Indicator bacteriophage for selecting and monitoring for efficacy of therapeutics and methods for using the same

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 661,739, filed April 24, 2018. The disclosures of U.S. Application Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, and 15 / 409,258, and U.S. Provisional Application No. 62 / 661,739, are incorporated herein by reference in their entireties.

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

[0003] background There is strong interest in improving the speed and sensitivity for the detection of bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples. Microbial pathogens can cause substantial morbidity in humans and livestock animals, as well as enormous economic losses. Microbial detection is also a high priority for the Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) in light of outbreaks of life-threatening or fatal illnesses caused by the ingestion of food contaminated with certain microorganisms (e.g., Staphylococcus spp.).

[0004] Traditional microbiological tests for detecting bacteria rely on nonselective and selective enrichment cultures, followed by plating on selective media and further testing to confirm suspect colonies. Such procedures can require several days. Various rapid methods have been investigated and introduced into practice to reduce the time requirements. However, these methods have drawbacks. For example, direct immunoassays or techniques involving gene probes generally require an overnight enrichment step to achieve adequate sensitivity. Polymerase chain reaction (PCR) tests also involve an amplification step and are therefore capable of both very high sensitivity and selectivity; however, economically, the sample size that can be subjected to PCR testing is limited. With dilute bacterial suspensions, most small subsamples are cell-free and therefore still require purification and / or lengthy enrichment steps.

[0005] The time required for traditional biological enrichment is dictated by the growth rate of the target bacterial population in the sample, by the effect of the sample matrix, and by the sensitivity required. In practice, most sensitive methods use overnight incubation, taking approximately 24 hours overall. Due to the time required for culture, these methods can take up to three days, depending on the organism to be identified and the source of the sample. This lag time is generally determined by the amount of time spent in contaminated food, water (or other product). This is inappropriate because the pathogens may have already made their way into livestock or humans. Furthermore, the rise of antibiotic-resistant bacteria and biodefense concerns has made rapid identification of bacterial pathogens in water, food, and clinical samples a critical priority worldwide.

[0006] Thus, there is a need for faster, simpler, and more sensitive detection and identification of microorganisms (eg, bacteria and other potentially pathogenic microorganisms). Summary of the Invention [Means for solving the problem]

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

[0008] In some aspects, the present invention includes a recombinant bacteriophage comprising an indicator gene inserted into the late gene region of the bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Staphylococcus-specific bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Staphylococcus aureus-specific bacteriophage genome. In further embodiments, the Staphylococcus aureus is methicillin-resistant S. aureus (MRSA). For example, in certain embodiments, the recombinant bacteriophage is a genetically modified bacteriophage genome. In some embodiments, the bacteriophage used to prepare the recombinant bacteriophage specifically infects Staphylococcus. In one embodiment, the recombinant bacteriophage is capable of distinguishing Staphylococcus in the presence of over 100 other types of bacteria.

[0009] In some embodiments of the recombinant indicator bacteriophage, the indicator gene may be codon-optimized and may 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 comprise an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late 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 an Oplophorus luciferase gene, a firefly luciferase gene, a Lucia luciferase gene, or a Renilla luciferase gene, or the luciferase gene may be a genetically engineered gene such as NanoLuc.

[0010] Also disclosed herein is a method for preparing a recombinant indicator bacteriophage. Some 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 homologous recombination plasmid / vector into the target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur 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 Staphylococcus-specific bacteriophage or a Staphylococcus aureus-specific bacteriophage. In some embodiments, the selected wild-type bacteriophage is T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or another naturally occurring phage having a genome with at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, or 70% homology to the phages disclosed above.

[0011] In some embodiments, the step of preparing a homologous recombination plasmid / vector comprises 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 gene of the selected bacteriophage, designing a sequence for homologous recombination downstream of the major capsid protein gene, the sequence comprising a codon-optimized indicator gene, and incorporating the sequence designed for homologous recombination into the plasmid / vector. The step of designing the sequence may further comprise inserting a non-translated region comprising a phage late gene promoter and a ribosome entry site upstream of the codon-optimized indicator gene. Thus, in some methods, the homologous recombination plasmid comprises a bacteriophage late gene promoter and a non-translated region comprising a ribosome entry site upstream of the codon-optimized indicator gene.

[0012] Some embodiments of the present invention are compositions comprising the recombinant indicator bacteriophages described herein. For example, the compositions 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 compositions may comprise a cocktail of different indicator phages that may encode and express the same or different indicator proteins.

[0013] In some embodiments, the present invention encompasses a method for detecting a microorganism of interest in a sample, comprising incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage contains 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 positive detection of the indicator protein product indicates the presence of the microorganism of interest in the sample.

[0014] In some embodiments of the method for preparing a recombinant indicator bacteriophage, the wild-type bacteriophage is a Staphylococcus-specific bacteriophage and the target pathogenic bacterium is Staphylococcus. In some embodiments, the step of isolating specific clones 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 includes a method for detecting bacteria such as Staphylococcus in a sample, comprising incubating the sample with a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates the presence of Staphylococcus in the sample. In some embodiments, the present invention includes a method for the detection of S. aureus using a recombinant bacteriophage derived from S. aureus. The sample can be a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

[0016] In some embodiments of methods for detecting bacteria, the sample is first incubated under conditions that favor growth for an enrichment time of 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, or 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. In some embodiments, the total time to result is less than 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, or 6 hours. In some embodiments, the signal to background ratio produced by detecting the indicator is at least 2.0 or at least 2.5. In some embodiments, the method detects as few as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample of standard size for the food safety industry.

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

[0018] In another aspect, the present invention may include a method for selecting a treatment for a subject, the method including the steps of: (i) obtaining a biological sample from the subject; (ii) detecting a specific microorganism or category of microorganisms in the biological sample using an indicator phage; and (iii) selecting a treatment based on the identity of the specific microorganism detected in the biological sample.

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

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

[0021] [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 the firefly luciferase gene and a T7 late promoter inserted into the late (class III) region of the bacteriophage. Also shown are sequences containing stop codons in all three reading frames and untranslated regions (UTRs) to prevent readthrough.

[0022] [Figure 2]Figure 2 shows the genome of bacteriophage SEA1, a myovirus (related to the T4 bacteriophage) with approximately 95% homology to the myovirus Salmonella phage S16, obtained from the lab of Francisco Diez-Gonzalez. The gene 57A chaperone for long-tail fiber formation is located near the late gene region, which consists of structural genes encoding virion proteins. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided the late gene promoter and / or other similar control elements are used.

[0023] [Figure 3] Figure 3 shows two homologous recombination plasmid constructs carrying luciferase genes for two different phages with approximately 500 bp of matching phage sequence upstream and downstream of the insertion site to facilitate homologous recombination. NANOLUC® luciferase is inserted into a pBAV1k-T5-GFP plasmid backbone with an upstream untranslated region containing the phage late gene promoter and ribosome entry site. S. aureus phage recombination plasmids were constructed to insert NANOLUC® within the late gene region, but at a distance from the major capsid protein (MCP) due to stability issues.

[0024] [Figure 4] FIG. 4 shows the isolation of recombinant phage from modified S. aureus bacteriophage using a plasmid construct such as that shown in FIG. 3 and a series of sequential infection and dilution steps to identify recombinant phage expressing an indicator gene.

[0025] [Figure 5]FIG. 5 illustrates the use of an indicator phage encoding a soluble luciferase to detect bacterial cells via detection of luciferase produced from replicating progeny phage during infection of the bacterial cells, according to one embodiment of the present invention.

[0026] [Figure 6] FIG. 6 shows a filter plate assay for detecting bacteria of interest using modified bacteriophages according to one embodiment of the present invention, in which bacteria and recombinant phages are incubated on a filter plate, and after production of progeny bacteriophages, the indicator protein is detected directly without removing the incubation medium.

[0027] [Figure 7] FIG. 7 shows a "No Concentration Assay" for detecting bacteria of interest using an engineered bacteriophage according to one embodiment of the present invention.

