Method and system for rapid detection of Cronobacter using infectious agents

Abstract

Disclosed herein are methods and systems for the rapid detection of microorganisms (e.g., Cronobacter spp.) in a sample. Genetically modified bacteriophages containing indicator genes in the late gene region are also disclosed. The specificity of bacteriophages, such as Cronobacter-specific bacteriophages, allows for the detection of specific microorganisms, such as Cronobacter spp., and the indicator signal can be amplified to optimize assay sensitivity.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority under U.S. Provisional Patent Application No. 62 / 616,956 filed January 12, 2018; U.S. Provisional Patent Application No. 62 / 628,616 filed February 9, 2018; and U.S. Provisional Patent Application No. 62 / 661,739 filed April 24, 2019. The disclosures of U.S. Patent Applications No. 13 / 773,339, No. 14 / 625,481, No. 15 / 263,619, No. 15 / 409,258, and U.S. Provisional Patent Applications No. 62 / 616,956, No. 62 / 628,616, and No. 62 / 661,739 are incorporated herein by reference in their entirety.

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

[0003] background Improving the speed and sensitivity for detecting bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples is of great importance. Microbial pathogens can cause substantial disease conditions in humans and domesticated animals, as well as enormous economic losses. Furthermore, the detection of microorganisms is a high priority for the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and the U.S. Department of Agriculture (USDA), given the life-threatening or fatal disease outbreaks caused by the consumption of food contaminated with certain microorganisms (e.g., Cronobacter spp., Salmonella spp., Listeria spp., or Staphylococcus spp.).

[0004] Traditional microbiological tests for detecting bacteria rely on non-selective and selective enrichment cultures, followed by plated cultures on selective media and further testing to identify suspicious colonies. Such procedures can take several days. Various rapid methods have been studied and introduced into practice to reduce time requirements. However, these methods have drawbacks. For example, direct immunoassays or techniques involving gene probes generally require an overnight enrichment step to obtain adequate sensitivity. Polymerase chain reaction (PCR) tests also involve an amplification step and therefore possess both very high sensitivity and selectivity; however, economically, the sample size that can be subjected to PCR testing is limited. When using dilute bacterial suspensions, most small by-product samples lack cells and therefore still require purification and / or lengthy enrichment steps.

[0005] The time required for traditional biological enrichment is determined by the growth rate of the target bacterial population in the sample, the effect of the sample matrix, and the required sensitivity. In practice, most highly sensitive methods use an overnight incubation, taking approximately 24 hours in total. Due to the time required for cultivation, these methods can take up to 3 days, depending on the organism to be identified and the source of the sample. This lag time is generally unsuitable because contaminated food, water (or other products) may have already entered livestock or humans. Furthermore, with increasing concerns about antibiotic-resistant bacteria and biological defenses, rapid identification of bacterial pathogens in water, food, and clinical samples has become a global priority.

[0006] Therefore, there is a need for faster, simpler, and more sensitive detection and identification of microorganisms (e.g., bacteria and other potentially pathogenic microorganisms). [Overview of the Initiative] [Means for solving the problem]

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

[0008] In some aspects, the present invention includes recombinant bacteriophages comprising an indicator gene inserted into the late gene region of a bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Cronobacter-specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified Cronobacter-specific bacteriophage genome derived from a bacteriophage that specifically recognizes Cronobacter spp. (formerly classified as Enterobacter sakazakii). In some embodiments, the bacteriophage used to prepare the recombinant bacteriophage specifically infects one or more Cronobacter spp. In one embodiment, the recombinant bacteriophage can distinguish Cronobacter spp. in the presence of other types of bacteria.

[0009] In some embodiments of recombinant indicator bacteriophages, the indicator gene may be codon-optimized and encode a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. Some recombinant bacteriophages further include an untranslated region upstream of the codon-optimized indicator gene, the untranslated region containing a late bacteriophage gene promoter and a ribosome entry site. In some embodiments, the indicator gene is a luciferase gene. The luciferase gene may be a naturally occurring gene, such as the Oplophorus luciferase gene, firefly luciferase gene, Lucia luciferase gene, or Renilla luciferase gene, or the luciferase gene may be a genetically engineered gene such as NANOLUC®.

[0010] Also disclosed herein are methods for preparing recombinant indicator bacteriophages. Several embodiments include the steps of: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector containing an indicator gene; transforming the target pathogenic bacterium with the homologous recombination plasmid / vector; infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage to induce homologous recombination between the plasmid / vector and the bacteriophage genome; and isolating a specific clone of the recombinant bacteriophage. In some embodiments, the selected wild-type bacteriophage is a Cronobacter-specific bacteriophage. In some embodiments, the selected wild-type bacteriophage is a myovirus (e.g., T4, T4-like, or Vil-like). In some embodiments, the selected wild-type bacteriophage infects Cronobacter spp. (e.g., bacteriophage Saka2 or Saka4). Cronobacter spp. phages Saka2 and Saka4 are newly isolated and sequenced phages, possibly myoviruses. In other embodiments, the selected wild-type bacteriophage is a podovirus (e.g., a T7-like virus or Sp6-like virus). In other embodiments, the selected wild-type bacteriophage is Saka10. Saka10 is a newly isolated and sequenced phage, possibly a podovirus associated with the T7 phage.

[0011] In some embodiments, the steps for preparing a homologous recombination plasmid / vector include determining the native nucleotide sequence in the late region of the genome of a selected bacteriophage, annotating the genome and identifying the major capsid protein gene of the selected bacteriophage, designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence includes a codon-optimization indicator gene, and incorporating the sequence designed for homologous recombination into a plasmid / vector. The steps for designing the sequence may include inserting a gene construct, which includes a non-translated region containing a phage late gene promoter and a ribosome entry site, upstream of the codon-optimization indicator gene. 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 homologous recombination plasmid includes a non-translated region containing a bacteriophage late gene promoter and a ribosome entry site upstream of the codon-optimization indicator gene.

[0012] Some embodiments of the present invention are compositions comprising recombinant indicator bacteriophages described herein. For example, a composition may comprise one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, a composition may comprise a cocktail of different indicator phages capable of encoding and expressing the same or different indicator proteins.

[0013] In some embodiments, the invention includes a method for detecting a target microorganism in a sample, the method comprising incubating the sample with a recombinant bacteriophage that infects the target microorganism, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target microorganism in the sample.

[0014] In some embodiments of a method for preparing a recombinant indicator bacteriophage, the wild-type bacteriophage is a Cronobacter spp.-specific bacteriophage and the target pathogenic bacterium is Cronobacter spp. In some embodiments, the wild-type bacteriophage specifically infects bacteria previously classified as Enterobacter sakazakii. In some embodiments, isolation of a particular clone of the recombinant bacteriophage comprises a limiting dilution assay to isolate clones that exhibit expression of the indicator gene.

[0015] Another aspect of the present invention is a method for detecting bacteria (e.g., Cronobacter spp.) in a sample, comprising the steps of: incubating the sample with a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage; and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates the presence of Cronobacter spp. in the sample. In some embodiments, the present invention includes a method for detecting Cronobacter spp. using a recombinant bacteriophage derived from a bacteriophage that targets Cronobacter spp. The sample may be a food sample or a water sample.

[0016] In some embodiments of the method for detecting bacteria, the sample is first incubated under conditions favorable for growth during 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 embodiments, the sample is not enriched prior to detection. In some embodiments, the total time to result is less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours or less than 2 hours. In some embodiments, the signal-to-background ratio generated by detecting the indicator is at least 2.0 or at least 2.5 or at least 3.0. In some embodiments, the method detects a low number of specific bacteria, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100, in a sample of standard size for the food safety industry.

[0017] Further embodiments include systems and kits for detecting Cronobacter spp., the systems or kits comprising recombinant bacteriophages derived from Cronobacter-specific bacteriophages. Some embodiments further include substrates for reacting with indicators for detecting soluble protein products expressed by the recombinant bacteriophages. These systems or kits may include features described with respect to the bacteriophages, compositions and methods of the present invention. In yet another embodiment, the present invention includes non-transient computer-readable media for use with the methods or systems according to the present invention. [Brief explanation of the drawing]

[0018] The present invention can be better understood by referring to the non-limiting drawings.

[0019] [Figure 1] Figure 1 shows an indicator phage construct according to one embodiment of the present invention, illustrating the insertion of a gene construct containing a luciferase gene, a bacteriophage late gene promoter, and a ribosome-binding site (RBS) into the late (class III) region of a bacteriophage. The promoter shown is either upstream of an endogenous late gene (e.g., a major capsid protein (MCP) gene) in addition to or separately from the endogenous late gene promoter.

[0020] [Figure 2]Figure 2 shows the genome of bacteriophage SAKA2 (a myovirus associated with the T4 bacteriophage) obtained from CRONOBACTER. A hypothetical gene homologous to the PG7 tail sheath protein surrounds the late gene region, which consists of structural genes encoding virion proteins. Other late genes shown are homologs of known head vertex proteins, ORF58.1, possibly the major capsid protein, and known outer head proteins. Since these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, insofar as late gene promoters and / or other similar regulatory elements are used.

[0021] [Figure 3] Figure 3 shows two homologous recombination plasmid construct designs for three different phages, each containing a luciferase gene along with approximately 500 bp of phage sequences matched upstream and downstream of the insertion site to promote homologous recombination. NANOLUC® luciferase is inserted into the pUC57.AmpR plasmid backbone, along with an upstream untranslated region containing a dedicated phage late gene promoter and ribosome entry site. Cronobacter phages Saka2 and Saka4 were newly isolated and sequenced phages, presumably myoviruses. Each construct consisted of a 500 bp homologous sequence comprising a fragment of the major capsid protein gene, followed by a T4 late gene promoter (which is an addition to the endogenous late gene promoter upstream of the major capsid protein in the phage genome), a luciferase gene, and approximately 500 bp of downstream matching sequences for homologous recombination. Saka9 and Saka10 are newly isolated and sequenced phages, possibly podoviruses (related to the T7 phage), that required a T7-like late gene promoter instead of a T4 late gene promoter.

[0022] [Figure 4]Figure 4 shows the isolation of recombinant phages from bacteriophage modifications using plasmid constructs (e.g., those shown in Figure 3) using a series of sequential infection and dilution steps to identify recombinant phages expressing indicator genes.

[0023] [Figure 5] Figure 5 shows the use of an indicator phage encoding soluble luciferase to detect bacterial cells during infection by detecting luciferase produced from the replication of progeny phages, according to one embodiment of the present invention.

[0024] [Figure 6] Figure 6 describes a filter plate assay for detecting a target bacterium using a modified bacteriophage according to one embodiment of the present invention, in which bacteria and recombinant phages are incubated on a filter plate and an indicator protein is directly detected after the generation of progeny bacteriophages without removal of the incubation medium.

[0025] [Figure 7] Figure 7 shows a "No Concentration Assay" for detecting a target bacterium using a modified bacteriophage according to an embodiment of the present invention.

[0026] [Figure 8] Figure 8 shows a hybrid immunophage (HIP) assay for detecting a target bacterium using a modified bacteriophage according to one embodiment of the present invention, which captures the target microorganism on the surface of the assay well before incubation with a recombinant infectious agent having an indicator gene, using an antibody against the target microorganism. [Modes for carrying out the invention]

[0027] Detailed description of the invention Compositions, methods, and systems exhibiting remarkable sensitivity in detecting target microorganisms (e.g., Cronobacter spp.) in test samples (e.g., biological samples, food samples, water samples, and environmental samples) are disclosed herein. Detection can be achieved in shorter timeframes than previously considered possible, using genetically modified infectivity factors, in assays performed without enrichment culture or, in some embodiments, with minimal incubation time (during which the microorganism may potentially grow). Furthermore, the success of using potentially high MOI, or high concentrations of plaque-forming units (PFU), for incubation with test samples is remarkable. Such high phage concentrations (PFU / mL) have previously been claimed to be detrimental in bacterial detection assays because they were claimed to cause "lysis from without infection." However, high concentrations of phages can facilitate the discovery, binding, and infection of a small number of target cells.

[0028] The compositions, methods, systems, and kits of the present invention may include infectious agents for use in the detection of microorganisms such as Cronobacter spp. In certain embodiments, the present invention may encompass compositions comprising a bacteriophage having an indicator gene inserted into the late gene region of the recombinant bacteriophage. In certain embodiments, the expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in the production of a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene (i.e., class III) region of the bacteriophage. The above bacteriophages may originate from podoviruses (e.g., T7, T7-like), myoviruses (e.g., T4, T4-like, ViI, ViI-like (or Vi1 virus according to GenBank / NCBI)), Cronobacter spp., specific bacteriophages, or other wild-type or engineered bacteriophages. In some embodiments, the above-selected wild-type bacteriophage is Saka2 or Saka4. Cronobacter spp. phages Saka2 and Saka4 are newly isolated and sequenced phages, possibly myoviruses. In other embodiments, the above-selected wild-type bacteriophage is a podovirus (e.g., a T7-like virus) or an Sp6-like virus. In other embodiments, the above-selected wild-type bacteriophage is Saka10. Saka10 is a newly isolated and sequenced phage, possibly a podovirus associated with the T7 phage.

[0029] In some aspects, the present invention includes a method for detecting a target microorganism. The method may use an infectious agent for the detection of the target microorganism (e.g., Cronobacter spp.). For example, in certain embodiments, the target microorganism is Cronobacter spp., and the infectious agent is a bacteriophage that specifically infects Cronobacter spp. Thus, in certain embodiments, the method may include the detection of the target bacterium in a sample by incubating the sample with a recombinant bacteriophage that infects the target bacterium. In certain embodiments, the recombinant bacteriophage includes an indicator gene. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage, and as a result, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in the production of an indicator protein product. The method may include the step of detecting the indicator protein product, where positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the indicator protein is soluble.

