Methods for producing mutant bacteriophages for detection of listeria

Abstract

To provide methods for producing mutant bacteriophages for detection of LISTERIA.SOLUTION: Methods for the production of mutant bacteriophages with altered host range are disclosed herein. Additionally, methods and systems for rapid detection of microorganisms, such as Listeria spp., in a sample are disclosed herein. A genetically modified bacteriophage comprising an indicator gene in the late gene region is also disclosed. The specificity of the bacteriophage, such as a Listeria-specific bacteriophage, allows detection of a specific microorganism, such as Listeria spp., and an indicator signal may be amplified to optimize assay sensitivity.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Patent Application No. 62 / 864,894, filed on June 21, 2019. The disclosures of U.S. Provisional Patent Application No. 62 / 864,894 and U.S. Patent Application No. 16 / 776,417 are hereby incorporated by reference in their entirety herein.

[0002] Field of the Invention The present invention relates to methods for the production of bacteriophages and the resulting bacteriophages.

Background Art

[0003] Background Improving the selectivity for the detection of bacteria, viruses, and other microorganisms in biological samples, food samples, water samples, and clinical samples is of great importance. Microbial pathogens can cause substantial morbidity in humans and livestock, as well as huge economic losses. Also, 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) considering the outbreak of life - threatening or fatal diseases caused by the ingestion of food contaminated with certain microorganisms (e.g., Listeria spp., Salmonella spp., or Staphylococcus spp.).

[0004] Traditional microbiological tests for the detection of bacteria rely on non-selective and selective enrichment cultures, followed by plating on selective media and further tests to confirm suspected colonies. Such procedures can take several days. A variety of rapid methods have been investigated and practiced to reduce the time requirements. However, these methods have drawbacks. For example, techniques that require direct immunoassays or gene probes generally require an overnight enrichment step to obtain appropriate sensitivity. Polymerase chain reaction (PCR) tests also include an amplification step and thus have the ability to be both very sensitive and selective; however, the sample size that can be economically subjected to PCR testing is limited. If the bacterial suspension is dilute, most small subsamples will be cell-free and thus a purification and / or lengthy enrichment step is still required.

[0005] The time required for traditional biological enrichment is defined by the growth rate of the target bacterial population in the sample, by the influence of the sample matrix, and by the sensitivity required. In practice, most sensitive methods employ an overnight incubation and take approximately 24 hours overall. Due to the time required for culturing, these methods can take up to 3 days, depending on the organism to be identified and the sample source. This lag time is generally inappropriate because contaminated food, water, or other products may already have entered livestock or humans. Furthermore, the increasing concerns about antibiotic-resistant bacteria and biodefense have made the rapid identification of bacterial pathogens in water samples, food samples, and clinical samples a top priority worldwide.

[0006] Bacteriophages (phages) can be used to detect pathogenic bacteria in food samples, environmental samples, and clinical samples due to their narrow range of host specificity. The narrow host range of phages can be used to detect potentially pathogenic bacteria while excluding the detection of non-harmful bacteria. However, phages are too specific and cannot detect each serotype or species of potentially harmful bacteria present in a sample. Detection of the target bacteria in a sample may require the use of phage cocktails. Therefore, it may be advantageous to expand the host range of phages specific to a particular host to include new target hosts.

[0007] Therefore, there is a need for methods to generate bacteriophages with an expanded host range and bacteriophages with an expanded host range. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0008] Abstract Embodiments of the present disclosure include methods for generating mutant bacteriophages (phages) with an expanded host range and the resulting phages obtained therefrom. The present disclosure can be embodied in various ways.

[0009] In one aspect, the present disclosure relates to a method for generating a mutant bacteriophage having an expanded host range. In some embodiments, the method includes (i) preparing a series of first co-culture mixtures in various ratios comprising a host bacterial strain and a target host bacterial strain; (ii) adding a phage strain to each of the first co-culture mixtures; (iii) incubating the first co-culture mixtures and the phage strain under bacterial culture conditions; (iv) collecting phage lysates from each of the plurality of first co-cultures; (v) pooling the phage lysates from each of the plurality of first co-cultures; (vi) assaying the phage lysate to determine whether the bacterial host range has been expanded; and (vii) isolating mutant phages having an expanded host range.

[0010] In another aspect, the present disclosure relates to a mutant bacteriophage having an expanded host range obtained thereby, wherein the mutant bacteriophage is capable of infecting a host bacterial strain and a target host bacterial strain.

[0011] In other cases, it is advantageous to utilize bacteriophages with a narrow host range. Accordingly, in another aspect, the present disclosure relates to a method for generating a mutant bacteriophage having a reduced host range. In some embodiments, the method comprises: (i) mutating a gene encoding a tail spike protein of a bacteriophage; (ii) generating a progeny phage lysate from the mutated bacteriophage; (iii) assaying the phage lysate to determine whether the bacterial host range is reduced; and (iv) isolating the mutant phage having a reduced host range.

[0012] In yet another aspect, the present disclosure relates to a recombinant bacteriophage comprising an indicator gene inserted into a late gene region of a mutant bacteriophage having an expanded host range genome. In some embodiments, the recombinant bacteriophage is a genetically modified Listeria - specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified bacteriophage genome derived from a bacteriophage that specifically recognizes Listeria spp. In some embodiments, the bacteriophage used to prepare the recombinant bacteriophage specifically infects one or more Listeria spp. In one embodiment, the recombinant bacteriophage can distinguish a host bacterial strain and a target host bacterial strain in the presence of other types of bacteria.

[0013] Methods for preparing recombinant indicator bacteriophages are also disclosed herein. Some embodiments include selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector containing an indicator gene; transforming the homologous recombination plasmid / vector into the target pathogenic bacterium; infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby enabling homologous recombination to occur between the plasmid / vector and the bacteriophage genome; and isolating specific clones of the recombinant bacteriophage. In some embodiments, the selected wild-type bacteriophage is a Listeria-specific bacteriophage. In some embodiments, the selected wild-type bacteriophage is a myovirus (e.g., T4, T4-like virus, Listeria phage LMTA-94, P100 virus or Vil-like). In some embodiments, the selected wild-type bacteriophage infects Listeria spp. In other embodiments, the selected wild-type bacteriophage is a podovirus (e.g., T7-like virus or Sp6-like virus). In other embodiments, the selected wild-type bacteriophage is LMA4 and LMA8. LMA4 and LMA8 are myoviruses, as in the genus P100 virus.

[0014] In some embodiments, the present invention is a method for detecting a target microorganism in a sample, the method comprising incubating the sample with a recombinant bacteriophage that infects the target microorganism, wherein the recombinant bacteriophage comprises an indicator gene inserted into a late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target microorganism in the sample.

Mode for Carrying Out the Invention

[0015] Detailed Description of the Invention Methods for generating bacteriophages with an expanded host range and the resulting mutant bacteriophages are provided herein, and the mutant bacteriophages can be used for the detection of target microorganisms (e.g., Listeria spp.) in test samples (e.g., biological samples, food samples, water samples, and environmental samples). Detection can be achieved in assays performed without culturing for enrichment or, in some embodiments, with a minimal incubation period during which the microorganism could potentially grow, using a genetically modified infectious agent, in a shorter time frame than previously thought possible.

[0016] In some aspects, the present disclosure relates to methods for detecting a target microorganism. The methods can use an infectious agent for detection of the target microorganism (e.g., Listeria spp.). For example, in certain embodiments, the target microorganism is Listeria spp. and the infectious agent is a bacteriophage that specifically infects Listeria spp. In some embodiments, the bacteriophage is mutated to have an expanded host range and can infect multiple serotypes of Listeria monocytogenes. Thus, in certain embodiments, the method can include detecting the target bacterium in the sample by incubating the sample with a recombinant mutant bacteriophage that infects the target bacterium. In certain embodiments, the recombinant mutant bacteriophage includes an indicator gene. The indicator gene can be inserted in certain embodiments into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in the production of an indicator protein product. The method can include the step of detecting the indicator protein product, where a positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In one embodiment, the indicator protein is soluble.

