METHODS AND SYSTEMS FOR THE RAPID DETECTION OF LISTERIA USING INFECTIOUS AGENTS.

Abstract

Methods and systems for the rapid detection of microorganisms such as Listeria spp. in a sample are disclosed herein. A genetically modified bacteriophage comprising a reporter gene in the late gene region is also disclosed. The specificity of the bacteriophage, such as the Listeria-specific bacteriophage, allows for the detection of a specific microorganism, such as Listeria spp., and a reporter signal can be amplified to optimize the sensitivity of the assay.

Description

METHODS AND SYSTEMS FOR THE RAPID DETECTION OF LISTERIA USING INFECTIOUS AGENTS Cross-reference to related applications This application claims priority over U.S. Provisional Application No. 62 / 798,248, filed on January 29, 2019. The disclosures in U.S. Applications Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, and 15 / 409,258 are incorporated herein by reference in their entirety. Field of invention This invention relates to compositions, methods, systems, and kits for the detection of microorganisms using infectious agents. Background of the invention There is strong interest in improving the speed and sensitivity of detection methods for bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples. Microbial pathogens can cause substantial morbidity in humans and domestic animals, as well as immense economic losses. Microorganism detection is also a high priority for the Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and the United States Department of Agriculture (USDA), given outbreaks of life-threatening or fatal illnesses caused by the consumption of food contaminated with certain microorganisms, such as Listeria spp., Salmonella spp., or Staphylococcus spp. In particular, Listeria species are known to cause a potentially serious infection called listeriosis. Listeria species, such as L. monocytogenes, are typically transmitted through the ingestion of contaminated food products. L. monocytogenes is a Gram-positive bacterium commonly associated with the contamination of food products, including milk, seafood, poultry, and meat. Foodborne illnesses such as listeriosis can be prevented by detecting contaminated food before it reaches consumers. Traditional microbiological tests for bacterial detection rely on non-selective and selective enrichment cultures followed by plating on selective media and further testing to confirm suspected colonies. These procedures can take up to seven days. For example, traditional tests for Listeria species in food products are complex and time-consuming, requiring enrichment periods of 24 to 48 hours, followed by lengthy additional testing, with a total detection time ranging from 5 to 7 days. A variety of rapid methods have been researched and implemented to reduce the time required. However, these methods have drawbacks.For example, polymerase chain reaction (PCR) assays, which also include an amplification step and are therefore capable of both very high sensitivity and selectivity, are economically limited to a small sample size. With dilute bacterial suspensions, most small subsamples will be cell-free, and therefore extensive purification and / or enrichment steps are still required. The time required for traditional biological enrichment is dictated by the growth rate of the target bacterial population in the sample, the effect of the sample matrix, and the required sensitivity. Due to the time required for culturing, these methods can take up to eight days, depending on the organism to be identified and the sample source. This delay is generally inadequate since contaminated food, water, or other products may have already reached livestock or humans. Furthermore, increases in antibiotic-resistant bacteria and biodefense considerations make rapid identification of bacterial pathogens in water, food, and clinical samples a clinical priority worldwide. Therefore, there is a need for faster, simpler, and more sensitive detection and identification of microorganisms, such as bacteria and other potentially pathogenic microorganisms. Brief description of the invention Modalities of the invention comprise compositions, methods, systems, and kits for the detection of microorganisms such as Listeria spp. The invention may also be modality in a variety of ways. In some aspects, the invention comprises a recombinant bacteriophage comprising a reporter gene inserted into a late gene region of a bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Listeria-specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified bacteriophage genome derived from a bacteriophage that specifically recognizes Listeria spp. In some embodiments, the bacteriophage used to prepare the recombinant bacteriophage specifically infects one or more Listeria spp. In one embodiment, the recombinant bacteriophage is able to distinguish Listeria spp. in the presence of other types of bacteria. In some embodiments, the recombinant bacteriophage specifically recognizes Listeria monocytogenes. In some recombinant marker bacteriophage forms, the marker gene may have optimized codons and may encode either a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates a signal after reaction with the substrate. Some recombinant bacteriophages further comprise an untranslated region upstream of a marker gene with optimized codons, where the untranslated region includes a bacteriophage late gene promoter and a ribosomal entry site. In some forms, the marker gene is a luciferase gene. The luciferase gene may be a naturally occurring gene, such as Oplophorus luciferase, Firefly luciferase, Lucia luciferase, or Renilla luciferase, or it may be a genetically modified gene such as NANOLUC®. Methods for preparing a recombinant reporter bacteriophage are also disclosed herein. Some methods include selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector comprising a reporter 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 the recombinant bacteriophage. In some methods, the selected wild-type bacteriophage is a Listeria-specific bacteriophage. In some embodiments, preparing a plasmid / vector for homologous recombination includes determining the natural nucleotide sequence in the late region of the selected bacteriophage genome; 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, wherein the sequence comprises a reporter gene with optimized codons; and incorporating the designed sequence for homologous recombination into a plasmid / vector. The step of designing a sequence may include inserting a genetic construct, comprising an untranslated region, including a phage late gene promoter and the ribosomal entry site, upstream of the reporter gene with optimized codons. In some embodiments, the phage late gene promoter is an exogenous promoter, distinct from any endogenous promoter in the phage genome.Therefore, in some methods, the plasmid for homologous recombination comprises an untranslated region that includes a bacteriophage late gene promoter and a ribosomal entry site upstream of the reporter gene with optimized codons. Some embodiments of the invention are compositions that include a recombinant indicator bacteriophage as described herein. For example, the compositions 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 compositions, methods, systems, and kits of the invention may comprise a cocktail of at least one recombinant bacteriophage for use in the detection of microorganisms such as Listeria spp. In some embodiments, the invention comprises a method for detecting a microorganism of interest in a sample comprising the steps of incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage comprises a reporter gene inserted into a late gene region of the bacteriophage such that expression of the reporter gene during bacteriophage replication followed by infection of the host bacterium results in a soluble reporter protein product, and detecting the reporter protein product, wherein positive detection of the reporter protein product indicates that the microorganism of interest is present in the sample. In some embodiments of the methods for preparing the recombinant indicator bacteriophage, the wild-type bacteriophage is a bacteriophage specific to Listeria spp. and the target pathogenic bacterium is Listeria spp. In some embodiments, the isolation of a particular clone of the recombinant bacteriophage comprises a limiting dilution assay to isolate a clone that shows expression of the indicator gene. Other aspects of the invention include methods for detecting bacteria, such as Listeria spp., in a sample. These methods involve incubating the sample with a recombinant bacteriophage derived from a Listeria-specific bacteriophage and detecting a marker protein product produced by the recombinant bacteriophage. Positive detection of the marker protein product indicates that Listeria spp. is present in the sample. In some embodiments, the invention includes methods for detecting Listeria spp. using a recombinant bacteriophage derived from a bacteriophage that targets Listeria spp. The sample may be a food or water sample. In some embodiments, the samples include environmental samples (e.g., sponges and swabs from surfaces or equipment used for bacterial control in factories and other processing facilities). In some methods for detecting bacteria, the sample is first incubated under growth-promoting conditions for an enrichment period of 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 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 methods, the sample is not enriched before detection. In some modalities, the total time for the results is less than 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours.In some modalities, the signal-to-background ratio generated when detecting the indicator is at least 2.0, 2.5, or 3.0. In some modalities, the method detects only 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 of the specific bacteria in a sample of a standard size for the food safety industry. Additional embodiments include systems and kits for detecting Listeria spp., wherein the systems or kits include a recombinant bacteriophage derived from a Listeria spp.-specific bacteriophage. Some embodiments further include a substrate for reacting with an indicator to detect the soluble protein product expressed by the recombinant bacteriophage. These systems or kits may include features described for the bacteriophage, compositions, and methods of the invention. In still other embodiments, the invention comprises a non-transient, computer-readable medium for use with methods or systems according to the invention. Brief description of the Figures The present invention can be better understood by referring to the following non-limiting figures. Figure 1 presents a reporter phage construct according to an embodiment of the invention that illustrates the insertion of a genetic construct comprising a luciferase gene, a bacteriophage late gene promoter, and a ribosomal binding site (RBS) inserted into the late (class III) region of a bacteriophage. The presented promoter is additional to and separate from the endogenous late gene promoter upstream of endogenous late genes, such as the gene for the major capsid protein (MCP). Figure 2 shows the genome of bacteriophage LMA4, a myovirus (related to Listeria phage LMTA94) obtained from wastewater. A hypothetical gene homologous to the putative pro-head protease p85 protein is located upstream of cps, the main capsid gene, within the late gene region, which consists of structural genes encoding virion proteins. Following cps is a transcriptional terminator, followed by a homolog to the LMTA-94 tail sheath protein (tsh). Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided that late gene promoters and / or other similar control elements are used. Figure 3 shows two plasmid construct designs for homologous recombination that carry the luciferase gene used to construct recombinant phages with approximately several hundred base pairs of matching phage sequence upstream and downstream of the insertion site to promote homologous recombination. The NANOLUC® luciferase is inserted into a core structure of the Gram-positive shuttle vector plasmid pCE104 with an upstream untranslated region containing a dedicated phage late gene promoter and a ribosomal entry site. pCE104.HR.A511.NanoLuc.v2 was used to construct recombinants for Pecentumvirus A511, LMA4, and LMA8. pCE104.HR.LP-ES1.NanoLuc was used to construct Homburvirus LP-ES1.Each construct consisted of 500 bp of homologous sequence consisting of a fragment of the major capsid protein (cps) gene followed by a late gene promoter, which was further appended to the upstream endogenous late gene promoter of the major capsid protein in the phage genome, the luciferase gene, and approximately 258 bp of downstream matched sequence for homologous recombination for pCE104.HR.A511.NanoLuc.v2 and 500 bp of downstream sequence for pCE104.HR.LPES1.NanoLuc. All recombinants used a PWOvirus late gene promoter instead of the T4 late gene promoter. Figure 4 shows a filtration plate assay for detecting bacteria of interest using a modified bacteriophage according to an embodiment of the invention where the bacteria and recombinant phages are incubated on filtration plates and after generation of the progeny bacteriophage the indicator protein is detected directly without removal of the incubation medium. Figures 5A and 5B show data from the Listeria detection assay modalities using recombinant bacteriophages specific to Listeria to detect Listeria in inoculated sponges, using 10 mL of added medium (Figure 5A) or 90 mL of added medium (Figure 5B). Figure 6 shows modality data for a Listeria detection assay that uses recombinant Listeria-specific bacteriophages to detect Listeria in environmental surface swab sponge samples. Detailed description of the invention This paper discloses compositions, methods, and systems that demonstrate remarkable sensitivity for the detection of a microorganism of interest, such as Listeria spp., in test samples (e.g., biological, food, water, and environmental samples). Detection can be achieved in a shorter time than previously thought possible using genetically modified infectious agents in assays performed with minimal enrichment times during which the microorganisms could potentially multiply. Also remarkable is the success of using a potentially high multiplicity of infection (MOI), or high concentrations of plaque-forming units (PFUs), for incubation with a test sample.Such high phage concentrations (PFU / mL) were previously thought to be detrimental in bacterial detection assays, as they were believed to cause lysis from the outside. However, a high phage concentration can facilitate finding, binding to, and infecting a low number of target cells. The compositions, methods, systems, and kits of the invention may comprise infectious agents for use in the detection of microorganisms such as Listeria spp. In certain embodiments, the invention may comprise a composition comprising a recombinant bacteriophage having a reporter gene inserted in a late gene region of the bacteriophage. In certain embodiments, expression of the reporter gene during bacteriophage replication followed by infection of a host bacterium results in the production of a soluble reporter protein product. In certain embodiments, the reporter gene may be inserted in a late gene region (i.e., class III) of the bacteriophage. The bacteriophage may be derived from a Listeria spp.