[0028] [Figure 8] FIG. 8 shows a hybrid immunophage (HIP) assay for detecting bacteria of interest using an engineered bacteriophage according to one embodiment of the present invention, in which antibodies against the microorganism of interest are used to capture the microorganism on the surface of the assay well prior to incubation with a recombinant infectious agent carrying an indicator gene. DETAILED DESCRIPTION OF THE INVENTION

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

[0030] The compositions, methods, systems, and kits of the present invention may include infectious agents for use in detecting such microorganisms. In certain embodiments, the present invention may include compositions comprising a recombinant bacteriophage having an indicator gene inserted into the late gene region of the bacteriophage. In certain embodiments, 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 bacteriophage may be derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or a Staphylococcus-specific bacteriophage, or another wild-type or engineered bacteriophage.

[0031] In some aspects, the present invention includes methods for detecting a microorganism of interest. The methods may use an infectious agent for the detection of the microorganism of interest. For example, in certain embodiments, the microorganism of interest is a bacterium and the infectious agent is a bacteriophage. Thus, in certain embodiments, the methods may include detecting a bacterium of interest in a sample by incubating the sample with a recombinant bacteriophage that infects the bacterium of interest. In certain embodiments, the recombinant bacteriophage includes an indicator gene. In certain embodiments, the indicator gene may be 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 production of an indicator protein product. The methods may include detecting the indicator protein product, where positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample. In some embodiments, the indicator protein is soluble.

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

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

[0034] Aspects of the present invention utilize the high specificity of binding agents capable of binding to particular microorganisms, such as binding components of infectious agents, as a means of detecting and / or quantifying specific microorganisms in a sample. In some embodiments, the present invention utilizes the high specificity of infectious agents (e.g., bacteriophages).

[0035] In some embodiments, detection is achieved through an indicator moiety associated with a binding agent specific for the microorganism of interest. For example, an 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 designated an indicator phage.

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

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

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

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

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

[0041] Use of the term "or" is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0042] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists from sample to sample.

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

[0044] The term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction 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 agent" refers to a binding agent that is not associated (i.e., covalently or non-covalently bound) to a solid support.

[0045] As used herein, "analyte" refers to a molecule, compound, or cell being measured. An analyte of interest may, in certain embodiments, interact with a binding agent. As described herein, the term "analyte" may refer to a protein or peptide of interest. An analyte may be an agonist, antagonist, or modulator. Alternatively, an analyte may have no biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, and the like.

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

[0047] As used herein, "bacteriophage" or "phage" includes one or more of several bacterial viruses. In this disclosure, the terms "bacteriophage" and "phage" refer to viruses that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and use them to replicate themselves, including viruses such as mycobacteriophage (e.g., for TB and paraTB), mycophage (e.g., for fungi), mycoplasma phage, and any other term. Here, "microscopic" means one millimeter or less in maximum dimension. Bacteriophages are viruses that naturally evolved to use bacteria as a means of replicating themselves. Phages do this by attaching themselves to bacteria, injecting their DNA (or RNA) into the bacteria, and inducing the bacteria to replicate the phage hundreds or even thousands of times. This is also called phage amplification.

[0048] As used herein, "late gene region" refers to a region of a viral genome that is transcribed late in the viral life cycle. The late gene region typically contains 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, from 8 minutes post-infection until lysis, class I (e.g., RNA polymerase) is transcribed early, at 4-8 minutes, and class II at 6-15 minutes, with the timing of II and III genes overlapping. A late promoter is a promoter that is naturally located and active in such a late gene region.

[0049] As used herein, "enrichment culture" refers to traditional culture (e.g., incubation in a medium favorable for microbial growth) and should not be confused with other possible uses of the term "enrichment" (e.g., enrichment by removing a liquid component of a sample and concentrating the microorganisms contained therein) or other forms of enrichment that do not involve traditional promotion of microbial growth. While very short-term enrichment cultures may be used in some embodiments of the methods described herein, they are not required, and are of much shorter duration than traditional enrichment cultures, if used at all.

[0050] As used herein, "recombinant" means a gene that is not found in any other way. "Modified" refers to genetic (i.e., nucleic acid) modification, usually done in a laboratory, to combine unmodified genetic material. This term is used interchangeably herein with the term "modified."

[0051] As used herein, "RLU" refers to the relative units of light emitted, as measured by a luminometer (e.g., GLOMAX® 96) or similar light-detecting instrument. For example, detection of the reaction between luciferase and an appropriate substrate (e.g., NANOLUC® and NANO-GLO®) is often reported in terms of the RLU detected.

[0052] As used herein, "time to result" refers to the total amount of time from the start of sample incubation to the generation of a result. The time to result does not include any confirmatory testing time. Data collection can occur any time after the result is generated.

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

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

[0055] Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are food- or water-borne pathogens. Bacterial cells detectable by the present invention include, but are not limited to, all species of Salmonella, Escherichia coli, Cronobacter, all strains of Staphylococcus, all species of Listeria (including, but not limited to, L. monocytogenes), and all species of Campylobacter. Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are pathogens of medical or veterinary importance. Such pathogens include, but are not limited to, Bacillus spp., Bordetella pertussis, Campylobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens, Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Shigella sonnei, Staphylococcus aureus, and Streptococcus spp. In some embodiments, bacterial cells detectable by the present invention include antibiotic-resistant bacteria (eg, methicillin-resistant Staphylococcus aureus (MRSA)).

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

[0057] The assay may include a variety of appropriate control samples, for example, a control sample containing no bacteriophage or a control sample containing bacteriophage without bacteria may be assayed as a control for background signal levels.

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

[0059] The recombinant indicator bacteriophage 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 results in a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage. Late genes encode structural proteins and are therefore generally expressed at higher levels than other phage genes. The late gene region may be a class III gene region and may include a gene for a major capsid protein.

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

[0061] In some embodiments, the indicator bacteriophage is derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or a Staphylococcus-specific bacteriophage, or another bacteriophage having a genome that is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, or a Staphylococcus-specific bacteriophage. In some embodiments, the indicator phage is derived from a bacteriophage that is highly specific for a particular pathogenic microorganism. Genetic modifications can avoid deletion of wild-type genes, so the modified phage can remain more similar to the wild-type infectious agent than many commercially available phages. Bacteriophage derived from the environment can be more specific for bacteria found in the environment and, thus, can be genetically distinct from commercially available phages.

[0062] Furthermore, phage genes considered non-essential may have unrecognized functions. For example, apparently non-essential genes may have important functions in increasing burst size, such as clever cleavage, adaptation, or trimming functions during assembly. Therefore, deleting genes and inserting indicators may be detrimental. Most phages can package DNA several percent larger than their native genome. In this regard, 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 (encoding approximately 500-600 bp), while FLuc is about 62 kDa (encoding approximately 1,700 bp). For comparison, the T7 genome is about 40 kbp, while the T4 genome is about 170 kbp and the genome of Staphylococcus-specific bacteriophages is about 157 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacteria (i.e., not be part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, NANOLUC® in combination with Promega's NANO-GLO®, an imidazopyrazinone substrate (furimazine), can provide a robust signal with low background.

[0063] In some indicator phage embodiments, the indicator gene may be inserted into an untranslated region to avoid disrupting a functional gene, leaving the wild-type phage gene intact, which may result in greater fitness when infecting non-laboratory strains of bacteria. Additionally, 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 levels of fusion protein being made, which appears as a background signal (e.g., luciferase) that cannot be separated from the phage.

[0064] The indicator gene can express a variety of 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 Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as NANOLUC®.

[0065] Thus, in some embodiments, the invention includes a genetically modified bacteriophage containing 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. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like viral capsid proteins, but is also not silenced, like endogenous bacterial genes or even early viral genes.

[0066] In some embodiments, the late promoter is a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 promoter, or another phage promoter similar to the promoter 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, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus, or S. aureus-specific bacteriophage, or another naturally occurring bacteriophage having a genome that is at least 70, 75, 80, 85, 90, or 95% homologous to a T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus, or S. aureus-specific bacteriophage.

[0067] Genetic modifications to an infectious agent can include the insertion, deletion, or substitution of small nucleic acid fragments, substantial portions of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acid comprises a non-native sequence. A non-native indicator gene can be inserted into the bacteriophage genome such that it is under the control of a bacteriophage promoter. In some embodiments, the non-native indicator gene is not part of a fusion protein. That is, in some embodiments, the genetic modification can be configured so 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 includes a method for detecting a bacterium of interest, the method comprising incubating a test sample with such a recombinant bacteriophage.