[0030] In certain embodiments, the present invention may encompass a system. The system may comprise at least some of the compositions of the present invention. The system may also comprise at least some of the components for carrying out the method. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present invention may encompass a system for the rapid detection of a target microorganism, such as Cronobacter spp., in a sample, the system comprising components for incubating the sample with an infectious agent specific to the target microorganism, wherein the infectious agent comprises components including an indicator portion; and components for detecting the indicator portion. In yet another embodiment, the present invention includes software for use in conjunction with the method or system.

[0031] Accordingly, some embodiments of the present invention address the requirements by using bacteriophage-based methods for amplifying a detectable signal indicating the presence of bacteria. In certain embodiments, a small number of bacteria, such as one, can be detected. The principles applied herein can be applied to the detection of various microorganisms. Because microorganisms have many binding sites for infectious agents on their surface, the ability to produce 100 or more progeny factors during infection, and the potential for high-level expression of encoded indicator moieties, the infectious agents or indicator moieties may be more readily detectable than the microorganisms themselves. In this way, embodiments of the present invention can achieve very large signal amplification from a single infected cell.

[0032] An aspect of the present invention is the utilization of the high specificity of binding factors that can bind to specific microorganisms, such as binding components of infectious agents, as a means for detecting and / or quantifying specific microorganisms in a sample. In some embodiments, the present invention utilizes the high specificity of infectious agents (e.g., bacteriophages).

[0033] In some embodiments, detection is achieved through an indicator moiety associated with a binding factor specific to the target microorganism. For example, the infectious agent may include an indicator moiety (e.g., a gene encoding a soluble indicator). In some embodiments, the indicator may be encoded by the infectious agent (e.g., a bacteriophage), and the bacteriophage is referred to as the indicator phage.

[0034] Some embodiments of the invention disclosed and described herein utilize the discovery that a single microorganism can bind a specific recognition factor (e.g., a phage). Following infection and replication by the phage, the progeny phages can be detected via an indicator moiety expressed during phage replication. This principle allows for the amplification of an indicator signal from one or more cells based on the specific recognition of a microbial surface receptor. For example, by exposing a single bacterial cell to multiple phages, and then allowing high levels of expression of an encoded indicator gene product during the amplification and replication of the phages, the indicator signal is amplified so that the single bacterium is detectable.

[0035] Embodiments of the methods and systems of the present invention include, but are not limited to, the detection and quantification of various microorganisms (e.g., bacteria) in various environments, including the detection of pathogens from food samples, water samples, and commercial samples. The methods of the present invention provide rapid detection and high detection sensitivity and specificity. In some embodiments, detection is possible within a single replication cycle of the bacteriophage, which is unexpected.

[0036] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention should have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by circumstances, singular terms should include plurals, and plural terms should include singulars. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, as well as the techniques described herein, are well known and commonly used in the art. Known methods and techniques are generally performed, unless otherwise indicated, by common methods well known in the art and as described in the various general and more specific references considered throughout this specification. Enzyme reactions and purification techniques are performed as generally achieved in the art or as specified by the manufacturer, as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is well known and commonly used in the art.

[0037] The following terms should be understood to have the meanings set forth below, unless otherwise indicated:

[0038] In this specification, the terms "a," "an," and "the" may refer to one or more unless otherwise noted.

[0039] The use of the term “or” is used to mean “and / or” unless it is explicitly indicated that only substitutes are being referred to, or that the substitutes are mutually exclusive; however, this disclosure supports the definitions that refer only to substitutes and to “and / or.” As used herein, “another” may mean at least the second or more.

[0040] Throughout this application, the term “approximately” is used to indicate that the value includes inherent error variations in the device or method used to determine the value, or variations present between samples.

[0041] The terms “solid support” or “support” refer to a structure that provides a substrate and / or surface upon which biomolecules can bind. For example, a solid support may be an assay well (i.e., a microtiter plate or multiwell plate), or a solid support may be a position on a filter, array, or mobile support (e.g., beads) or membrane (e.g., a filter plate, latex particles, paramagnetic particles, or lateral flow strip).

[0042] The term "binding factor" refers to a molecule that can specifically and selectively bind to a second (i.e., different) target molecule. The interaction may be non-covalent as a result of, for example, hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction may be covalent. The term "soluble binding factor" refers to a binding factor that does not associate (i.e., covalently or non-covalently) with a solid support.

[0043] As used herein, “analyte” means the molecule, compound, or cell being measured. The analyte of interest may interact with binding factors in certain embodiments. As used herein, the term “analyte” may mean the protein or peptide of interest. The analyte may be an agonist, antagonist, or modulator. Alternatively, the analyte may not have any biological effect. Examples of analytes include small molecules, sugars, oligosaccharides, lipids, peptides, peptide mimes, and organic compounds.

[0044] The terms “detectable portion,” “detectable biomolecule,” “reporter,” “indicator,” or “indicator portion” refer to molecules that can be measured in a quantitative assay. For example, the indicator portion may include an enzyme that can be used to convert a substrate into a product that can be measured. The indicator portion may be an enzyme that catalyzes a reaction that produces bioluminescent emission (e.g., luciferase). Alternatively, the indicator portion may be a radioactive isotope that can be quantified. Alternatively, the indicator portion may be a fluorophore. Alternatively, other detectable molecules may be used.

[0045] As used herein, “bacteriophage” or “phage” includes one or more types of bacterial viruses. In this disclosure, the terms “bacteriophage” and “phage” refer to viruses that can invade living bacteria, fungi, mycoplasmas, protozoa, yeasts, and other microscopic living organisms, and that use such organisms to replicate themselves, including viruses such as mycobacteriophages (e.g., with respect to TB and paraTB), mycophages (e.g., with respect to fungi), mycoplasma phages, and any other terms. Here, “microscopic” means having a maximum dimension of 1 millimeter or less. Bacteriophages are viruses that have evolved in nature to use bacteria as a means of replication. Phages do this by attaching the phage itself to a bacterium, injecting its DNA (or RNA) into the bacterium, and inducing the bacterium to replicate the phage hundreds or even thousands of times. This is also known as phage amplification.

[0046] As used herein, “late gene region” refers to a region of the viral genome that is transcribed late in the viral life cycle. Typical late gene regions include 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, in phage T7, the late genes (synonymous with class III) are transcribed from, for example, 8 minutes after infection until lysis, while class I genes (e.g., RNA polymerase) are transcribed early, between 4 and 8 minutes, and class II genes are transcribed between 6 and 15 minutes, so the timing of II and III genes overlaps. A late promoter is a promoter that is naturally located and active in such late gene regions.

[0047] As used herein, “culturing for enrichment” means conventional culturing (e.g., incubation in a medium favorable to microbial growth) and should not be confused with other possible uses of the term “enrichment” (e.g., enrichment by taking out the liquid components of a sample and concentrating the microorganisms contained therein) or other forms of enrichment that do not involve conventional promotion of microbial growth. Culturing for enrichment over a period of time may be used in some embodiments of the methods described herein.

[0048] As used herein, “recombinant” means a genetic (i.e., nucleic acid) modification, typically performed in a laboratory, to combine genetic material that would not otherwise be found. This term is used herein as interchangeable with the term “modified.”

[0049] As used herein, "RLU" refers to the relative luminescence measured by a luminometer (e.g., GLOMAX® 96) or a similar light-detecting instrument. For example, the detection of a reaction between luciferase and a suitable substrate (e.g., NANOLUC® and NANO-GLO®) is often reported by the detected RLU.

[0050] As used herein, “time to results” refers to the total time from the start of sample incubation until results are generated. Time to results does not include any confirmation test time. Data collection may be performed at any time after results have been generated.

[0051] sample Each embodiment of the method and system of the present invention can enable rapid detection and quantification of microorganisms in a sample. For example, a method according to the present invention can be performed in a shortened period of time with excellent results.

[0052] The bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are pathogens originating from food or water.

[0053] The sample may be a liquid, solid, or semi-solid. The sample may be a swab of a solid surface. Examples of samples include environmental materials (e.g., water samples) or filters derived from air or aerosol samples from a cyclone collector. Samples may include vegetables, meat, fish, poultry, peanut butter, processed foods, infant formula, milk powder, tea, starch, eggs, milk, cheese, or other dairy products.

[0054] In some embodiments, samples may be used directly in the detection method of the present invention without preparation, concentration, or dilution. For example, liquid samples (including, but not limited to, milk and juice) may be directly assayed. Samples may be diluted or suspended in buffered solutions or bacterial culture media, but not limited to these. Solid or semi-solid samples may be suspended in a liquid by finely chopping, mixing, or macerating the solid in the liquid. Samples should be maintained within a pH range that promotes bacteriophage adhesion to the host bacterial cells. Samples may also contain divalent and monovalent cations (Na + Mg2+ , and Ca 2+ The sample should contain an appropriate concentration of (but not limited to) the following. Preferably, the sample is maintained at a temperature that preserves the viability of any pathogen cells contained in the sample.

[0055] In some embodiments of the detection assay, the sample is maintained at a temperature that preserves the viability of any pathogen cells present in the sample. For example, it is preferable to maintain the sample at a temperature that promotes bacteriophage attachment during the process in which bacteriophages are attaching to bacterial cells. It is also preferable to maintain the sample at a temperature that promotes bacteriophage replication and host lysis during the process in which bacteriophages are replicating within infected bacterial cells or lysing such infected cells. Such temperatures are at least about 25 degrees Celsius, more preferably about 45°C or less, and most preferably about 37°C.

[0056] The assay may include various appropriate control samples. For example, a control sample without bacteriophages or a control sample containing bacteriophages without bacteria may be assayed as a control for background signal levels.

[0057] Indicator Bacteriophage As described in more detail herein, the compositions, methods, systems, and kits of the present invention may include infectious agents for use in the detection of pathogenic microorganisms. In certain embodiments, the present invention includes a recombinant indicator bacteriophage, where the bacteriophage genome is genetically modified to include an indicator or reporter gene. In some embodiments, the present invention may include a composition comprising a recombinant bacteriophage having an indicator gene incorporated into the bacteriophage genome.

[0058] Recombinant indicator bacteriophages may contain a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium produces a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage. Since late genes encode structural proteins, they are generally expressed at higher levels than other phage genes. The late gene region may be a class III gene region and may contain a gene for a major capsid protein.

[0059] Some embodiments include designing (and optionally preparing) a sequence for homologous recombination downstream of the major capsid protein gene. Other embodiments include designing (and optionally preparing) a sequence for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequence includes a codon-optimized reporter gene after an untranslated region. The untranslated region may include a late-stage phage gene promoter and a ribosome entry site.

[0060] In some embodiments, the indicator bacteriophage is derived from T7, T4, or another similar phage. Indicator bacteriophages may also originate from other bacteriophages having genomes with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to T4-like, T7-like, T4, T4-like, Cronobacter spp.-specific, ViI, or ViI-like (or Vi1 virus-like according to GenBank / NCBI) bacteriophages. In some embodiments, the selected wild-type bacteriophage is Saka2 or Saka4. Cronobacter spp. phages Saka2 and Saka4 are newly isolated and sequenced phages, possibly myoviruses. In other embodiments, the selected wild-type bacteriophage is a podovirus (e.g., a T7-like virus) or an Sp6-like virus. In other embodiments, the selected wild-type bacteriophage is Saka10. Saka10 is a newly isolated and sequenced phage, possibly a podovirus associated with the T7 phage. In some embodiments, the indicator phage is derived from a bacteriophage highly specific to a particular pathogenic microorganism. Genetic modification can avoid deletion of wild-type genes, and therefore the modified phage can remain more similar to the wild-type infectivity factor than many commercial phages. Bacteriophages derived from the environment may be more specific to bacteria found in the environment, and thus may be genetically different from commercial phages.

[0061] Furthermore, phage genes that appear non-essential may possess unrecognized functions. For example, seemingly non-essential genes may have crucial functions in increasing burst size, such as sophisticated cleavage, fitting, or trimming functions in assembly. Therefore, deleting genes and inserting indicators may be detrimental. Most phages can package DNA several percent larger than their native genome. In this consideration, smaller indicator genes may be a more appropriate choice for modifying bacteriophages (especially those with smaller genomes). OpLuc and NANOLUC® proteins are only about 20 kDa (coding about 500-600 bp), while FLuc is about 62 kDa (coding about 1,700 bp). For comparison, the T7 genome is about 40 kbp, the T4 genome is about 170 kbp, and the genome of a Cronobacter-specific bacteriophage is about 157 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacterium (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be timely and easily detectable. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, Promega's NANO-GLO®, combined with an imidazopyrazinon substrate (flimazine), can provide a robust signal with low background.

[0062] In some indicator phage embodiments, the indicator gene may be inserted into the untranslated region to avoid disruption of the functional gene while leaving the wild-type phage gene intact, which may result in a better fit when infecting non-laboratory bacterial strains. Furthermore, including stop codons in all three reading frames may help increase expression by reducing read-through (also known as leaky expression). This strategy may also eliminate the possibility of low-level production of fusion proteins that appear as background signals (e.g., luciferase) that cannot be isolated from the phage.

[0063] An indicator gene can express various biomolecules. The indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. In alternative embodiments, and as described in more detail herein, the luciferase is one of the following: Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as NANOLUC®.

[0064] Accordingly, in some embodiments, the present invention includes a genetically modified bacteriophage containing a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-natural indicator gene is under the control of a late promoter. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like a viral capsid protein, but is also not arrested, like an endogenous bacterial gene or even an early viral gene.

[0065] In some embodiments, the late promoter is a T4-like, T7-like, or ViI-like promoter, or another phage promoter similar to one 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 a T7, T4, T4-like, ViI, ViI-like, or Cronobacter spp. specific bacteriophage, or another wild-type bacteriophage having a genome with at least 70%, 75%, 80%, 85%, 90%, or 95% homology to a T7, T4, T4-like, ViI, ViI-like, or Cronobacter specific bacteriophage.