[0017] Embodiments of the methods and systems of the present invention can be applied to the detection and quantification of various microorganisms (e.g., bacteria) in various environments, including but not limited to the detection of pathogens from food samples, water samples, and environmental samples. The methods of the present invention rapidly provide high detection sensitivity and specificity. In some embodiments, detection is possible within a single replication cycle of the bacteriophage, which is unexpected.

[0018] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall 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 described herein, as well as these techniques, are well known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods well known in the art and as described in various general and more specific references discussed throughout this specification, unless otherwise indicated. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with the experimental procedures and techniques described herein is well known and commonly used in the art.

[0019] The following terms shall be understood to have the following meanings, unless otherwise indicated:

[0020] As used herein, the terms "a", "an", and "the" can refer to one or more, unless otherwise noted.

[0021] The use of the term "or" is used to mean "and / or" unless it is clearly indicated that only alternatives are being referred to or the alternatives are mutually exclusive, but the present disclosure supports definitions that refer only to alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0022] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method used to determine that value, or the variation that exists between samples.

[0023] The term "solid support" or "support" means a structure that provides a substrate and / or surface to which a biomolecule can bind. For example, the solid support can be an assay well (i.e., for example, a microtiter plate or multi-well plate), or the solid support can 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 a lateral flow strip).

[0024] The term "binding factor" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction can be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction can be covalent. The term "soluble binding factor" refers to a binding factor that is not associated (i.e., covalently or non-covalently) with a solid support.

[0025] As used herein, "analyte" refers to the molecule, compound or cell being measured. The analyte of interest can interact with a binding factor in certain specific embodiments. As described herein, the term "analyte" can refer to a protein or peptide of interest. The analyte can be an agonist, antagonist, or modulator. Alternatively, the analyte may not have a biological effect. Examples of analytes can include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, and organic compounds.

[0026] The terms "detectable moiety", "detectable biomolecule", "reporter", "indicator", or "indicator moiety" refer to a molecule that can be measured in a quantitative assay. For example, the indicator moiety can include an enzyme that is used to convert a substrate into a measurable product. The indicator moiety can be an enzyme (e.g., luciferase) that catalyzes a reaction that produces bioluminescence emission. Alternatively, the indicator moiety can be a radioisotope that can be quantified. Alternatively, the indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0027] As used herein, "bacteriophage" " or "phage" includes one or more of a plurality of bacterial viruses. In the present disclosure, the terms "bacteriophage" and "phage" refer to viruses that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic-level living organisms, and that use the above organisms to replicate the virus itself, including mycobacteriophages (e.g., related to TB and para-TB), mycophages (e.g., related to fungi), mycoplasma phages, and any other terms, etc. Here, "microscopic-level" means that the maximum dimension is 1 millimeter or less. Bacteriophages are viruses that have evolved to use bacteria in nature as a means of replicating themselves. Phages do this by attaching the phage itself to bacteria, injecting its DNA (or RNA) into the bacteria, and inducing the bacteria to replicate the phage hundreds or even thousands of times. This is also referred to as phage amplification.

[0028] As used herein, the term "late gene region" refers to the region of the viral genome that is transcribed late in the viral life cycle. The late gene region typically includes the most abundantly expressed genes (e.g., the structural proteins that assemble into the bacteriophage particles). The late genes are synonymous with class III genes and include genes that have structural and assembly functions. For example, in phage T7, the late genes (synonymous with class III) are transcribed at a time when class I (e.g., RNA polymerase) is transcribed early, from 4 - 8 minutes after infection, and class II is transcribed from 6 - 15 minutes, so the timings of classes II and III overlap, and the late promoter is a promoter that is naturally located and active in such a late gene region.

[0029] As used herein, "culturing for enrichment" refers to traditional culturing (e.g., incubation in a medium convenient for microbial propagation), and should not be confused with other possible uses of the term "enrichment" (e.g., enrichment by removing the liquid components of a sample and concentrating the microorganisms contained therein), or other forms of enrichment that do not include the traditional promotion of microbial propagation. Culturing for enrichment over a period of time can be used in some embodiments of the methods described herein.

[0030] As used herein, "host range" refers to the number of host species used by a pathogen. The host range describes the breadth of organisms that a bacteriophage can infect, along with any restrictions on the host range that are due to bacteriophage, host, or environmental characteristics.

[0031] As used herein, "recombinant" refers to genetic (i.e., nucleic acid) modification as typically performed in experiments to bring together genetic material not found together otherwise. This term is used interchangeably herein with the term "modified".

[0032] As used herein, "RLU" refers to relative light units, measured by a luminometer (e.g., GLOMAX® 96) or similar device that detects light. For example, detection of a reaction product between luciferase and an appropriate substrate (e.g., NANOLUC® and NANO-GLO®) is often reported as the detected RLU.

[0033] As used herein, "time to results" refers to the total amount of time from the start of sample incubation until results are generated. Time to results does not include any confirmation test time. Data collection can be performed at any time after results are generated.

[0034] Generation of Mutant Bacteriophages Embodiments of methods for generating mutant bacteriophages with an expanded host range begin with the selection of a bacteriophage for genetic modification. Some bacteriophages are highly specific for a target bacterium. This presents an opportunity for highly specific detection. In some cases, it may be advantageous to expand the host range of a highly specific phage to enable detection of multiple strains of potentially harmful bacteria in a single assay.

[0035] The host range of a bacteriophage is considered to be the breadth of bacteria (i.e., genus, species, or strain) that the bacteriophage can infect productively. The host range of some bacteriophages is quite narrow, having the ability to infect only a few strains within the same species. Other phages can infect many bacterial species, sometimes across different genera. However, most bacteriophages are thought to have a relatively narrow host range. This can be due, in part, to the specificity of the phage's host-binding proteins, biochemical interactions during infection, the presence of related prophages or certain plasmids, and the phage resistance mechanisms of the bacteria.

[0036] In some cases, it is advantageous to utilize phages with a very broad host range. Thus, in one aspect, the present disclosure relates to methods for generating mutant bacteriophages with an expanded host range. In some embodiments, the method comprises: (i) preparing a series of first co-culture mixtures at various ratios comprising a host bacterial strain and a target host bacterial strain; (ii) adding a phage strain to each of the first co-culture mixtures; (iii) incubating the first co-culture mixtures and the phage strain under bacterial culture conditions; (iv) collecting phage lysates from each of the plurality of first co-cultures; (v) pooling the phage lysates from each of the plurality of first co-cultures; (vi) assaying the phage lysates to determine whether the bacterial host range has been expanded; and (vii) isolating mutant phages with an expanded host range.

[0037] In other cases, it is advantageous to utilize bacteriophages with a narrow host range. Thus, in another aspect, the present disclosure relates to methods for generating mutant bacteriophages with a reduced host range. In some embodiments, the method comprises: (i) mutating a gene encoding a tail spike protein of a bacteriophage; (ii) generating progeny phage lysates from the mutated bacteriophage; (iii) assaying the phage lysates to determine whether the bacterial host range has been reduced; and (iv) isolating mutant phages with a reduced host range.