[0068] In some embodiments, expression of the indicator gene in progeny bacteriophage after infection of the 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 result in a fusion protein. Unlike systems that use fusion of a detection moiety to a capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., a soluble luciferase). In some embodiments, the indicator or reporter ideally does not comprise the bacteriophage structure; i.e., the indicator or reporter is not attached to the phage structure. Thus, the gene for the indicator or reporter is not fused to other genes in the recombinant phage genome. This can greatly increase the sensitivity of the assay (down to a single bacterium) and simplify the assay, allowing the assay to be completed in less than one hour for some embodiments, as opposed to the several hours required for additional purification steps required with constructs that generate detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to protein folding constraints that may, for example, alter the conformation of the enzyme active site or access to substrates.

[0069] Furthermore, fusion proteins by definition limit the number of moieties attached to a protein subunit on the bacteriophage. For example, using a commercially available system designed to serve as a platform for fusion proteins, approximately 415 copies of the fusion moiety are generated in each T7 bacteriophage particle, corresponding to approximately 415 copies of the gene 10B capsid protein. Without this constraint, infected bacteria would be expected to express more copies of the detection moiety (e.g., luciferase) than can fit on the bacteriophage. Furthermore, large fusion proteins (e.g., capsid-luciferase fusions) may inhibit assembly of the bacteriophage particle, thus resulting in fewer bacteriophage progeny. Therefore, a soluble, non-fused indicator gene product may be preferred.

[0070] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a color change. A variety of suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes may serve as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0071] In some embodiments, the use of soluble detection moieties eliminates the need to remove contaminating parent phage from lysates of the infected sample cells. With a fusion protein system, any bacteriophage used to infect sample cells has an attached detection moiety and is indistinguishable from daughter bacteriophage that also contain the detection moiety. Because detection of sample bacteria relies on detection of newly created (de novo synthesized) detection moieties, the use of fusion constructs requires additional steps to separate the old (parent) 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 parent phage after infection by physical or chemical means, and / or chemically modifying the parent bacteriophage with a binding moiety (e.g., biotin), which can then be bound and separated (e.g., by streptavidin-coated Sepharose beads). However, even with all these attempts at removal, parent phage can persist if a high concentration of parent phage is used to ensure infection of a small number of sample cells, creating a background signal that can obscure the detection of signal from progeny phage in infected cells.

[0072] In contrast, with the soluble detector moieties expressed in some embodiments of the invention, purification of the parent phage from the final lysate is not necessary because the parent phage does not have any detector moieties attached. Therefore, any detector moieties present after infection must be newly produced, indicating the presence of the infected bacterium or bacteria. To take advantage of this benefit, the generation and preparation of parent phage can include purifying the phage from any free detector moieties generated during parent bacteriophage generation in bacterial culture. Standard bacteriophage purification techniques can be used to purify some embodiments of phage according to the invention (e.g., sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography, etc.). chromatographies, and dialysis or derivative techniques (e.g., Amicon brand concentrators - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as part of the preparation of recombinant phage of the invention to separate parent phage particles from contaminating luciferase protein produced during propagation of the phage in the bacterial host. In this way, the parent recombinant bacteriophage of the invention are substantially free of any luciferase produced during production in the bacteria. Removal of residual luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with a test sample.

[0073] In some modified bacteriophage embodiments, the late promoter (a class III promoter, e.g., from T7, T4, or ViI) has high affinity for the RNA polymerase of the same bacteriophage that transcribes the genes for structural proteins assembled into the bacteriophage particle. These proteins are the most abundant proteins made by the phage, as each bacteriophage particle contains tens or even hundreds of copies of these molecules. Use of a viral late promoter can optimally ensure high levels of expression of the luciferase detection moiety. Use of a late viral promoter derived from, specific for, or active under the original wild-type bacteriophage from which the indicator phage is derived (e.g., the T4 or T7 late promoters associated with T4-, T7-, or ViI-based systems) can further ensure optimal expression of the detection moiety. Use of standard bacterial (non-viral / non-bacteriophage) promoters can be detrimental to expression in some cases. Because these promoters are often downregulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production), in some embodiments, the phage is engineered to encode and express high levels of a soluble (free) indicator moiety, preferably using a location in the genome that does not limit expression to the number of subunits of the phage structural components.

[0074] Compositions of the invention may include one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may include a cocktail of different indicator phages that may 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.

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

[0076] Thus, the methods of the present invention utilize the high specificity of binding agents associated with infectious agents that recognize and bind to specific microorganisms of interest as a means of amplifying the signal 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 on specific microorganisms and therefore specifically infect those microorganisms. Thus, these infectious agents can be suitable binding agents for targeting microorganisms of interest.

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

[0078] Some embodiments of the present invention utilize the binding specificity and high-level gene expression capabilities of recombinant bacteriophages for rapid and sensitive targeting to infect and facilitate detection of bacteria of interest. In some embodiments, Staphylococcus or S. aureus-specific bacteriophages are genetically modified to contain a reporter gene. In some embodiments, the late gene region of the bacteriophage is genetically modified to contain the 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.

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

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

[0081] Thus, some embodiments of methods for preparing indicator bacteriophages include selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, determining the native sequence in the late region of the genome of the selected bacteriophage, annotating the genome and identifying the major capsid protein gene of the selected bacteriophage, designing sequences for homologous recombination adjacent to the major capsid protein gene, where the sequences comprise a codon-optimized reporter gene, incorporating the sequences designed for homologous recombination into a plasmid / vector, transforming the plasmid / vector into the target pathogenic bacterium, selecting for the transformed bacteria, infecting the transformed bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur 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 titering steps as necessary after the first limiting dilution assay until recombinant bacteriophage represent a detectable proportion of the mixture. For example, in some embodiments, the limiting dilution and titering steps can be repeated until at least 1 / 30 of the bacteriophage in the mixture are recombinant before isolating specific clones of recombinant bacteriophage. A recombinant:wild-type ratio of 1:30 is predicted in some embodiments to yield an average of 3.2 transducing units (TU) per 96 plaques (e.g., in a 96-well plate). The initial ratio of recombinant phage to wild-type phage can be determined by performing a limiting dilution assay based on TCID50 (50% tissue culture infectious dose), as previously described in U.S. Application No. 15 / 409,258. Due to the Poisson distribution, a 1:30 ratio results in a 96% chance of observing at least 1 TU in any of the 96 wells.

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

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

[0084] For example, Figure 2 shows the genome of bacteriophage SEA1, a naturally occurring phage with approximately 95% sequence identity to the T4-related myovirus bacteriophage S16. The SEA1 bacteriophage was obtained from Francisco Martinez's lab, and whole-genome sequencing was performed using an Illumina MiSeq and de novo sequence assembly. As discussed in the Examples, the major capsid protein 220 and various other structural genes are located within the late gene region 210, which consists of structural genes encoding virion proteins. Gene 57A 230, encoding a chaperone for long-tail fiber formation, is located at the border of the late gene region. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided the late gene promoter and / or other similar regulatory elements are used.

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

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

[0087] The major capsid protein segment 416-915 is part of the structural gene encoding the virion proteins. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided that a late gene promoter and / or other similar control elements are used.

[0088] In some embodiments, indicator phage according to the present invention comprise a Staphylococcus-specific bacteriophage genetically engineered to contain a reporter gene, such as a luciferase gene. For example, the indicator phage may be a Staphylococcus-specific bacteriophage whose genome comprises the sequence of the NANOLUC® gene. The recombinant Staphylococcus-specific NanoLuc bacteriophage genome may further comprise a promoter such as T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1, Staphylococcus-specific, ViI, or another late promoter. In some embodiments, the Staphylococcus-specific bacteriophage is a Staphylococcus aureus-specific bacteriophage.

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

[0090] FIG. 4 shows the isolation of recombinant phages from a mixture of wild-type bacteriophages and recombinant bacteriophages resulting from homologous recombination.