[0066] Genetic modifications to infectious agents may include insertions, deletions, or substitutions of small nucleic acid fragments, substantial parts of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acids contain non-natural sequences. Non-natural indicator genes may be inserted into the bacteriophage genome so as to be under the control of the bacteriophage promoter. Thus, in some embodiments, the non-natural indicator gene is not part of the fusion protein. That is, in some embodiments, the genetic modification may be configured such that the indicator protein product does not contain polypeptides of the wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present invention encompasses a method for detecting a bacterium of interest, the method comprising the step of incubating a test sample with such recombinant bacteriophage.

[0067] In some embodiments, the expression of an indicator gene in progeny bacteriophages after infection with a host bacterium results in a free soluble protein product. In some embodiments, the non-native indicator gene is not linked to a gene encoding a phage structural protein, and therefore does not produce a fusion protein. Unlike systems that use a fusion of the detection portion to a capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., soluble luciferase). In some embodiments, the indicator or reporter ideally does not contain the bacteriophage structure; that is, the indicator or reporter is not bound to the phage structure. Thus, the gene of the indicator or reporter is not fused with other genes in the recombinant phage genome. This can greatly increase the sensitivity of the assay (down to a single bacterium), simplify the assay, and, in some embodiments, allow the assay to be completed in 2 hours or less, in contrast to several hours due to the further purification steps required for constructs that produce a detectable fusion protein. Furthermore, fusion proteins may be less active than soluble proteins due to constraints on protein folding, which can alter, for example, the conformation of the enzyme active site or access to the substrate. If the above concentration is 10 bacterial cells / mL sample, for example, less than 2 hours may be sufficient for the assay.

[0068] Furthermore, a fusion protein by definition limits the number of regions attached to the protein subunits in the bacteriophage. For example, using a commercially available system designed to act as a platform for fusion proteins, approximately 415 copies of the fusion region are produced in each T7 bacteriophage particle, corresponding to approximately 415 copies of the gene 10B capsid protein. Without this constraint, it could be predicted that infected bacteria would express more copies of the detection region (e.g., luciferase) that could fit into the bacteriophage. Moreover, a large fusion protein (e.g., a capsid-luciferase fusion) may inhibit the assembly of the bacteriophage particle, thus producing fewer bacteriophage offspring. Therefore, soluble non-fusion indicator gene products may be preferred.

[0069] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a change in color. Various suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes can function as the indicator portion. In some embodiments, firefly luciferase is the indicator portion. In some embodiments, Oplophorus luciferase is the indicator portion. In some embodiments, NANOLUC® is the indicator portion. Luciferases fabricated by other engineering techniques or other enzymes that produce a detectable signal may also be suitable indicator portions.

[0070] In some embodiments, the use of a soluble detection portion eliminates the need to remove contaminated parental phages from the lysates of the infected sample cells. With a fusion protein system, any bacteriophage used to infect the sample cells has an attached detection portion and is indistinguishable from daughter bacteriophages that also include the detection portion. Since the detection of sample bacteria relies on the detection of a newly created (de novo synthesized) detection portion, the use of a fusion construct requires an additional step to separate the old (parental) portion from the newly created (daughter bacteriophage) portion. This can be achieved by washing the infected cells multiple times before the completion of the bacteriophage life cycle, inactivating excess parental phages after infection by physical or chemical means, and / or chemically modifying the parental bacteriophage with a binding portion (e.g., biotin) which can then be bound and separated (e.g., by streptavidin-coated Sepharose beads). However, even if all these attempts are made during removal, parental phages can still remain, especially if high concentrations are used to ensure infection of a small number of sample cells, creating background signals that can obscure the detection of signals from the progeny phages of infected cells.

[0071] In contrast, using the soluble detection portion expressed in some embodiments of the present invention eliminates the need to purify the parent phage from the final lysate, because the parent phage does not have any detection portion to which it can be attached. Therefore, any detection portion present after infection must be newly generated to indicate the presence of one or more infected bacteria. To take advantage of this benefit, the generation and preparation of the parent phage may include the purification of the phage from any free detection portion generated during the generation of the parent bacteriophage in the bacterial culture. Standard bacteriophage purification techniques can be used to purify some embodiments of the phages according to the present invention (e.g., sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography, and dialysis or derived techniques (e.g., Amicon brand concentrators - Millipore, Inc.)). Cesium chloride isopycnic ultracentrifugation may be used as part of the preparation of the recombinant phage of the present invention to separate parent phage particles from contaminating luciferase proteins produced during phage growth in the bacterial host. In this way, the parent recombinant bacteriophage of the present invention is substantially free of any luciferase produced during generation in the bacteria. Removal of any remaining luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with a test sample.

[0072] In some embodiments of the modified bacteriophage, the late promoter (class III promoter, e.g., derived from T7, T4, ViI, or Saka) has high affinity for the RNA polymerase of the same bacteriophage that transcribes the genes for structural proteins assembled into bacteriophage particles. These proteins are the most abundant proteins produced by the phage, because each bacteriophage particle contains tens or hundreds of copies of these molecules. The use of a viral late promoter can, optimally, ensure high levels of expression of the luciferase detection region. The use of a late viral promoter derived from, specific to, or active under the original wild-type bacteriophage from which the indicator phage originates (e.g., T4-based, T7-based, ViI-based, or Saka-based systems of the T4, T7, ViI, or Saka late promoter) can further ensure optimal expression of the detection region. The use of standard bacterial (non-viral / non-bacteriophage) promoters may, in some cases, be detrimental to expression. This is because these promoters are often downregulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production). Therefore, in some embodiments, the phage is engineered to encode a soluble (free) indicator moiety and be expressed at high levels, preferably using an in-genomic arrangement that does not limit expression to the number of subunits of the phage structural components.

[0073] The compositions of the present invention may comprise one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the composition may comprise a cocktail of different indicator phages capable of encoding and expressing the same or different indicator proteins. In some embodiments, the bacteriophage cocktail comprises at least two different types of recombinant bacteriophages.

[0074] Method for preparing indicator bacteriophages Embodiments of methods for producing indicator bacteriophages begin with the selection of wild-type bacteriophages for genetic modification. Some bacteriophages are highly specific to target bacteria. This provides an opportunity for highly specific detection.

[0075] Accordingly, the method of the present invention utilizes the high specificity of binding factors associated with infectious agents that recognize and bind to specific microorganisms of interest, as a means of amplifying signals and thereby detecting low levels of microorganisms (e.g., single microorganisms) present in a sample. For example, infectious agents (e.g., bacteriophages) specifically recognize surface receptors of specific microorganisms and therefore specifically infect those microorganisms. Thus, these infectious agents can be suitable binding factors for targeting microorganisms of interest.

[0076] Some embodiments of the present invention utilize the binding specificity and high level of gene expression ability of recombinant bacteriophages for rapid and highly sensitive targeting to facilitate the detection of the bacteria of interest. In some embodiments, a Cronobacter-specific bacteriophage is genetically modified to include a reporter gene. In some embodiments, the late gene region of the bacteriophage is genetically modified to include a reporter gene. In some embodiments, the reporter gene is located downstream of the major capsid gene. In other embodiments, the reporter gene is located upstream of the major capsid gene. In some embodiments, the inserted gene construct further includes an exogenous, dedicated promoter to drive the expression of the indicator gene. The exogenous promoter is present in addition to any endogenous promoter in the phage genome. Since bacteriophages produce polycistronic mRNA transcripts, only a single promoter is required upstream of the first gene / cistron in the transcript. Conventional recombinant constructs simply use the endogenous bacteriophage promoter to drive the inserted gene. In contrast, adding an additional promoter upstream of the reporter gene and ribosome binding site can increase gene expression by acting as a second start site for transcription. The complex and small genomes of viruses often have duplicate genes in different frames, sometimes in two different directions.

[0077] Some embodiments of a method for preparing recombinant indicator bacteriophages include the steps of: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium such as Cronobacter spp.; preparing a homologous recombination plasmid / vector containing an indicator gene; transforming the target pathogenic bacterium with the homologous recombination plasmid / vector; infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage to induce homologous recombination between the plasmid / vector and the bacteriophage genome; and isolating a specific clone of the recombinant bacteriophage.

[0078] Various methods for designing and preparing homologous recombination plasmids are known. Various methods for transforming bacteria with plasmids are known, including heat shock, F-piliate-mediated bacterial conjugation, electroporation, and other methods. Various methods for isolating specific clones after homologous recombination are also known. Some embodiments of the methods described herein utilize specific strategies.

[0079] Accordingly, some embodiments of a method for preparing an indicator bacteriophage include the steps of: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; determining a native sequence in the late region of the genome of the selected bacteriophage; annotating the genome and identifying the major capsid protein gene of the selected bacteriophage; designing a sequence for homologous recombination adjacent to the major capsid protein gene, wherein the sequence includes a codon-optimized reporter gene; incorporating the sequence designed for homologous recombination into a plasmid / vector; transforming the plasmid / vector into a target pathogenic bacterium; selecting the transformed bacteria; infecting the transformed bacteria with the selected wild-type bacteriophage, thereby inducing homologous recombination between the plasmid and the bacteriophage genome; determining the titer of the resulting recombinant bacteriophage lysate; and performing a limiting dilution assay to enrich and isolate the recombinant bacteriophage. Some embodiments further include repeating the limiting dilution and titration steps as needed after the first limiting dilution assay until recombinant bacteriophages represent a detectable proportion of the mixture. For example, in some embodiments, the limiting dilution and titration steps may be repeated until at least 1 / 30 of the bacteriophages in the mixture are recombinant before isolating a specific clone of the recombinant bacteriophage. A 1:30 recombinant:wild-type ratio is expected to produce an average of 3.2 transduction units (TU) per 96 plaques (e.g., in a 96-well plate) in some embodiments. The initial recombinant phage:wild-type phage ratio can be determined by performing a limiting dilution assay based on TCID50 (50% tissue culture infectious dose), as previously described in U.S. Patent Application No. 15 / 409,258. By Poisson distribution, a 1:30 ratio gives a 96% probability of observing at least 1 TU somewhere in the 96 wells.

[0080] Figure 1 shows a schematic representative diagram of the genome structure of the recombinant indicator bacteriophage of the present invention. In the embodiment shown in Figure 1, the detection region is encoded by a luciferase gene 100 inserted into a late (class III) gene 110, which is expressed late in the viral life cycle. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. Therefore, in the embodiment of the recombinant phage shown in Figure 1, the indicator gene (i.e., luciferase) is a construct containing the luciferase gene 100, inserted into the late gene region immediately after the major capsid protein (MCP) gene 120. In some embodiments, the construct shown in Figure 1 may include stop codons in all three reading frames to ensure that luciferase is not incorporated into the MCP gene product via the creation of a fusion protein. As also shown in Figure 1, the construct may include a further dedicated late promoter 130 to drive the transcription and expression of the luciferase gene. The above construct also includes a ribosome-binding site (RBS) 140. This construct ensures that soluble luciferase is produced, and as a result, its expression is limited to the number of unique capsid proteins in the phage display system.

[0081] As noted herein, in certain embodiments, it may be preferable to utilize infectious agents isolated from the environment for the generation of the infectious agents of the present invention. In this way, infectious agents specific to naturally occurring microorganisms can be generated.

[0082] For example, Figure 2 shows the genome of bacteriophage SAKA2 (a wild-type bacteriophage that specifically infects Cronobacter spp.). As discussed in the examples, the major capsid protein and various other structural genes are located within a late gene region consisting of structural genes encoding virion proteins. A hypothetical gene homologous to the PG7 tail sheath protein gene (nucleotides 74424-77221) is located around the late gene region consisting of structural genes encoding virion proteins. Other late genes shown are homologs of known parietal proteins (nucleotides 77115-77653), ORF58.1 (nucleotides 77693-78610), possibly the major capsid protein, and homologs of known outer head proteins (nucleotides 80248-81840). Since these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, insofar as late gene promoters and / or other similar regulatory elements are used.

[0083] Numerous known methods and 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 be used to selectively edit bacteriophage genomes, or the CRISPR / CAS9 system can also be used. Several embodiments of methods for preparing recombinant indicator bacteriophages involve designing plasmids that can be readily recombinant with wild-type bacteriophage genomes to generate recombinant genomes. In plasmid design, some embodiments include the addition of a codon-optimized reporter gene, such as a luciferase gene. Some embodiments further include the addition of elements to an upstream untranslated region. For example, in designing a plasmid for recombinant with a Cronobacter-specific bacteriophage genome, an upstream untranslated region may be added between the sequence encoding the C-terminus of the gp23 / major capsid protein and the start codon of the NANOLUC® reporter gene. The untranslated region may contain promoters, such as T4, T4-like, T7, T7-like, Cronobacter-specific bacteriophage, ViI, or ViI-like promoters. The untranslated region may also contain ribosome entry / binding sites (RBS), also known as "Schein-Dalgarno sequences" in bacterial systems. One or both of these elements, or other untranslated elements, may be embedded within a short upstream untranslated region consisting of random sequences with roughly the same GC content as the rest of the phage genome. The random region should not contain an ATG sequence because it would act as a start codon.

[0084] The compositions of the present invention may include various infectious factors and / or indicator genes. For example, Figure 3 shows two homologous recombination plasmid constructs used to produce indicator phages specific to Cronobacter spp. The constructs were prepared and used in recombination with Cronobacter spp. phage Saka2, Cronobacter spp. phage Saka4, or Cronobacter spp. phage Saka10 to generate recombinant bacteriophages of the present invention. The first construct in Figure 3 is shown as a general schematic diagram of the recombinant plasmid used for homologous recombination insertion of NANOLUC® luciferase into both Cronobacter spp. phages Saka2 and Saka4, each with 500 bp of upstream and downstream homologous sequences corresponding to the respective phage:homologous recombination plasmids pUC57.HR.Saka2.NANOLUC® and pUC57.HR.Saka4.NANOLUC®.

