indicator portion. In some embodiments of both the system and kit of the present invention, the infectious agent is a recombinant bacteriophage that infects the target bacterium, and the recombinant bacteriophage comprises an indicator gene inserted into a late gene region of the bacteriophage as the indicator portion, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. Some systems further comprise components for capturing the target microorganism on a solid support.

[0160] In other embodiments, the present invention is a method, system, or kit for rapidly detecting a target microorganism in a sample, the method, system, or kit comprising an infectious agent component specific to the target microorganism, wherein the infectious agent comprises an indicator portion, and a component for detecting the indicator portion. 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 bacterium. 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 type of specific bacterium in a sample. In some embodiments, the system or kit detects specific bacteria in an amount of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 in the sample.

[0161] In certain embodiments, the system and / or kit may further comprise a component for washing the captured microorganism sample. Additionally or alternatively, the system and / or kit may further comprise a component for determining the amount of the indicator portion, wherein the amount of the indicator portion detected corresponds to the amount of the microorganism in the sample. For example, in certain embodiments, the system or kit may include a luminometer or other device for measuring luciferase enzyme activity.

[0162] 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 via computer input by a user and / or a liquid handling robot performs at least one step.

[0163] Accordingly, in certain embodiments, the present invention may include a system or kit for rapid detection of a target microorganism in a sample, the system or kit including components for incubating the sample with an infectious agent specific for the target microorganism, wherein the infectious agent includes an indicator portion; components for capturing the microorganism from the sample onto a solid support; components for washing the captured microorganism sample to remove unbound infectious agent; and components for detecting the indicator portion. In some embodiments, the same component can be used for the capturing step and / or the incubating step and / or the washing step (e.g., a filter component). Some embodiments further include components for determining the amount of the target microorganism in the sample, wherein the amount of the indicator portion detected corresponds to the amount of microorganism in the sample. Such a system may include various embodiments and sub-embodiments similar to those described above with respect to the method for rapid detection of microorganisms. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage. In a computerized system, the system may be fully automated, semi-automated, or directed by a user via a computer (or some combination thereof).

[0164] In some embodiments, the system may include components for isolating the target microorganism from other components in the sample.

[0165] In one embodiment, the present invention is a system or kit comprising components for detecting a target microorganism, the components including: components for isolating at least one microorganism from other components in a sample; components for infecting at least one microorganism with a plurality of parental infectious agents; components for lysing at least one infected microorganism to release progeny infectious agents present in the microorganism; and components for detecting the progeny infectious agents, or, with higher sensitivity, soluble proteins encoded and expressed by the infectious agents, wherein detection of the infectious agent or the soluble protein product of the infectious agent indicates the presence of the microorganism in the sample. The infectious agent may include a Staphylococcus-specific NANOLUC bacteriophage.

[0166] The system or kit may include various components for detecting the progeny infectious agents. For example, in one embodiment, the progeny infectious agent (e.g., bacteriophage) may include an indicator moiety. In one embodiment, the indicator moiety in the progeny infectious agent (e.g., bacteriophage) may be a detectable moiety (e.g., a soluble luciferase protein) that is expressed during replication.

[0167] In other embodiments, the present invention may include a kit for rapid detection of a target microorganism in a sample, the system comprising components for incubating the sample with an infectious agent specific to the target microorganism, wherein the infectious agent comprises an indicator portion; components for capturing the microorganism from the sample onto a solid support; components for washing the captured microorganism sample to remove unbound infectious agent; and components for detecting the indicator portion. In some embodiments, the same components may be used for the capturing step and / or the incubating step and / or the washing step. Some embodiments further include components for determining the amount of the target microorganism in the sample, wherein the amount of the indicator portion detected corresponds to the amount of the microorganism in the sample. Such kits may include various embodiments and sub-embodiments similar to those described above with respect to the method of rapid detection of microorganisms. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

[0168] In some embodiments, the kit may include components for isolating the target microorganism from other components in the sample.