[0091] In a first step 402, S. aureus bacteria transformed with the homologous recombination plasmid are infected with S. aureus bacteriophage K, resulting in approximately 186 wild type:1 Progeny phage are generated, with a mixture of parental and recombinant phage having a ratio of recombinant phage 434. The resulting recombinant phage mix is ​​diluted 404 into a 96-well plate 406 to give an average of 5 recombinant transducing units (TU) / plate (9.3 PFU / well). The 96-well plate is assayed for luciferase activity to identify wells 436 containing recombinant phage compared to wells 440 containing wild-type bacteriophage. Bacteria 438 are added 408; for example, each well may contain approximately 50 μL of turbid S. aureus culture. This allows the phage to replicate and produce luciferase enzyme 442. After a 2-hour incubation at 37° C., as shown in 410, the wells can be screened for the presence of luciferase 442. Any positive wells may have been inoculated with a single recombinant phage; at this stage, the mixture may contain a ratio of approximately 9.3 wild-type phage:1 recombinant, an enrichment over the original 186:1 ratio. In one embodiment, one of the five wells was found to contain soluble luciferase via luciferase assay and contained phage at an approximate ratio of 2.4 total:1 recombinant. If necessary (i.e., if the recombinant:total ratio is lower than 1:30), progeny from this enriched culture 412 may be subjected to further limiting dilution assay(s) 414 to increase the ratio and determine the actual concentration of recombinant phage transducing units. For example, if the ratio is 1:384 recombinant:PFU, approximately 5 recombinant TU per 96-well plate 416 may be aliquoted from the previous positive well, along with 1920 (5 x 384 = 1920) total contaminating phage 414, resulting in an approximate seeding of 20 predominantly wild-type phage per well of the second dilution assay plate 420 (1920 PFU / 96 well = 20 PFU / well). Any positive luciferase wells may have seeded a single recombinant, along with 19 wild-type phage. These wells can be analyzed for the presence of luciferase 442.

[0092] After addition of bacteria and incubation (e.g., 2 hours at 37°C) 418, soluble luciferase and phage are present at approximately 20 total:1 recombinant 420. This ratio may be verified by TU50 titration for recombinants and plaque assay for total PFU. Finally, a plaque assay may be performed 422 to screen for recombinants expressing luciferase 446. A small number of individual plaques (e.g., n=48) may be individually picked and screened in a third multiwell plate 426 for luciferase activity 436. In one embodiment, this approach should ensure that enough plaques are screened so that there are approximately three recombinants in the mix of plaques screened, based on the known ratio of recombinants to total phage. One plaque may be picked 424 from the plate into each well of a 96-well plate, and a luciferase assay may be performed 426 to determine which wells contained phage exhibiting luciferase activity 442. Wells 428 that show luciferase activity represent pure recombinant phage 434 , while wells 430 without luciferase activity represent pure wild-type phage 432 .

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

[0094] Thus, as illustrated in Figure 4, recombinant phage generated by homologous recombination of a plasmid designed for recombination with a wild-type phage genome can be isolated from a mixture containing as little as 0.005% of the total phage genome. After isolation, large-scale production can be performed to obtain high-titer recombinant indicator phage stocks suitable for use in S. aureus detection assays. Furthermore, cesium chloride isopycnic density gradient centrifugation can be used to separate phage particles from contaminating luciferase protein to reduce background.

[0095] Methods of using infectious agents to detect microorganisms As noted herein, in certain embodiments, the present invention may include methods for using infectious particles to detect microorganisms. The methods of the present invention may be embodied in a variety of ways.

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

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

[0098] In certain embodiments, assays can be performed utilizing a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments using recombinant bacteriophages of the invention (i.e., indicator bacteriophages) can be performed in the following time periods depending on the sample type, sample size, and assay format: 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. The assay may allow for rapid detection of specific bacterial strains in total assay times of less than 10.0, 10.5, 11.0, 11.5, 12, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0 hours. For example, the amount of time required may be slightly shorter or longer depending on the strain of bacteriophage and the strain of bacteria being detected in the 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 contaminant bacteria.

[0099] Figure 5 illustrates a strategy for using indicator phage to produce soluble luciferase according to one embodiment of the present invention. In this method, phage (e.g., T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, Rambo, SAPJV1 phage) can be engineered to express soluble luciferase during phage replication. Luciferase expression is driven by a viral capsid promoter (e.g., the bacteriophage T7 or T4 late promoter), resulting in high expression. Because the parent phage does not contain luciferase, any luciferase detected in the assay must arise from the replication of progeny phage during infection of bacterial cells. Therefore, it is generally not necessary to separate the parent phage from the progeny phage.

[0100] In these experiments, at least a portion of the sample 500 containing the bacteria 502 to be quantified is placed in a spin column filter and centrifuged to remove the LB broth, followed by the addition of an appropriate multiplicity of phage 504 engineered to express soluble luciferase 503. The infected cells are incubated for a sufficient time (e.g., 30-90 minutes at 37°C) to allow progeny phage replication and cell lysis to occur. The parental phage 504 and progeny phage 516 in the lysate, plus the free luciferase 503, 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 can be employed in which the bacterial sample is applied to a 96-well filter plate, and after all of the procedures listed above, luciferase can be assayed directly in the original 96-well filter plate without the final centrifugation step.

[0101] Figure 6 illustrates a filter plate assay for detecting bacteria of interest using engineered bacteriophage according to one embodiment of the present invention. Briefly, a sample 616 containing bacteria of interest 618 is added to a well 602 of a multi-well filter plate 604, and the sample may be concentrated by spinning 606 to remove liquid from the sample. Genetically engineered phage 620 is added to the well and incubated with additional media for a time sufficient for adsorption 608, followed by infection of the target bacteria and progression of the phage life cycle 610 (e.g., approximately 45 minutes). Finally, a luciferase substrate is added and reacts with any luciferase 624 present. The resulting light emission is measured in a luminometer 614, which detects luciferase activity 626.

[0102] In certain embodiments, the assay can be performed without concentrating bacteria on or near the capture surface. Figure 7 illustrates a "No Concentration Assay" for detecting bacteria of interest using modified bacteriophage according to one embodiment of the present invention. Aliquots of indicator phage 714 are dispensed into individual wells 702 of a multiwell plate 704, followed by the addition of a test sample aliquot containing bacteria 712 and incubation 706 for a period sufficient for the phage to replicate and produce a soluble indicator 716 (e.g., luciferase) (e.g., 45 minutes at 37°C). The plate wells 708 containing the soluble indicator and phage may then be assayed 710 (e.g., luciferase assay) to measure indicator activity 718 in the plate. In this embodiment, the test sample is not concentrated (e.g., by centrifugation) but is simply incubated directly with the indicator phage for a period of time and then assayed for luciferase activity.

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

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

[0105] In one embodiment, the present invention may comprise a method for detecting a bacterium of interest in a sample, comprising incubating the sample with a recombinant bacteriophage that infects the bacterium of interest, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the bacterium of interest in the sample. In some embodiments, the amount of indicator moiety detected corresponds to the amount of the bacterium of interest present in the sample.

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

[0107] In certain embodiments, the recombinant infectious agent can be purified to be free of any residual indicator protein that may be produced during 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 added benefit of removing bacteriophage that do not have DNA (i.e., empty phage or "ghosts").

[0108] In some embodiments of the methods of the present invention, the microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or several target microorganisms can be applied directly to an assay vessel (e.g., a spin column, a microtiter well, or a filter), and the assay is performed in the assay vessel. Various embodiments of such assays are disclosed herein.

[0109] Test sample aliquots can be dispensed directly into wells of a multiwell plate, indicator phage can be added, and after a sufficient period for infection, lysis buffer can be added as well as a substrate for the indicator moiety (e.g., luciferase substrate for a luciferase indicator) and assayed for detection of the indicator signal. Some embodiments of the method can be performed on a filter plate. Some embodiments of the method can be performed with or without concentration of the sample prior to infection with indicator phage.

[0110] For example, in many embodiments, multi-well plates are used to perform the assay. The choice of plate (or any other container in which the detecting step can be performed) can affect the detecting 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 higher background signals. Other colors of plates may produce lower background signals but may have slightly lower sensitivity. Furthermore, one reason for 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 well but prevents light leakage from well to well, thus reducing background. Thus, the choice of plate or other assay container can affect the sensitivity and background signal for the assay.