[0085] The lower construct in Figure 3 shows the recombinant plasmid used for homologous recombination insertion of NANOLUC® luciferase into Saka10 (podovirus) to target Cronobacter strains that are not sufficiently infected by either Saka2 or Saka4: homologous recombination plasmid pUC57.HR.Saka10.NANOLUC®.

[0086] In certain embodiments, the plasmid is referred to as pUC57.HR.Saka2.NanoLuc. The detection / indicator portion is encoded by the NANOLUC® reporter gene 300. The insert (represented by a series of squares) is located within the standard AmpR version 310 of pUC57. The upstream homologous recombination region consists of a 500 bp major capsid protein C-terminal fragment 320, a T4-like phage late promoter consensus sequence in the 5' untranslated region, and the Shine-Dalgarno ribosome entry / binding site 330. The codon-optimized NANOLUC® reporter gene 300 follows immediately. The downstream homologous recombination region (UTR) consisting of 340 and the hypothetical protein N-terminal fragment is located at the end of the homologous recombination region.

[0087] In certain embodiments, the plasmid is referred to as pUC57.HR.Saka10.NanoLuc. The detection / indicator portion is encoded by the NanoLuc® reporter gene 380. The insert (represented by a series of squares) is located within the standard AmpR version 360 of pUC57. The upstream homologous recombination region consists of the 500 bp major capsid protein C-terminal fragment 360, the T7 phage late promoter consensus sequence within the 5' untranslated region, and the Shine-Dalgarno ribosome entry / binding site 370. The codon-optimized NANOLUC® reporter gene 350 follows immediately. The downstream homologous recombination region (UTR) consisting of 380 and the hypothetical protein N-terminal fragment are located at the end of the homologous recombination region. The major capsid protein fragment is part of a structural gene encoding a virion protein. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be predicted to have similar expression levels, insofar as late gene promoters and / or other similar regulatory elements are used.

[0088] In some embodiments, the indicator phage according to the present invention comprises a Cronobacter-specific bacteriophage genetically engineered to contain a reporter gene (e.g., a luciferase gene). For example, the indicator phage may be a Cronobacter spp.-specific bacteriophage whose genome contains the sequence of the above-mentioned NANOLUC® gene. The recombinant Cronobacter-specific NanoLuc bacteriophage genome may further comprise the consensus promoters for T4, T7, Cronobacter-specific, ViI, Saka bacteriophages, or another late promoter. In further embodiments, the promoter is an exogenous promoter. Insertion of an exogenous promoter to drive the expression of the indicator gene is advantageous in that the expression is restricted by the expression of other phage proteins (e.g., major capsid protein).

[0089] Accordingly, in embodiments of the recombinant phage produced as a result of recombination, the indicator gene (i.e., NANOLUC®) is inserted into a late gene region immediately downstream of the gene encoding the major capsid protein, thereby creating a recombinant bacteriophage genome containing the NANOLUC® gene. The construct further comprises a consensus promoter of T4, T7, Cronobacter-specific bacteriophage, ViI, or another late promoter or another suitable promoter to drive the transcription and expression of the luciferase gene. The construct may also include a complex untranslated region synthesized from several UTRs. This construct ensures that soluble luciferase is produced and, as a result, its expression is not limited to the number of specific capsid proteins in the phage display system.

[0090] Figure 4 shows the isolation of recombinant phages from a mixture of wild-type and recombinant bacteriophages obtained from homologous recombination. In the first step 402, Cronobacter spp. bacteria transformed with homologous recombination plasmids are infected with Cronobacter spp. bacteriophage Saka2, producing progeny phages having a mixture of parental and recombinant phages in a very low ratio of wild-type to recombinant phages 434. The resulting recombinant phage mixture is diluted into a 96-well plate 406 404, giving an average of 5 recombinant transduction units (TU) / plate (9.3 PFU / well). The 96-well plate is assayed for luciferase activity to identify wells 436 containing recombinant phages compared to wells 440 containing wild-type bacteriophages 438. Bacteria 438 are added 408; for example, each well may contain approximately 50 μL of turbid Cronobacter culture. This allows the phage to replicate and produce the luciferase enzyme 442. After incubation at 37°C for 5 hours as shown in 410, wells can be screened for the presence of luciferase 442. Any positive well is likely inoculated with a single recombinant phage, at which stage the mixture may contain approximately 10 wild-type phage:1 recombinant ratio (enriched beyond the original ratio). If necessary (i.e., if the recombinant:total ratio is lower than 1:30), offspring from this enriched culture 412 can be subjected to a further limiting dilution assay 414 to increase the ratio and determine the actual concentration of recombinant phage transduction units. For example, if the above ratio was 1:384 recombinant:PFU, then approximately 1920 total contaminated phages (5 × 384 = 1920) / 96 well plate 416 may be aliquoted from the previously positive wells 414, resulting in an approximate inoculation of wild-type phage / well (1920 PFU / 96 wells = 20 PFU / well) for the majority of the 20 second dilution assay plates 420. Any positive luciferase well will likely be inoculated with a single recombinant along with 19 wild-type phages 442. These wells can be analyzed for the presence of luciferase 442.

[0091] After bacterial addition and incubation (e.g., 5 hours at 37°C)418, soluble luciferase and phages are present in approximately 20 total:1 recombinants420. This ratio can be verified by TU50 titer measurement of recombinants, limiting dilution assays based on tissue culture infection dose 50 (TCID50) assays that score luciferase activity instead of cell killing, and plaque assays against total PFU. Finally, plaque assays can be performed422 to screen for recombinants expressing luciferase446. A small number of individual (e.g., n=48) plaques may be picked individually and screened against luciferase activity436 in a third multiwell plate426. In one embodiment, this approach should ensure that approximately 3 recombinants are present in the mixture of plaques being screened based on a known ratio of recombinants to total phages, since a sufficient number of plaques are screened. A single plaque can be removed from the plate described above and placed into each well of a 96-well plate 424, and a luciferase assay can be performed to determine which wells contained phages exhibiting luciferase activity 442 426. Wells exhibiting luciferase activity 428 represent pure recombinant phage 434, while wells lacking luciferase activity 430 represent pure wild-type phage 432.

[0092] Next, individual plaques may be suspended in buffer (e.g., 100 μL TMS) or culture medium, and aliquots (e.g., about 5 μL) may be added to wells containing turbid Cronobacter cultures, which can then be assayed after incubation (e.g., about 45 minutes to 1 hour at 37°C). Positive wells are expected to contain pure cultures of recombinant phages. Certain embodiments may include a further round of plaque purification.

[0093] Therefore, as illustrated in Figure 4, recombinant phages produced by homologous recombination of plasmids designed for recombination in wild-type phage genomes can be isolated from a mixture containing a very small percentage (e.g., 0.005%) of the entire phage genome. After isolation, large-scale production can be performed to obtain a high-titer recombinant indicator phage stock suitable for use in Cronobacter spp. detection assays. Furthermore, isodensity gradient centrifugation using cesium chloride can be used to separate phage particles from contaminating luciferase proteins and reduce background noise.

[0094] How to use infectious agents to detect Cronobacter spp. As noted herein, in certain embodiments, the present invention may encompass a method for using infectious particles to detect microorganisms. The method of the present invention can be embodied in a variety of ways.

[0095] In one embodiment, the present invention may include a method for detecting a target bacterium in a sample, comprising the steps of: incubating the sample with a bacteriophage that infects the target bacterium, wherein the bacteriophage comprises an indicator gene, and as a result, the expression of the indicator gene during bacteriophage replication after infection with the target bacterium produces a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the target bacterium in the sample.

[0096] In certain embodiments, assays may be performed using a general concept that can be modified to suit different sample types or sizes and assay formats. Embodiments using the recombinant bacteriophage (i.e., indicator bacteriophage) of the present invention may be performed for 1.5 hours, 2.0 hours, 2.5 hours, 3.0 hours, 3.5 hours, 4.0 hours, 4.5 hours, 5.0 hours, 5.5 hours, 6.0 hours, 6.5 hours, 7.0 hours, 7.5 hours, 8.0 hours, 8.5 hours, 9.0 hours, 9.5 hours, 10.0 hours, 10.5 hours, 11.0 hours, 11.5 hours, and 12 hours, depending on the sample type, sample size, and assay format. Total assay times of less than 12.5 hours, 13.0 hours, 13.5 hours, 14.0 hours, 14.5 hours, 15.0 hours, 15.5 hours, 16.0 hours, 16.5 hours, 17.0 hours, 17.5 hours, 18.0 hours, 18.5 hours, 19.0 hours, 19.5 hours, 20.0 hours, 21.0 hours, 21.5 hours, 22.0 hours, 22.5 hours, 23.0 hours, 23.5 hours, 24.0 hours, 24.5 hours, 25.0 hours, 25.5 hours, or 26.0 hours may enable rapid detection of certain bacterial strains, such as Cronobacter spp. For example, the required time may be slightly shorter or longer depending on the bacteriophage strain and the bacterial strain detected in this assay, the type and size of the sample being tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target contaminating bacteria.

[0097] Figure 5 shows a strategy using an indicator phage that generates soluble luciferase according to an embodiment of the present invention. In this method, a phage (e.g., T7, T4, Saka2, Saka4, Saka9, or Saka10 phage) can be manipulated to express soluble luciferase during phage replication. Luciferase expression is driven by a viral capsid promoter (e.g., a late promoter for bacteriophage T7 or T4) to result in high expression. Since the parental phage does not contain luciferase, the luciferase detected in the assay must arise from the replication of the progeny phage during infection of the bacterial cell. Therefore, generally speaking, it is not necessary to isolate the parental phage from the progeny phage.

[0098] In these experiments, at least a portion of the sample 500 containing the bacterium 502 to be quantified is placed in a spin column filter, centrifuged to remove LB broth, and appropriate multiplicity phage 504, genetically engineered to express soluble luciferase 503, is added. The infected cells can be incubated for a sufficient time (e.g., 30–120 minutes at 37°C) for progeny phage replication and cell lysis to occur. The parent phage 504 and progeny phage 516 + free luciferase 503 in the lysate can then be collected, for example, by centrifugation, and the level of luciferase in the filtrate can be quantified using a luminometer 518. Alternatively, a high-throughput method (where the bacterial sample is applied to a 96-well filter plate) can be used, and after all the operations listed above have been performed, the assay can be performed directly for luciferase in the original 96-well filter plate without a final centrifugation step.

[0099] Figure 6 shows a filter plate assay for detecting a target bacterium using a modified bacteriophage according to one embodiment of the present invention. Briefly, a sample 616 containing the target bacterium 618 is added to well 602 of a multiwell filter plate 604, and the sample is concentrated by centrifugation 606 to remove the liquid from the sample. A genetically modified phage 620 is added to the well and incubated with further medium for a sufficient time for adsorption 608, followed by infection of the target bacterium and advancement of the phage life cycle 610 (e.g., about 45 minutes). Finally, a luciferase substrate is added and reacted with any present luciferase 624. The resulting luminescence is measured with a luminometer that detects luciferase activity 626 614.

[0100] In certain embodiments, the assay may be performed without concentrating the bacteria on or near the capture surface. Figure 7 illustrates a “No Concentration Assay” for detecting a target bacterium using a modified bacteriophage according to one embodiment of the present invention. Aliquots of indicator phage 714 are distributed into individual wells 702 of a multiwell plate 704, and then aliquots of the test sample containing bacteria 712 are added and incubated for a sufficient period of time (e.g., 45 minutes at 37°C) for the phage to replicate and produce a soluble indicator 716 (e.g., luciferase). The plate wells 708 containing the soluble indicator and phage can then be assayed 710 to measure the indicator activity 718 on the plate (e.g., luciferase assay). In this embodiment, the test sample may not be concentrated (e.g., by centrifugation) but simply incubated directly with the indicator phage for a certain period of time and then assayed for luciferase activity.

[0101] In some embodiments, the sample can be enriched before testing by incubation under growth-promoting conditions. In such embodiments, the enrichment period may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours or longer, depending on the type and size of the sample.

[0102] In some embodiments, the indicator bacteriophage includes a detectable indicator portion, and infection by a single pathogenic cell (e.g., a bacterium) can be detected by the amplified signal produced via the indicator portion. Thus, the method may include detecting an indicator portion produced during phage replication, where detection of the indicator indicates the presence of the bacterium of interest in the sample.

[0103] In one embodiment, the present invention may include a method for detecting a target bacterium in a sample, comprising the steps of: incubating the sample with a recombinant bacteriophage that infects the target bacterium, wherein the recombinant bacteriophage includes an indicator gene inserted into the late gene region of the bacteriophage, and as a result, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium produces a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the amount of the indicator portion detected corresponds to the amount of the target bacterium present in the sample.

[0104] As described in more detail herein, the methods and systems of the present invention can utilize a range of concentrations of parent indicator bacteriophages to infect bacteria present in a sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly locate, bind to, and infect target bacteria present in very small numbers in the sample, such as single cells. In some embodiments, the phage concentration may be sufficient to locate, bind to, and infect target bacteria in less than one hour. In other embodiments, these events may occur in less than two hours or less than three hours after the addition of the indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubation step may be 1 × 10⁻⁶. 5 Higher than PFU / mL, or 1×10 6 Higher than PFU / mL, or 1 × 10 7 Higher than PFU / mL

[0105] In certain embodiments, the recombinant infectious agent can be purified so as to be free from any residual indicator proteins that may be generated during the production of the infectious agent stock. Accordingly, in certain embodiments, the recombinant bacteriophage can be purified using isodensity gradient centrifugation with cesium chloride before incubation with the sample. If the infectious agent is a bacteriophage, this purification may have the additional benefit of removing bacteriophages that do not possess DNA (i.e., empty phages or "ghosts").

[0106] In some embodiments of the method of the present invention, the microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or more target microorganisms may be applied directly to an assay vessel (e.g., a spin column, a microtiter well, or a filter), and the assay is performed in that assay vessel. Various embodiments of such assays are disclosed herein.