[0038] In some embodiments, the selected wild-type bacteriophage is a Listeria-specific bacteriophage. In certain embodiments, the selected wild-type bacteriophage is derived from a phage of the order Caudovirales. Caudovirales is an order of tailed bacteriophages with double-stranded DNA (dsDNA) genomes. Each virion of the order Caudovirales has an icosahedral head containing the viral genome and a flexible tail. The order Caudovirales contains five bacteriophage families: Myoviridae (long contractile tails), Siphoviridae (long non-contractile tails), Podoviridae (short non-contractile tails), Ackermannviridae, and Herelleviridae. The term myovirus can be used to describe any bacteriophage having an icosahedral head and a long contractile tail and encompassing bacteriophages in both the Myoviridae and Herelleviridae families. In some embodiments, the selected wild-type bacteriophage is a member of the Myoviridae family (e.g., Listeria phage B054, Listeria phage LipZ5, Listeria phage PSU-VKH-LP041, and Listeria phage WIL-2). In other embodiments, the selected wild-type bacteriophage is a member of the Herelleviridae family. The genus Pecentumvirus under the Herelleviridae family includes bacteriophages such as Listeria phage LMSP-25, Listeria phage LMTA-148, Listeria phage LMTA-34, Listeria phage LP-048, Listeria phage LP-064, Listeria phage LP-083-2, Listeria phage LP-125, Listeria virus P100, Listeria phage List-36, Listeria phage WIL-1, Listeria phage vB_LmoM_AG20, and Listeria virus A511.LMA4 and LMA8 are also, similarly, within the Pecentumvirus genus under the Herelleviridae family. In other embodiments, the selected wild-type bacteriophage is LMA4 or LMA8. In certain cases, the selected wild-type bacteriophage is LP-ES3A, which is derived from A511 but is adapted to infect serotype 3A of Listeria monocytogenes. In yet other embodiments, the selected wild-type bacteriophage is a member of the Ackermannviridae family. In yet other embodiments, the selected wild-type bacteriophage is a member of the Siphoviridae family, which includes Listeria phages A006, A118, A500, B025, LP-026, LP-030-2, LP-030-3, LP-037, LP-101, LP-110, LP-114, P35, P40, P70, PSA, vB_LmoS_188, and vB_Lmos_293. In other embodiments, the selected wild-type bacteriophage is LP-ES1. LP-ES1 is also, similarly, within the Homburgvirus genus under the Siphoviridae family.

[0039] In some embodiments, the method of generating a mutant bacteriophage having an expanded host range includes identifying and selecting a bacteriophage that is specific for a target bacterium. In some embodiments, the target bacterium is a potentially harmful bacterium. In further embodiments, the selected bacteriophage can infect a host bacterial strain but cannot infect a target host bacterial strain. A host bacterial strain is any bacterial strain that the selected phage can infect. A target host bacterial strain is any bacterial strain that the selected phage cannot infect. In some embodiments, the selected phage is mutated to be able to infect the target host bacterial strain.

[0040] In other embodiments, the method of generating a mutant bacteriophage having a reduced host range includes identifying and selecting a bacteriophage that is specific for a target bacterium. In some embodiments, the target bacterium is a potentially harmful bacterium. In further embodiments, the selected bacteriophage can infect a host bacterial strain, but can also infect a non-target host bacterial strain. A host bacterial strain is any bacterial strain that the selected phage can infect. A non-target host bacterial strain is any bacterial strain that is disadvantageous for a desired use and that the selected phage can infect. In some embodiments, the selected phage is mutated so that it cannot infect non-target host bacterial strains.

[0041] The genus Listeria includes seven species (monocytogenes, ivanovii, seeligeri, innocua, welshimeri, martii, and grayi). Only two species are known to be pathogenic: L. monocytogenes and L. ivanovii (formerly, L. monocytogenes serotype 5). However, L. ivanovii mainly infects animals and rarely causes disease in humans. Serotyping differentiates Listeria isolates at levels below the species level. L. monocytogenes strains are serotyped according to variations in the somatic antigen (O) and flagellar antigen (H). The surface of the bacterium is covered with lipopolysaccharide (LPS), and the outermost portion of the LPS is known as the O antigen. Somatic antigens are found in both Gram-positive and Gram-negative bacteria. Flagella are tail-like structures that assist in the movement of bacteria. The long, thread-like portion of the flagellum is called the H antigen.

[0042] L. monocytogenes has a high degree of genetic diversity and a clonal population structure. Twelve serotypes of L. monocytogenes (1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7) have been recognized. Two major phylogenetic divisions within L. monocytogenes have been described. The first division consists of serotypes 1 / 2b, 3b, 4b, 4d, and 4e, and the second division consists of serotypes 1 / 2a, 1 / 2c, 3a, and 3c. Additionally, the less common serotypes 4a and 4c have also been described and constitute a third division. Three serotypes (1 / 2a, 1 / 2b, and 4b) are responsible for the majority of clinical cases. More than 50% of L. monocytogenes isolated from food and the environment are serotype 1 / 2 (particularly 1 / 2a and 1 / 2b). However, the serotype 4b lineage is the most predominant cause of foodborne outbreaks of human listeriosis.

[0043] Some bacteriophages have a narrow host range and can infect some serotypes of L. monocytogenes but not others. For example, some bacteriophages are specific to Listeria monocytogenes 19115 (serotype 4b) and cannot detect other serotypes of Listeria monocytogenes (e.g., Listeria monocytogenes 51782 (serotype 3a)). In some embodiments, the selected bacteriophage is specific to one serotype of L. monocytogenes and cannot infect another serotype of L. monocytogenes. In some cases, the selected bacteriophage is specific to Listeria monocytogenes 19115 (serotype 4b) and cannot infect Listeria monocytogenes 51782 (serotype 3a).

[0044] Some bacteriophages are known to have a broad host range and can infect bacterial species across multiple genera. For example, bacteriophage Mu can infect species of Escherichia coli, Citrobacter freundii, Shigella sonnei, and Enterobacter. Similarly, some bacteriophages are known to have a broad host range within a particular bacterial genus. For example, a number of Listeria - specific bacteriophages (including A511 and P100) can infect several serotypes of Listeria monocytogenes and other Listeria spp.

[0045] For certain applications, it may be advantageous to use a Listeria - specific bacteriophage with a broad host range. For other applications, it may be advantageous to use a Listeria - specific phage that can infect multiple species of Listeria. Thus, in some embodiments, the bacteriophage is mutated to be able to infect at least 2, 3, 4, 5, 6, and 7 species of Listeria. In other embodiments, the bacteriophage is mutated to be able to infect at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 serotypes of L. monocytogenes.

[0046] In some embodiments, the method includes the step of preparing a series of first co-culture mixtures at various ratios, including a host bacterial strain and a target host bacterial strain. In further embodiments, the host bacterial strain and the target host bacterial strain are cultured separately to obtain respective stock cultures. In certain embodiments, the host bacterial strain relates to Listeria monocytogenes 19115 (serotype 4b). In further embodiments, the target bacterial strain is Listeria monocytogenes 51782 (serotype 3a). In other embodiments, the target bacterial strain is Listeria monocytogenes serotype 1 / 2a. In some embodiments, the target bacterial strain is Listeria monocytogenes serotype 1 / 2b. In still further embodiments, the target bacterial strain includes one or more of the following Listeria monocytogenes serotypes: 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7.

[0047] After separately culturing the volumes of the host and target host bacterial strains that have never been in the presence of the selected phage of interest, the bacterial cultures are combined with each other at various ratios to create a series of co-cultures. In one case, the series of first co-cultures comprises the ratios of host bacterial strain:target host bacterial strain of 1:0, 9:1, 1:1, 1:9, and 0:1. In other cases, the plurality of first co-cultures comprises any suitable ratio between 1:0 and 0:1.

[0048] In a further embodiment, the method includes adding a phage strain to each of the first co-culture mixtures. The co-culture to phage ratio varies depending on the minimum multiplicity of infection (MOI) of phage / host to induce productive culture infection. In some cases, the MOI is at least 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0. In certain embodiments, the MOI is 1.0. In some embodiments, the phage strain is specific for Listeria spp. In other embodiments, the phage strain is specific for Listeria monocytogenes. In yet other embodiments, the phage is specific for one serotype of Listeria monocytogenes. For example, the selected phage can be specific for Listeria monocytogenes 19115 (serotype 4b). In certain cases, the phage is selected from A511, P100, LMTA-94, LMA4, LMA8, P70, LP-ES1, LP-ES3A.

[0049] In certain cases, the method includes incubating the first co-culture mixture and the phage strain under bacterial culture conditions. A growth medium suitable for the host and / or target host organism is made with 1.2 - 1.5% agar, poured into a sterile Petri dish, and allowed to solidify to create a "base layer". 300 μL of a bacterial culture in mid-logarithmic growth phase is mixed with 100 μL of phage and incubated at room temperature for 15 - 20 minutes with shaking. The infected culture is then mixed with 4 mL of melted (50 - 55 °C) medium made with 0.7 - 1.0% agar, suitable for bacterial growth, and poured onto the base agar layer in the Petri dish. The "top layer" is cooled to room temperature, solidified, and then incubated for 12 - 15 hours under optimal growth conditions for culturing the bacterial strain.