[0169] These systems and kits of the present invention include various components. As used herein, the term "component" is broadly defined and includes any suitable device or collection of suitable devices for performing the described method. The components need not be integrally connected or installed with respect to each other in any particular manner. The present invention includes any suitable arrangement of the components with respect to each other. For example, the components need not be present in the same space. However, in some embodiments, the components are connected to each other in an integrated unit. In some embodiments, the same component may perform multiple functions.

[0170] Computer Systems and Computer-Readable Media The above system can be embodied in the form of a computer system, as described in either the current technology or any of its components. Representative examples of computer systems include general-purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices capable of implementing the steps that make up the method of the present technology.

[0171] The computer system may include a computer, an input device, a display unit, and / or the Internet. The above computer may further include a microprocessor. The above microprocessor may be connected to a communication bus. The above computer may also include a memory. The above memory may include random access memory (RAM) and read-only memory (ROM). The above computer system may further include a storage device. The above storage device may be a hard disk drive or a removable storage device (e.g., a floppy (registered trademark) disk drive, an optical disk drive, etc.). The above storage device may also be other similar means for loading a computer program or other instructions into the above computer system. The above computer system may also include a communication unit. The above communication unit enables the above computer to connect to other databases and the Internet through an I / O interface. The above communication unit enables transfer to other databases and receipt of data from other databases. The above communication unit may include a modem, an Ethernet (registered trademark) card, or any other similar device that enables the above computer system to connect to databases and networks (e.g., LAN, MAN, WAN, and the Internet). The above computer system can thus facilitate input from a user through an input device accessible to the above system via an I / O interface.

[0172] A computing device typically includes an operating system that provides executable program instructions for the general management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., hard disk, random access memory, read-only memory, etc.) that stores instructions that, when executed by a processor of the server, cause the computing device to perform its intended functions. Implementations suitable for the operating system and the general functionality of the computing device are known or commercially available and can be readily implemented by those skilled in the art, particularly in view of the disclosure herein.

[0173] The computer system executes a set of instructions stored in one or more storage elements to process input data. The storage elements may also hold data or other information as described. The storage elements may be in the form of an information source or a physical memory element present in the processing machine.

[0174] The environment can include various data stores as well as other memories and storage media as considered above. These can exist in various locations (e.g., local to (and / or within) one or more of the above computers, or in a storage medium remote from any or all of the computers across the network). In a particular group of embodiments, the information can exist in a storage area network ("SAN") with which those skilled in the art are familiar. Similarly, any essential files for performing the functions attributed to the above computers, servers, or other network devices can be stored locally and / or remotely where appropriate. If the system includes computing devices, each such device can include hardware elements that can be electrically connected via a bus, and the elements can include, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touch screen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system can also include one or more storage devices (e.g., disk drives, optical storage devices, and solid state storage devices (e.g., random access memory ("RAM") or read only memory ("ROM"), and can include removable media devices, memory cards, flash cards, etc.).

[0175] Such a device may also include a computer-readable storage media reader, a communication device (e.g., a modem, a network card (wireless or wired), an infrared communication device, etc.), and a working memory as described above. The computer-readable storage media reader can be connected to or configured to receive computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information. The system and various devices may also typically include many software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs (e.g., a client application or a web browser). It should be recognized that alternative embodiments may have many variations from those described above. For example, customized hardware may also be used, and / or certain elements may be implemented in hardware, software (including portable software such as an applet), or both. Additionally, connections to other computing devices (e.g., network input / output devices) may be used.

[0176] Non-transitory storage media and computer-readable media for containing a code or a part of a code may include any suitable media (including storage media and communication media (e.g., any method or technology for storing and / or transmitting information such as computer-readable instructions, data structures, program modules, or other data), volatile and non-volatile, removable and non-removable media implemented in, but not limited to, these) known or used in the art. These include RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other media that can be used to store the desired information and can be accessed by the above system devices. Based on the above disclosure and the teachings provided in this specification, those skilled in the art will recognize other ways and / or methods for implementing the above various embodiments.