[0107] Test sample aliquots may be directly distributed into the wells of a multiwell plate, an indicator phage may be added, and after a sufficient period for infection, lysis buffer may be added as well as a substrate for the indicator portion (e.g., a luciferase substrate for a luciferase indicator), and the assay may be performed for detection of the indicator signal. Some embodiments of this method can be performed on a filter plate. Some embodiments of this method may be performed with or without concentration of the sample prior to infection with the indicator phage.

[0108] For example, in many embodiments, multi-well plates are used to perform the assay described above. The choice of plate (or any other container in which the detection step may be performed) can affect the detection step. For example, some plates may contain a colored or white background, which can affect the detection of light emission. Generally, white plates have higher sensitivity but also produce a higher background signal. Other colors of plates may produce a lower background signal but may have slightly lower sensitivity. Furthermore, one reason for the background signal is light leakage from one well to another adjacent well. Some plates have white wells, while the rest of the plate is black. This allows for a high signal within the wells but prevents light leakage from well to well, and thus can reduce the background. Therefore, the choice of plate or other assay container can affect the sensitivity and background signal for the assay described above.

[0109] The method of the present invention may include various other steps to increase sensitivity. For example, as will be discussed in more detail herein, the method may include a step of washing the captured and infected bacteria after adding the bacteriophage but before incubation, in order to remove excess parent bacteriophage and / or luciferase or other reporter protein that contaminates the bacteriophage preparation.

[0110] In some embodiments, the detection of the microorganisms of the above purpose can be completed without the need to culture the sample as a method for increasing the population of the microorganisms. For example, in certain embodiments, the total time required for detection is 26.0, 25.0, 24.0, 23.0, 22.0, 21.0, 20.0, 19.0, 18.0, 17.0, 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, less than 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, 1.0 hour, 45 minutes, or less than 30 minutes. Minimizing the time to results is critical in testing food and the environment for pathogens.

[0111] In contrast to assays known in the art, the method of the present invention can detect individual microorganisms. Therefore, in certain embodiments, the method can detect ≤10 cells of the microorganism present in a sample (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9 microorganisms). For example, in certain embodiments, the recombinant bacteriophage is highly specific to Cronobacter spp. In one embodiment, the recombinant bacteriophage can distinguish Cronobacter spp. in the presence of other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a specific type in a sample. In certain embodiments, the recombinant bacteriophage can detect specific bacteria in small numbers, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 individuals, in a sample.

[0112] Accordingly, an aspect of the present invention provides a method for detecting microorganisms in a test sample via an indicator portion. In some embodiments, if the microorganism of interest is a bacterium, the indicator portion may be associated with an infectious agent (e.g., an indicator bacteriophage). The indicator portion may react with a substrate to emit a detectable signal or an endogenous signal (e.g., a fluorescent protein). In some embodiments, the detection sensitivity may reveal the presence of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the microorganism of interest in the test sample. In some embodiments, as few as one cell of the microorganism of interest may produce a detectable signal. In some embodiments, the bacteriophage is a T4-like or ViI-like bacteriophage. In some embodiments, the recombinant bacteriophage is derived from a Cronobacter-specific bacteriophage. In certain embodiments, recombinant Cronobacter-specific bacteriophages are highly specific to Cronobacter spp.

[0113] In some embodiments, the indicator portion encoded by the infectious agent may be detectable during or after replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicator portions are known in the art, and many are commercially available. In some embodiments, the indicator phage comprises an enzyme which acts as the indicator portion. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator may be detectable by a change in color in a substrate that can emit light and / or is converted. Various suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes may act as the indicator portion. In some embodiments, firefly luciferase is the indicator portion. In some embodiments, Oplophorus luciferase is the indicator portion. In some embodiments, NANOLUC® is the indicator portion. Luciferases or other enzymes that produce a detectable signal, fabricated by other engineering techniques, can also be suitable indicator portions.

[0114] Accordingly, in some embodiments, the recombinant bacteriophage of the Method, System, or Kit is prepared from a wild-type Cronobacter-specific bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an endogenous signal, such as a fluorescent protein (e.g., green fluorescent protein). The indicator may emit light and / or be detectable by a change in color. In some embodiments, the indicator gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to generate a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is one of the following: Oplophorus luciferase, firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase, or an engineered luciferase, such as NANOLUC®, Rluc8.6-535, or Orange Nano-Lantern.

[0115] The step of detecting the indicator described above may include the step of detecting light emission. In some embodiments, a luminometer may be used to detect the reaction of an indicator (e.g., luciferase) with a substrate. RLU detection can be achieved with a luminometer, or other machines or devices may also be used. For example, a spectrophotometer, CCD camera, or CMOS camera may detect color changes and other light emission. Absolute RLU is important for detection, but a high signal-to-background ratio (e.g., >2.0, >2.5, or >3.0) is also necessary for reliable detection of single cells or a small number of cells.

[0116] In some embodiments, the indicator phage is genetically engineered to include a gene for an enzyme (e.g., luciferase) that is produced only during infection by a bacterium specifically recognized and infecting the phage. In some embodiments, the indicator portion is expressed later in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused with a phage structural protein that limits its copy number.

[0117] Accordingly, in some embodiments utilizing indicator phages, the present invention encompasses a method for detecting a target microorganism, the method comprising the steps of: capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phages; giving time for infection and replication to generate progeny phages and express a soluble indicator portion; and detecting the progeny phages, preferably the indicator, wherein the detection of the indicator indicates the presence of the bacterium in the sample.

[0118] For example, in some embodiments, test sample bacteria may be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a spin filter or plate filter with a pore size of 0.45 μm). In one embodiment, the infectious agent (e.g., an indicator phage) is added in a minimal volume to the sample directly captured on the filter. In one embodiment, the microorganisms captured on the filter or plate surface are then washed once or multiple times to remove excess unbound infectious agent. In one embodiment, a culture medium (e.g., Luria-Bertani broth (also referred to herein as LB), buffered peptone water (also referred to herein as BPW), or Tryptic Soy broth or Tryptone Soy broth (also referred to herein as TSB)) may be added for a further incubation time to allow replication of bacterial cells and phages, as well as high-level expression of the gene encoding the indicator portion. However, a surprising aspect of some embodiments of the test assay is that the incubation step with the indicator phage only needs to be long enough for a single phage life cycle. The amplification power of using bacteriophages was previously thought to require more time for the phage to replicate over several cycles. A single replication cycle of the indicator phage may be sufficient to facilitate highly sensitive and rapid detection according to some embodiments of the present invention.

[0119] In some embodiments, aliquots of a test sample containing bacteria may be applied to a spin column, and after infection with recombinant bacteriophages and optional washing to remove any excess bacteriophages, the amount of soluble indicator detected is proportional to the amount of bacteriophages produced by the infected bacteria.

[0120] The soluble indicator (e.g., luciferase) released into the surrounding liquid during the lysis of the above-mentioned bacteria can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter, the filtrate is collected in a new container for an assay (e.g., in a luminometer), and then a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

[0121] In various embodiments, the purified parental indicator phage does not contain the detectable indicator itself, because the parental phage can be purified before it is used for incubation with the test sample. Late (class III) gene expression occurs late in the viral life cycle. In some embodiments of the present invention, the parental phage can be purified to eliminate any present indicator proteins (e.g., luciferase). In some embodiments, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium produces a soluble indicator protein product. Therefore, in many embodiments, it is not essential to separate the parental phage from the progeny phage before the detection step. In one embodiment, the microorganism is a bacterium, and the indicator phage is a bacteriophage. In one embodiment, the indicator portion is a soluble luciferase, which is released upon lysis of the host microorganism.

[0122] Therefore, in an alternative embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with a portion of the sample that remains on the filter or bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a regular 96-well plate), and the substrate reaction can be detected by placing the plate directly into the luminometer.

[0123] For example, in one embodiment, the present invention may encompass a method for detecting Cronobacter spp., the method comprising the steps of: infecting cells captured on a 96-well filter plate with a plurality of parent indicator phages capable of expressing luciferase upon infection; washing away excess phages; adding LB broth to give the phages time to replicate and lyse the specific Cronobacter spp. targets (e.g., 30-120 minutes); and detecting the indicator luciferase by adding a luciferase substrate and directly measuring the luciferase activity in the 96-well plate, wherein the detection of luciferase activity indicates the presence of Cronobacter spp. in the sample.

[0124] In another embodiment, the present invention may encompass a method for detecting Cronobacter spp., the method comprising: infecting cells in a liquid solution or suspension in a 96-well plate with a plurality of parent indicator phages capable of expressing luciferase upon infection; giving the phages time (e.g., 30-120 minutes) to replicate and lyse the specific Cronobacter spp. targets; and detecting the indicator luciferase by adding a luciferase substrate and directly measuring the luciferase activity in the 96-well plate, wherein the detection of luciferase activity indicates the presence of Cronobacter spp. in the sample. In such embodiments, the capture step is not essential. In some embodiments, the liquid solution or suspension may be a consumable test sample (e.g., vegetable wash liquid). In some embodiments, the liquid solution or suspension may be vegetable wash liquid fortified with concentrated LB broth, Tryptic / Tryptone Soy broth, peptone water, or nutrient broth. In some embodiments, the above liquid solution or suspension may be bacteria diluted in LB broth.

[0125] In some embodiments, lysis of the bacteria can occur before, during, or after the detection step. Experiments suggest that infected but unlysed cells can be detectable in some embodiments upon addition of the luciferase substrate. Perhaps luciferase can exit the cell and / or the luciferase substrate can enter the cell without complete cell lysis. Thus, for embodiments utilizing a spin filter system, lysis is required if only the luciferase released into the lysate (and no luciferase remains in the intact bacteria) is analyzed in the luminometer. However, for embodiments utilizing a filter plate or 96-well plate with a sample of the solution or suspension, lysis is not essential for detection if the original plate filled with intact and lysed cells is assayed directly in the luminometer.

[0126] In some embodiments, the reaction between the indicator moiety (e.g., luciferase) and the substrate can continue for 30 minutes or longer, and detection at various time points can be desirable to optimize sensitivity. For example, in embodiments using a 96-well filter plate as the solid support and luciferase as the indicator, the luminometer readings can be taken initially and at intervals of 10 or 15 minutes until the reaction is complete.

[0127] Surprisingly, high concentrations of phage utilized to infect the test sample achieved successful detection of a very small number of target microorganisms in a very short time frame. Incubating the phage with the test sample in some embodiments requires only a time sufficient for a single phage life cycle. In some embodiments, the concentration of bacteriophage for this incubating step is 7×10 6 、8×10 6 、9×10 6 、1.0×10 7 、1.1×107 , 1.2 × 10 7 , 1.3 × 10 7 , 1.4×10 7 , 1.5×10 7 , 1.6×10 7 , 1.7×10 7 , 1.8×10 7 , 1.9×10 7 , 2.0×10 7 , 3.0×10 7 , 4.0×10 7 , 5.0×10 7 , 6.0×10 7 , 7.0×10 7 , 8.0×10 7 , 9.0×10 7 , or 1.0 × 10 8 Higher than PFU / mL

[0128] The success of phages at such high concentrations is remarkable because many phages are previously associated with "non-infectious lysis," which kills target cells and thereby prevents the generation of useful signals from earlier phage assays. The clean-up of the prepared phage stock described herein is thought to help mitigate this problem (e.g., clean-up by isodensity gradient ultracentrifugation with cesium chloride). This is because, in addition to removing any contaminating luciferase associated with the phages, this clean-up can also remove ghost particles (particles that have lost DNA). These ghost particles can lyse bacterial cells via "non-infectious lysis," killing the cells prematurely and thereby preventing the generation of indicator signals. Electron microscopy clearly shows that crude phage lysates (i.e., before cesium chloride clean-up) can contain more than 50% ghost particles. These ghost particles may contribute to the premature death of the above microorganisms through the action of many phage particles that create holes in the cell membrane. Therefore, ghost particles may have been a contributing factor to the previous problem in which high PFU concentrations were reported to be harmful. Furthermore, very clean phage preparations allow the above assay to be performed without a washing step, which in turn allows the assay to be performed without an initial enrichment step. Some embodiments include an initial enrichment step, and in some embodiments, this enrichment step allows for a shorter enrichment incubation time.

[0129] Some embodiments of the test method may further include confirmatory assays. Various assays are known in the art to confirm initial results, usually at a later point in time. For example, the sample may be cultured (e.g., the CHROMAGAR® and DYNABEADS® assays described in the examples), PCR may be used to confirm the presence of microbial DNA, or other confirmatory assays may be used to confirm the initial results.

[0130] In certain embodiments, the method of the present invention may combine the use of a binder (e.g., an antibody) with the detection of an infectious agent to purify and / or concentrate the target microorganism (e.g., Cronobacter spp.) from the sample. For example, in certain embodiments, the present invention encompasses a method for detecting the target microorganism in a sample, the method comprising the steps of: capturing the microorganism from the sample onto a support using a capture antibody specific to the target microorganism (e.g., Cronobacter spp.); incubating the sample with a recombinant bacteriophage that infects Cronobacter spp., wherein the recombinant bacteriophage includes an indicator gene inserted into the late gene region of the bacteriophage, and as a result, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium produces a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of Cronobacter spp. in the sample.

[0131] For example, Figure 8 shows a hybrid immunophage (HIP) assay for detecting a target bacterium using a modified bacteriophage according to one embodiment of the present invention. First, the sample is applied to a microtiter plate well coated with a bacterium-specific antibody 802. The plate is then centrifuged to promote the binding of the bacteria to the capture antibody 804. After a sufficient time to allow complete bacterial capture, a solution containing the bacterium-specific NANOLUC® phage is added to each sample 806. Incubation with the phage results in the binding and attachment of one or more phages to the captured bacteria 808. Finally, the sample is incubated to promote phage replication and luciferase expression. This results in cell lysis and the release of soluble luciferase 810.