[0111] The methods of the invention may include various other steps to increase sensitivity. For example, as discussed in more detail herein, the methods may include washing the captured and infected bacteria after adding the bacteriophage but before incubation to remove excess parent bacteriophage and / or luciferase or other reporter proteins that contaminate the bacteriophage preparation.

[0112] In some embodiments, detection of the microorganism of interest can be completed without the need to culture the sample as a method of increasing the population of the microorganism. For example, in certain embodiments, the total time required for detection is less than 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, 1.0 hour, 45 minutes, or less than 30 minutes. Minimizing time to results is critical in food and environmental testing for pathogens.

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

[0114] As noted herein, in certain embodiments, the invention may include methods of using recombinant bacteriophages to detect resistance of microorganisms to antibiotics, or to detect the effectiveness of antibiotics against microorganisms as otherwise noted. In another embodiment, the invention includes methods for selecting antibiotics for the treatment of infectious diseases. Additionally, the methods may include methods for detecting antibiotic-resistant bacteria in a sample. The methods of the invention may be embodied in a variety of ways.

[0115] The method may include contacting a sample containing a microorganism with an antibiotic and an infectious agent as described above. In some embodiments, the present disclosure provides a method for determining an effective dose of an antibiotic in killing or inhibiting the growth of a microorganism, the method comprising: (a) separately incubating one or more antibiotic solutions with one or more samples containing a microorganism, wherein the concentrations of the one or more antibiotic solutions are different and define a range; (b) incubating the microorganisms in the one or more samples with an infectious agent containing an indicator gene, wherein the infectious agent is specific to the microorganism of interest; and (c) detecting an indicator protein product produced by the infectious agent in the one or more samples, wherein detection of the indicator protein product in one or more of the multiple samples indicates that the concentration of the antibiotic solution used to treat one or more of the one or more samples is ineffective, and lack of detection of the indicator protein indicates that the antibiotic is effective, thereby determining an effective dose of the antibiotic.

[0116] In some embodiments, the antibiotic and the infectious agent are added to the sample simultaneously so that the sample is in contact with both the antibiotic and the infectious agent. In other embodiments, the antibiotic and the infectious agent are added sequentially, for example, the sample is contacted with the antibiotic and then the sample is contacted with the infectious agent. In certain embodiments, the method may include incubating the sample with the antibiotic for a certain period of time and then contacting the sample with the infectious agent. The incubation time may vary depending on the properties of the antibiotic and the microorganism, for example, based on the doubling time of the microorganism. In some embodiments, the incubation time is less than 24 hours, less than 18 hours, less than 12 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. The incubation time of the microorganism with the infectious agent may also vary depending on the life cycle of the specific infectious agent; in some cases, the incubation time is less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. Microorganisms resistant to antibiotics can survive and grow, and the infectious agent specific to the microorganism is replicated; conversely, microorganisms sensitive to antibiotics are killed, and therefore the infectious agent is not replicated. The infectious agent detected by this method contains an indicator moiety, the amount of which corresponds to the amount of the microorganism present in the antibiotic-treated sample. Therefore, positive detection of the indicator moiety indicates that the microorganism is resistant to the antibiotic.

[0117] In some embodiments, the method can be used to determine whether antibiotic-resistant microorganisms are present in a clinical sample. For example, the method can be used to determine whether a patient is infected with Staphylococcus aureus that is resistant or susceptible to a particular antibiotic. A clinical sample obtained from the patient can then be incubated with an antibiotic specific to S. aureus. The sample can then be incubated for a period of time with a recombinant phage specific to S. aureus. In samples containing S. aureus that are resistant to the antibiotic, detection of the indicator protein produced by the recombinant phage will be positive. In samples containing S. aureus that are susceptible to the antibiotic, detection of the indicator protein will be negative. In some embodiments, the method for detecting antibiotic resistance can be used to select effective therapeutic agents to which pathogenic bacteria are susceptible.

[0118] In certain embodiments, the total time required for detection is less than 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, or less than 1.0 hour. The total time required for detection will vary depending on the bacterium of interest, the type of phage, and the antibiotic being tested.

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

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

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

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

[0123] In some embodiments, detection of a signal is used to determine antibiotic resistance. In some embodiments, the signal generated by a sample is compared to an experimentally determined value. In further embodiments, the experimentally determined value is the signal generated by a control sample. In some embodiments, the background threshold is determined using a control that does not contain microorganisms. In some embodiments, the experimentally determined value is a background threshold calculated from the average background signal plus one to three or more standard deviations of the average background signal. In some embodiments, the background threshold may be calculated from the average background signal plus two standard deviations of the average background signal. In other embodiments, the background threshold may be calculated from several multiples (e.g., two or three) of the average background signal. Detection of a sample signal above the background threshold indicates the presence of one or more antibiotic-resistant microorganisms in the sample. For example, the average background signal may be 250 RLU. The threshold background value may be calculated by multiplying the average background signal (e.g., 250) by three to calculate a value of 750 RLU. Samples having bacteria with signal values ​​above 750 RLU are determined to be positive for containing antibiotic-resistant bacteria.

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

[0125] In some embodiments, the sample signal is compared to a control signal to determine whether an antibiotic-resistant microorganism is present in the sample. A lack of change in signal detection compared to a control sample contacted with an infectious agent but not an antibiotic indicates that the microorganism is resistant to the antibiotic, while a decrease in indicator moiety detection compared to a control sample contacted with an infectious agent but not an antibiotic indicates that the microorganism is susceptible to the antibiotic. A lack of change in detection refers to a signal detected from a sample treated with an antibiotic and an infectious agent that is at least 80%, at least 90%, or at least 95% of the signal from a control sample not treated with an antibiotic. A decrease in detection refers to a signal detected from a sample treated with an antibiotic and an infectious agent that is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or at least 30% of the signal from a control sample not treated with an antibiotic.

[0126] Optionally, the sample containing the microorganism of interest is an uncultivated sample. Optionally, the infectious agent is a phage, and includes an indicator gene inserted into the late gene region of the phage, such that expression of the indicator gene during phage replication after infection of the host bacterium results in a soluble indicator protein product. As noted above, each of the features of the compositions used in the present methods can also be utilized in methods for detecting antibiotic resistance in a microorganism of interest.

[0127] Also provided herein are methods for determining an effective dose of an antibiotic for killing a microorganism. In some embodiments, the antibiotic is effective for killing Staphylococcus species. For example, the antibiotic can be cefoxitin, which is effective against most methicillin-susceptible S. aureus (MSSA). Typically, one or more antibiotic solutions having different concentrations are prepared to define a range of different concentrations of the solutions. In some cases, the concentration ratio of the least concentrated antibiotic solution to the most concentrated antibiotic solution ranges from 1:2 to 1:50, e.g., 1:5 to 1:30, or 1:10 to 1:20. In some cases, the minimum concentration of the one or more antibiotic solutions is at least 1 μg / mL, e.g., at least 2 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 20 μg / mL, at least 40 μg / mL, at least 80 μg / mL, or at least 100 μg / mL. Each of the one or more antibiotic solutions is incubated with an aliquot of a sample containing the microorganism of interest. In some cases, the infectious agent, which is specific to the microorganism and includes an indicator moiety, is added simultaneously with the antibiotic solution. In some cases, an aliquot of the sample is incubated with the antibiotic solution for a period of time before adding the infectious agent. The indicator moiety can be detected, with a positive detection indicating that the antibiotic solution is not effective, and a negative detection indicating that the antibiotic solution is effective and that the concentration of the antibiotic solution is at an effective dose.Thus, in some embodiments, a method for determining an effective dose of an antibiotic in killing a microorganism of interest includes the steps of separately incubating each of one or more antibiotic solutions with the microorganism of interest in a sample, wherein the concentrations of the one or more antibiotic solutions are different and define a range; incubating the microorganism in the one or more samples with an infectious agent comprising an indicator moiety; and detecting the indicator moiety of the infectious agent in the one or more samples, wherein positive detection of the indicator moiety in one or more of the one or more samples indicates that the concentration of the antibiotic solution used to treat one or more of the one or more samples is ineffective, and lack of detection of the indicator protein indicates that the antibiotic is effective, thereby determining an effective dose of the antibiotic. In some embodiments, two or more antibiotic solutions are tested, and the concentration ratio of the least concentrated solution to the most concentrated solution in the one or more antibiotic solutions ranges from 1:2 to 1:50, e.g., 1:5 to 1:30, or 1:10 to 1:20. In some cases, the minimum concentration of the one or more antibiotic solutions is at least 1 μg / mL, e.g., at least 2 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 20 μg / mL, at least 40 μg / mL, at least 80 μg / mL, or at least 100 μg / mL.