-specific bacteriophage or another wild-type or genetically modified bacteriophage. In some embodiments, the recombinant bacteriophage is constructed from at least one of the bacteriophages LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A. In some embodiments, the compositions, methods, systems, and kits of the invention may comprise a cocktail of at least one recombinant bacteriophage for use in the detection of microorganisms such as Listeria spp. In some aspects, the invention comprises a method for detecting a microorganism of interest. The method may utilize an infectious agent for the detection of the microorganism of interest, such as Listeria spp. For example, in certain embodiments, the microorganism of interest is Listeria spp., and the infectious agent is a bacteriophage that specifically infects Listeria spp. Therefore, in certain embodiments, the method may comprise detecting a bacterium of interest in a sample by incubating the sample with a recombinant bacteriophage that infects the bacterium of interest. In certain embodiments, the recombinant bacteriophage comprises a reporter gene. The reporter gene may, in certain embodiments, be inserted into a late gene region of the bacteriophage such that expression of the reporter gene during bacteriophage replication followed by infection of the host bacteria results in the production of a reporter protein product.The method may involve detecting the indicator protein product, where positive detection of the indicator protein product indicates that the bacteria of interest is present in the sample. In some embodiments, the indicator protein is soluble. In certain embodiments, the invention may comprise a system. The system may contain at least some of the compositions of the invention. Also, the system may comprise at least some of the components for carrying out the method. In certain embodiments, the system is formulated as a kit. Therefore, in certain embodiments, the invention may comprise a system for the rapid detection of a microorganism of interest, such as Listeria spp., in a sample, comprising: a component for incubating the sample with an infectious agent specific to the microorganism of interest, wherein the infectious agent comprises an indicator group; and a component for detecting the indicator group. In still other embodiments, the invention includes software for use with the methods or systems. Therefore, some embodiments of the present invention address a need by using bacteriophage-based methods to amplify a detectable signal indicating the presence of bacteria. In certain embodiments, as few as 10 bacteria are detected. The principles applied herein can be applied to the detection of a variety of microorganisms. Due to numerous binding sites for an infectious agent on the surface of a microorganism, the ability to produce progeny during infection, and the potential for high expression of an encoded indicator group, the infectious agent or an indicator group may be more readily detectable than the microorganism itself. In this way, the embodiments of the present invention can achieve tremendous signal amplification from even a single infected cell. Aspects of the present invention utilize the high specificity of binding agents that can bind to particular microorganisms, such as the binding component of infectious agents, as a means to detect and / or quantify the specific microorganism in a sample. In some embodiments, the present invention utilizes the high specificity of infectious agents such as bacteriophages. In some methods, detection is achieved through a reporter group associated with the binding agent specific to the microorganism of interest. For example, an infectious agent may comprise a reporter group, such as a gene encoding a soluble reporter. In some methods, the reporter may be encoded by the infectious agent, such as a bacteriophage, and the bacteriophage is designated as a reporter phage. Some embodiments of the invention described herein utilize the discovery that a single microorganism is capable of binding to specific recognition agents, such as a phage. Following phage infection and replication, progeny phages can be detected through a reporter group expressed during phage replication. This principle allows the amplification of a reporter signal from one or a few cells based on the specific recognition of surface receptors on the microorganism. For example, by exposing as few as 10 bacterial cells to multiple phages, subsequently allowing phage amplification and high-level expression of a reporter gene product encoded during replication, the reporter signal is amplified to make the bacteria detectable. Embodiments of the methods and systems of the invention can be applied for the detection and quantification of a variety of microorganisms (e.g., bacteria) in a variety of circumstances, including, but not limited to, the detection of pathogens from food, water, and commercial samples. The methods of the present invention provide high sensitivity and specificity for rapid detection. In some embodiments, detection is possible within a single bacteriophage replication cycle, which is unexpected. Definitions Unless otherwise defined herein, the scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by the context, singular terms shall include plurals, and plural terms shall include the singular. In general, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, described herein, are those well known and commonly used in the art. Known methods and techniques are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references discussed throughout this specification, unless otherwise indicated.Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly practiced in the field, or as described herein. The nomenclature used in relation to the laboratory procedures and techniques described herein is that which is well-known and commonly used in the field. Unless otherwise stated, the following terms are understood to have the following meanings: As used herein, the terms “a”, “an” and “the” may refer to one or more, unless specifically stated otherwise. The term “or” is used to mean “and / or” unless explicitly stated to refer to alternatives only or that the alternatives are mutually exclusive, although the disclosure supports a definition that refers to alternatives only and “and / or”. As used herein, “other” may mean at least one other or more. Throughout this application, the term “approximately” is used to indicate that a value includes the inherent error variation for the device, the method being employed to determine the value, or the variation that exists between samples. The term “solid support” or “support” means a structure that provides a substrate and / or surface to which biomolecules can bind. For example, a solid support can be a test well (i.e., such as a microtiter plate or a multi-well plate), or the solid support can be a location on a filter, array, or movable support, such as a bead or membrane (e.g., a filter plate, latex particles, paramagnetic particles, or a lateral flow band). The term “binding agent” refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction may be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or it may be covalent. The term “soluble binding agent” refers to a binding agent that is not associated with (i.e., covalently or non-covalently bound to) a solid support. As used herein, an “analyte” refers to a molecule, compound, or cell that is being measured. The analyte of interest may, in certain modalities, interact with a binding agent. As described herein, the term “analyte” may refer to a protein or peptide of interest. An analyte may be an agonist, an antagonist, or a modulator. Or, an analyte may have no biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, and the like. The term “detectable group,” “detectable biomolecule,” “reporter,” “indicator,” or “indicator group” refers to a molecule that can be measured in a quantitative assay. For example, an indicator group might be an enzyme that can be used to convert a substrate into a measurable product. An indicator group might be an enzyme that catalyzes a reaction that generates diluminescent emissions (e.g., luciferase). Or, an indicator group might be a radioisotope that can be quantified. Or, an indicator group might be a fluorophore. Or, other detectable molecules might be used. As used herein, “dactyliophage” or “phage” includes one or more of multiple dacteriophilic viruses. In this disclosure, the terms “dactyliophage” and “phage” include viruses such as microdactyliophage (as for TB and paraTB), mycophage (as for fungi), mycoplasma phage, and any other term that refers to a virus that can invade bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and those it uses to replicate itself. Here, “microscopic” means that the largest dimension is one millimeter or less. Bacteriophages are viruses that have evolved in nature to use bacteria as a means to replicate themselves. A phage does this by attaching itself to a bacterium and injecting its DNA (or RNA) into that bacterium, inducing it to replicate the phage hundreds or even thousands of times. This is referred to as phage amplification. As used herein, “late gene region” refers to a region of a viral genome that is transcribed late in the viral replication cycle. The late gene region typically includes the most abundantly expressed genes (e.g., structural proteins assembled into the bacteriophage particle). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, late genes (synonymous with class III) are transcribed in phage T7, for instance, from 8 minutes post-infection until lysis. Class I genes (e.g., RNA polymerase) are early, from 4 to 8 minutes, and class II genes are early, from 6 to 15 minutes, so there is a temporal overlap between class II and class III genes. A late promoter is one that is naturally located and active in such a late gene region. As used herein, “enrichment culture” refers to traditional culture, such as incubation in a medium conducive to the growth of microorganisms, and should not be confused with other possible uses of the word “enrichment,” such as enrichment by removing the liquid component of a sample to concentrate the microorganism contained therein, or other forms of enrichment that do not include the traditional facilitation of microorganism growth. Enrichment culture for periods of time may be employed in some of the methods described herein. As used herein, “recombinant” refers to genetic modifications (i.e., nucleic acid) typically performed in a laboratory to combine genetic material that could not otherwise be found. This term is used interchangeably with the term “modified” herein. As used herein, “RLU” refers to relative light units (RLUs) measured by a luminometer (e.g., GLOMAX® 96) or similar light-detecting instrument. For example, the detection of the reaction between luciferase and an appropriate substrate (e.g., NANOLUC® with NANO-GLO®) is frequently reported in detected RLUs. As used herein, “time to results” refers to the total amount of time from the start of sample incubation to the generation of results. Time to results does not include any confirmatory test time. Data collection can be performed at any time after a result has been generated. Samples Each of the embodiments of the methods and systems of the invention can enable the rapid detection and quantification of microbes in a sample. For example, the methods according to the present invention can be performed in a shorter period of time with superior results. The bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are food- or water-borne pathogens. Samples can be liquid, solid, or semi-solid. Samples can be swabs taken from solid surfaces. Samples can include environmental materials, such as water samples, or air sample filters or aerosol samples from cyclone collectors. Samples can be of vegetables, meat, fish, poultry, peanut butter, processed foods, powdered infant formula, powdered milk, teas, starches, eggs, milk, cheese, or other dairy products. In some embodiments, samples can be used directly in the detection methods of the present invention without preparation, concentration, or dilution. For example, liquid samples, including but not limited to milk and juices, can be evaluated directly. Samples can be diluted or suspended in a solution, including but not limited to a buffer solution or a bacterial culture medium. A sample that is a solid or semi-solid can be suspended in a liquid by chopping, mixing, or macerating the solid in the liquid. A sample must be maintained within a pH range that promotes bacteriophage binding to the host bacterial cell. A sample must also contain appropriate concentrations of divalent and monovalent cations, including but not limited to Na+, Mg2+, and Ca2+.Preferably, a sample is kept at a temperature that maintains the viability of any pathogenic cells contained within the sample. In some detection assay modalities, the sample is maintained at a temperature that preserves the viability of any pathogenic cells present in the sample. For example, during the steps in which bacteriophages bind to bacterial cells, it is preferable to maintain the sample at a temperature that facilitates bacteriophage binding. During the steps in which bacteriophages replicate within an infected bacterial cell or lyse such an infected cell, it is preferable to maintain the sample at a temperature that promotes bacteriophage replication and host lysis. Such temperatures are at least approximately 25 degrees Celsius (C), more preferably no higher than approximately 35 degrees C, and more preferably approximately 30 degrees C. The assays may include several appropriate control samples. For example, control samples that do not contain bacteriophages or control samples that contain bacteriophages without bacteria can be evaluated as controls for background signal levels. Indicator Bacteriophage As described in more detail herein, the compositions, methods, systems, and kits of the invention may comprise infectious agents for use in the detection of pathogenic microorganisms. In certain embodiments, the invention comprises a recombinant reporter bacteriophage, wherein the bacteriophage genome is genetically modified to include a reporter gene. In some embodiments, the invention may include a composition comprising a recombinant bacteriophage having a reporter gene incorporated into the bacteriophage genome. A recombinant reporter bacteriophage may include a reporter gene. In certain variants of the infectious agent, the reporter gene does not encode a fusion protein. For example, in some variants, expression of the reporter gene during bacteriophage replication followed by infection of a host bacterium results in a soluble reporter protein product. In other variants, the reporter gene may be inserted into a late gene region of the bacteriophage. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. The late gene region may be a class III gene region and may include a gene for a major capsid protein. Some approaches include designing (and optionally preparing) a sequence for homologous recombination downstream of the major capsid protein gene. Other approaches include designing (and optionally preparing) a sequence for homologous recombination upstream of the major capsid protein gene. In some approaches, the sequence comprises a reporter gene with optimized codons preceded by an untranslated region. The untranslated region may include a phage late gene promoter and ribosomal entry site. In some formulations, a reporter bacteriophage is derived from a Listeria-specific phage. In some formulations, the selected wild-type bacteriophage or wild-type bacteriophage cocktail is capable of infecting at least one target Listeria spp. Listeria species are ubiquitous in the environment and are often found in water, wastewater, and soil. In some formulations, the selected wild-type bacteriophage or wild-type bacteriophage cocktail is capable of infecting one or more, two or more, or three or more target Listeria spp. In certain instances, the target Listeria species are selected from L. monocytogenes, L. ivanovii, and L. grayi. In some cases, the selected wild-type bacteriophage belongs to the order Caudovirales. Caudovirales are an order of tailed bacteriophages with double-stranded DNA (dsDNA) genomes. Each virion of the order Caudovirales has an icosahedral head containing the viral genome and a flexible tail. The order Caudovirales comprises five families of bacteriophages: Myoviridae (long contractile tails), Siphoviridae (long non-contractile tails), Podoviridae (short non-contractile tails), Ackermannviridae, and Herelleviridae. The term myovirus can be used to describe any bacteriophage with an icosahedral head and a long contractile tail, encompassing bacteriophages of the families Myoviridae and Herelleviridae. In some modalities, the selected wild-type bacteriophage is a member of the Myoviridae family such as Listeria phage B054, Listeria phage LipZ5, Listeria phage PSUVKH-LP041, and Listeria phage WIL-2.In other models, the selected wild-type bacteriophage is a member of the Herelleviridae family. The genus Pecentumvirus, of the Herelleviridae family, includes bacteriophages such as Listeria phage LMSP-25, Listeria phage LMTA-148, Listeria phage LMTA-34, Listeria phage LP-048, Listeria phage LP-064, Listeria phage LP-0832, Listeria phage LP-125, Listeria P100 virus, Listeria phage List-36, Listeria phage WIL-1, Listeria phage vB_LmoM_AG20, and Listeria A511 virus. LMA4 and LMA8 are also likely to belong to the genus Pecentumvirus, of the Herelleviridae family. In other models, the selected wild-type bacteriophage is either LMA4 or LMA8. In certain cases, the selected wild-type bacteriophage is LP-ES3A, which is derived from A511 but has been adapted to be able to infect serotype 3A of Listeria monocytogenes.In other configurations, the selected wild-type bacteriophage is a member of the Ackermannviridae family. In still other configurations, the selected wild-type bacteriophage is a member of the Siphoviridae family, which includes Listeria phages A006, A118, A500, B025, LP-026, LP-030-2, LP-030-3, LP-037, LP-101, LP-110, LP-114, P35, P40, P70, PSA, vB_LmoS_188, and vB_Lmos_293. In other configurations, the selected wild-type bacteriophage is LP-ES1. It is also likely that LP-ES1 belongs to the genus Homburgvirus, of the Siphoviridae family. In some modalities, a marker bacteriophage is derived from a Listeria-specific phage. An indicator bacteriophage can be constructed from a Pecentumvirus, Tequatravirus, Vil, Kuttervirus, Homburgvirus, LMTA-94, LMA4, LMA8, P70, LP-ES1, LP-ES3A, or another bacteriophage that has 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 with the Listeria phage LMTA-94, P70, T7, T7 type, T4, T4 type, Listeria spp. specific bacteriophages, bacteriophages Vil, or Vil-type (Kuttervirus, according to GenBank / NCBI). In other modalities, the selected wild-type bacteriophage is LP-ES1, LP-ES3A, LMA4, or LMA8. In some modalities, the reporter phage is obtained from a bacteriophage that is highly specific for a particular pathogenic microorganism.Genetic modifications can prevent deletions of wild-type genes, and therefore the modified phage may remain more similar to the wild-type infectious agent than many commercially available phages. The bacteriophage obtained from the environment may be more specific to bacteria found in that environment and, as such, genetically distinct from commercially available phages. In another aspect of the invention, a cocktail composition comprises at least one type of recombinant bacteriophage. In some embodiments, the cocktail composition comprises at least one type of recombinant bacteriophage constructed from LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A. In other embodiments, the cocktail composition comprises at least one type of recombinant bacteriophage constructed from LMA8, LP-ES1, and LP-ES3A. Furthermore, phage genes thought to be non-essential may have an unrecognized function. For example, a seemingly non-essential gene may play an important role in increasing cleavage size, such as subtle cutting, fitting, or regulatory functions in assembly. Therefore, deleting genes to insert a marker gene can be detrimental. Most phages can pack DNA that is a few percent larger than their natural genome. With this in mind, a smaller marker gene may be a more appropriate choice for modifying a bacteriophage, especially one with a smaller genome. The OpLuc and NANOLUC® proteins are only about 20 kDa (approximately 500 to 600 bp to encode), while FLuc is about 62 kDa (approximately 1,700 bp to encode).Furthermore, the reporter gene must not be endogenously expressed by the bacteria (i.e., it is not part of the bacterial genome), it must generate a high signal-to-background ratio, and it must be readily detectable in a timely manner. In some modalities, the reporter gene is a luciferase. In other modalities, the reporter gene is an active subunit of a luciferase. Promega's NANOLUC® is a modified luciferase from Oplophorus gracilirostris (deep-sea shrimp). In some modalities, NANOLUC® combined with Promega's NANOGLO®, an imidazopyrazine (furimazine) substrate, can provide a robust signal with low background. In some reporter phage variants, the reporter gene can be inserted into an untranslated region to avoid disruption of functional genes, leaving the wild-type phage genes intact. This can lead to greater fitness when infecting non-laboratory bacterial strains. Additionally, including stop codons in all three reading frames can help increase expression by reducing the overreading, also known as permeable expression. This strategy can also eliminate the possibility of a fusion protein being produced at low levels, which would manifest as a background signal (e.g., luciferase) that cannot be separated from the phage. A reporter gene can express a variety of biomolecules. A reporter gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one modality, the reporter gene encodes a luciferase enzyme. Several types of luciferase can be used. In alternative modalities, and as described in more detail herein, the luciferase is either Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or a genetically modified luciferase. In some modalities, the luciferase gene is derived from Oplophorus. In some modalities, the reporter gene is a genetically modified luciferase gene, such as NANOLUC®. Therefore, in some embodiments, the present invention comprises a genetically modified bacteriophage comprising a non-bacteriophage reporter gene in the late gene region (class III). In some embodiments, the non-native reporter 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, as viral capsid proteins, but is also not inhibited as endogenous bacterial genes or even early viral genes. In some forms, the late promoter is a promoter from Pecentumvirus, Tequatravirus, Homburgvirus, or Kuttervirus, or another phage promoter similar to that found in the selected wild-type phage, i.e., without genetic modification. The late gene region may be a class III gene region, and the bacteriophage may be derived from Listeria phage LMTA-94, P70, A511, LP-ES1, LP-ES3A, LMA4, LMA8, Pecentumvirus, Tequatravirus, Homburgvirus, Kuttervirus, T7, T4, T4-type, Vil, or Listeria spp.-specific bacteriophages. or another wild-type bacteriophage having a genome with at least 70, 75, 80, 85, 90, or 95% homology with LMTA-94, LMA4, LMA8, Pecentumvirus, Tequatravirus, Homburgvirus, Kuttervirus, T7, T4, Vil, or Listeria-specific bacteriophages. The Pecentumvirus late gene promoter differs from the T4 or Tequatravirus promoter in that it consists not only of the -10 region but also of a -35 region.This -35 region differs from the standard -35 region found in most bacterial promoters. Genetic modifications to infectious agents may include insertions, deletions, or substitutions of a small fragment of nucleic acid, a substantial part of a gene, or an entire gene. In some embodiments, inserted or substituted nucleic acids comprise non-native sequences. A non-native reporter gene may be inserted into a bacteriophage genome such that it is under the control of a bacteriophage promoter. Therefore, in some embodiments, the non-native reporter gene is not part of a fusion protein. That is, in some embodiments, a genetic modification may be configured such that the reporter protein product does not comprise polypeptides of the wild-type bacteriophage. In some embodiments, the reporter protein product is soluble. In some embodiments, the invention comprises a method for detecting a bacterium of interest comprising the step of incubating a test sample with such a recombinant bacteriophage.In some embodiments, expression of the reporter gene in progeny bacteriophages followed by infection of a host bacterium results in a free, soluble protein product. In some embodiments, the non-native reporter gene is not contiguous with a gene encoding a structural phage protein and therefore does not generate a fusion protein. Unlike systems employing fusion of a detection group with the capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble reporter (e.g., soluble luciferase). In some embodiments, the reporter is ideally free from the bacteriophage structure. That is, the reporter is not bound to the phage structure. As such, the gene for the reporter is not fused with other genes in the recombinant phage genome.This can greatly increase the assay's sensitivity (down to a single bacterium) and simplify the assay, allowing it to be completed in two hours or less for some modalities, unlike the many hours required with constructs that produce detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding restrictions that can alter the conformation of the enzyme's active site or access to the substrate. If the concentration is 1,000 bacterial cells / mL of the sample, for example, less than four hours of infection may be sufficient for detection of the target bacterium. Furthermore, fusion proteins, by definition, limit the number of groups attached to protein subunits in the bacteriophage. For example, using a commercially available system designed to serve as a platform for a fusion protein would result in approximately 415 copies of the fusion group, corresponding to the approximately 415 copies of the capsid protein 10B gene in each bacteriophage T7 particle. Without this restriction, infected bacteria might be expected to express many copies of the detection group (e.g., luciferase) that can fit into the bacteriophage. Moreover, large fusion proteins, such as a capsid-luciferase fusion, can inhibit bacteriophage particle assembly, thus generating fewer bacteriophage progeny. Therefore, a soluble, non-fused reporter gene product may be preferable. In some embodiments, the reporter phage encodes a reporter, such as a detectable enzyme. The reporter gene product may generate light and / or be detectable by a color change. Several suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can serve as the reporter group. In some embodiments, Firefly luciferase is the reporter group. In some embodiments, Oplophorus luciferase is the reporter group. In some embodiments, NANOLUC® is the reporter group. Other engineered luciferases or other enzymes that generate detectable signals may also be suitable reporter portions. In some formulations, the use of a soluble detection group eliminates the need to remove a contaminating parental phage from the lysate of infected sample cells. With a fusion protein system, any bacteriophage used to infect sample cells would have the detection group attached and would be indistinguishable from the descendant bacteriophage that also contains the detection group. Because the detection of sample bacteria depends on the detection of a newly created (de novo) detection group, using fusion constructs requires additional steps to separate old (parental) groups from newly created (descendant) groups.This can be achieved by washing infected cells multiple times before the bacteriophage completes its life cycle, inactivating excess parental phage after infection using physical or chemical means, and / or chemically modifying the parental bacteriophage with a binding group (such as biotin), which can then be attached and detached (using streptavidin-coated sepharose beads). However, even with all these attempts at removal, parental phage can remain when a high concentration of parental phage is used to ensure infection of a small number of sample cells, creating a background signal that can obscure the detection of progeny phage signals from infected cells. In contrast, with the soluble detection group expressed in some embodiments of the present invention, purification of the parental phage from the final lysate is not necessary, since the parental phage does not have any detection group attached. Therefore, any detection group present after infection must have been created de novo, indicating the presence of an infected bacterium or bacteria. To take advantage of this benefit, the production and preparation of the parental phage can include the purification of the phage from any free detection group produced during the production of the parental bacteriophage in bacterial culture.Standard bacteriophage purification techniques can be employed to purify some phage modalities according to the present invention, such as sucrose density gradient centrifugation, cesium chloride density gradient isopycnic centrifugation, HPLC, size exclusion chromatography, and dialysis or derived technologies (such as Amicon-Millipore, Inc. brand concentrators). Cesium chloride isopycnic ultracentrifugation can be employed as part of the preparation of the recombinant phage of the invention to separate parental phage particles from contaminating luciferase protein produced after phage propagation in the bacterial host. In this way, the parental recombinant bacteriophage of the invention is substantially free of any luciferase generated during production in the bacteria.Removing residual luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with a test sample. In some modified bacteriophage variants, the late promoter (a class III promoter, for example, from Pecentumvirus, Homburgvirus, T7, T4, Vil, or LMA4 / 8) has a high affinity for the RNA polymerase of the same bacteriophage that transcribes genes for structural proteins assembled in the bacteriophage particle. These proteins are the most abundant proteins made by the phage, as each bacteriophage particle comprises dozens or hundreds of copies of these molecules. The use of a viral late promoter can ensure an optimally high level of expression of the luciferase detection group. The use of a viral late promoter derived from, specific for, or active under the original wild-type bacteriophage from which the indicator phage is derived (for example, a late promoter from Pecentumvirus, Homburgvirus, T4, T7, Vil, or LMA4 / 8 with a Pecentumvirus, T4, T7, Vil, or LMA-based system) can further ensure optimal expression of the detection group.The use of a standard bacterial (non-viral / non-bacteriophage) promoter can, in some cases, be detrimental to expression, as these promoters are frequently downregulated during bacteriophage infection (to ensure the bacteriophage prioritizes bacterial resources for phage protein production). Therefore, in some modalities, the phage is preferably genetically modified to encode and express at a high level a soluble (free) reporter group, using a genomic placement that does not limit expression to the number of subunits of a phage structural component. The compositions of the invention may comprise one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more reporter genes. In some embodiments, the compositions may include cocktails of different reporter phages that could encode and express the same or different reporter proteins. In some embodiments, the bacteriophage cocktail comprises at least two different types of recombinant bacteriophages. Methods for Preparing Indicator Bacteriophage Methods for preparing a marker bacteriophage begin with selecting a wild-type bacteriophage for genetic modification. Some bacteriophages are highly specific for a target bacterium. This presents an opportunity for highly specific detection. bAn^nn / Lznz / E / Yi Therefore, the methods of the present invention utilize the high specificity of binding agents associated with infectious agents that recognize and bind to a particular