[0050] In some embodiments, the method includes collecting phage lysates from each of the plurality of first co-cultures. In certain embodiments, after incubation, the phage lysates are separated from each of the first co-culture mixing ratios. In some embodiments, each co-culture mixture is centrifuged and the supernatant is filtered to obtain the phage lysate. For example, each co-culture can be centrifuged at 3220×g for 20 minutes at room temperature. In some embodiments, the filter is less than 0.75 μm, 0.65 μm, 0.55 μm, 0.45 μm, 0.35 μm, 0.25 μm, or 0.15 μm. In certain embodiments, the filter is 0.45 μm. Optionally, the phage lysates from each of the plurality of first co-cultures are pooled to obtain a single volume of phage lysate.

[0051] In some embodiments, the method includes assaying the phage lysate to determine whether the bacterial host range has been expanded. Optionally, the pooled phage lysate is plated for single plaques on the identified host strain and on the selected target host strain. For example, a double-layer assay can be used to check for bacteriophage specificity against the host and target host organisms. Cultures of the host and target host organisms in the mid-logarithmic growth phase of untreated cultures can be grown and used in the double-layer assay. When plaque-forming units (PFUs) are formed, the infectivity of the organisms used in the assay is detected. This step ensures that the infectivity against the original host is lost and that mutant phages that have undergone the desired host expansion event are exposed.

[0052] In some embodiments, the pooled phage lysates are then passaged, isolated, and assayed again into the plurality of first co-cultures as described in detail herein. The passage is repeated until mutant phages that infect the target host cell strain are detected. In some embodiments, the phage is passaged at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0053] In some embodiments, the method includes isolating mutant phages having an expanded host range. After confirmation of activity against both the host and the target host organism, the method includes isolating the mutant phages. In some embodiments, the isolated mutant phage population is then expanded to generate a stock of mutant phages.

[0054] The disclosed method presents a procedure by which the phage infects a given bacterial host that would not be infected by other methods.

[0055] Accordingly, the method of the present invention utilizes the high specificity of the binding factor associated with the infectious agent that recognizes and binds to a particular target microorganism.

[0056] In other applications, it may be advantageous to use bacteriophages with a narrower host range. For example, it may be advantageous to use Listeria-specific phages that are capable of infecting only clinically relevant serotypes (1 / 2a, 1 / 2b, and 4b) and not non-pathogenic Listeria serotypes. Accordingly, in some embodiments, the bacteriophage is mutated to be capable of infecting pathogenic serotypes of L. monocytogenes and not non-pathogenic serotypes of L. monocytogenes.

[0057] In some embodiments, the host range of a bacteriophage can be reduced or altered by modifying the proteins responsible for host specificity. Bacterial host specificity can be defined by the cell wall binding domain (CBD) or other proteins present in the bacteriophage. The CBD can be important for specific cell wall recognition and can be present in endolysin, or spanin, or tail fiber, or tail spike protein. For example, the tail spike protein binds to the cell surface of the bacterial host and mediates bacterial host recognition. Thus, mutations in the tail spike protein can be used to alter the ability of the mutated bacteriophage to recognize a particular bacterial strain.

[0058] Thus, in some embodiments, the gene encoding the tail fiber or tail spike protein can be mutated to change or reduce the specificity of a particular bacteriophage. For example, a bacteriophage can have two tail spike proteins (the first providing specificity for the first L. monocytogenes serotype (e.g., 4b) and the second providing specificity for the second L. monocytogenes serotype (e.g., 7)). Thus, in some embodiments, one of the tail spike proteins can be mutated to change the specificity of the bacteriophage. Any molecular method generally known in the art for mutating a bacteriophage can be used to change the tail fiber / tail spike protein, and as a result, the specificity of the bacteriophage is narrowed. For example, the bacteriophage can be subjected to a random mutagenesis protocol.

[0059] In some cases, the bacteriophage genome can be fragmented into overlapping DNA fragments. Fragments encoding the desired tail spike protein (i.e., the tail spike protein specific for a non-desired serotype) can be amplified using error-prone PCR to introduce targeted mutagenesis within the gene. Fragments of genomic DNA can then be reassembled to generate a library of phage genomes. The pooled phage genomes can then be transfected into Listeria, thereby generating mutant progeny phage. The progeny phage can then be assayed for plaque formation on the desired Listeria strain. Surviving phage mutants with the desired specificity can then be isolated.

[0060] Accordingly, in some embodiments, the disclosure relates to a method for generating mutant bacteriophages with a reduced host range. In some embodiments, the method comprises: (i) mutating a gene encoding a tail spike protein of a bacteriophage; (ii) generating a progeny phage lysate from the mutated bacteriophage; (iii) assaying the phage lysate to determine whether the bacterial host range is reduced; and (iv) isolating a mutant phage having a reduced host range.

[0061] Indicator mutant bacteriophage As described in more detail herein, the compositions and methods of the present disclosure can include infectious agents for use in the detection of pathogenic microorganisms. In certain embodiments, the present disclosure includes recombinant indicator mutant bacteriophages, wherein the bacteriophage genome is genetically modified to include an indicator or reporter gene. In some embodiments, the present disclosure can include compositions comprising recombinant mutant bacteriophages in which an indicator gene has been incorporated into the genome of a previously mutated bacteriophage. In some embodiments, the mutant bacteriophages are generated according to methods described in detail herein. In some embodiments, the mutant bacteriophages have an expanded and / or reduced host range.

[0062] Recombinant indicator mutant bacteriophages can include a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during bacteriophage replication after infection of host bacteria results in a soluble indicator protein product. In certain embodiments, the indicator gene can be inserted into the late gene region of the mutant bacteriophage. Since late genes encode structural proteins, they are generally expressed at higher levels than other phage genes. The late gene region can be a class III gene region and can include the gene for the major capsid protein.

[0063] Some embodiments include the step of designing (and optionally preparing) a sequence for homologous recombination downstream of the major capsid protein gene. Other embodiments include the step of 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 following an untranslated region. The untranslated region can include a phage late gene promoter and a ribosome entry site.

[0064] In some embodiments, the selected wild-type bacteriophage is derived from a phage of the order Caudovirales. Caudovirales is an order of tailed bacteriophages that have a double-stranded DNA (dsDNA) genome. Each virion of the order Caudovirales has an icosahedral head containing the viral genome and a flexible tail. The order Caudovirales includes five bacteriophage families: Myoviridae (long contractile tails), Siphoviridae (long non-contractile tails), Podoviridae (short non-contractile tails), Ackermannviridae, and Herelleviridae. The term myovirus can be used to describe any bacteriophage having an icosahedral head and a long contractile tail and encompassing bacteriophages in both the families Myoviridae and Herelleviridae. In some embodiments, the selected wild-type bacteriophage is a member of the family Myoviridae (e.g., Listeria phage B054, Listeria phage LipZ5, Listeria phage PSU-VKH-LP041, and Listeria phage WIL-2). In other embodiments, the selected wild-type bacteriophage is a member of the family Herelleviridae. The genus Pecentumvirus under the family Herelleviridae includes bacteriophages such as Listeria phage LMSP-25, Listeria phage LMTA-148, Listeria phage LMTA-34, Listeria phage LP-048, Listeria phage LP-064, Listeria phage LP-083-2, Listeria phage LP-125, Listeria virus P100, Listeria phage List-36, Listeria phage WIL-1, Listeria phage vB_LmoM_AG20, and Listeria virus A511. LMA4 and LMA8 are also, likewise, within the genus Pecentumvirus under the family Herelleviridae.In other embodiments, the selected wild-type bacteriophage is LMA4 or LMA8. In certain cases, the selected wild-type bacteriophage is LP-ES3A, which is derived from A511 but adapted to infect serotype 3A of Listeria monocytogenes. In still other embodiments, the selected wild-type bacteriophage is a member of the family Ackermannviridae. In still other embodiments, the selected wild-type bacteriophage is a member of the family Siphoviridae, which includes Listeria phages A006, A118, A500, B025, LP-026, LP-030-2, LP-030-3, LP-037, LP-101, LP-110, LP-114, P35, P40, P70, PSA, vB_LmoS_188, and vB_Lmos_293. In other embodiments, the selected wild-type bacteriophage is LP-ES1. LP-ES1 is also, likewise, within the genus Homburgvirus under the family Siphoviridae.