[0128] In some embodiments, the present invention includes methods for detecting antibiotic-resistant microorganisms in the presence of antibiotic-susceptible microorganisms. In certain cases, detection of antibiotic-resistant bacteria can be used to prevent the spread of infection in healthcare settings. In some embodiments, patients in healthcare settings can be monitored for the establishment of antibiotic-resistant bacteria. Preventive measures can then be implemented to prevent the spread of antibiotic-resistant bacteria.

[0129] In some embodiments of the method for detecting antibiotic-resistant microorganisms, the sample may contain both antibiotic-resistant and antibiotic-susceptible bacteria. For example, the sample may contain both MRSA and MSSA. In some embodiments, MRSA can be detected in the presence of MSSA without the need to isolate MRSA from the sample. In the presence of antibiotics, MSSA does not generate a signal above the threshold, but MRSA present in the sample can generate a signal above the threshold. Therefore, if both are present in the sample, a signal above the threshold indicates the presence of an antibiotic-resistant strain (e.g., MRSA).

[0130] Thus, aspects of the present invention provide methods for detecting microorganisms in a test sample via an indicator moiety. In some embodiments, when the microorganism of interest is a bacterium, the indicator moiety can be associated with an infectious agent (e.g., an indicator bacteriophage). The indicator moiety can react with a substrate to emit a detectable signal or can emit an intrinsic signal (e.g., a fluorescent protein). In some embodiments, the detection sensitivity can reveal the presence of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the microorganism of interest in the test sample. In some embodiments, as few as one cell of the microorganism of interest can 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 Staphylococcus-specific bacteriophage. In certain embodiments, the recombinant Staphylococcus-specific bacteriophage is highly specific for Staphylococcus.

[0131] In some embodiments, the indicator moiety 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 contains an enzyme, which serves as the indicator moiety. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator may emit light and / or be detectable by a color change. Various suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may serve as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that produce a detectable signal may also be suitable indicator moieties.

[0132] Thus, in some embodiments, the recombinant bacteriophage of the present methods, systems, or kits is prepared from a wild-type Staphylococcus-specific bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an intrinsic signal, such as a fluorescent protein (e.g., green fluorescent protein, etc.). The indicator may emit light and / or be detectable by a color change. 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 an engineered luciferase, such as NANOLUC®, Rluc8.6-535, or orange nano-lantern.

[0133] Detecting the indicator may include detecting light emission. In some embodiments, a luminometer may be used to detect the reaction of an indicator (e.g., luciferase) with a substrate. Detection of RLUs may 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 emissions. While absolute RLUs are important for detection, a high signal-to-background ratio (e.g., >2.0, >2.5, or >3.0) is also required to reliably detect single cells or small numbers of cells.

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

[0135] Thus, in some embodiments utilizing indicator phage, the present invention includes a method for detecting a microorganism of interest, comprising capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phage; allowing time for infection and replication to produce progeny phage and express a soluble indicator moiety; and detecting the progeny phage, preferably the indicator, wherein detection of the indicator indicates the presence of the bacterium in the sample.

[0136] For example, in some embodiments, test sample bacteria can be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a 0.45 μm pore size spin filter or plate filter). In one embodiment, the infectious agent (e.g., 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 excess unbound infectious agent. In one embodiment, medium (e.g., Luria-Bertani broth, also referred to herein as LB, or Tryptic Soy Broth or Tryptone Soy Broth, also referred to herein as TSB) can be added for an additional incubation period to allow bacterial cell and phage replication and high-level expression of the gene encoding the indicator moiety. However, a surprising aspect of some embodiments of the test assay is that the incubation step with indicator phage only needs to be long enough for a single phage life cycle. The amplification power of using bacteriophages was previously thought to be more time consuming as the phage replicates over several cycles. A single replication cycle of an indicator phage may be sufficient to facilitate sensitive and rapid detection according to some embodiments of the present invention.

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

[0138] Soluble indicators (e.g., luciferase) released into the surrounding liquid upon lysis of the bacteria 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) after which a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

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

[0140] Thus, in an alternative embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with the portion of the sample that remains on the filter or that remains 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.

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

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

[0143] In some embodiments, the reaction between the indicator moiety (e.g., luciferase) and the substrate can continue 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.

[0144] Surprisingly, the high concentrations of phage utilized to infect the test sample successfully achieved detection of very small numbers of target microorganisms in a very short time frame. Incubating the phage with the test sample, in some embodiments, only needs to be long enough for a single phage life cycle. In some embodiments, the concentration of bacteriophage for this incubation step is 7 x 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 1.0×10 8 Higher than PFU / mL.

[0145] The success of phage at such high concentrations is surprising because many of the phage were previously associated with "non-infectious lysates," which killed target cells and thereby prevented the generation of useful signals from earlier phage assays. Purification of prepared phage stocks as described herein (e.g., clarification by cesium chloride isopycnic density gradient ultracentrifugation) is believed to help alleviate this problem because, in addition to removing any contaminating luciferase associated with the phage, this clarification can also remove ghost particles (particles that have lost their DNA). These ghost particles can lyse bacterial cells via "non-infectious lysates," prematurely killing the cells and thereby preventing the generation of indicator signals. Electron microscopy clearly shows that crude phage lysates (i.e., before cesium chloride clarification) can have more than 50% ghosts. These ghost particles may contribute to the early death of the microorganism through the action of many phage particles puncturing the cell membrane. Therefore, ghost particles may have contributed to previous problems where high PFU concentrations were reported to be harmful. Furthermore, the extremely clean phage preparation allows the assay to be performed without a washing step, which allows the assay to be performed without an initial enrichment step. Some embodiments include an initial enrichment step, which in some embodiments allows for a shorter enrichment incubation time.

[0146] Some embodiments of the testing method may further include a confirmatory assay. Various assays are known in the art to confirm initial results, usually at a later time. For example, the sample may be cultured (e.g., the CHROMAGAR® / DYNABEADS® assay described in Example 4), PCR may be used to confirm the presence of microbial DNA, or other confirmatory assays may be used to confirm the initial results.

[0147] In certain embodiments, the methods of the invention combine detection with the infectious agent and the use of a binding agent (e.g., an antibody) to purify and / or concentrate a microorganism of interest from a sample. For example, in certain embodiments, the invention includes a method for detecting a microorganism of interest in a sample, comprising capturing the microorganism from the sample on a support using a capture antibody specific for the microorganism of interest, incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage contains 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 positive detection of the indicator protein product indicates the presence of the microorganism of interest in the sample.

[0148] For example, Figure 8 illustrates a Hybrid Immuno-Phage (HIP) assay for detecting bacteria of interest using engineered bacteriophages according to one embodiment of the present invention. Samples are first applied to a microtiter plate that has been thoroughly coated with bacteria-specific antibodies 802. The plate is then centrifuged to allow bacteria to bind to the capture antibodies 804. After a sufficient time has passed to allow complete bacterial capture, a solution containing bacteria-specific NANOLUC® phage is added to each sample 806. Incubation with the phage results in the binding and attachment of single or multiple phages to the captured bacteria 808. Finally, the samples are incubated to allow phage replication and luciferase expression, leading to cell lysis and release of soluble luciferase 810.