microorganism of interest as a means of amplifying a signal and thus detecting low levels of a microorganism (e.g., a single microorganism) present in a sample. For example, infectious agents (e.g., bacteriophages) specifically recognize receptors on the surface of particular microorganisms and therefore specifically infect those microorganisms. As such, these infectious agents can be appropriate binding agents for targeting a microorganism of interest. Some embodiments of the invention utilize the binding specificity and high-level gene expression capability of the recombinant bacteriophage for rapid and sensitive targeting to infect and facilitate the detection of a bacterium of interest. In some embodiments, a Listeria-specific bacteriophage is genetically modified to include a reporter gene. In some embodiments, the late gene region of a bacteriophage is genetically modified to include a reporter gene. In some embodiments, a reporter gene is positioned downstream of the main capsid gene. In other embodiments, a reporter gene is positioned upstream of the main capsid gene. In some embodiments, the inserted genetic construct further comprises its own exogenous promoter, dedicated to directing the expression of the reporter gene. The exogenous promoter is in addition to any endogenous promoter in the phage genome.Because bacteriophages produce polycistronic mRNA transcripts, only a single promoter is required upstream of the first gene / cistron in the transcript. Conventional recombinant constructs use only the endogenous bacteriophage promoter to target inserted genes. In contrast, adding an additional promoter upstream of the promoter gene and ribosomal binding site can increase gene expression by acting as a secondary initiation site for transcription. The complex and compact genomes of viruses often have genes that overlap in different frames, sometimes in two different directions. Some methods for preparing a recombinant reporter bacteriophage include selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium such as Listeria spp.; preparing a homologous recombination plasmid / vector comprising a reporter 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 the recombinant bacteriophage. Several methods for designing and preparing a plasmid for homologous recombination are known. Several methods for transforming bacteria with a plasmid are known, including heat shock, F pili-mediated bacterial conjugation, electroporation, and others. Several methods for isolating a particular clone followed by homologous recombination are also known. Some of the methodological approaches described herein utilize specific strategies. Therefore, some modalities of methods for preparing the indicator bacteriophage include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; determining the natural sequence in the late region of the selected bacteriophage genome; annotate the genome and identify the major capsid protein gene of the selected bacteriophage; design a sequence for homologous recombination adjacent to the major capsid protein gene, wherein the sequence comprises a reporter gene with optimized codons; incorporate the designed sequence for homologous recombination into a plasmid / vector; transform the plasmid / vector into target pathogenic bacteria; select the transformed bacteria; infect the transformed bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid and the bacteriophage genome; determine the titer of the resulting recombinant bacteriophage lysate; and perform a limiting dilution assay to enrich and isolate the recombinant bacteriophage.Some methods also involve repeating the limiting dilution and titration steps, followed by the first limiting dilution assay, as many times as necessary until the recombinant bacteriophage represents a detectable fraction of the mixture. For example, in some methods, the limiting dilution and titration steps may be repeated until at least 1 / 30 of the bacteriophage in the mixture is recombinant before isolating a particular recombinant bacteriophage clone. A 1:30 recombinant:wild-type ratio is expected, in some methods, to generate an average of 3.2 transduction units (TU) per 96 plates (e.g., in a 96-well plate). The initial ratio of recombinant to wild-type phage can be determined by performing limiting dilution assays based on TCID50 (tissue culture infectious dose of 50%) as previously described in U.S. Application No. 15 / 409,258.Using a Poisson distribution, a ratio of 1:30 generates a 96% probability of observing at least one TU somewhere in the 96 wells. Figure 1 shows a schematic representation of the genomic structure of a recombinant indicator bacteriophage of the invention. For the embodiment depicted in Figure 1, the detection group is encoded by a luciferase gene 100 inserted within the late gene region (class III) 110, which is expressed late in the viral replication cycle. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. Therefore, in the recombinant phage embodiment depicted in Figure 1, the indicator gene (i.e., luciferase) is inserted in the late gene region, immediately following the gene for the major capsid protein (cps) 120, and is a construct comprising the luciferase gene 100.In some embodiments, the construct depicted in Figure 1 may include stop codons in all three reading frames to ensure that luciferase is not incorporated into the cps gene product through the creation of a fusion protein. Also, as depicted in Figure 1, the construct may comprise an additional dedicated late promoter 130 to direct transcription and expression of the luciferase gene. The construct also includes a ribosome binding site (RBS) 140. This construct ensures that soluble luciferase is produced in such a way that expression is not limited by the number of capsid proteins inherent in the phage presentation system. As noted here, in certain applications, it may be preferable to use infectious agents that have been isolated from the environment for the production of the infectious agents of the invention. In this way, infectious agents that are specific to naturally occurring microorganisms can be generated. For example, Figure 2 shows the genome of bacteriophage LMA4, a wild-type bacteriophage that specifically infects Listeria spp. As discussed in the Examples, the major capsid protein (cps) 240 and several other structural genes are within the late gene region 210, which consists of structural genes encoding virion proteins. The genes encoding tRNA 220 represent the adjacent genomic sequence, but outside the late gene region. A hypothetical gene homologous to the putative pro-head protease of Listeria phage LMTA-94 230 is located upstream of cps 240, which consists of structural genes encoding virion proteins. Other late genes represented are homologs of the putative major capsid protein (cps) of Listeria phage LMTA-94 240, followed by a transcriptional terminator 250, and a homolog of the tail sheath protein (tsh) of Listeria phage LMTA-94 260.Because these virion proteins are expressed at a very high level, any gene inserted into this region can be expected to have similar expression levels, provided that late gene promoters and / or other similar control elements are used. Numerous methods and commercial products exist for preparing plasmids. For example, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques can be used in combination to prepare plasmids. Synthetic plasmids can also be ordered commercially (e.g., GeneWiz). Cosmids can also be employed, or the CRISPR / Cas9 system could be used to selectively edit a bacteriophage genome. Some methods for preparing a recombinant reporter bacteriophage include designing a plasmid that can readily recombine with the wild-type bacteriophage genome to generate recombinant genomes. In designing a plasmid, some approaches include adding a reporter gene with optimized codons, such as a luciferase gene. Some approaches also include adding elements to the upstream untranslated region.For example, in designing a plasmid to recombine with the Listeria-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 may include a promoter, such as the T4, Tequatravirus, Homburgvirus, T7, T7-like, Pecentumvirus, Listeria-specific bacteriophage, Vil, or Kuttervirus promoter. The untranslated region may also include a Ribosomal Entry / Binding Site (RBS), also known as a “Shine-Dalgarno sequence” with bacterial systems. Either or both of these elements, or other untranslated elements, may be incorporated within a short upstream untranslated region made of random sequences comprising approximately the same GC content as the rest of the phage genome.The random region must not include an ATG sequence, as it will act as a start codon. The compositions of the invention may comprise several infectious agents and / or reporter genes. For example, Figure 3 shows a plasmid construct for homologous recombination used in the preparation of the reporter phage specific to Listeria spp. The constructs were made and used in recombination with the Listeria spp. phage LMA4, the Listeria spp. phage LMA8, and the LP bAn^nn / Lznz / B / Yi phage. ES3A of Listeria spp., the LP-ES1 phage of Listeria spp., or other Listeria-specific phages to generate the recombinant bacteriophage of the invention. The construct in Figure 3 shows a general scheme for the recombination plasmid used for homologous recombination insertion of the NANOLUC® luciferase into both the LMA4 and LMA8 phages of Listeria spp. Pecentumvirus, each with 500 bp of upstream and downstream homologous sequence: homologous recombination plasmid pCE104.HR.ListeriaFago.NANOLUC.v2. Pecentumv / rus.NANOLUC.v2 and the recombination plasmid used for homologous recombination insertion of the NANOLUC® luciferase into the Listeria spp. Homburgvirus phage LP-ES1, pCE104.HR.LP-ES1 .NanoLuc In certain modalities, a plasmid is designated pCE104.HR.Pecentumv / r(js.NanoLuc.v2). The detection / indicator group is encoded by the reporter gene NANOLUC® 300. The insert, represented by the series of rectangles, is in the Gram-positive shuttle vector, pCE104 310. The upstream homologous recombination region consists of 500 bp of the C-terminal fragment 320 of the major capsid protein. A consensus sequence of the late Pecentumvirus promoter and a Shine-Dalgarno ribosomal entry / binding site are located within the 5' untranslated region 330. The NANOLUC® reporter gene with optimized codons 300 immediately follows. The endogenous transcriptional terminator then follows, along with the untranslated region (UTR) and the hypothetical N-terminal fragment of the protein consisting of the recombination. downstream homologue 340 that are found at the end of the homologue recombination region. The Major Capsid Protein fragment is part of a structural gene that encodes a virion protein. Because these virion proteins are expressed at a very high level, any gene inserted into this region can be expected to have similar expression levels, provided that late gene promoters and / or other similar control elements are used. In some embodiments, the reporter phage according to the invention comprises a Listeria-specific bacteriophage genetically modified to include a reporter gene, such as a luciferase gene. For example, a reporter phage may be a Listeria spp.-specific bacteriophage wherein the genome comprises the NANOLUC® gene sequence. A Listeria-specific bacteriophage genome with recombinant NanoLuc may further comprise a consensus promoter from the Pecentumvirus bacteriophage, T4, T7, Listeria-specific bacteriophage, Vil, LMA4, or LMA8, or another late promoter. In additional embodiments, the promoter is an exogenous promoter. The insertion of an exogenous promoter to direct the expression of a reporter gene is advantageous in that the expression is not limited by the expression of other phage proteins (e.g., the major capsid protein). Therefore, in the recombinant phage modality generated as a result of recombination, the reporter gene (i.e., NANOLUC®) is inserted into the late gene region, just downstream of the gene encoding the major capsid protein, thereby creating recombinant bacteriophage genomes comprising the NANOLUC® gene. The construct may further comprise the Listeria phage LMTA-94 consensus promoter, T4, T7, a Listeria-specific bacteriophage, Vil, or another late promoter or other promoter suitable for driving transcription and expression of the luciferase gene. The construct may also comprise a composite untranslated region synthesized from several bAn^nn / Lznz / B / Yi UTR. This construct ensures that soluble luciferase is produced in such a way that expression is not limited to the number of capsid proteins inherent in the phage presentation system. The recombinant phage generated by homologous recombination of a plasmid engineered for recombination with the wild-type phage genome can be isolated from a mixture comprising a very small percentage (e.g., 0.005%) of total phage genomes. Following isolation, large-scale production can be carried out to obtain stocks of high-titer recombinant indicator phages suitable for use in the Listeria spp. detection assay. Furthermore, cesium chloride density gradient centrifugation can be used to separate the phage particles from contaminating luciferase protein to reduce background. Methods for Using Infectious Agents to Detect Listeria spp. As noted herein, in certain embodiments, the invention may comprise methods for using infectious particles to detect microorganisms. The methods of the invention may be implemented in a variety of ways. In one embodiment, the invention may comprise a method for detecting a bacterium of interest in a sample comprising the steps of: incubating the sample with the bacteriophage that infects the bacterium of interest, wherein the bacteriophage comprises a reporter gene such that expression of the reporter gene during replication of the bacteriophage after infection of the bacterium of interest results in the production of a soluble reporter protein product; and detecting the reporter protein product, wherein positive detection of the reporter protein product indicates that the bacterium of interest is present in the sample. In certain instances, the invention comprises a method for detecting Listeria spp.in a sample comprising: incubating the sample with a cocktail composition comprising at least one recombinant bacteriophage specific for Listeria; and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Listeria spp. is present in the sample. In some modality, at least one type of recombinant bacteriophage is constructed from LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A. In other modality, at least one type of recombinant bacteriophage is constructed from LMA8, LP-ES1, and LP-ES3A. In certain modalities, the test can be performed using a general concept that can be modified to accommodate different sample types or sizes and test formats. The modalities employing the recombinant bacteriophage of the invention (i.e., indicator bacteriophage) can enable the rapid detection of specific bacterial strains such as Listeria spp., with total assay times below 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, or 26.0 hours, depending on the sample type, sample size, and test format.For example, the amount of time required may be somewhat shorter or longer depending on the bacteriophage strain and the strain of bacteria to be detected in the assay, the type and size of the sample to be tested, the conditions required for the viability of the target, the complexity of the physical / chemical environment, and the concentration of “endogenous” non-target bacterial contaminants. The bacteriophage (e.g., phage T7, T4, P70, P100, A511, LP-ES3A, LP-ES1, LMA4, or LMA8) can be genetically modified to express soluble luciferase during phage replication. Luciferase expression is driven by a viral capsid promoter (e.g., the late promoter of bacteriophage Pecentumvirus or T4), resulting in high expression. Parent phages are prepared to be luciferase-free, so the