[0177] A computer-readable medium can include, but is not limited to, an electronic, optical, magnetic, or other storage device that can provide computer-readable instructions to a processor. Other examples include, but are not limited to: floppy (registered trademark) disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROM, RAM, SRAM, DRAM, associative memory (“CAM”), DDR, flash memory (e.g., NAND flash or NOR flash, ASICs, configured processors, optical storage media, magnetic tape or other magnetic storage media, or any other medium that a computer processor can read instructions from. In one embodiment, the computing device can include a single type of computer-readable medium (e.g., random access memory (RAM)). In other embodiments, the computing device can include two or more types of computer-readable media (e.g., random access memory (RAM), disk drive, and cache). The computing device can be in communication with one or more external computer-readable media (e.g., an external hard disk drive, or an external DVD or Blu-ray drive).

[0178] As discussed above, the above-described embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in a memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language (such as C, C++, C#, Visual Basic, Java®, Python, Perl, JavaScript®, and ActionScript (Adobe Systems, Mountain View, Calif.)). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and state machines. Such processors may 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.

[0179] The computing device includes a network interface. In some embodiments, the network interface is configured to communicate via a wired or wireless communication link. For example, the network interface may enable communication on a network via Ethernet®, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth®, infrared, etc. As another example, the network interface may enable communication on a network (e.g., CDMA, GSM®, UMTS, or other cellular communication network). In some embodiments, the network interface may enable a point-to-point connection with another device, for example, via a Universal Serial Bus (USB), 1394 FireWire, serial connection, or parallel connection, or a similar interface. Some embodiments of a suitable computing device may include two or more network interfaces for communication on one or more networks. In some embodiments, the computing device may include a data store in addition to or instead of the network interface.

[0180] Some embodiments of a suitable computing device may include or communicate with a number of external or internal devices (e.g., a mouse, CD-ROM, DVD, keyboard, display, audio speaker, one or more microphones, or any other input or output device). For example, the computing device may communicate with various user interface devices and a display. The display may use any suitable technology (including, but not limited to, LCD, LED, CRT, etc.).

[0181] The instruction set for execution by the computer system may include various commands that instruct a processing machine to perform a specific task (e.g., the steps constituting the method of the present technology). The instruction set may be in the form of a software program. Furthermore, the software may be in the form of a collection of separate programs, a program module having a larger program, or a part of a program module, as in the present technology. The software may also include modular programming in the form of object-oriented programming. The processing of input data by the processing machine may be responsive to user commands, the results of previous processing, or requests created by another processing machine.

[0182] While the present invention has been disclosed with reference to certain embodiments, many modifications, variations, and changes to the described embodiments are possible without departing from the scope and spirit of the present invention, as defined by the appended claims. Accordingly, it is intended that the present invention not be limited to the embodiments described above, but rather have the full scope defined by the language of the following claims and their equivalents.

Example

[0183] The results shown in the following examples demonstrate the detection of a small number of cells and even single bacteria with a shortened time to result. (Example 1) Bacterial detection using the Staphylococcus aureus - specific bacteriophage NanoLuc indicator phage after incubation of the sample in the medium

[0184] Samples of methicillin-resistant S. aureus (MRSA) were primed by adding 135 μl of a sample containing S. aureus to rich medium. The rich medium included a sub-inhibitory antibiotic (cefoxitin) to enable specific enrichment and induction of MRSA. The samples were incubated in the rich medium for 1 - 2 hours. After priming of the samples, additional cefoxitin was added to the medium and the samples were incubated for a further 2 hours. Staphylococcus aureus-specific bacteriophage NanoLuc indicator phage was added to the samples and incubated for 2 hours. Lysis buffer and NANO-GLO® reagent were added. After a 5-minute incubation, bioluminescence was measured using a GLOMAX® 96 instrument. A signal / background ratio ≧ 750 RLU indicated a positive detection of S. aureus. A signal / background ratio < 750 RLU indicated that the sample was negative for S. aureus.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0185] A clinical sample is obtained from a patient. The clinical sample is incubated with a Staphylococcus-specific bacteriophage NanoLuc® indicator phage for 2 hours. After incubation, lysis buffer and NANO-GLO® reagent are added. After a 5-minute incubation with the lysis buffer and NANO-GLO® reagent, bioluminescence is measured using a GLOMAX® 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 that the sample is negative for Staphylococcus.