[0132] In some embodiments, synthetic phages are designed to optimize desirable traits for use in pathogen detection assays. In some embodiments, bioinformatics and prior analysis of genetic modifications are used to optimize the desired traits. For example, in some embodiments, the gene encoding the phage tail protein may be optimized to recognize and bind to a specific species of bacteria. In other embodiments, the gene encoding the phage tail protein may be optimized to recognize and bind to an entire genus of bacteria, or a specific group of species within a genus. In this way, the phage may be optimized to detect a broader or narrower group of pathogens. In some embodiments, the synthetic phage may be designed to improve the expression of the reporter gene. Furthermore and / or alternatively, in some cases, the synthetic phage may be designed to increase the burst size of the phage to improve detection.

[0133] In some embodiments, the stability of the phage can be optimized to improve its shelf life. For example, enzymatic solubility can be increased to enhance subsequent phage stability. Furthermore and / or alternatively, phage thermal stability can be optimized. Thermally stable phages better preserve their functional activity during storage, thereby increasing their shelf life. Therefore, in some embodiments, thermal stability and / or pH tolerance can be optimized.

[0134] In some embodiments, the genetically modified phage or the synthetically derived phage includes a detectable indicator. In some embodiments, the indicator is luciferase. In some embodiments, the phage genome includes an indicator gene (e.g., a luciferase gene or another gene encoding a detectable indicator).

[0135] System and Kit of the Present Invention In some embodiments, the present invention includes a system (e.g., an automated system or kit) comprising components for carrying out the methods disclosed herein. In some embodiments, the system or kit according to the present invention includes an indicator phage. The methods described herein may also utilize such an indicator phage system or kit. Some embodiments described herein are particularly suited to automation or kits, given the minimum amounts of reagents and materials required to carry out the above methods. In certain embodiments, each component of the kit may include a self-compacting unit that can be delivered from a first site to a second site.

[0136] In some embodiments, the present invention includes a system or kit for the rapid detection of a target microorganism in a sample. In certain embodiments, the system or kit comprises components for incubating the sample with an infectious agent specific to the target microorganism, wherein the infectious agent may comprise components including an indicator portion and components for detecting the indicator portion. In some embodiments of both the system and the kit of the present invention, the infectious agent is a recombinant bacteriophage that infects the target bacterium, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage as the indicator portion, and as a result, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium produces a soluble indicator protein product. Some systems further include components for capturing the target microorganism on a solid support.

[0137] In other embodiments, the present invention includes a method, system, or kit for the rapid detection of a target microorganism in a sample, comprising an infectious factor component specific to the target microorganism, wherein the infectious factor comprises an indicator portion and a component for detecting the indicator portion. In some embodiments, the bacteriophage is a T4-like, ViI, ViI-like, or Cronobacter spp.-specific bacteriophage. In one embodiment, the recombinant bacteriophage is derived from a Cronobacter spp.-specific bacteriophage. In certain embodiments, the recombinant bacteriophage is highly specific to a particular bacterium. For example, in certain embodiments, the recombinant bacteriophage is highly specific to Cronobacter spp. In embodiments, the recombinant bacteriophage can distinguish Cronobacter spp. in the presence of other types of bacteria. In certain embodiments, the system or kit detects specific bacteria in small numbers, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100, in the sample.

[0138] In certain embodiments, the system and / or kit may further include components for washing the captured microbial sample. Alternatively, the system and / or kit may further include components for determining the amount of the indicator portion, the amount of the indicator portion detected herein corresponds to the amount of microorganisms in the sample. For example, in certain embodiments, the system or kit may include a luminometer or other device for measuring luciferase enzyme activity.

[0139] In some systems and / or kits, the same components may be used for multiple steps. In some systems and / or kits, the steps are automated or controlled by the user via computer input, and / or a liquid handling robot performs at least one step.

[0140] Accordingly, in certain embodiments, the present invention may encompass a system or kit for the rapid detection of a target microorganism in a sample, the system or kit comprising components for incubating the sample with an infectious agent specific to the target microorganism, where the infectious agent comprises a component including an indicator portion; components for capturing the microorganism from the sample onto a solid support; components for washing the captured microorganism sample to remove unbound infectious agents; and components for detecting the indicator portion. In some embodiments, the same components may be used for the capturing and / or incubation and / or washing steps (e.g., a filter component). In some embodiments, the system or kit comprises components for determining the amount of the target microorganism in the sample, where the amount of the indicator portion detected further comprises a component corresponding to the amount of the microorganism in the sample. Such systems may include various embodiments and subordinate embodiments similar to those described above with respect to methods for the rapid detection of microorganisms. In one embodiment, the microorganism is a bacterium, and the infectious agent is a bacteriophage. In a computerized system, the system may be fully automated, semi-automated, or controlled by a user via a computer (or a combination of these).

[0141] In some embodiments, the system may include components for isolating the target microorganism from other components in the sample.

[0142] In one embodiment, the present invention comprises a system or kit comprising components for detecting a target microorganism, the system comprising: components for isolating at least one microorganism from other components in the sample; components for infecting the at least one microorganism with a plurality of parental infectivity factors; components for lysing the at least one infected microorganism to release progeny infectivity factors present in the microorganism; and components for detecting the progeny infectivity factors, or, with higher sensitivity, components for detecting soluble proteins encoded and expressed by the infectivity factors, wherein the infectivity factor or the soluble protein product of the infectivity factor comprises components indicating the presence of the microorganism in the sample. The infectivity factor may include a Cronobacter-specific NANOLUC® bacteriophage having a NANOLUC® indicator gene.

[0143] The above system or kit may include various components for the detection of progeny-infectious factors. For example, in one embodiment, the progeny-infectious factor (e.g., bacteriophage) may include an indicator portion. In one embodiment, the indicator portion in the progeny-infectious factor (e.g., bacteriophage) may be a detectable portion expressed during replication (e.g., a soluble luciferase protein).

[0144] In other embodiments, the present invention may encompass a kit for the rapid detection of a target microorganism in a sample, the system comprising components for incubating the sample with an infectious agent specific to the target microorganism, wherein the infectious agent comprises a component including an indicator portion; components for capturing the microorganism from the sample onto a solid support; components for washing the captured microorganism sample to remove unbound infectious agents; and components for detecting the indicator portion. In some embodiments, the same components may be used for the capturing step and / or the incubation step and / or the washing step. Some embodiments further include components for determining the amount of the target microorganism in the sample, wherein the amount of the indicator portion detected corresponds to the amount of the microorganism in the sample. Such a kit may include various embodiments and subordinate embodiments similar to those described above with respect to a method for the rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium, and the infectious agent is a bacteriophage.

[0145] In some embodiments, the kit may include components for isolating the target microorganism from other components in the sample.

[0146] These systems and kits of the present invention include various components. As used herein, the term “component” is broadly defined and includes any suitable apparatus or collection of suitable apparatus for carrying out the methods described herein. The components do not need to be integrally connected or installed with respect to one another in any particular way. The present invention includes any suitable arrangement of the components with respect to one another. For example, the components do not need to be in the same space. However, in some embodiments, the components are connected to each other in an integrated unit. In some embodiments, the same component may perform multiple functions.

[0147] Computer systems and computer-readable media The above system may be embodied in the form of a computer system, as described in any of the current technologies or their components. Typical examples of computer systems include general-purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices that can implement the processes constituting the methods of this technology.

[0148] The computer system may include a computer, input devices, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further include storage devices. The storage devices may be hard disk drives or removable storage devices (e.g., floppy disk drives, optical disk drives, etc.). The storage devices may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit enables the computer to connect to other databases and the Internet through an I / O interface. The communication unit enables transfers to and from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that enables the computer system to connect to databases and networks (e.g., LAN, MAN, WAN, and the Internet). The above computer system can therefore facilitate user input through input devices that are accessible to the system via an I / O interface.

[0149] A computing device typically includes an operating system that provides executable program instructions for the general management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., a hard disk, random-access memory, read-only memory, etc.) that stores instructions causing the computing device to perform its intended functions when executed by a server processor. Suitable implementations of the operating system and the general functionality of the computing device are known or commercially available and can be readily implemented by those skilled in the art, particularly in view of the disclosure herein.

[0150] The above computer system executes a set of instructions stored in one or more memory elements in order to process input data. The memory elements may also hold data or other information as described. The memory elements may be in the form of information sources present in the processing machine or physical memory elements.

[0151] The environment may include various data stores and other memory and storage media as considered above. These may reside in various locations (e.g., in storage media local to (and / or located within) one or more of the computers, or remotely from any or all of the computers across a network). In a particular group of embodiments, information may reside in a storage area network ("SAN") as is familiar to those skilled in the art. Similarly, any essential files for performing functions attributable to the computers, servers, or other network devices may be stored locally and / or remotely, where appropriate. Where the system includes computing devices, each such device may include hardware elements that can be electrically connected via a bus, such as, for example, at least one central processing unit (CPU), at least one input device (e.g., mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., display device, printer, or speaker). Such a system may also include one or more storage devices (e.g., disk drives, optical storage devices, and solid-state storage devices (e.g., random-access memory ("RAM") or read-only memory ("ROM")), as well as removable media devices, memory cards, flash cards, etc.).

[0152] Such devices may also include a computer-readable storage medium reader, a communication device (e.g., a modem, a network card (wireless or wired), an infrared communication device, etc.), and working memory as described above. The computer-readable storage medium reader may be connected to or configured to receive computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for storing, transmitting, and retrieving computer-readable information temporarily and / or more permanently. The system and various devices also typically include many software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs (e.g., a client application or a web browser). It should be recognized that alternative embodiments may have many variations from those described above. For example, customized hardware may also be used, and / or certain elements may be implemented in hardware, software (including portable software (e.g., applets)), or both. Furthermore, connections to other computing devices (e.g., network input / output devices) may be used.

[0153] Non-temporary storage media and computer-readable media for containing code or a portion of code may include any suitable media known or used in the art (including, but not limited to, volatile and non-volatile, removable and non-removable media implemented in any method or technique for storing and / or transmitting information such as computer-readable instructions, data structures, program modules, or other data), such as RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital multipurpose disc (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other media that may be used to store desired information and can be accessed by the system devices. Based on the above disclosure and the teachings provided herein, a person skilled in the art will recognize other ways and / or methods for implementing the various embodiments described above.

[0154] Computer-readable media may include, but are not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, SRAMs, DRAMs, associative memory ("CAM"), DDRs, flash memory (e.g., NAND flash or NOR flash), ASICs, configured processors, optical storage media, magnetic tapes or other magnetic storage media, or any other media from which a computer processor can read instructions. In one embodiment, the computing device may include a single type of computer-readable media (e.g., random access memory (RAM)). In other embodiments, the computing device may include two or more types of computer-readable media (e.g., random access memory (RAM), disk drives, and caches). The computing device may be in communication with one or more external computer-readable media (e.g., external hard disk drives, or external DVD or Blu-ray drives).

[0155] As discussed above, the embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. These instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language (e.g., C, C++, C#, Visual Basic, Java®, Python, Perl, JavaScript®, and ActionScript (Adobe Systems, Mountain View, Calif.)). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and state machines. Such processors may further include programmable electronic devices (e.g., PLCs), programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memory (PROMs), electronically programmable read-only memory (EPROMs or EEPROMs), or other similar devices.

[0156] The computing device described above includes a network interface. In some embodiments, the network interface is configured to communicate via a wired or wireless communication link. For example, the network interface may enable communication over a network via Ethernet®, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth®, infrared, etc. As another example, the network interface may enable communication over a network (e.g., CDMA, GSM®, UMTS, or other cellular communication network). In some embodiments, the network interface may enable point-to-point connections with other devices via interfaces such as Universal Serial Bus (USB), 1394 FireWire, serial or parallel connections, or similar interfaces. Some embodiments of a suitable computing device may include two or more network interfaces for communication over one or more networks. In some embodiments, the computing device may include a data store in addition to or instead of a network interface.

[0157] Some embodiments of a suitable computing device may include, or be in communication with, numerous external or internal devices (e.g., a mouse, CD-ROM, DVD, keyboard, display, audio speakers, one or more microphones, or any other input or output devices). For example, the computing device may be in communication with various user interface devices and displays. The display may use any suitable technology (including, but not limited to, LCD, LED, CRT, etc.).

[0158] The set of instructions for execution by the computer system described above may include a variety of commands that instruct a processing machine to perform a specific task (e.g., a step constituting the method of this 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 with a larger program, or part of a program module, as in this 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, the results of previous processing, or requests created by another processing machine.

[0159] While the present invention has been disclosed with reference to certain embodiments, many modifications, changes, and alterations to the embodiments described are possible without departing from the scope and spirit of the invention, as defined in the appended claims. Thus, the present invention is not intended to be limited to the embodiments described above, but has the entire scope as defined by the following claims and their equivalents. [Examples]

[0160] The results shown in the following examples demonstrate reduced time to results, detection of a small number of cells, and even single bacteria.

[0161] Example 1. Preparation and isolation of indicator phages from Cronobacter-specific bacteriophages Indicator phage Cronobacter-specific bacteriophages Saka2.NANOLUC, Saka4.NANOLUC, and Saka10.NANOLUC were prepared via homologous recombination using the procedure described previously. Cronobacter phages Saka2, Saka4, and Saka10 were isolated from sewage samples.

[0162] The genome sequences of these phages were obtained using de novo sequence assembly and whole-genome sequencing with the Illumina MiSeq system. Based on previously known and annotated genomes of the relevant phages, the late gene regions and major capsid protein genes were located on the novel phage genomes described above. Plasmids were designed and synthesized to insert NANOLUC®, along with appropriate late gene promoters and ribosome binding sites, approximately 500 bp adjacent to the matching phage sequence, to promote homologous recombination.