luciferase detected in the assay must originate from progeny phage replication during bacterial cell infection. Therefore, there is generally no need to separate the parent phage from the progeny phage. Figure 4 depicts a filter plate assay for detecting Listeria using a modified bacteriophage according to one embodiment of the invention. Briefly, samples 416 containing a bacterium of interest 418 can be added to wells 402 of a multi-well filter plate 404 and centrifuged 406 to concentrate the samples by removing sample liquid. Genetically modified phages 420 are added to the wells and incubated with additional medium added for a sufficient time for uptake 408 followed by infection of target bacteria and advancement of the phage replication cycle 410 (e.g., ~240 minutes). Finally, luciferase substrate is added and reacts with any luciferase present 424. The resulting emission is measured in a luminometer 414, which detects luciferase activity 426. In some methods, bacterial enrichment of the sample is not required before testing. In others, the sample can be enriched before testing by incubation under growth-promoting conditions. In such methods, the enrichment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more, depending on the sample type and size. In some embodiments, the indicator bacteriophage comprises a detectable indicator group, and infection of a single pathogenic cell (e.g., bacteria) can be detected by an amplified signal generated through the indicator group. Therefore, the method may involve detecting an indicator group produced during phage replication, where detection of the indicator group indicates that the bacteria of interest are present in the sample. In one embodiment, the invention may comprise a method for detecting a bacterium of interest in a sample comprising the steps of: incubating the sample with a recombinant bacteriophage that infects the bacterium of interest, wherein the recombinant bacteriophage comprises a reporter gene inserted into a late gene region of the bacteriophage such that expression of the reporter gene during bacteriophage replication after infection of host bacteria results in the production of a soluble reporter protein product; and detecting the reporter protein product, wherein positive detection of the reporter protein product indicates that the bacterium of interest is present in the sample. In some embodiments, the amount of reporter group detected corresponds to the amount of bacterium of interest present in the sample. As described in more detail herein, the methods and systems of the invention can utilize a range of concentrations of parental indicator bacteriophage to infect bacteria present in the sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly find, bind to, and infect target bacteria present in very low numbers in the sample, such as ten cells. In some embodiments, the phage concentration may be sufficient to find, bind to, and infect the target bacteria in less than one hour. In other embodiments, these events may occur in less than two, three, or four hours following the addition of the indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubation step is greater than 1 x 10⁵ PFU / mL, greater than 1 x 10⁶ PFU / mL, or greater than 1 x 10⁷ PFU / mL. In certain methods, the recombinant infectious agent can be purified to remove any residual indicator proteins that may be generated after the production of the stock of infectious agent. Therefore, in certain methods, the recombinant bacteriophage can be purified using cesium chloride density gradient centrifugation before incubation with the sample. When the infectious agent is a bacteriophage, this purification can have the added benefit of removing bacteriophages that lack DNA (i.e., empty phages or “ghosts”). In some embodiments of the methods of the invention, the microorganism can be detected without any isolation or purification of the microorganisms from a sample. For example, in certain embodiments, a sample containing one or a few microorganisms of interest can be applied directly to a test container such as a centrifuge column, a microtiter well, or a filter, and the assay is carried out in that test container. Several embodiments of such assays are disclosed herein. Aliquots of a test sample can be dispensed directly into wells of a multi-well plate, the indicator phage can be added, and after a sufficient time for infection, a buffer solution of systolic acid can be added, as well as a substrate for the indicator group (e.g., luciferase substrate for a luciferase indicator), and the sample is evaluated for the detection of the indicator signal. Some variations of the method can be performed on filter plates. Some variations of the method can be performed with or without pre-concentration of the sample before infection with an indicator phage. For example, in many modalities, multi-well plates are used to conduct the tests. The choice of plates (or any other container in which the detection can be performed) can affect the detection step. For example, some plates may have a colored or white background, which can affect the detection of light emissions. In general, white plates have higher sensitivity but also generate a higher background signal. Other colors or plates may generate a lower background signal but also have slightly lower sensitivity. Additionally, one reason for the background signal is light leakage from one well to an adjacent well. Some plates have white wells, but the rest of the plate is black. This allows for a high signal within the well but prevents light leakage from one well to another and can therefore reduce the background signal.Therefore, the choice of plate or other test container can influence the sensitivity and background signal for the test. The methods of the invention may comprise several other steps to increase the sensitivity. For example, as discussed in more detail herein, the method may comprise a step of washing the captured and infected bacteria, after adding the bacteriophage, but before incubation, removing excess parental bacteriophage and / or luciferase or other reporter protein that contaminates the bacteriophage preparation. In some modalities, the detection of the microorganism of interest can be completed without the need to culture the sample as a way to increase the population of microorganisms. For example, in certain modalities, the total time required for detection is less than 28.0 hours, 27.0 hours, 26.0 hours, 25.0 hours, 24.0 hours, 23.0 hours, 22.0 hours, 21.0 hours, 20.0 hours, 19.0 hours, 18.0 hours, 17.0 hours, 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, or less than 1.0 hour. Minimizing the time to results is critical for testing pathogens in food and the environment. In contrast to known assays in the art, the method of the invention can detect individual microorganisms. Therefore, in certain embodiments, the method can detect as few as 10 cells of the microorganism present in a sample. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Listeria spp. In one embodiment, the recombinant bacteriophage can distinguish Listeria spp. in the presence of other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of the specific type in the sample. In certain embodiments, the recombinant bacteriophage detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 of the specific bacteria in the sample. Therefore, aspects of the present invention provide methods for detecting microorganisms in a test sample using an indicator group. In some embodiments, where the microorganism of interest is a bacterium, the indicator group may be associated with an infectious agent such as an indicator bacteriophage. The indicator group may react with a substrate to emit a detectable signal or may emit an intrinsic signal (e.g., fluorescent protein). In some embodiments, the detection sensitivity may reveal the presence of as few as 50, 40, 30, 20, 10, 5, or 2 cells of the microorganism of interest in a test sample. In some embodiments, even a single cell of the microorganism of interest may generate a detectable signal. In some embodiments, the bacteriophage is a Pecentumvirus, Tequatravirus, Homburgvirus, or Kuttervirus.In some formulations, the recombinant bacteriophage is derived from a Listeria-specific bacteriophage. In certain formulations, a recombinant Listeria-specific bacteriophage is highly specific for Listeria spp. In some embodiments, the indicator group encoded by the infectious agent can be detectable during or after the agent's replication. Many different types of detectable biomolecules suitable for use as indicator groups are known in the field, and many are commercially available. In some embodiments, the indicator phage comprises an enzyme, which serves as the indicator group. In some embodiments, the indicator phage genome is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator may emit light and / or may be detectable by a color change in an added substrate. Several suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can serve as the indicator group.In some formulations, firefly luciferase is the indicator group. In some formulations, oplophorus luciferase is the indicator group. In some formulations, NANOLUC® is the indicator group. Other luciferases or other genetically modified enzymes that generate detectable signals may also be appropriate indicator groups. Therefore, in some embodiments, the recombinant bacteriophage for these methods, systems, or kits is prepared from the wild-type Listeria-specific bacteriophage. In some embodiments, the reporter gene encodes a protein that emits an intrinsic signal, such as a fluorescent protein (e.g., green fluorescent protein or others). The reporter may emit light and / or be detectable by a color change. In some embodiments, the reporter gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to generate a signal. In some embodiments, the reporter gene is a luciferase gene. In some embodiments, the luciferase gene is one of Oplophorus luciferase, Firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase, or an engineered luciferase such as NANOLUC®, Rluc8.6-535, or Nano-Orange Lantern. Detecting the indicator may involve detecting light emissions. In some modalities, a luminometer can be used to detect the indicator's reaction (e.g., luciferase) with a substrate. RLU detection can be achieved with a luminometer, or other machines or devices can also be used. For example, a spectrophotometer, CCD camera, or CMOS camera can detect color changes and other light emissions. Absolute RLUs are important for detection, but the signal-to-background ratio also needs to be high (e.g., >2.0, >2.5, or >3.0) in order for single cells or low cell numbers to be reliably detected. In some variants, the reporter phage is genetically engineered to contain the gene for an enzyme, such as luciferase, that is produced only after infection of bacteria that the phage specifically recognizes and infects. In some variants, the reporter group is expressed late in the viral life cycle. In some variants, as described herein, the reporter is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits its copy number. Therefore, in some embodiments using a reporter phage, the invention comprises a method for detecting a microorganism of interest comprising the steps of capturing at least one bacterium from the sample; incubating the at least one bacterium with a plurality of reporter phages; allowing time for infection and replication to generate progeny phages and express a soluble reporter group; and detecting the progeny phage, or preferably the reporter, wherein detection of the reporter demonstrates that the bacterium is present in the sample. For example, in some methods, the bacteria in the test sample can be captured by adhering to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a centrifuge filter or a 0.45 µm pore size plate filter). In one method, the infectious agent (e.g., a reporter phage) is added in a minimal volume to the captured sample directly onto the filter. In another method, the microorganism captured on the filter or plate surface is subsequently washed one or more times to remove excess unbound infectious agent.In one modality, a medium (e.g., Luria-Bertani (LB) broth, buffered peptone water (BPW), tryptic soy broth, tryptone soy broth (TSB), Listeria enrichment broth (BLEB) buffered with brain heart infusion (BHI), University of Vermont (UVM) broth, or Fraser broth) may be added for an additional incubation time to allow for bacterial cell and phage replication and high-level expression of the gene encoding the reporter group. However, a surprising aspect of some assay modalities is that the reporter phage incubation step only needs to be long enough for a single phage life cycle.The amplification power of using the bacteriophage was previously thought to require more time, with the phage replicating over several cycles. A single cycle of indicator phage replication may be sufficient to facilitate sensitive and rapid detection according to some embodiments of the present invention. In some embodiments, aliquots of a test sample comprising bacteria can be applied to a centrifuge column and after infection with a recombinant bacteriophage and optional washing to remove any excess bacteriophage, the amount of soluble indicator detected will be proportional to the amount of bacteriophages produced by infected bacteria. The soluble indicator (e.g., luciferase) released into the surrounding liquid after bacterial lysis can then be measured and quantified. In one method, the solution is centrifuged through the filter, and the filtrate is collected for assay in a new receptacle (e.g., a luminometer) followed by the addition of a substrate for the indicator enzyme (e.g., luciferase substrate). Alternatively, the indicator signal can be measured directly on the filter. In several embodiments, the purified parental reporter phage does not comprise the detectable reporter itself, as the parental phage can be purified before incubation with a test sample. Late gene expression (Class III) occurs late in the viral replication cycle. In some embodiments of the present invention, the parental phage can be purified to exclude any existing reporter protein (e.g., luciferase). In some embodiments, reporter gene expression during bacteriophage replication after infection of host bacteria results in a soluble reporter protein product. Therefore, in many embodiments, it is not necessary to separate the parental phage from the progeny phage before the detection step. In one embodiment, the microorganism is a bacterium, and the reporter phage is a bacteriophage.In one modality, the indicator group is soluble luciferase, which is released after lysis of the host microorganism. Therefore, in an alternative modality, the indicator substrate (e.g., luciferase substrate) can be incubated with the portion of the sample remaining on a filter or bound to a plate surface. Consequently, in some modalities, the solid support is a 96-well filtration plate (or regular 96-well plate), and the substrate reaction can be detected by placing the plate directly in the luminometer. For example, in one embodiment, the invention may comprise a method for detecting Listeria spp. comprising the steps of: infecting captured cells onto a 96-well filtration plate with a plurality of parental indicator phage capable of expressing luciferase after infection; washing off excess phage; adding BHI broth and allowing time for the phage to replicate and lyse the specific target Listeria spp. (e.g., 60-240 minutes); and detecting the indicator luciferase by adding luciferase substrate and measuring luciferase activity directly on the 96-well plate, wherein the detection of luciferase activity indicates that Listeria spp. is present in the sample. In another embodiment, the invention may comprise a method for detecting Listeria spp. comprising the steps of: infecting cells in liquid solution or suspension in a 96-well plate with a plurality