[0149] Recent advances in synthetic biology have increased interest in bacteriophage-based therapeutic approaches to treat pathogenic diseases (see, e.g., Phage Therapy in the Era of Synthetic Biology, Cold Spring Harb Perspect Biol 2016; 8:a023879; and U.S. Patent No. 9,597,407, and U.S. Patent Application No. 2017026 See, e.g., Nos. 6306 and 20160331804, the contents of which are incorporated by reference herein as if set forth in their entireties. Bacteriophages designed and engineered to detect pathogens in medical samples from patients for the presence of specific microorganisms, such as specific types of bacteria, can enhance the utility of such therapeutic phages.

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

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

[0152] In some embodiments, synthetic phage are designed to optimize desired traits for use in pathogen detection assays. In some embodiments, bioinformatics and prior analysis of genetic modifications are used to optimize desired traits. For example, in some embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to specific species of bacteria. In other embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to entire genuses of bacteria or specific groups of species within a genus. In this way, phage can be optimized to detect broader or narrower groups of pathogens. In some embodiments, synthetic phage can be designed to improve reporter gene expression. Additionally and / or alternatively, in some cases, synthetic phage can be designed to increase the burst size of the phage to improve detection.

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

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

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

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

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

[0158] Systems and kits 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, a 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 suitable for automation or kits given the minimal amount of reagents and materials required to carry out the methods. In certain embodiments, each of the components of the kit may comprise a self-contained unit deliverable from a first site to a second site.

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

[0160] In other embodiments, the present invention includes a method, system, or kit for rapidly detecting a microorganism of interest in a sample, the method, system, or kit including an infectious agent component specific for the microorganism of interest, the infectious agent comprising an indicator moiety, and a component for detecting the indicator moiety. In some embodiments, the bacteriophage is a T4-like, ViI, ViI-like, or Staphylococcus-specific bacteriophage. In one embodiment, the recombinant bacteriophage is derived from a Staphylococcus-specific bacteriophage. In certain embodiments, the recombinant bacteriophage is highly specific for a particular bacterium. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Staphylococcus. In one embodiment, the recombinant bacteriophage is capable of distinguishing Cronobacter in the presence of over 100 other types of bacteria. In another embodiment, the recombinant bacteriophage is capable of distinguishing Staphylococcus in the presence of over 100 other types of bacteria. In certain embodiments, the system or kit detects a single bacterium of a particular type in a sample, hi some embodiments, the system or kit detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample.

[0161] In certain embodiments, the system and / or kit may further include a component for washing the captured microbial sample. Additionally or alternatively, the system and / or kit may further include a component for determining the amount of the indicator moiety, where the amount of detected indicator moiety 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.

[0162] In some systems and / or kits, the same components may be used for multiple steps. Thus, the steps are automated or controlled by a user via computer input and / or a liquid handling robot performs at least one step.

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

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

[0165] In one embodiment, the invention includes a system or kit that includes components for detecting a microorganism of interest, including components for isolating at least one microorganism from other components in a sample, components for infecting at least one microorganism with multiple parent infectious agents, components for lysing at least one infected microorganism to release progeny infectious agents present in the microorganism, and components for detecting the progeny infectious agents, or more sensitively, soluble proteins encoded and expressed by the infectious agents, wherein detection of the infectious agent or soluble protein products of the infectious agent indicates the presence of the microorganism in the sample. The infectious agent may include a Staphylococcus-specific NANOLUC bacteriophage.

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

[0167] In other embodiments, the present invention may include a kit for rapid detection of a microorganism of interest in a sample, the system including components for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety; a component for capturing the microorganism from the sample onto a solid support; a component for washing the captured microorganism sample to remove unbound infectious agent; and a component for detecting the indicator moiety. In some embodiments, the same components may be used for the capturing, incubating, and / or washing steps. Some embodiments further include a component for determining the amount of the microorganism of interest in the sample, where the amount of indicator moiety detected corresponds to the amount of the microorganism in the sample. Such a kit may include various embodiments and subembodiments similar to those described above with respect to the method for rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

[0168] In some embodiments, kits may include components for isolating the microorganism of interest from other components in the sample.

[0169] These systems and kits of the present invention include various components. As used herein, the term "component" is broadly defined and includes any suitable device or collection of suitable devices for carrying out the described method. The components need not be integrally connected or mounted relative to one another in any particular way. The present invention encompasses any suitable arrangement of the components relative to one another. For example, the components need not reside in the same space. However, in some embodiments, the components are connected to one another in an integral unit. In some embodiments, the same component may perform multiple functions.

[0170] Computer System and Computer-Readable Medium The system may be embodied in the form of a computer system, as described in the present technology or any of its components. Representative examples of computer systems include general purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices capable of implementing the steps comprising the methods of the present technology.

[0171] The computer system may include a computer, an input device, 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 a storage device. The storage device may be a hard disk drive or a removable storage device (e.g., a floppy disk drive, an optical disk drive, etc.). The storage device 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 allows the computer to connect to other databases and the Internet through an I / O interface. The communication unit allows data to be transferred to and received from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that allows the computer system to connect to databases and networks (e.g., LANs, MANs, WANs, and the Internet). The computer system may therefore facilitate input from a user through input devices accessible to the system via an I / O interface.

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

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

[0174] The environment may include various data stores and other memory and storage media, as discussed above. These may reside in a variety of locations (e.g., in storage media local to (and / or residing in) one or more of the computers, or remote from any or all of the computers across a network). In a particular group of embodiments, information may reside on a storage area network ("SAN"), familiar to those skilled in the art. Similarly, any files necessary to perform the functions ascribed to the computers, servers, or other network devices may be stored locally and / or remotely, as appropriate. Where the system includes computing devices, each such device may include hardware elements that may be electrically coupled via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system may also include one or more storage devices (e.g., disk drives, optical storage devices, and solid-state storage devices such as random access memory ("RAM") or read-only memory ("ROM"), as well as removable media devices, memory cards, flash cards, etc.

[0175] 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 a working memory as described above. The computer-readable storage medium reader may be connected to or configured to accept computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information. The systems 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., client applications or web browsers). 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 particular elements may be implemented in hardware, software (including portable software (e.g., applets)), or both. Additionally, connections to other computing devices (e.g., network input / output devices) may be used.

[0176] Non-transitory storage media and computer-readable media for containing code or portions of code may include any suitable media known or used in the art, including storage media and communication media (e.g., volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage and / or transmission of information such as computer-readable instructions, data structures, program modules, or other data), including RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage device, or any other medium that can be used to store the desired information and that can be accessed by the system device. Based on the above disclosure and the teachings provided herein, one skilled in the art will recognize other ways and / or methods to implement the various embodiments.

[0177] 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, ROM, RAM, SRAM, DRAM, content addressable memory ("CAM"), DDR, flash memory (e.g., NAND flash or NOR flash), ASICs, configured processors, optical storage media, magnetic tape or other magnetic storage media, or any other medium from which a computer processor can read instructions. In one embodiment, the computing device may include a single type of computer-readable medium (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 cache). The computing device may be in communication with one or more external computer-readable media (e.g., an external hard disk drive, or an external DVD or Blu-ray drive).

[0178] As discussed above, the embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, 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 include programmable electronic devices (e.g., PLCs), programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electronically programmable read-only memories (EPROMs or EEPROMs), and the like. or other similar devices.

[0179] The computing device includes a network interface. In some embodiments, the network interface is configured to communicate over 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 communication over a network with another device, e.g., a Universal Serial Bus (USB), 1394 It may allow point-to-point connections via FireWire, serial or parallel connections, or similar interfaces. Some embodiments of suitable computing devices 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.

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

[0181] The set of instructions for execution by the computer system may include various commands that instruct a processing machine to perform particular tasks (e.g., steps constituting the methods of the present technology). The set of instructions may be in the form of a software program. Moreover, the software may be in the form of a collection of separate programs, a program module with a larger program, or a portion of a program module, as in the present technology. The software may also include modular programming in the form of object-oriented programming. The processing of input data by the processing machine may be in response to user commands, results of previous processing, or a request made by another processing machine.