[0186] A patient having a sample positive for Staphylococcus is treated with a therapeutic phage specific for Staphylococcus. After treatment with the therapeutic phage specific for Staphylococcus for 72 hours, a second clinical sample is obtained from the patient. The clinical sample is then incubated with a Staphylococcus-specific bacteriophage NanoLuc® indicator phage for 2 hours. After incubation, lysis buffer and NANO-GLO® reagent are added and incubated for 5 minutes. Next, bioluminescence is measured using a GLOMAX® 96 instrument to monitor the change in the presence of Staphylococcus in the treated clinical sample. A signal / background ratio ≧750 RLU indicates a positive detection of Staphylococcus. A signal / background ratio <750 RLU indicates that the sample is negative for Staphylococcus. If the sample is positive for Staphylococcus, treatment with the therapeutic phage specific for Staphylococcus is continued, and the effectiveness of the treatment is continuously monitored using the Staphylococcus-specific bacteriophage NanoLuc® indicator phage. In certain embodiments, for example, the following items are provided. (Item 1) A recombinant bacteriophage comprising an indicator gene inserted into the late gene region of a bacteriophage genome. (Item 2) The recombinant bacteriophage according to item 1, which specifically infects Staphylococcus. (Item 3) The recombinant bacteriophage according to item 2, wherein the Staphylococcus is Staphylococcus aureus. (Item 4) The recombinant bacteriophage according to item 3, wherein the Staphylococcus aureus is methicillin-resistant. (Item 5) The recombinant bacteriophage according to item 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. (Item 6) The recombinant bacteriophage according to item 5, further comprising an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region comprises a bacteriophage late gene promoter and a ribosome entry site. (Item 7) A cocktail composition comprising at least two different types of recombinant bacteriophages, wherein at least one of the recombinant bacteriophages comprises the indicator gene according to item 1. (Item 8) A method for preparing a recombinant indicator bacteriophage, comprising: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector comprising an indicator gene; transforming the target pathogenic bacterium with the homologous recombination plasmid / vector; Infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby causing homologous recombination between the plasmid / vector and the bacteriophage genome, and isolating a specific clone of the recombinant bacteriophage A method comprising the steps of: (Item 9) The step of preparing the homologous recombination plasmid / vector is determining the natural nucleotide 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 for homologous recombination downstream of the major capsid protein gene, the sequence including a codon optimization indicator gene, and incorporating the sequence designed for homologous recombination into a plasmid / vector The method according to item 8, comprising the steps of: (Item 10) The method according to item 9, wherein the step of designing the sequence further includes inserting an untranslated region including a phage late gene promoter and a ribosome entry site upstream of the codon optimization indicator gene. (Item 11) The method according to item 8, wherein the homologous recombination plasmid includes an untranslated region including a bacteriophage late gene promoter and a ribosome entry site upstream of the codon optimization indicator gene. (Item 12) The method according to item 8, wherein the wild-type bacteriophage is a Staphylococcus-specific bacteriophage and the target pathogenic bacterium is Staphylococcus. (Item 13) The method according to item 12, wherein the Staphylococcus is Staphylococcus aureus. (Item 14) The method according to item 13, wherein the Staphylococcus aureus is methicillin-resistant (MRSA). (Item 15) The method according to item 8, wherein the step of isolating a specific clone of the recombinant bacteriophage comprises a limiting dilution assay for isolating a clone that exhibits expression of the indicator gene. (Item 16) A method for detecting Staphylococcus in a sample, comprising: incubating the sample with a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage comprising an indicator gene inserted into a late gene region of the bacteriophage genome; and detecting an indicator protein product produced by the recombinant bacteriophage, wherein a positive detection of the indicator protein product indicates the presence of Staphylococcus in the sample. A method comprising: (Item 17) The method according to item 16, wherein the Staphylococcus is Staphylococcus aureus. (Item 18) The method according to item 17, wherein the Staphylococcus aureus is methicillin-resistant. (Item 19) The method according to item 16, wherein the sample is a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample. (Item 20) The method according to item 16, for detecting bacteria in a standard-sized sample for the food safety industry at a level of as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. (Item 21) The method according to item 19, wherein the food sample comprises meat, fish, vegetables, eggs, or powdered milk for infants. (Item 22) The method according to item 16, wherein the sample is incubated with a cocktail composition comprising at least two different types of recombinant bacteriophages, and at least one of the recombinant bacteriophages comprises the indicator gene described in item 16. (Item 23) The method according to item 16, wherein the sample is first incubated under conditions favorable for 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. (Item 24) The method according to item 16, wherein the total time to result 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. (Item 25) The method according to item 16, wherein the ratio of signal to background generated by the step of detecting the indicator is at least 2.0 or at least 2.5. (Item 26) A kit for detecting Staphylococcus, comprising a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage. (Item 27) The kit according to item 26, wherein the Staphylococcus is Staphylococcus aureus and the Staphylococcus-specific bacteriophage is a Staphylococcus aureus-specific bacteriophage. (Item 28) The kit according to item 27, wherein the Staphylococcus aureus is methicillin-resistant and the Staphylococcus aureus-specific bacteriophage is a methicillin-resistant Staphylococcus aureus-specific bacteriophage. (Item 29) The kit according to item 26, further comprising a substrate for reacting with an indicator for detecting a soluble protein product expressed by the recombinant bacteriophage. (Item 30) A system for detecting Staphylococcus, comprising a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage. (Item 31) The system according to item 30, wherein the Staphylococcus is Staphylococcus aureus. (Item 32) The system according to item 31, wherein the Staphylococcus aureus is methicillin-resistant. (Item 33) A method for selecting a treatment for a subject, comprising: (i) obtaining a biological sample from the subject; (ii) detecting a specific microorganism or category of microorganisms in the biological sample using an indicator phage; and (iii) selecting a treatment based on the identity of the specific microorganism detected in the biological sample. A method comprising the steps of: (Item 34) The method according to item 33, wherein the indicator phage is a phage prepared by synthesis. (Item 35) The method according to item 33, wherein the indicator phage is a genetically modified, naturally occurring phage. (Item 36) A method for monitoring the effectiveness of a treatment for a subject having a pathogenic medical condition, comprising: (i) obtaining a biological sample from the subject; (ii) detecting a specific microorganism or category of microorganisms in the biological sample using an indicator phage; (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) using the indicator phage to detect the specific microorganism or category of microorganisms in the second biological sample; and (vi) determining a decrease, increase, or steady level of the specific microorganism or category of microorganisms in the subject based on the amounts detected in the first and second biological samples. A method comprising.