of parental indicator phage capable of expressing luciferase after infection; allowing time for the phage to replicate and lyse the specific target Listeria spp. (e.g., 60-240 minutes); and detecting the indicator luciferase by adding luciferase substrate and measuring luciferase activity directly in the 96-well plate, wherein the detection of luciferase activity indicates that Listeria spp. is present in the sample. In such an embodiment, a capture step is not required. In some embodiments, the liquid solution or suspension may be a consumable test sample, such as a vegetable wash.In some formulations, the liquid solution or suspension may be a vegetable wash fortified with concentrated Luria-Bertani (LB) broth, buffered peptone water (BPW), tryptic soy broth, tryptone soy broth (TSB), Listeria enrichment broth (BLEB) buffered with brain and heart infusion (BHI), University of Vermont (UVM) broth, or Fraser broth. In some formulations, the liquid solution or suspension may consist of bacteria diluted in BHI broth. In some assays, bacterial lysis can occur before, during, or after the detection step. Experiments suggest that unused infected cells may be detectable after the addition of a luciferase substrate in some assays. Presumably, luciferase can exit the cells and / or the luciferase substrate can enter the cells without complete cell lysis. Therefore, for assays using the centrifuge filter system, where only luciferase released in the lysate (and not luciferase still within intact bacteria) is tested in the luminometer, lysis is required for detection. However, for assays using filter plates or 96-well plates with sample in solution or suspension, where the original plate filled with intact and used cells is tested directly in the luminometer, lysis is not required for detection. In some modalities, the reaction of the indicator group (e.g., luciferase) with the substrate may continue for 60 minutes or more, and detection at multiple time points may be desirable to optimize sensitivity. For example, in modalities using 96-well filter plates as the solid support and luciferase as the indicator, luminometer readings may initially be taken at 10- or 15-minute intervals until the reaction is complete. Surprisingly, high concentrations of phage used to infect test samples have successfully detected very low numbers of a target microorganism in a very short time. In some methods, phage incubation with a test sample only needs to be long enough for one phage life cycle. In some modalities, the bacteriophage concentration for this incubation step is greater than 7 x 10®, 8 x 106, 9 x 106, 1.0 x 107, 1.1 x 107, 1.2 x 107, 1.3 x 107, 1.4 x 107, 1.5 x 107, 1.6 x 107, 1.7 x 107, 1.8 x 107, 1.9 x 107, 2.0 x 107, 3.0 x 107, 4.0 x 107, 5.0 x 107, 6.0 x 107, 7.0 x 107, 8.0 x 107, 9.0 x 107, or 1.0 x 108PFU / mL. The success with such high phage concentrations is surprising because large phage numbers were previously associated with “lysis from the outside,” which eliminated target cells and thus prevented the generation of a useful signal from earlier phage assays. It is possible that the cleaning of the prepared phage stock described herein (e.g., cleaning by isopycnic ultracentrifugation in a cesium chloride density gradient) could help alleviate this problem, since in addition to removing any contaminating luciferase associated with the phage, this cleaning can also remove ghost particles (particles that have lost DNA). Ghost particles can lyse bacterial cells prematurely, thus preventing the generation of the indicator signal. Electron microscopy demonstrates that a sample of crude phage (i.e., before cesium chloride cleaning) can contain more than 50% ghost particles.These ghost particles can contribute to the premature death of the microorganism through the action of numerous phage particles piercing the cell membrane. Ghost particles may have contributed to previous problems where high concentrations of PFU were reported to be detrimental. Furthermore, a very clean phage preparation allows the assay to be performed without washing steps, making it possible to run the assay without an initial concentration step. Some modalities include an initial concentration step, and in some of these, this concentration step allows for a shorter enrichment incubation time. Some methods for testing may also include confirmatory assays. A variety of assays are known in the technique to confirm an initial result, usually at a later time point. For example, samples may be cultured (e.g., on selective chromogenic plates) and PCR may be used to confirm the presence of microbial DNA, or other confirmatory assays may be used to confirm the initial result. In certain embodiments, the methods of the present invention combine the use of a binding agent (e.g., antibody) to purify and / or concentrate a microorganism of interest, such as Listeria spp., from the sample with detection using an infectious agent. For example, in certain embodiments, the present invention comprises a method for detecting a microorganism of interest in a sample comprising the steps of: capturing the microorganism from the sample onto a pre-coated support using a capture antibody specific for the microorganism of interest, such as Listeria spp.; and incubating the sample with a recombinant bacteriophage that infects Listeria spp.wherein the recombinant bacteriophage comprises a reporter gene inserted into a late gene region of the bacteriophage such that expression of the reporter gene during bacteriophage replication followed by infection of host bacteria results in a soluble reporter protein product; and detecting the reporter protein product, wherein positive detection of the reporter protein product indicates that Listeria spp. is present in the sample. In some approaches, synthetic phages are engineered to optimize desirable traits for use in pathogen detection assays. In some approaches, bioinformatics and prior genetic modification analyses are employed to optimize these traits. For example, in some approaches, the genes encoding phage tail proteins can be optimized to recognize and bind to particular bacterial species. In other approaches, the genes encoding phage tail proteins can be optimized to recognize and bind to an entire genus of bacteria, or a particular group of species within a genus. In this way, the phage can be optimized to detect broader or narrower groups of pathogens. In some approaches, the synthetic phage can be engineered to enhance reporter gene expression.Additionally, and / or alternatively, in some instances, the synthetic phage can be designed to increase the phage rupture size to improve detection. In some embodiments, phage stability can be optimized to improve shelf life. For example, enzyme solubility can be increased to enhance subsequent phage stability. Additionally and / or alternatively, phage thermostability can be optimized. Thermostable phages retain functional activity better during storage, thus increasing their shelf life. Therefore, in some embodiments, thermostability and / or pH tolerance can be optimized. In some embodiments, the genetically modified or synthetically derived phage includes a detectable marker. In some embodiments, the marker is a luciferase. In some embodiments, the phage genome includes a marker gene (for example, a luciferase gene and another gene encoding a detectable marker). Systems and kits of the invention In some embodiments, the invention comprises systems (e.g., automated systems or kits) that include components for performing the methods described herein. In some embodiments, the indicator phage is included in systems or kits according to the invention. Methods described herein may also utilize such indicator phage systems or kits. Some embodiments described herein are particularly suitable for automation and / or kits, given the minimal quantity of reagents and materials required to perform the methods. In certain embodiments, each component of a kit may comprise a self-contained unit that is deliverable from a first site to a second site. In some embodiments, the invention comprises systems or kits for the rapid detection of a microorganism of interest in a sample. The systems or kits may, in certain embodiments, comprise a component for incubating the sample with an infectious agent specific to the microorganism of interest, wherein the infectious agent comprises an indicator group and a component for detecting the indicator group. In some embodiments of both the systems and kits of the invention, the infectious agent is a recombinant bacteriophage that infects the bacterium of interest, and the recombinant bacteriophage comprises a indicator gene inserted in a late gene region of the bacteriophage as the indicator group, such that expression of the indicator gene during bacteriophage replication followed by infection of host bacteria results in a soluble indicator protein product.Some systems also include a component for capturing the microorganism of interest on a solid support. In other embodiments, the invention comprises a method, system, or kit for the rapid detection of a microorganism of interest in a sample, comprising a component of the infectious agent that is specific to the microorganism of interest, wherein the infectious agent comprises an indicator group, and a component for detecting the indicator group. In some embodiments, the bacteriophage is a Tequatravirus, Vil, Kuttervirus, Homburgvirus, Pecentumvirus, or a bacteriophage specific to Listeria spp. In one embodiment, the recombinant bacteriophage is derived from a bacteriophage specific to Listeria spp. 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 Listeria spp. In one embodiment, the recombinant bacteriophage can distinguish Listeria spp. in the presence of other types of bacteria.In certain modalities, a system or kit detects only 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in the sample. In certain embodiments, the systems and / or kits may also include a component for washing the captured microorganism sample. Additionally or alternatively, the systems and / or kits may also include a component for determining the quantity of the indicator group, where the quantity of the indicator group detected corresponds to the quantity of 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. 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 user-controlled via computer input and / or where a liquid-handling robot performs at least one step. Therefore, in certain embodiments, the invention may comprise a system or kit for the rapid detection of a microorganism of interest in a sample, comprising: a component for incubating the sample with an infectious agent specific to the microorganism of interest, wherein the infectious agent comprises an indicator group; a component for capturing the microorganism from the sample onto a solid support; a component for washing the sample with the captured microorganism to remove unbound infectious agent; and a component for detecting the indicator group. In some embodiments, the same component may be used in steps for capturing, incubating, and / or washing (e.g., a filter component). Some embodiments further comprise a component for determining the quantity of the microorganism of interest in the sample, wherein the quantity of the indicator group detected corresponds to the quantity of microorganism in the sample.Such systems may include various modes and sub-modes analogous to those described earlier for rapid microorganism detection methods. In one mode, 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 user-controlled via a computer (or some combination thereof). In some modalities, the system may comprise a component to isolate the microorganism of interest from the other components in the sample. In one embodiment, the invention comprises a system or kit comprising components for detecting a microorganism of interest, comprising: a component for isolating at least one microorganism from other components in the sample; a component for infecting at least one microorganism with a plurality of a parental infectious agent; a component for lysing at least one infected microorganism to release progeny infectious agents present in the microorganism; and a component for detecting the progeny infectious agents, or, more sensitively, a soluble protein encoded and expressed by the infectious agent, wherein the detection of the infectious agent or a soluble protein product of the infectious agent indicates that the microorganism is present in the sample. The infectious agent may comprise a Listeria-specific bacteriophage with NANOLUC® carrying the NANOLUC® reporter gene. Systems or kits may comprise a variety of components for the detection of progeny infectious agents. For example, in one modality, the progeny infectious agent (e.g., bacteriophage) may comprise a reporter group. In another modality, the reporter group in the progeny infectious agent (e.g., bacteriophage) may be a detectable group that is expressed during replication, such as a soluble luciferase protein. In other embodiments, the invention may comprise a kit for the rapid detection of a microorganism of interest in a sample. The kit comprises: a component for incubating the sample with an infectious agent specific to the microorganism of interest, wherein the infectious agent comprises an indicator group; a component for capturing the microorganism from the sample onto a solid support; a component for washing the sample with the captured microorganism to remove unbound infectious agent; and a component for detecting the indicator group. In some embodiments, the same component may be used in the capture, incubation, and / or washing steps. Some embodiments further comprise a component for determining the quantity of the microorganism of interest in the sample, wherein the quantity of the indicator group detected corresponds to the quantity of microorganism in the sample.Such kits may include several modes and sub-modes analogous to those described earlier for rapid microorganism detection methods. In one mode, the microorganism is a bacterium and the infectious agent is a bacteriophage. In some forms, the kit may include a component to isolate the microorganism of interest from the other components in the sample. These systems and kits of the invention include various components. As used herein, the term “component” is broadly defined and includes any suitable apparatus or collections of apparatus suitable for carrying out the described method. The components need not be connected or integrally situated with each other in any particular way. The invention includes any suitable arrangement of the components with each other. For example, the components need not be in the same room. But in some embodiments, the components are connected to each other in an integral unit. In some embodiments, the same components may perform multiple functions. Computer Systems and Computer Readable Media The system, as described in this technique, or any of its components, can be configured as a computing system. Typical examples of a computing system include a general-purpose computer, a programmed microprocessor, a microcontroller, a peripheral integrated circuit element, and other devices or arrangements of devices capable of implementing the steps that constitute the method of this technique. A computer system may comprise a computer, an input device, a display unit, and / or the Internet. The computer may also comprise a microprocessor. The microprocessor may be connected to a communication channel. The computer may also include memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further comprise a storage device. The storage device may be a hard disk or a removable storage unit such as a floppy disk drive, optical disc drive, etc. The storage device may also be another similar medium for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet through an I / O interface.The communication unit allows the transfer of data to, as well as the