[0065] In some embodiments, the indicator bacteriophage is derived from a Listeria - specific phage. The indicator bacteriophage may be from Pecentumvirus, Tequatravirus, ViI, Kuttervirus, Homburgvirus, A511, P100, P70, LMTA - 94, LMA4, LMA8, P70, LP - ES1, LP - ES3A or Listeria phage LMTA - 94, P70, T7, T7 - like, T4, T4 - like, Listeria spp. - specific bacteriophage, ViI, or a ViI - like (Kuttervirus according to GenBank / NCBI) bacteriophage and can be constructed from another bacteriophage having a genome with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In other embodiments, the selected wild - type bacteriophage is A511, P100, P70, LP - ES1, LP - ES3A, LMA4 or LMA8. In some embodiments, the indicator phage is derived from a bacteriophage that is highly specific for a particular pathogenic microorganism. The genetic modification may avoid deletion of wild - type genes, and thus the modified phage may remain more similar to wild - type infectious agents than many commercially available phages. Bacteriophages derived from the environment may be more specific for bacteria found in the environment and thus may have a distinct gene from commercially available phages.

[0066] In another aspect of the present invention, the cocktail composition comprises at least one type of recombinant bacteriophage. In some embodiments, the cocktail composition comprises at least one type of recombinant bacteriophage constructed from LMA4, LMA8, A511, P70, LP-ES1, and LP-ES3A. In other embodiments, the cocktail composition comprises at least one type of recombinant bacteriophage constructed from LMA8, LP-ES1, and LP-ES3A.

[0067] Furthermore, phage genes that are considered non-essential may have unrecognized functions. For example, genes that appear non-essential may have important functions in increasing burst size, such as sophisticated cleavage, fitting, or trimming functions in assembly. Thus, deleting a gene and inserting an indicator can be harmful. Most phages can package DNA that is several percent larger than their native genome. In this consideration, smaller indicator genes may be a more appropriate choice for modifying bacteriophages (especially bacteriophages with smaller genomes). The OpLuc and NANOLUC® proteins are only about 20 kDa (encoding about 500 - 600 bp), while FLuc is about 62 kDa (encoding about 1,700 bp). For comparison, the genome of T7 is about 40 kbp, while the T4 genome is about 170 kbp, and the genome of a Listeria-specific bacteriophage is about 157 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacterium (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be readily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, Promega's NANO-GLO® NANOLUC® in combination with the imidazopyrazinone substrate (furimazine) can provide a robust signal with low background.

[0068] In some indicator mutant phage embodiments, the indicator gene can be inserted into the untranslated region to avoid disruption of functional genes while leaving the wild-type phage gene intact, which can result in greater fitness when infecting non-laboratory strain bacteria. Further, including stop codons in all three reading frames can help increase expression by reducing read-through (also known as leaky expression). This strategy can also eliminate the possibility that a fusion protein that appears as a background signal (e.g., luciferase) that cannot be separated from the phage is produced at low levels.

[0069] The 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 Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or 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®.

[0070] Accordingly, in some embodiments, the invention includes a genetically modified bacteriophage comprising a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. Using a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like a viral capsid protein, but also not shut off like an endogenous bacterial gene or even an early viral gene.

[0071] In some embodiments, the late promoter is a Pecentumvirus, Tequatravirus, Homburgvirus, or Kuttervirus promoter, or another phage promoter found in a selected wild-type phage, i.e., a promoter similar to those found in wild-type phages without genetic modification. The late gene region can be a class III gene region, and the bacteriophage can be derived from Listeria phage LMTA-94, P70, A511, LP-ES1, LP-ES3A, LMA4, LMA8, Pecentumvirus, Tequatravirus, Homburgvirus, Kuttervirus, T7, T4, T4-like, ViI, Listeria spp.-specific bacteriophage, or another wild-type bacteriophage having a genome with at least 70%, 75%, 80%, 85%, 90%, or 95% homology to LMTA-94, LMA4, LMA8, Pecentumvirus, Tequatravirus, Homburgvirus, Kuttervirus, T7, T4, ViI, or a Listeria-specific bacteriophage. The Pecentumvirus late gene promoter consists of not only the -10 region but also the -35 region, making it different from the T4 or Tequatravirus promoter. This -35 region is different from the standard -35 region found in most bacterial promoters.

[0072] Genetic modifications to the infectious agent can include the insertion, deletion, or substitution of small fragments of nucleic acid, substantial portions of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acid comprises a non-natural sequence. The non-natural indicator gene can be inserted into the bacteriophage genome such that it is under the control of a bacteriophage promoter. Thus, in some embodiments, the non-natural indicator gene is not part of a fusion protein. That is, in some embodiments, the genetic modification can be configured such that the indicator protein product does not include the polypeptide of the wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the invention encompasses a method for detecting a target bacterium, the method including the step of incubating a test sample with such a recombinant bacteriophage.

[0073] In some embodiments, the expression of the indicator gene in the progeny bacteriophage after infection of the host bacterium results in a free soluble protein product. In some embodiments, since the non-natural indicator gene is not linked to a gene encoding a phage structural protein, no fusion protein is produced. Unlike systems that use fusions of the detection moiety to the capsid protein (i.e., fusion proteins), some embodiments of the present invention express a soluble indicator or reporter (e.g., soluble luciferase). In some embodiments, the indicator or reporter ideally does not include the bacteriophage structure. That is, the indicator or reporter is not bound to the phage structure. Thus, the gene for the indicator or reporter is not fused to 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 contrast to the several hours required for additional purification steps required for constructs that produce detectable fusion proteins, for some embodiments, allows the assay to be completed in 2 hours or less. Additionally, fusion proteins may be less active than soluble proteins due to protein folding constraints that can alter, for example, the conformation of the enzyme active site or access to the substrate. For example, when the concentration is 10 bacterial cells / mL sample, less than 2 hours may be sufficient for the assay.

[0074] Furthermore, by definition, the fusion protein limits the number of portions attached to the protein subunits in the bacteriophage. For example, using a commercially available system designed to act as a platform for the fusion protein, approximately 415 copies of the fusion portion, corresponding to approximately 415 copies of the gene 10B capsid protein, occur in each T7 bacteriophage particle. Without this constraint, the infected bacteria could be predicted to express more copies of the indicator protein product (e.g., luciferase) that can conform to the bacteriophage. Furthermore, large fusion proteins (e.g., capsid-luciferase fusions) can inhibit the assembly of the bacteriophage particles, and thus result in fewer bacteriophage progeny. Therefore, a soluble non-fusion indicator gene product may be preferred.

[0075] In some embodiments, the indicator phage encodes a reporter such as a detectable enzyme. The indicator gene product can produce light and / or be detectable by a color change. Various suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes can act as the indicator protein product. In some embodiments, firefly luciferase is the indicator protein product. In some embodiments, Oplophorus luciferase is the indicator protein product. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal can also be suitable indicator protein products.

[0076] In some embodiments, the use of soluble indicator proteins obviates the need to remove contaminating parental phage from the lysate of the infected sample cells. Using a fusion protein system, any bacteriophage used to infect the sample cells has an attached detection moiety and cannot be distinguished from the daughter bacteriophage that also contains the detection moiety. Since detection of the sample bacteria relies on detection of the newly created (de novo synthesized) detection moiety, use of the fusion construct requires additional steps to separate the old (parental) moiety from the newly created (daughter bacteriophage) moiety. This can be accomplished by washing the infected cells multiple times prior to completion of the bacteriophage life cycle, inactivating excess parental phage after infection by physical or chemical means, and / or chemically modifying the parental bacteriophage with a binding moiety (e.g., biotin) which can then be bound and separated (e.g., by streptavidin-coated sepharose beads). However, even using all of these attempts at removal, parental phage can remain when high concentrations of parental phage are used to ensure infection of a small number of sample cells, creating a background signal that can obscure detection of the signal from the progeny phage of the infected cells.