Claims

1. A method for preparing a recombinant indicator bacteriophage, comprising: selecting a Staphylococcus - specific bacteriophage that specifically infects methicillin - resistant Staphylococcus aureus (MRSA); preparing a homologous recombination plasmid / vector containing an indicator gene, wherein the indicator gene does not encode a fusion protein; transforming the homologous recombination plasmid / vector into MRSA; infecting the transformed MRSA with the Staphylococcus - specific bacteriophage, thereby causing homologous recombination between the plasmid / vector and the genome of the bacteriophage; and isolating a specific clone of the recombinant bacteriophage A method comprising the above steps.

2. The step of preparing a homologous recombination plasmid / vector comprises: determining the native nucleotide sequence in the late region of the genome of the Staphylococcus - specific bacteriophage; annotating the genome and identifying the major capsid protein gene of the Staphylococcus - specific bacteriophage; designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence contains a codon - optimized indicator gene; and incorporating the sequence designed for homologous recombination into a plasmid / vector The method according to claim 1, comprising the above steps.

3. The method according to claim 2, wherein the step of designing the sequence further comprises inserting an untranslated region containing a phage late - gene promoter and a ribosome entry site upstream of the codon - optimized indicator gene.

4. The method according to claim 1, wherein the homologous recombination plasmid contains an untranslated region containing a phage late - gene promoter and a ribosome entry site upstream of the codon - optimized indicator gene.

5. The method according to claim 1, wherein the step of isolating a specific clone of the recombinant bacteriophage comprises a limiting dilution assay for isolating a clone showing the expression of the indicator gene.

6. The method according to claim 1, wherein the Staphylococcus - specific bacteriophage is SEA1.

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