reception of data from, other databases. This unit may include a modem, an Ethernet card, or any similar device that allows the computer system to connect to databases and networks such as LANs, MANs, WANs, and the Internet. The computer system can therefore facilitate user input through an input device, accessible to the system via an I / O interface. A computing device will typically include an operating system that provides executable program instructions for the general management and operation of that computing device, and will typically include a computer-readable storage medium (e.g., a hard disk, random-access memory, read-only memory, etc.) that stores instructions which, when executed by a server processor, enable the computing device to perform its intended functions. Suitable implementations for the operating system and general functionality of the computing device are either known or commercially available, and are readily implemented by those skilled in the art, particularly in light of the disclosure herein. The computer system executes a set of instructions stored in one or more storage elements in order to process input data. The storage elements can also hold other data or information as desired. The storage element can be in the form of a data source or a physical memory element within the processing machine. The environment may include a variety of data stores and other memory and storage media, as discussed earlier. These may reside in a variety of locations, such as on local storage for (and / or residing on) one or more of the computers, or remotely from any or all of the computers across the network. In a particular set of modalities, the information may reside on a storage area network (“SAN”) familiar to those skilled in the art. Similarly, any files necessary to perform the functions assigned to the computers, servers, or other network devices may be stored locally and / or remotely, as appropriate.Where a system includes computing devices, each of these devices may include hardware elements that can be electrically coupled via a bus. These elements include, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touchscreen, or numeric keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system may also include one or more storage devices, such as disk drives, optical storage devices, and solid-state storage devices, such as random-access memory (RAM) or read-only memory (ROM), as well as removable media devices, memory cards, flash cards, etc. Such devices may also include a computer-readable storage media reader, a communications device (e.g., a modem, a network card (wireless or wired), an infrared communications device, etc.), and functional memory as described earlier. The computer-readable storage media reader may connect to, or be configured to receive, a computer-readable storage medium, which represents 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 will also typically include a number of software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs, such as a client application or web browser. It should be noted that alternative configurations may have numerous variations from those described above. For example, custom hardware may also be used, and / or specific elements may be implemented in hardware, software (including portable software, such as applets), or both. Furthermore, connections to other computing devices, such as network input / output devices, may be employed. Non-transient storage media and computer-readable media for containing code, or portions of code, may include any appropriate medium known or used in the art, including storage media and communication media, such as but not limited to volatile and non-volatile, removable and non-removable media implemented in any method or technology for the storage and / or transmission of information as computer-readable instructions, data structures, program modules, or other data, including RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disc (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information and that can be accessed by a system device.Based on the disclosure and techniques provided herein, an expert in the technique will appreciate other ways and / or methods to implement the various bAn^nn / i ¡wp / yl modalities. A computer-readable medium may include, but is not limited to, any electronic, optical, magnetic, or other storage device capable of providing a processor with computer-readable instructions. Other examples include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, SRAM, DRAM, content-addressable memory (CAM), DDR, flash memory such as NAND flash or NOR flash, an ASIC, a configured processor, optical storage, magnetic tape, or other magnetic storage, or any other medium from which a computer's processor can read instructions. In one embodiment, the computing device may comprise only one type of computer-readable medium, such as random-access memory (RAM).In other configurations, the computing device may comprise two or more types of computer-readable media, such as random access memory (RAM), a disk drive, and cache. The computing device may communicate with one or more external computer-readable media, such as an external hard disk drive or an external DVD or Blu-ray drive. As discussed earlier, the modality comprises a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter of code written in any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one modality, the computing device comprises a single processor. In other modality, the device comprises two or more processors. Such processors may include a microprocessor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), field-programmable gate arrays (FPGAs), and state machines.These processors may also include programmable electronic devices such as PLCs, programmable interrupt controllers (PIOs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electronically programmable read-only memories (EPROMs or EEPROMs), or other similar devices. The computing device includes a network interface. In some configurations, the network interface is configured to communicate via wired or wireless communication links. For example, the network interface may enable communication across networks using Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (WiMAX), Bluetooth, infrared, etc. Alternatively, the network interface may enable communication across networks such as CDMA, GSM, UMTS, or other cellular communication networks. In some configurations, the network interface may allow point-to-point connections with another device, such as via the Universal Serial Bus (USB), FireWire 1394, serial or parallel connections, or similar interfaces. Some suitable computing devices may include two or more network interfaces for communication across one or more networks.In some configurations, the computing device may include data storage in addition to or instead of a network interface. Some types of suitable computing devices may include or communicate with a number of external or internal devices such as a mouse, CD-ROM drive, DVD drive, keyboard, display, audio speakers, one or more microphones, or any other input or output device. For example, the computing device may communicate with several user interface devices and a display. The display may use any suitable technology, including, but not limited to, LCD, LED, CRT, and similar technologies. The set of instructions for execution by the computer system may include various commands that instruct the processing machine to perform specific tasks, such as the steps that constitute the method of the present technique. The set of instructions 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 within a larger program, or a portion of a program module, as in the present technique. 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 in response to user commands, results of previous processing, or a request made by another processing machine. While the present invention has been described with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the scope and substance of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention is not limited to the described embodiments, but rather has the full scope defined by the language of the following claims and their equivalents. Examples The results represented in the following examples demonstrate the detection of a low number of cells, only 1 Listeria bacteria, in a shortened time for the results. Example 1. Creation and Isolation of an Indicator Phage from a Listeria-Specific Bacteriophage The Listeria-specific indicator phages LMA4.NANOLUC, LMA8.NANOLUC, and other Listeria bacteriophages were created through homologous recombination using the procedures described above. See Figures 1–3, which depict and describe the recombinant Listeria phages derived from LMA4 and LMA8. The genomic sequences of these phages were obtained through whole-genome sequencing using the Illumina MiSeq system with de novo sequence assembly. Based on previously known and annotated genomes of related phages, late gene regions and Major Capsid Protein genes were located in the new phage genomes. Plasmids were designed and synthesized to insert NANOLUC® along with the appropriate late gene promoter and ribosomal binding site, flanked by approximately 200–500 bp of matched phage sequence to promote homologous recombination. Target bacteria were transformed with the Plasmids for Homologous Recombination under appropriate antibiotic selection and infected with their respective wild-type phage to allow bAn^nn / Lznz / E / Yi homologous recombination with the plasmid. After homologous recombination to generate recombinant bacteriophage genomes, a series of titration and enrichment steps was used to isolate specific recombinant bacteriophages expressing NANOLUC® as previously described. Finally, large-scale production was carried out to obtain stocks with high titers suitable for use in Listeria spp. detection assays. Isopycnic density gradient centrifugation in cesium chloride can be used to separate phage particles from the contaminating luciferase protein to reduce background signal. In other methods, isopycnic density gradient centrifugation in sucrose can be used to separate phage particles from the contaminating luciferase protein to reduce background signal. Example 2 Inoculated sponge sample - Sponge test for Listeria EZ Reach polyurethane sponge samplers were pre-moistened with Dey / Engley broth and inoculated with <1 CFU of Listeria monocytogenes, which was diluted from an overnight culture or with 100 CFU of challenge bacteria (Cronobacter sakazakii). The loop of the sponge was broken off, and the sponge was placed in a bag containing the medium. Buffered Listeria enrichment broth (BLEB) medium (Remel) (10 or 90 mL) was added to cover as much of the sponge as possible. The sponge was then gently massaged to release bacteria into the medium, and enrichment continued at 35 °C for 16–18 hours or 24 hours. After enrichment, the sponges were gently massaged / squeezed to remove the liquid and then removed from the medium in the bag. The bag was then gently massaged to mix the contents. 150 pL aliquots were transferred to a 96-well plate. The sponge was then placed back into the medium for further enrichment at 35 °C, if required. Sponge samples were tested with a Listeria phage cocktail after 1-hour and 4-hour infection. Briefly, phage reagent (10 pL) was added to the samples, and the samples were incubated at 30 °C for either 1 hour or 4 hours. Finally, 65 pL of Luciferase Master Mix reagent was added to each well and gently mixed by pipetting up and down. The samples were read (i.e., luminescence was detected) using a luminometer (GloMax96) 3 minutes after substrate addition. Sponges / swabs were then returned to the bag / tube, and enrichment continued at 35 °C for a total of 24 hours. Optionally, aliquots could be taken, further enriched, and retested. The results are shown in Figures 5A and 5B. A signal-to-background (S / B) ratio greater than 3 was considered positive. The background level was determined to be 100 RLU based on previous phage characterizations. These experiments indicate that the Listeria phage assay can detect an addition of 1 CFU of L. monocytogenes ATCC 19115 after 16–18 hours of enrichment and 1 hour of infection. Figure 5A (10 mL of medium) shows that sponge samples 1 and 4 were negative for Listeria detection (i.e., S / B < 3). Sponge samples 2, 3, 5, and the 10 CFU control were all positive for all enrichment and infection times. Figure 5B (90 mL of medium) shows that sponge 1 was negative for Listeria detection, while all other samples were positive.These data indicate that sponges with 10 mL of added medium generated a better signal with a higher relative S / B than samples with 90 mL of added medium. Therefore, the experiment demonstrates that it is possible to detect an addition of 1 CFU to a culture overnight under all conditions evaluated (i.e., 16-18 hours enrichment - 1 hour infection, 16-18 hours enrichment - 4 hours infection, 24 hours enrichment - 1 hour infection, and 24 hours enrichment - 4 hours infection). Example 3. Environmental surface sample - sponge test for Listeria Stainless steel surfaces were inoculated with the specified number of cells in the medium. The cells were allowed to dry on the surface and kept at room temperature for 18–24 hours before being cleaned with EZ Reach polyurethane sponge samplers that had been pre-moistened with Letheen medium (World BioProducts). Listeria monocytogenes 19115 was used as a blank and Staphylococcus aureus 12600 as a challenge strain. The sponge handle was broken off, and the sponge was placed back into the bag. Listeria buffered enrichment broth (BLEB) medium (Remel) (20 mL) was added to cover as much of the sponge as possible. The sponge was gently massaged to release the bacteria into the medium, and enrichment continued at 35 °C for 20 hours. After enrichment, the sponges were gently massaged, squeezed to remove the liquid, and removed from the bag. The bag was then gently massaged to mix the contents. 150 pL aliquots were transferred to a 96-well plate. The sponge was placed back into the medium and incubated at 35 °C if further enrichment was required. Sponge samples were tested with the Listeria phage cocktail after 1 and 4 hours of infection. Briefly, phage reagent (10 pL) was added to the samples and incubated at 30 °C for 4 hours. Finally, 65 pL of Luciferase Master Mix reagent was added to each well and gently mixed by pipetting up and down. The samples were read (i.e., luminescence was detected) on a GloMax96 instrument 3 minutes after substrate addition. Phage reagent (10 pL) was added to the samples and incubated at 30 °C for 4 hours. Finally, 65 pL of luciferase Master Mix reagent was added to each well and gently mixed by pipetting up and down. The samples were read (i.e., luminescence was detected) on a GloMax96 instrument 3 minutes (180 seconds) after substrate addition. Figure 6 shows the RLUs generated from the stainless steel surface swabs. Samples that generated RLUs >300 were considered positive. These data show that the Listeria phage assay can detect a surface inoculation of 100 CFU of L. monocytogenes with a 20-hour enrichment and 4-hour infection in the presence of a non-Listeria bacterium present at a CFU level 10 times higher than the target Listeria bacterium. Sample 2 and the negative control were negative for L. monocytogenes detection. All other samples, including the positive control, were positive. Compared to experiments with inoculated sponges, the stainless steel surface swabs required a longer enrichment period and a higher CFU level for positive detection.This can be attributed to several variables, including greater damage to cells inoculated onto the surface compared to those from an overnight culture. Cells inoculated onto the surface often experience a significant loss of viability. Furthermore, cell recovery from a sponge-coated surface can be highly variable.