[0077] In contrast, when using the soluble indicator protein products expressed in some embodiments of the present invention, purification of the parental phage from the final lysate is not required. This is because the parental phage does not have any attached indicator protein. Thus, any indicator protein present after infection must be newly created, indicating the presence of one or more infected bacteria. To take advantage of this benefit, the production and preparation of the parental phage can include purification of the phage from any free indicator protein produced during the production of the parental 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 derivatization techniques (e.g., Amicon brand concentrators - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as part of the preparation of the recombinant phages of the present invention to separate the parental phage particles from contaminating luciferase proteins produced during the growth of the phage in the bacterial host. In this way, the parental recombinant bacteriophages of the present invention are substantially free of any luciferase produced during production in the bacteria. Removal of the remaining luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with test samples.

[0078] In some embodiments of the modified bacteriophage, the late promoter (class III promoter, e.g., those derived from Pecentumvirus, Homburgvirus, T7, T4, ViI, or LMA4 / 8) has a high affinity for the RNA polymerase of the same bacteriophage that transcribes the genes of the structural proteins that assemble into the bacteriophage particles. These proteins are the most abundant proteins made by the phage, as each bacteriophage particle contains dozens or hundreds of copies of these molecules. The use of a viral late promoter can optimally ensure a high level of expression of the luciferase indicator protein. The use of a late viral promoter that is derived from, specific to, or active under the original wild-type bacteriophage from which the indicator phage is derived (e.g., Pecentumvirus, T4, T7-based, ViI-based, or LMA-based systems, with Pecentumvirus, Homburgvirus, T4, T7, ViI, or LMA4 / 8 late promoters) can further ensure optimal expression of the indicator protein. The use of standard bacterial (non-viral / non-bacteriophage) promoters can, in some cases, be detrimental to expression because these promoters are often downregulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production). Thus, in some embodiments, the phage is preferably engineered to encode a soluble (free) indicator protein and express it at high levels using an arrangement within the genome that does not limit expression to the number of subunits of the phage structural components.

[0079] The compositions of the present disclosure can include one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the composition can include a cocktail of different indicator phages that can encode and express the same or different indicator proteins. In some embodiments, the cocktail of bacteriophages includes at least two different types of recombinant bacteriophages.

[0080] Method of using mutant bacteriophages to detect Listeria spp. As noted herein, in certain embodiments, the present disclosure relates to methods of using infectious particles to detect microorganisms.

[0081] In another aspect, the present disclosure is a method for detecting a target bacterium in a sample, the method comprising incubating the sample with a bacteriophage that infects a host and the target host bacterium, wherein the bacteriophage includes an indicator gene, such that expression of the indicator gene during bacteriophage replication after infection of the target bacterium results in the production of a soluble indicator protein product; and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of the target bacterium in the sample.

[0082] In some embodiments, the selected bacteriophage is mutated as described in detail herein to expand and / or reduce its host range. In some embodiments, the selected bacteriophage can be specific to one serotype of Listeria monocytogenes. For example, some bacteriophages are specific to Listeria monocytogenes 19115 (serotype 4b), but cannot detect other serotypes of Listeria monocytogenes (e.g., Listeria monocytogenes 51782 (serotype 3a)). In some embodiments, the selected bacteriophage is specific to one serotype of Listeria monocytogenes but cannot infect another serotype of Listeria monocytogenes. In some cases, the selected bacteriophage is specific to Listeria monocytogenes 19115 (serotype 4b) but cannot infect Listeria monocytogenes 51782 (serotype 3a).

[0083] In some embodiments, the mutated bacteriophage infects the host and the target host bacteria of interest. In certain embodiments, the host bacterial strain relates to Listeria monocytogenes 19115 (serotype 4b). In further embodiments, the target bacterial strain is Listeria monocytogenes 51782 (serotype 3a).

[0084] In some embodiments, the mutant bacteriophage can be engineered to express soluble luciferase during replication of the phage. Expression of the luciferase is driven by a viral capsid promoter (e.g., bacteriophage T7 or T4 late promoter), resulting in high expression. Since the parent phages are prepared such that they do not contain luciferase, the luciferase detected in the assay must be derived from the replication of progeny phages during infection of bacterial cells. Thus, it is generally not necessary to separate the parent phages from the progeny phages.

[0085] In some embodiments, enrichment of bacteria in the sample is not required prior to testing. In some embodiments, the sample can be enriched prior to testing by incubation under conditions that promote growth. In such embodiments, the enrichment period can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or longer, depending on the type and size of the sample.

[0086] In one embodiment, the present invention is a method for detecting a target bacterium in a sample, the method comprising incubating the sample with 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, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in the production of 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 bacterium in the sample. In some embodiments, the amount of indicator protein detected corresponds to the amount of target bacterium present in the sample.

[0087] As described in more detail herein, the method can utilize the concentration range in which the parental indicator bacteriophage infects bacteria present in the sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly find, bind to, and infect target bacteria that are present in very low numbers in the sample, such as single cells. In some embodiments, the phage concentration may be sufficient to find, bind to, and infect the target bacteria in less than 1 hour. In other embodiments, these events may occur in less than 2 hours, less than 3 hours, or less than 4 hours after addition of the indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubating step is higher than 1×10 5 PFU / mL, higher than 1×10 6 PFU / mL, or higher than 1×10 7 PFU / mL.

[0088] In certain embodiments, the recombinant infectious agent can be purified so as not to contain any residual indicator protein that may be generated during the production of the infectious agent stock. Thus, in certain embodiments, the recombinant bacteriophage can be purified using cesium chloride isopycnic density gradient centrifugation prior to incubation with the sample. When the infectious agent is a bacteriophage, this purification can have the additional advantage of removing bacteriophages that do not have DNA (i.e., empty phages or "ghosts").

[0089] In some embodiments of the methods of the present invention, the microorganism can be detected without any isolation or purification of the microorganism from the sample. For example, in certain embodiments, a sample containing one or several target microorganisms can be applied directly to an assay vessel (e.g., a spin column, a microtiter well, or a filter), and the assay is performed within that assay vessel. Various embodiments of such assays are disclosed herein.

[0090] Test sample aliquots can be dispensed directly into the wells of a multiwell plate, an indicator phage can be added, and after a sufficient period for infection, a lysis buffer can be added, as well as a substrate for the indicator protein (e.g., a luciferase substrate for a luciferase indicator), and assayed for detection of the indicator signal. Some embodiments of the above methods can be performed on a filter plate. Some embodiments of the above methods can be performed with or without concentration of the sample prior to infection with the indicator phage.

[0091] For example, in many embodiments, a multi-well plate is used to perform the above assay. The choice of plate (or any other container in which the detection step can be performed) can affect the detection step. For example, some plates may include a colored or white background that can affect the detection of light emission. Generally, white plates have higher sensitivity but also produce a higher background signal. Other colors of plates may produce a lower background signal but may have slightly lower sensitivity. Additionally, one reason for the background signal is light leakage from one well to another adjacent well. There are some plates with white wells, while the rest of the plates are black. This allows for a high signal inside the wells but prevents light leakage from well to well and thus can reduce the background. Therefore, the choice of plate or other assay container can affect the sensitivity and background signal for the assay.

[0092] The method of the present invention may include various other steps for increasing sensitivity. For example, as discussed in more detail herein, the method may include a step of washing the captured and infected bacteria after the addition of the bacteriophage but before incubation to remove excess parent bacteriophage and / or luciferase or other reporter proteins that contaminate the bacteriophage preparation.

[0093] In some embodiments, the detection of the microorganisms for the above purposes can be accomplished without the need to culture the sample as a method of increasing the population of the microorganisms. For example, in certain embodiments, the total time required for detection is less than 28.0 hours, 27.0 hours, 26.0 hours, 25.0 hours, 24.0 hours, 23.0 hours, 22.0 hours, 21.0 hours, 20.0 hours, 19.0 hours, 18.0 hours, 17.0 hours, 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, 1.0 hour, 45 minutes or 30 minutes. Minimizing the time to results is critical in food and environmental testing for pathogens.