[0163] Target bacteria were transformed with the homologous recombination plasmid described above under appropriate antibiotic selection, and their respective wild-type phages were infected to enable homologous recombination with the plasmid. Following homologous recombination to generate the recombinant bacteriophage genome, a series of titration and enrichment steps were used to isolate specific recombinant bacteriophages expressing NANOLUC® as previously described.

[0164] Finally, large-scale generation was performed to obtain high-titer stocks suitable for use in Cronobacter spp. detection assays. Cesium chloride isodense gradient centrifugation was used to separate the phage particles from the luciferase protein contaminating them and reduce background noise.

[0165] Example 2. Infant formula protocol - Cronobacter Cronobacter bacterial suspensions were cultured overnight in culture medium. After overnight incubation, the cultures were diluted to the desired concentration in sterile water. The diluted cells were cultured on non-selective plates, and the CFU was quantified. Cronobacter dilutions were added to infant formula (PIF). The inoculated PIF was dried at a high temperature using a vacuum pump. The dried and added PIF samples were then inoculated with 10g, 100g, or 300g samples of PIF at various CFU levels. The inoculated PIF samples were stored at room temperature for 2–4 weeks and then evaluated.

[0166] 10g, 100g, and 300g samples of PIF were prepared. 10g of PIF sample was mixed with 90mL of pre-warmed (37°C) buffered peptone water (BPW) medium in a 1:9 sample:volume ratio; 100g of PIF was mixed with 300mL of pre-warmed BPW medium in a 1:3 sample:volume ratio; and 300g of PIF was mixed with 900mL of pre-warmed BPW medium in a 1:3 sample:volume ratio. The samples were homogenized for 120 seconds at the highest setting using a STOMACHER® or equivalent device (peristaltic blender). The homogenized samples were incubated at 37°C for 16–18 hours without shaking. After incubation, the samples were thoroughly mixed, and 1mL of the sample was taken and transferred to a centrifuge tube. The above samples were diluted 1:10 in BPW (100 μl sample: 900 μl BPW). 150 μl of the diluted samples were transferred to a 96-well plate. 10 μl of a bacteriophage cocktail of Cronobacter-specific bacteriophage solution was added to each well and incubated at 37°C for 2 hours. 10 μl of lysis buffer was added to each well, followed by 50 μl of the prepared luciferase substrate, and the readings were measured with a luminometer. Samples positive for Cronobacter had a relative light unit (RLU) reading of 500 or greater, while negative samples had a reading of less than 500 RLU.

[0167] Positive detection of Cronobacter was confirmed. Samples were enriched at 37°C for 24 hours. 50 μl of each enriched sample was placed in a Brilliance plate (Oxoid). TM Brilliance TM Cronobacter sakazakii (Agar cat# OXCM0129R) was streaked and incubated at 37°C for 24 hours. Positive detection was confirmed by the growth of blue-green colonies. [Table 1]

[0168] 100g PIF samples inoculated with different levels and different Cronobacter strains (Table 1) were tested for Cronobacter using an infant formula assay. For 100g samples, a 1:3 dilution of PIF versus BPW (buffered peptone water) was used. Samples with readings greater than 500 RLU / s or 5:1 signal / background were considered positive. 24-hour enriched samples were plated onto Brilliance plates for confirmation.

[0169] The matrices tested included: infant formula (milk-based), infant formula (soy-based), infant formula supplemented with prebiotics and probiotics, infant formula fortified with rice starch, or skim milk powder.

[0170] Example 3. Inclusion and Exclusion Study Inclusion strains (Cronobacter) were obtained from academic research institutions, government agencies, and commercially available sources (Table 2). Each strain was grown overnight in TSB medium at 37±1°C until it reached the steady phase. Cells were diluted to 100 CFU in 0.1 mL of TSB and mixed with recombinant phage for 2 hours at 37±1°C. After infection, samples were mixed with lysis buffer and luciferase substrate and then read using a luminometer. Samples with an RLU value greater than 150 were considered positive. Exclusion strains were also obtained from commercially available sources and grown overnight until they reached the steady phase. Assays with exclusion strains were performed in the same manner as with inclusion strains, except that the overnight cultures were directly assayed (Table 3).

[0171] The above Cronobacter assay showed 100% inclusion of the 75 Cronobacter strains tested (Table 2). The above Cronobacter assay also showed exclusion of 38 of the 41 non-Cronobacter strains tested (Table 3). The three non-Cronobacter strains detected by the above Cronobacter assay were derived from closely related Enterobacter species. Any presumptive positivity from the above phage assay could be enriched for a total of 24 ± 2 hours at 37 ± 1°C, followed by OXOID TM BRILLIANCE TM Cronobacter Sakazakii can be streaked on agar and further incubated at 37±1°C for 24±2 hours for confirmation. The presence of colonies that have grown sufficiently (1mm-3mm) and appear blue-green indicates a positive sample. [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2]

[0172] Example 4. Strength study Assay intensity was demonstrated by varying three parameters: enrichment time (14 hours and 24 hours), recombinant phage concentration (±20%), and luciferase substrate weight (±10%). Briefly, 10 g milk-based infant formula samples were either left unenriched or dried in infant formula with 0.2–2 CFU / 10 g C. muytjensii FSL-F6-031 added, and stored at room temperature for 2–4 weeks (20–25°C). The Cronobacter assay protocol described above was followed using variations in enrichment time, recombinant phage concentration, and substrate weight, as shown in Table 4. Samples with an RLU value greater than 500 were considered positive. Samples were enriched for a total of 24 ± 2 hours, followed by OXOID. TM BRILLIANCE TM Samples were identified by plating them onto Cronobacter Sakazakii agar. The plates were incubated at 37±1°C for a further 24±2 hours. The presence of well-growthed blue-green colonies indicated a positive sample. A summary of the above tests is shown in Table 4.

[0173] Strength tests of the Cronobacter assay showed that variations in enrichment time, recombinant phage concentration, and luciferase substrate volume did not alter the results compared to the standard protocol. Enrichment times of 14 and 24 hours, recombinant phage volumes of 8 μL and 12 μL, and luciferase substrate volumes of 45 μL and 55 μL produced identical results to the standard protocol of 16-hour enrichment, 10 μL of recombinant phage, and 50 μL of luciferase substrate in both uninoculated and low-inoculum test samples (Table 4). These results indicate that these deviations from the Cronobacter assay protocol did not alter the final results. [Table 4]

[0174] Example 5. Matrix study Matrix studies compared Cronobacter (10g test portion) with ISO 22964:2006 (10g test portion), and Cronobacter (100g and 300g test portions) with FDA BAM Ch. 29 Cronobacter:2012 (100g test portion). The above Cronobacter 10g portion was compared with ISO 22964:2006 using a paired study design. The above Cronobacter 100g and 300g portions were compared with the FDA BAM Ch. 29 100g portion using a non-paired study design. For each matrix and comparison, the above study included five replicated test portions of the uninoculated matrix (0 CFU / test portion), 20 replicated test portions at low levels to obtain fractionally positive results (0.2–2 CFU / test portion), and five replicated test portions at high levels to obtain consistently positive results (2–10 CFU / test portion).

[0175] Both milk-based and soy-based PIFs were purchased from local retailers and pre-screened for routine contamination using the ISO 22964:2006 method. To prepare the inoculum, Cronobacter was grown in tryptic soya broth for 18–24 hours at 37±1°C. The cultures were diluted in BPW, reconstituted in PIFs, and placed under rapid vacuum for 4–8 hours until the samples were completely dry. After drying, the dried inoculum was diluted in the PIF matrix to be used in each study to obtain low levels expected to yield stepwise positive results and high levels expected to yield all positive results, and allowed to stand at room temperature (20–25°C) for 2–4 weeks to allow equilibrium in the matrix. Bulk lots of the matrix were inoculated with the diluted inoculum before testing.

[0176] On the day of analysis, the total aerobic bacterial count was determined according to FDA BAM Ch. 3, and the levels of Cronobacter in low- and high-level inoculum were determined by most probable number (MPN) analysis. For paired samples, MPN analysis was performed using the ISO 22694:2006 method. For low-level inoculum, five 25g test portions, five 4g test portions, and 20 10g test portions from the matrix study described above were analyzed. For high-level inoculum, five 10g test portions, five 4g test portions, and five 1.5g test portions from the matrix study described above were analyzed.

[0177] For unpaired samples, MPN analysis was performed using the FDA BAM Ch. 29 method. For low-level indoctrines, five 200g test portions, five 50g test portions, and 20 100g test portions from the matrix study described above were analyzed. For high-level indoctrines, five 100g test portions, five 50g test portions, and five 25g test portions from the matrix study described above were analyzed. The number of positives was used to calculate MPN using the LCF MPN calculator provided by AOAC RI.

[0178] The Cronobacter test portion was processed according to the instructions for use. Briefly, 90 mL (10 g test portion), 300 mL (100 g test portion), or 900 mL (300 g test portion) of pre-warmed BPW (37±1°C) was added to the PIF test portion. The sample was homogenized and enriched at 37±1°C for 16-18 hours. The enriched sample was thoroughly mixed, and then aliquots were taken for analysis. The sample was diluted 1:10 (100 μl sample: 900 μl BPW) in pre-warmed BPW (37±1°C), and 150 μl of the diluted sample was transferred to a 96-well plate. The sample was then infected with recombinant phage for 2 hours at 37±1°C. Lysis buffer and luciferase substrate were added to the sample. The sample was then read using a luminometer. A reading of ≥500 RLU was considered positive. To confirm each Cronobacter assay, samples were enriched at 37±1°C for a total of 24±2 hours. The enriched samples were thoroughly mixed, and then aliquots were taken for analysis. 50 μL was oxydized. TM BRILLIANCE TM Cronobacter Sakazakii was soaked into agar and incubated at 37±1°C for 24±2 hours. The presence of colonies that have grown sufficiently (1mm-3mm) and appear bluish-green indicates a positive sample.

[0179] Sections E and F were performed to confirm the findings of FDA BAM Ch. 29. Briefly, 2 × 40 mL aliquots were centrifuged at 3,000 × g for 10 minutes after 24 hours of enrichment. The supernatant was discarded, and the resulting pellet was resuspended in 200 μl of sterile phosphate-buffered saline. 100 μl aliquots from the resuspended pellet were plated onto two DFI dye-producing agar plates and two R&F Cronobacter dye-producing agar plates. Furthermore, one loop of each enrichment was used to soak two DFI dye-producing agar plates and two R&F Cronobacter dye-producing agar plates. All plates were incubated at 36 ± 1°C for 18–24 hours. Presumed positive colonies were confirmed by PCR, as outlined in Section F of BAM Ch. 29.

[0180] ISO 22964:2006 (the latest version at the time of testing) was used in the method developer's laboratory for matrix evaluation. Briefly, 90 mL of BPW was added to 10 g of PIF. The above sample was incubated at 37 ± 1 °C for 18 ± 2 hours. Next, 0.1 mL was transferred from the BPW culture to 10 mL of mLST / vancomycin medium and incubated at 44 ± 1 °C for 24 ± 2 hours. One loop of the mLST / vancomycin culture was soaked into Enterobacter sakazakii Isolation Agar and incubated at 44 ± 1 °C for 24 ± 2 hours. Next, 1 to 5 presumed positive colonies were soaked into tryptic soya agar (TSA) plates and incubated at 25 °C for 48 ± 4 hours. Colonies that turned yellow were selected for further biochemical confirmation.

[0181] For the FDA BAM Ch. 29 Cronobacter method, 900 mL of sterile BPW was added to 100 g PIF in a sterile 2 L Erlenmeyer flask, and the mixture was gently stirred by hand until the PIF was uniformly suspended. The test samples were incubated at 36 ± 1 °C for 24 ± 2 hours. After enrichment, the above samples were thoroughly mixed, and 4 × 40 mL from each sample were transferred to 50 mL centrifuge tubes. The above aliquots were centrifuged at 3,000 × g for 10 minutes, and the supernatant was discarded. The resulting pellet was resuspended in 200 μl of phosphate-buffered saline. Two aliquots were used for PCR to determine presumptive positivity, and two aliquots were used for culture confirmation, if necessary. For PCR screening, two aliquots were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 3,000 × g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 400 μL of PREPMAN ULTRA® sample preparation reagent and mixed at maximum vortex speed until the pellet was completely resuspended. The sample was heated in a dry bath incubator at 100°C for 10 minutes and then cooled to room temperature. Once the sample had reached room temperature, it was centrifuged at 15,000 × g for 2 minutes, and a 50 μL aliquot of the supernatant was transferred to a new microcentrifuge tube for PCR analysis. PCR analysis was performed for each sample with or without an internal control (InC). The PCR reaction components and PCR protocol were followed as outlined in the FDA BAM Chapter 29 reference method. Presumed positives were confirmed using FDA BAM Ch. 29, sections E and F. In short, 100 μL aliquots from the resuspended pellet were plated onto two DFI dye-producing agar plates and two R&F Cronobacter dye-producing agar plates. Furthermore, one loopful of each enrichment was streaked onto two DFI dye-producing agar plates and two R&F Cronobacter dye-producing agar plates. All plates were incubated at 36±1°C for 18–24 hours. Colonies were confirmed by PCR, as outlined in section F of BAM Ch. 29.

[0182] All trial results were analyzed using POD statistical analysis for 95% confidence intervals (CI). POD analysis is described in the AOAC INTERNATIONAL guidelines in Appendix J. The data from the above analysis are shown in Tables 5-8.