Claims

1. A recombinant bacteriophage comprising a reporter gene inserted into a late gene region of the bacteriophage genome, wherein the recombinant bacteriophage specifically infects Listeria spp.

2. The recombinant bacteriophage according to claim 1, wherein the recombinant bacteriophage is constructed from an LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A bacteriophage 3. The recombinant bacteriophage according to claim 1, wherein the reporter gene is optimized in its codons and encodes a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates a signal after reaction with a substrate.

4. The recombinant bacteriophage according to claim 1, further comprising an untranslated region upstream of the reporter gene with optimized codons, wherein the untranslated region includes a bacteriophage late gene promoter and a ribosomal entry site.

5. A cocktail composition comprising at least one recombinant bacteriophage according to claim 1.

6. The cocktail composition according to claim 5, wherein at least one recombinant bacteriophage is constructed from one of LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A.

7. The cocktail composition according to claim 5, comprising at least two recombinant bacteriophages constructed from two LMA8, LP-ES1, and LP-ES3A.

8. A method for preparing a recombinant reporter bacteriophage, comprising: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector comprising a reporter 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 the recombinant bacteriophage.

9. The method according to claim 8, wherein preparing a plasmid / vector for homologous recombination comprises: determining the natural nucleotide sequence in the late region of the selected bacteriophage genome; 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, wherein the sequence comprises a reporter gene with optimized bAn^nn / ίζηζ / E / γι codons; and incorporating the designed sequence for homologous recombination into a plasmid / vector.

10. The method according to claim 9, wherein designing a sequence further comprises inserting an untranslated region that includes a late gene promoter of the phage and the ribosomal entry site upstream of the reporter gene with optimized codons.

11. The method according to claim 8, wherein the homologous recombination plasmid comprises an untranslated region including a bacteriophage late gene promoter and a ribosomal entry site upstream of the reporter gene with optimized codons.

12. The method according to claim 8, wherein the wild-type bacteriophage is a Listeria-specific bacteriophage and the target pathogenic bacteria is Listeria monocytogenes or another Listeria spp.

13. The method according to claim 8, wherein isolating a particular clone of the recombinant bacteriophage comprises a limiting dilution assay to isolate a clone that demonstrates expression of the reporter gene.

14. A method for detecting Listeria spp. in a sample comprising: incubating the sample with a cocktail composition comprising at least one recombinant bacteriophage specific for Listeria according to claim 1; and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Listeria spp. is present in the sample.

15. The method according to claim 14, wherein at least one recombinant bacteriophage is constructed from one of LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A.

16. The method according to claim 14, comprising at least two recombinant bacteriophages constructed from at least two of LMA8, LP-ES1, and LP-ES3A.

17. The method according to claim 14, wherein the sample is a food, environmental, water or commercial sample.

18. The method according to claim 14, wherein the method detects only 10, 9, 8, 7, 6, 5, 4, 3, 2 or a single bacterium in a sample of a standard size for the food safety industry.

19. The method according to claim 17, wherein the food sample comprises meat, fish, vegetables, eggs, dairy products, dried food products or powdered infant formula.

20. The method according to claim 14, wherein the sample is first incubated under growth-promoting conditions for an enrichment period of less than 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours.

21. The method according to claim 14, wherein the total time for the results is less than 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours.

22. The method according to claim 14, wherein the signal-to-background ratio generated upon detecting the indicator is at least 2.0 or at least 2.5 or at least 3.

0.

23. A kit for detecting Listeria spp. comprising a recombinant bacteriophage derived from a Listeria-specific bacteriophage.

24. The kit according to claim 23, further comprising a substrate for reacting with an indicator for detecting the soluble protein product expressed by the recombinant bacteriophage.

25. A system for detecting Listeria spp. comprising recombinant bacteriophages derived from a Listeria-specific bacteriophage.