[0094] In contrast to assays known in the art, the method of the present invention can detect individual microorganisms. Thus, in certain embodiments, the method can detect ≦10 cells of the microorganisms present in the sample (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 microorganisms). For example, in certain embodiments, the recombinant bacteriophage is highly specific for Listeria spp. In one embodiment, the recombinant bacteriophage can distinguish Listeria spp. in the presence of other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a specific type in the sample. In certain embodiments, the recombinant bacteriophage detects specific bacteria in the sample in amounts as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100.

[0095] Accordingly, aspects of the present invention provide a method for the detection of microorganisms in a test sample via an indicator protein. In some embodiments, where the microorganism of interest is a bacterium, the indicator protein may be associated with an infectious agent (e.g., an indicator bacteriophage). The indicator protein may react with a substrate to emit a detectable signal or may emit an endogenous signal (e.g., a fluorescent protein). In some embodiments, the detection sensitivity may be able to reveal the presence of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the microorganism of interest in the test sample. In some embodiments, as few as 1 cell of the microorganism of interest may produce a detectable signal. In some embodiments, the bacteriophage is a P100 virus, a T4-like or ViI-like bacteriophage. In some embodiments, the recombinant bacteriophage is derived from a Listeria-specific bacteriophage. In certain embodiments, the recombinant Listeria-specific bacteriophage is highly specific for Listeria spp.

[0096] In some embodiments, the indicator protein 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 moieties are known in the art and many are commercially available. In some embodiments, the indicator phage comprises an enzyme, which functions as the indicator protein. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator may be detectable by emitting light and / or by a change in color in an added substrate. Various suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes may function as the indicator protein. In some embodiments, firefly luciferase is the indicator protein. In some embodiments, Oplophorus luciferase is the indicator protein. In some embodiments, NANOLUC® is the indicator protein. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator proteins.

[0097] Thus, in some embodiments, the recombinant bacteriophage of the above method, system or kit is prepared from a wild-type Listeria-specific bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an endogenous signal, such as a fluorescent protein (e.g., green fluorescent protein, etc.). The indicator can emit light and / or be detectable by a change in color. In some embodiments, the indicator gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to generate a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is one of Oplophorus luciferase, firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase or engineered luciferase, such as NANOLUC®, Rluc8.6-535 or orange nano-lantern.

[0098] The step of detecting the indicator may include the step of detecting the emission of light. In some embodiments, a luminometer can be used to detect the reaction of an indicator (e.g., luciferase) with a substrate. Detection of RLU can be achieved with a luminometer, or other machines or devices can also be used. For example, a spectrophotometer, a CCD camera, or a CMOS camera can detect color changes and other light emissions. Absolute RLU is important for detection, but a high signal-to-background ratio (e.g., >2.0, >2.5 or >3.0) is also required to ensure detection of single cells or a small number of cells.

[0099] In some embodiments, the indicator phage is genetically engineered to contain a gene for an enzyme (e.g., luciferase) that is produced only upon infection of bacteria that the phage specifically recognizes and infects. In some embodiments, the indicator protein is expressed late in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits its copy number.

[0100] Accordingly, in some embodiments utilizing an indicator phage, the invention encompasses a method for detecting a target microorganism, the method comprising the steps of: capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phages; allowing time for infection and replication to produce progeny phages and express a soluble indicator moiety; and detecting the progeny phages, preferably the indicator, wherein detection of the indicator comprises an indication that the bacterium is present in the sample.

[0101] For example, in some embodiments, the test sample bacteria can be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a spin filter or plate filter with a 0.45 μm pore size). In one embodiment, the infectious agent (e.g., an indicator phage) is added in a minimal volume to the sample captured directly on the filter. In one embodiment, the microorganisms captured on the filter or plate surface are then washed one or more times to remove any unbound excess infectious agent. In one embodiment, a medium (e.g., Luria-Bertani broth (also referred to herein as LB), buffered peptone water (also referred to herein as BPW)), or Tryptic Soy broth or Tryptone Soy broth (also referred to herein as TSB) is added for further incubation time to allow replication of bacterial cells and phages and high-level expression of the gene encoding the indicator protein. However, a surprising aspect of some embodiments of the test assay is that only the incubation step with the indicator phage needs to be long enough for a single phage life cycle. The amplification power of using bacteriophages was previously thought to require more time for the phages to replicate over several cycles. A single replication cycle of the indicator phage can be sufficient to facilitate sensitive and rapid detection according to some embodiments of the present invention.

[0102] In some embodiments, an aliquot of a test sample containing bacteria can be applied to a spin column, and after infection with a recombinant bacteriophage and optional washing to remove any excess bacteriophage, the amount of soluble indicator detected is proportional to the amount of bacteriophage produced by the infected bacteria.

[0103] The soluble indicator protein (e.g., luciferase) released into the surrounding liquid upon lysis of the bacterium can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter, and the filtrate is collected in a new container for assay (e.g., in a luminometer), and then a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

[0104] In various embodiments, the purified parental indicator phage does not contain the detectable indicator itself. This is because the parental phage can be purified before it is used to incubate with the test sample. Expression of late (class III) genes occurs late in the viral life cycle. In some embodiments of the invention, the parental phage can be purified to remove any existing indicator protein (e.g., luciferase). In some embodiments, expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. Thus, in many embodiments, it is not essential to separate the parental phage from the progeny phage prior to the detection step. In one embodiment, the microorganism is a bacterium and the indicator phage is a bacteriophage. In one embodiment, the indicator moiety is soluble luciferase, which is released upon lysis of the host microorganism.

[0105] Thus, in an alternative embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with a portion of the sample that remains on the filter or remains bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a normal 96-well plate), and the substrate reaction can be detected by placing the plate directly into the luminometer.

[0106] For example, in one embodiment, the present invention may include a method for detecting Listeria spp., the method comprising infecting cells captured on a 96-well filter plate with a plurality of parental indicator phages capable of expressing luciferase upon infection; washing away excess phages; adding LB broth and allowing time for the phages to replicate and lyse the specific Listeria spp. target (e.g., 30 - 120 minutes); and adding a luciferase substrate and detecting the indicator luciferase by measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity includes indicating the presence of the Listeria spp. in the sample.

[0107] In another embodiment, the present invention may include a method for detecting Listeria spp., the method comprising infecting cells in a liquid solution or suspension in a 96-well plate with a plurality of parental indicator phages capable of expressing luciferase upon infection; allowing time for the phages to replicate and lyse the specific Listeria spp. target (e.g., 30 - 120 minutes); and adding a luciferase substrate and detecting the indicator luciferase by measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity includes indicating the presence of the Listeria spp. in the sample. In such an embodiment, the step of capturing is not essential. In some embodiments, the liquid solution or suspension can be a consumable test sample (e.g., vegetable wash). In some embodiments, the liquid solution or suspension can be a vegetable wash fortified with concentrated LB broth, Tryptic / Tryptone Soy broth, buffered peptone water, or nutrient broth. In some embodiments, the liquid solution or suspension can be bacteria diluted in LB broth.

[0108] 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. Presumably, luciferase can exit the cells and / or the luciferase substrate can enter the cells without complete cell lysis. Thus, for embodiments utilizing a spin filter system, lysis is required if only the luciferase released into the lysate (and the luciferase is not further present within intact bacteria) is analyzed in the luminometer. However, for embodiments utilizing a filter plate or 96-well plate with a sample in 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.

[0109] In some embodiments, the reaction of the indicator protein product (e.g., luciferase) with 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, luminometer readings can be taken initially and at 10 or 15 minute intervals until the reaction is complete.