[0183] The developer study of the above method showed no difference between the above assay and the ISO 22964:2006 and FDA BAM Ch. 29 Cronobacter reference methods for all matrices tested (Tables 5-8). All test portions that were presumably positive by the Cronobacter assay were confirmed to contain Cronobacter by their respective reference methods. There were no false negative results. The above POD analysis showed no significant difference between the Cronobacter assay and the ISO 22964:2006 reference method in paired studies (Table 5). There was no statistically significant difference between the number of Cronobacter assay estimates and BAM Ch. 29 confirmation results (Table 6). Comparisons between Cronobacter assay results and BAM Ch. 29 confirmations, as well as between estimated and confirmed results, were also not statistically significant (Tables 6 and 7). Comparisons between the Cronobacter assay and FDA BAM Ch. 29 unpaired studies showed no statistically significant difference in the performance of the two methods (Table 8). One exception was the comparison of the 100g milk-based assay versus the BAM Ch. 29 method (Table 8). The difference between the above-mentioned graded positives was statistically significant, with a dPOD of 0.35 and a CI of (0.04, 0.58) in this case. The aerobic bacterial plate count of the PIF used in the above study was 0 CFU / g, which indicates that the PIF had either no background bacterial flora present at the start of the enrichment process or was at a very low level.

[0184] Independent experimental evaluations included matrix studies comparing the Cronobacter assay to the ISO 22964:2017 and FDA BAM Chapter 29 reference methods for milk-based PIFs. For method comparisons against ISO 22964:2017, 30 pairs of 10g test portions were evaluated. For method comparisons against FDA BAM Chapter 29, 100g and 300g test portions of the Cronobacter assay were compared to the 100g test portion of the reference method. Within each sample set, there were 5 uninoculated samples (0 CFU / test portion), 20 low-level inoculated samples (0.2–2 CFU / test portion), and 5 high-level inoculated samples (2–10 CFU / test portion). The low inoculation levels were designed to produce a gradual positive result (the candidate or reference method would produce 5–15 positive results (25–75%)).

[0185] The above PIF was purchased from a local distributor and pre-screened for routine contamination of the analyte according to ISO 22964:2017, and analyzed for total aerobic bacterial count according to FDA BAM Chapter 3. After screening, the matrix was inoculated with Cronobacter strains. For validation, lyophilized cultures were used to inoculate the above PIF. The lyophilized cultures were prepared by transferring a single Cronobacter sakazakii colony from tryptic soy agar containing 5% sheep blood to brain-heart infusion (BHI) broth, and incubating the cultures at 35±2°C for 18-24 hours. After incubation, the cultures were diluted in sterile cryoprotective material (reconstituted nonfat-based milk powder) (NFDM) and placed on a lyophilization system for 48-72 hours. After removing the cultures from the lyophilization system, the lyophilized cultures were diluted in NFDM to low levels expected to produce stepwise positive results and high levels expected to produce overall positive results. A bulk lot of the above matrix was inoculated. After inoculation, the matrix was kept at room temperature (24±2℃) for two weeks to allow for the equilibrium of organisms within the matrix.

[0186] The total aerobic bacterial count was determined according to FDA BAM Ch. 3. Cronobacter levels in low-level and high-level inoculum were determined by MPN on the day of analysis. For paired sample analysis, the low-level MPN was determined by evaluating the 5 × 25 g test portion, the 20 × 10 g test portion from the above test, and the 5 × 4 g test portion. Cronobacter levels in high-level inoculum were determined by evaluating the 5 × 10 g test portion, the 5 × 4 g test portion, and the 5 × 1.5 g test portion from the above test.

[0187] For unpaired analysis, the low-level MPN was determined by evaluating the 5 × 200 g test portion, the 20 × 100 g reference method test portion from the above study, and the 5 × 50 g test portion. The Cronobacter levels of the high-level inoculum were determined by evaluating the 5 × 100 g reference method test portion, the 5 × 50 g test portion, and the 5 × 25 g test portion from the above study. Each test portion was enriched with BPW and analyzed according to the reference method procedure. The number of positives from the three test levels was used to calculate the MPN using the LCF MPN calculator (version 1.6).

[0188] For ISO 22964:2017, 10g of the PIF test portion was enriched with 90mL of BPW (ISO formulation) and incubated at 37±1℃ for 18±2 hours. After incubation, 0.1mL of the primary enrichment was transferred to 10mL of Cronobacter Selective Broth (CSB) and incubated at 41.5±1℃ for 24±2 hours. After incubation, one loop of the CSB was soaked into Chromogenic Cronobacter Isolation (CCI) agar and incubated at 41.5±1℃ for 24±2 hours. After incubation of the CCI plate, 1 to 5 representative Cronobacter colonies (medium-sized, 1mm to 3mm blue-green to blue colonies) were transferred to tryptone soya agar (TSA) and incubated at 35±1℃ for 18 to 24 hours. After incubation, oxidase tests were performed on representative colonies (1mm-3mm, colored yellow), and final biochemical confirmation was carried out using VITEK® 2 GN Biochemical Identification cards in accordance with AOAC Official Method 2011.17.

[0189] For BAM Ch. 29, a 100g PIF test portion was added to a 2L Erlenmeyer flask, enriched with 900mL of pre-warmed (37°C) BPW, and incubated at 37±1°C for 24±2 hours. After incubation, 4×40mL aliquots were transferred to 4×50mL conical vials. The aliquots were centrifuged at 3,000×g for 10 minutes. For each conical tube, the supernatant was aspirated, and lipid precipitates were removed using a sterile cotton swab. The remaining pellet was resuspended by adding 200μL of phosphate-buffered saline and vortexing the suspension at maximum speed for 20 seconds. For each sample, two of the aliquots were used for Cronobacter PCR screening, and two of the aliquots were used for culture confirmation.

[0190] For PCR screening, two aliquots were transferred to separate 1.5 mL microcentrifuge tubes and centrifuged at 3,000 × g for 5 minutes. The supernatant and lipid layer were removed, and the pellet was resuspended by adding 400 μL of PREPMAN ULTRA® sample preparation reagent and mixing by vortexing at maximum speed until suspension was achieved. The above samples were heat-treated in a dry bath incubator at 100 °C for 10 minutes and then cooled to room temperature. Once the above samples had reached room temperature, the above samples were centrifuged at 15,000 × g for 2 minutes, and a 50 μL aliquot of the supernatant was transferred to a new microcentrifuge tube for PCR analysis. PCR analysis was performed for each sample with and without an internal control (InC). The above PCR reaction components and PCR protocol followed the reference method outlined in FDA BAM Ch. 29.

[0191] Regardless of the estimated PCR results, 100 μL of suspended cells from each sample were infused into two DFI dye-producing agar plates and two R&F agar plates. The DFI dye-producing agar plates and R&F agar plates were incubated at 36±1°C for 18–24 hours. After incubation, representative Cronobacter colonies from the DFI dye-producing agar plates (light green to dark green-brownish colonies, or colonies with a green center and white to yellow edges) and representative Cronobacter colonies from the R&F agar plates (colonies with a red background, blue to black, or blue to gray) were biochemically identified using VITEK® 2 GN Biochemical Identification cards (AOAC Official Method 2011.17) and PCR analysis.

[0192] For all three levels, POD analysis between the Cronobacter assay and the reference method showed no 5% statistically significant difference in the number of positive results obtained by the above method (Tables 5-8). For all three levels, POD analysis between the estimated and confirmed results of the Cronobacter assay showed no 5% statistically significant difference for all analyzed test portions. The study showed no statistically significant difference at each level (Tables 5-8). The aerobic bacterial plate count of PIF used in the above study was 40 CFU / g, which corresponds to approximately 400 CFU (10g), 4,000 CFU (100g), or 12,000 CFU of the above PIF. The CFU (300g) demonstrated the presence of the background bacterial flora present at the start of the entire process. [Table 5] [Table 6] [Table 7] [Table 8] The present invention provides, for example, the following items: (Item 1) A recombinant bacteriophage comprising an indicator gene inserted into the late gene region of the bacteriophage genome, wherein the recombinant bacteriophage is a recombinant bacteriophage that specifically infects Cronobacter spp. (Item 2) The recombinant bacteriophage is a recombinant bacteriophage as described in item 1, derived from wild-type Saka2, Saka4, or Saka10 bacteriophage. (Item 3) The recombinant bacteriophage described in item 1, wherein the indicator gene is codon-optimized and encodes a soluble enzyme that generates a signal upon reaction with a soluble protein product or substrate that generates an endogenous signal. (Item 4) The recombinant bacteriophage according to item 1, further comprising an untranslated region upstream of the codon optimization indicator gene, wherein the untranslated region comprises a late-stage bacteriophage gene promoter and a ribosome entry site. (Item 5) A cocktail composition comprising at least two different types of recombinant bacteriophages, wherein at least one of the recombinant bacteriophages comprises the indicator gene described in item 1. (Item 6) A method for preparing recombinant indicator bacteriophages, A step of selecting wild-type bacteriophages that specifically infect target pathogenic bacteria, A step of preparing homologous recombination plasmids / vectors containing indicator genes, A step of transforming target pathogenic bacteria with the homologous recombinant plasmid / vector, The process involves infecting the transformed target pathogenic bacterium with a selected wild-type bacteriophage, thereby inducing homologous recombination between the plasmid / vector and the bacteriophage genome, and The process of isolating a specific clone of a recombinant bacteriophage. Methods that include... (Item 7) The process of preparing homologous recombination plasmids / vectors is as follows: Determining the native nucleotide sequence in the late region of the genome of the selected bacteriophage; Annotating the genome and identifying the major capsid protein genes of the selected bacteriophage; Designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence includes a codon optimization indicator gene; and Incorporating the sequence designed for homologous recombination into a plasmid / vector. The method described in item 6, including the method described in item 6. (Item 8) The method according to item 7, further comprising designing a sequence to insert an untranslated region containing a phage late gene promoter and a ribosome entry site upstream of the codon optimization indicator gene. (Item 9) The method according to item 8, wherein the homologous recombination plasmid includes an untranslated region containing a bacteriophage late gene promoter and a ribosome entry site upstream of the codon optimization indicator gene. (Item 10) The method according to item 8, wherein the wild-type bacteriophage is a Cronobacter-specific bacteriophage and the target pathogenic bacterium is Cronobacter spp. (Item 11) The method according to item 8, wherein the step of isolating a specific clone of a recombinant bacteriophage includes a limiting dilution assay for isolating a clone exhibiting expression of the indicator gene. (Item 12) A method for detecting Cronobacter spp. in a sample, wherein the method is: The steps include: incubating the sample with a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage containing an indicator gene inserted into the late gene region of the bacteriophage genome; and A step of detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates the presence of Cronobacter spp. in the sample. A method that includes this. (Item 13) The method according to item 12, wherein the sample is a food sample, an environmental sample, a water sample, or a commercial sample. (Item 14) The method according to item 12 for detecting small amounts of bacteria, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, in a standard-sized sample for the food safety industry. (Item 15) The method according to item 14, wherein the food sample includes meat, fish, vegetables, eggs, dairy products, dry food products, or infant formula. (Item 16) The method according to item 12, wherein the sample is incubated with a cocktail composition comprising at least two different types of recombinant bacteriophages, and at least one of the recombinant bacteriophages comprises the indicator gene described in item 12. (Item 17) The method according to item 16, wherein the sample is first incubated under conditions favorable to growth for an enrichment period of less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours. (Item 18) The method used for item 16 where the total time to the result is less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours. (Item 19) The method according to item 12, wherein the signal-to-background ratio generated by the step of detecting the indicator is at least 2.0 or at least 2.5. (Item 20) A kit for detecting Cronobacter spp., containing recombinant bacteriophages derived from Cronobacter-specific bacteriophages. (Item 21) The kit according to item 20, further comprising an indicator and a substrate for reaction, for detecting soluble protein products expressed by the recombinant bacteriophage. (Item 22) A system for detecting Cronobacter spp., including recombinant bacteriophages derived from Cronobacter-specific bacteriophages.

Claims

1. A method for detecting Cronobacter spp. in a sample, the method being: A step of incubating the sample with a cocktail composition comprising at least two different types of recombinant bacteriophages, wherein each of the at least two different types of recombinant bacteriophages is a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage comprising an indicator gene inserted into the late gene region of a bacteriophage genome and an exogenous phage late gene promoter upstream of the indicator gene, wherein the indicator gene is not contiguous with a gene encoding a bacteriophage structural protein, does not produce a fusion protein, and expression of the indicator gene yields an indicator protein product; and A step of detecting the indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates the presence of Cronobacter spp. in the sample. The sample includes, and the sample is a sample of infant formula. A method wherein the at least two different types of recombinant bacteriophages include at least one first recombinant bacteriophage derived from a podovirus Cronobacter-specific bacteriophage and at least one second recombinant bacteriophage derived from a myovirus Cronobacter-specific bacteriophage.

2. The method according to claim 1, for detecting bacteria in small quantities of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 in a sample for the food safety industry.

3. The method according to claim 1, wherein the sample is initially incubated under conditions favorable to growth for an enrichment period of less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours.

4. The combined time to the result is less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours, according to claim 1.

5. The method according to claim 1, wherein the signal-to-background ratio generated by the step of detecting the indicator protein product is at least 2.0 or at least 2.

5.

6. A kit for detecting Cronobacter spp. comprising a cocktail composition comprising at least two different types of recombinant bacteriophages, wherein each of the at least two different types of recombinant bacteriophages is a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage comprising the indicator gene described in Claim 1, which is inserted into the late gene region of the bacteriophage genome, and the at least two different types of recombinant bacteriophages comprises at least one first recombinant bacteriophage derived from a podovirus Cronobacter-specific bacteriophage and at least one second recombinant bacteriophage derived from a myovirus Cronobacter-specific bacteriophage.

7. The kit according to claim 6, further comprising a substrate for reacting with the indicator protein product for detecting the indicator protein product expressed by the recombinant bacteriophage.

8. A system for detecting Cronobacter spp. comprising a cocktail composition comprising at least two different types of recombinant bacteriophages, wherein each of the at least two different types of recombinant bacteriophages is a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage comprising the indicator gene described in Claim 1, which is inserted into the late gene region of the bacteriophage genome, and the at least two different types of recombinant bacteriophages comprises at least one first recombinant bacteriophage derived from a podovirus Cronobacter-specific bacteriophage and at least one second recombinant bacteriophage derived from a myovirus Cronobacter-specific bacteriophage.

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