[0110] Surprisingly, the high concentration of phage utilized to infect the test sample achieved successful detection of a very small number of target microorganisms within a very short time frame. Incubating the phage with the test sample in some embodiments requires only a time sufficient for a single phage life cycle. In some embodiments, the concentration of bacteriophage for this incubating step is 7×10 6 , 8×10 6 , 9×10 6 , 1.0×10 7 , 1.1×10 7, 1.2×10 7 , 1.3×10 7 , 1.4×10 7 , 1.5×10 7 , 1.6×10 7 , 1.7×10 7 , 1.8×10 7 , 1.9×10 7 , 2.0×10 7 , 3.0×10 7 , 4.0×10 7 , 5.0×10 7 , 6.0×10 7 , 7.0×10 7 , 8.0×10 7 , 9.0×10 7 , or higher than 1.0×10 8 PFU / mL.

[0111] Such success of phages at such high concentrations is surprising because many of the phages were previously associated with "non-infectious lysis", which killed the target cells, thereby preventing the generation of useful signals from earlier phage assays. The clean-up of the prepared phage stocks described herein is thought to help mitigate this problem (e.g., purification by cesium chloride isopycnic density gradient ultracentrifugation). Because in addition to removing any contaminating luciferase associated with the phages, this purification can also remove ghost particles (particles that have lost their DNA). The ghost particles can lyse bacterial cells via "non-infectious lysis", killing the cells early and thereby preventing the generation of an indicator signal. Electron microscopy clearly shows that crude phage lysates (i.e., before cesium chloride purification) can have more than 50% ghosts. These ghost particles can contribute to the early death of the microorganisms through the action of many phage particles that puncture the cell membrane. Thus, the ghost particles may have been a factor in the previous problem where high PFU concentrations were reported to be detrimental. Furthermore, very clean phage preparations allow the assay to be performed without a washing step, which allows the assay to be performed without an initial concentration step. Some embodiments include an initial concentration step, and in some embodiments, this concentration step allows for a shorter enrichment incubation time.

[0112] Some embodiments of the test method may further include a confirmation assay. Various assays are known in the art for confirming initial results at a later time point. For example, a sample can be cultured (e.g., the CHROMAGAR® / DYNABEADS® assay described in the examples), PCR can be utilized to confirm the presence of microbial DNA, or other confirmation assays can be used to confirm the initial results.

[0113] In certain embodiments, the methods of the invention may combine the use of a binding agent (e.g., an antibody) to purify and / or concentrate the microorganism of interest (e.g., Listeria spp.) from the sample, in addition to the detection of the infectious agent. For example, in certain embodiments, the invention includes a method for detecting a microorganism of interest in a sample, the method comprising: capturing the microorganism (e.g., Listeria spp.) from the sample onto a support, using a capture antibody specific for the microorganism of interest (e.g., Listeria spp.); incubating the sample with a recombinant bacteriophage that infects Listeria spp., wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product; and detecting the indicator protein product, wherein a positive detection of the indicator protein product indicates the presence of Listeria spp. in the sample.

[0114] In some embodiments, the synthetic phage is designed to optimize desirable traits for use in pathogen detection assays. In some embodiments, genetic modification bioinformatics and previous analyses are used to optimize desirable traits. For example, in some embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to specific species of bacteria. In other embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to an entire genus of bacteria, or a specific group of species within a genus. In this way, the phage can be optimized to detect a broader or narrower group of pathogens. In some embodiments, the synthetic phage can be designed to improve the expression of the reporter gene. Additionally and / or alternatively, in some cases, the synthetic phage can be designed to increase the burst size of the phage to improve detection.

[0115] In some embodiments, the stability of the phage can be optimized to improve the storage period. For example, enzybiotic solubility can be increased to increase subsequent phage stability. Additionally and / or alternatively, phage thermal stability can be optimized. Thermostable phages better preserve functional activity during storage, thereby increasing shelf life. Thus, in some embodiments, the thermal stability and / or pH tolerance can be optimized.

[0116] 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).

Example

[0117] Example 1. Listeria phage evolution for recognizing serotype 3A of Listeria monocytogenes Passage #1: In one example of phage evolution of a mixture of 19115 (serotype 4b) / 51782 (serotype 3a), Listeria monocytogenes was prepared in 2 mL Brain Heart Infusion (BHI) with 1 mM CaCl2 (total of 5 tubes - 1 set for each phage) at the following ratios (% host / target: 100 / 0, 90 / 10, 50 / 50, 10 / 90, and 0 / 100).

[0118] The phage was added individually to each tube at an MOI of 1.0. The tubes were incubated overnight at 30 °C with shaking. The tubes were centrifuged and the supernatant (phage lysate) was filtered through a 0.45 μm filter.

[0119] All phage lysates (from all host / target ratio tubes) were pooled and plated for 1 plaque against the host and target strains. The plaque lysate was prepared from the plaque formed on the target strain by picking up the plaque with a pipette tip and adding it to Luria Broth (LB) MOPS buffer. The plaque lysate was plated on the host and target strains for a single plaque. These steps were repeated at least a total of 4 times to isolate mutant phages.

[0120] Passages #2 - #3: The phage lysate (250 μL) from step 4 above was added to 4 mL of BHI with 1 mM CaCl2. The phage lysate was incubated at 30 °C for 10 hours with shaking (about 160 rpm). The tubes were centrifuged and the supernatant (phage lysate) was filtered through a 0.45 μm filter. The phage lysate was plated on the host and target strains for a single plaque.

[0121] The plaque lysate was prepared from plaques formed on the target strain by picking up the plaques with a pipette tip and adding them to Luria Broth (LB) MOPS buffer. The plaque lysate was plated on the host and target strains for a single plaque. These steps were repeated at least a total of 4 times to isolate the mutant phage.

[0122] If no plaques were observed on the target strain in the first two passages, all steps were repeated. The present invention provides, for example, the following items. (Item 1) A method for generating a mutant bacteriophage having an expanded host range, the method comprising: (i) preparing a series of first co-culture mixtures in various ratios comprising a host bacterial strain and a target host bacterial strain; (ii) adding a phage strain to each of the first co-culture mixtures; (iii) incubating the first co-culture mixtures and the phage strain under bacterial culture conditions; (iv) collecting phage lysates from each of the plurality of first co-cultures; (v) pooling the phage lysates from each of the plurality of first co-cultures; (vi) assaying the phage lysate to determine whether the bacteriophage host range has been expanded; and (vii) isolating a mutant phage having an expanded host range, a method comprising. (Item 2) The method according to item 1, wherein the selected phage strain infects the host bacterial strain and does not infect the target host bacterial strain. (Item 3) The method according to item 1, wherein the selected phage strain is selected from A511, P100, LMA4, and LMA8. (Item 4) The method according to item 1, wherein the host bacterial strain is Listeria monocytogenes 19115. (Item 5) The method according to item 1, wherein the target host bacterial strain is Listeria monocytogenes 51782. (Item 6) The method according to item 1, wherein the series of plurality of first co-cultures comprises ratios of the host bacterial strain: the target host bacterial strain of 1:0, 9:1, 1:1, 1:9, and 0:1. (Item 7) The method according to item 1, wherein the steps are repeated until the assayed phage shows evidence of an expanded host range. (Item 8) The method according to item 1, further comprising the step of passaging the obtained phage cultured at each co-culture ratio. (Item 9) A recombinant mutant bacteriophage having an expanded host range, comprising an indicator gene inserted into the late gene region of the bacteriophage genome, wherein the recombinant bacteriophage is mutated to infect a host bacterium and a target host bacterium, the recombinant mutant bacteriophage. (Item 10) The recombinant bacteriophage is the recombinant mutant bacteriophage according to item 9, derived from wild-type A511, P100, LMA4 or LMA8 bacteriophage. (Item 11) The indicator gene is the recombinant mutant bacteriophage according to item 9, which is codon-optimized and encodes a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. (Item 12) The recombinant mutant bacteriophage according to item 9, further comprising an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region comprises a bacteriophage late gene promoter and a ribosome entry site. (Item 13) A cocktail composition comprising at least two different types of recombinant mutant bacteriophages, wherein at least one of the recombinant bacteriophages comprises the indicator gene according to item 9.

Claims

【Claim 1】 The invention described in the specification.