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

[0183] The results presented in the Examples below demonstrate detection of low numbers of cells, even single bacteria, with reduced time to results. Example 1 Bacterial detection using the Staphylococcus aureus-specific bacteriophage NanoLuc indicator phage after incubation of samples in culture medium

[0184] Samples for methicillin-resistant S. aureus (MRSA) were primed by adding 135 μl of sample containing S. aureus to rich nutrient medium. The rich nutrient medium contained a sub-inhibitory antibiotic (cefoxitin) to allow specific enrichment and induction of MRSA. The samples were incubated in rich nutrient medium for 1-2 hours. After priming the sample, additional cefoxitin was added to the medium, and the sample was incubated for an additional 2 hours. Staphylococcus aureus-specific bacteriophage NanoLuc indicator phage was added to the sample and incubated for 2 hours. Lysis buffer and NANO-GLO® reagent were added. After a 5-minute incubation, bioluminescence measurements were performed using a GLOMAX® 96 instrument. A signal-to-background ratio of ≥750 RLU indicated positive detection of S. aureus. A signal-to-background ratio of <750 RLU indicated the sample was negative for S. aureus. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] Example 2 Diagnosis and monitoring of Staphylococcus infections using the Staphylococcus-specific bacteriophage NanoLuc® indicator phage after incubation of the sample in culture medium

[0185] A clinical sample is obtained from a patient. The clinical sample is incubated with the Staphylococcus-specific bacteriophage NanoLuc® indicator phage for 2 hours. After incubation, lysis buffer and the NANO-GLO® reagent are added. After a 5-minute incubation with the lysis buffer and the NANO-GLO® reagent, bioluminescence is measured using a GLOMAX® 96 instrument to determine the presence of Staphylococcus in the clinical sample. A signal / background ratio of ≥ 750 RLU indicates positive detection of Staphylococcus. A signal / background ratio of < 750 RLU indicates the sample is negative for Staphylococcus.

[0186] Patients with Staphylococcus-positive samples are treated with a Staphylococcus-specific therapeutic phage. After 72 hours of treatment with the Staphylococcus-specific therapeutic phage, a second clinical sample is obtained from the patient. The clinical sample is then incubated with a Staphylococcus-specific bacteriophage NanoLuc® indicator phage for 2 hours. After incubation, lysis buffer and NANO-GLO® reagent are added and incubated for 5 minutes. Bioluminescence is then measured using a GLOMAX® 96 instrument to monitor changes in the presence of Staphylococcus in the clinical sample after treatment. A signal / background ratio of ≥ 750 RLU indicates positive detection of Staphylococcus. A signal / background ratio of < 750 RLU indicates the sample is negative for Staphylococcus. If the sample is positive for Staphylococcus, treatment with the Staphylococcus-specific therapeutic phage continues, and the effectiveness of treatment continues to be monitored using the Staphylococcus-specific bacteriophage NanoLuc® indicator phage. In certain embodiments, for example, the following items are provided: (Item 1) A recombinant bacteriophage comprising an indicator gene inserted into the late gene region of the bacteriophage genome. (Item 2) 2. The recombinant bacteriophage according to item 1, which specifically infects Staphylococcus. (Item 3) 3. The recombinant bacteriophage according to item 2, wherein the Staphylococcus is Staphylococcus aureus. (Item 4) 4. The recombinant bacteriophage according to item 3, wherein the Staphylococcus aureus is methicillin-resistant. (Item 5) 2. The recombinant bacteriophage of item 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. (Item 6) 6. The recombinant bacteriophage according to item 5, further comprising an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late gene promoter and a ribosome entry site. (Item 7) A cocktail composition comprising at least two different types of recombinant bacteriophages, at least one of which comprises the indicator gene described in item 1. (Item 8) 1. A method for preparing a recombinant indicator bacteriophage, comprising: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector 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 allowing homologous recombination to occur between the plasmid / vector and the bacteriophage genome; and Isolating specific clones of recombinant bacteriophage A method comprising: (Item 9) The step of preparing a homologous recombination plasmid / vector comprises: determining the native nucleotide sequence in the late region of the genome of said selected bacteriophage; annotating the genome and identifying the major capsid protein gene of the selected bacteriophage; designing a sequence for homologous recombination downstream of the major capsid protein gene, the sequence comprising a codon-optimized indicator gene; and Incorporating said sequences designed for homologous recombination into a plasmid / vector Item 9. The method according to item 8, comprising: (Item 10) 10. The method of claim 9, wherein the step of designing a sequence further comprises inserting an untranslated region comprising a phage late gene promoter and a ribosome entry site upstream of the codon-optimized indicator gene. (Item 11) 9. The method of claim 8, wherein the homologous recombination plasmid comprises a bacteriophage late gene promoter and an untranslated region comprising a ribosome entry site upstream of the codon-optimized indicator gene. (Item 12) 9. The method of claim 8, wherein the wild-type bacteriophage is a Staphylococcus-specific bacteriophage and the target pathogenic bacterium is Staphylococcus. (Item 13) Item 13. The method according to item 12, wherein the Staphylococcus is Staphylococcus aureus. (Item 14) Item 14. The method of item 13, wherein the Staphylococcus aureus is methicillin-resistant (MRSA). (Item 15) 9. The method according to item 8, wherein the step of isolating specific clones of recombinant bacteriophage comprises a limiting dilution assay to isolate clones that exhibit expression of the indicator gene. (Item 16) 1. A method for detecting Staphylococcus in a sample, comprising: incubating the sample with a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage containing an indicator gene inserted into the late gene region of the bacteriophage genome; and detecting an indicator protein product produced by said recombinant bacteriophage, wherein positive detection of said indicator protein product indicates that Staphylococcus is present in said sample. A method comprising: (Item 17) Item 17. The method according to item 16, wherein the Staphylococcus is Staphylococcus aureus. (Item 18) 18. The method of claim 17, wherein the Staphylococcus aureus is methicillin-resistant. (Item 19) 17. The method of claim 16, wherein the sample is a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample. (Item 20) 17. The method of item 16, which detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bacteria in a sample of standard size for the food safety industry. (Item 21) 20. The method of claim 19, wherein the food sample comprises meat, fish, vegetables, eggs, or infant formula. (Item 22) 17. The method of claim 16, wherein the sample is incubated with a cocktail composition comprising at least two different types of recombinant bacteriophage, at least one of the recombinant bacteriophage comprising the indicator gene of claim 16. (Item 23) 17. The method of claim 16, wherein the sample is first incubated under conditions that favor growth for an enrichment time of 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. (Item 24) 17. The method of claim 16, wherein the total time to result is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, or less than 6 hours. (Item 25) 17. The method of claim 16, wherein the signal to background ratio produced by the step of detecting the indicator is at least 2.0 or at least 2.5. (Item 26) A kit for detecting Staphylococcus, comprising a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage. (Item 27) 27. The kit according to item 26, wherein the Staphylococcus is Staphylococcus aureus and the Staphylococcus-specific bacteriophage is a Staphylococcus aureus-specific bacteriophage. (Item 28) 28. The kit according to item 27, wherein the Staphylococcus aureus is methicillin-resistant and the Staphylococcus aureus-specific bacteriophage is a methicillin-resistant Staphylococcus aureus-specific bacteriophage. (Item 29) 27. The kit of claim 26, further comprising a substrate for reacting with an indicator to detect a soluble protein product expressed by the recombinant bacteriophage. (Item 30) A system for detecting Staphylococcus comprising a recombinant bacteriophage derived from a Staphylococcus-specific bacteriophage. (Item 31) Item 31. The system according to item 30, wherein the Staphylococcus is Staphylococcus aureus. (Item 32) 32. The system of claim 31, wherein the Staphylococcus aureus is methicillin-resistant. (Item 33) 1. A method for selecting a treatment for a subject, comprising: (i) obtaining a biological sample from said subject; (ii) using the indicator phage to detect a specific microorganism or category of microorganism in said biological sample; and (iii) selecting a treatment based on the identity of the specific microorganism detected in the biological sample. A method comprising: (Item 34) 34. The method of claim 33, wherein the indicator phage is a synthetically prepared phage. (Item 35) 34. The method of claim 33, wherein the indicator phage is a genetically modified, naturally occurring phage. (Item 36) 1. A method for monitoring the effectiveness of a treatment for a subject having a pathogenic medical condition, comprising: (i) obtaining a biological sample from said subject; (ii) using indicator phage to detect specific microorganisms or categories of microorganisms in said biological sample; (iii) initiating treatment for said subject; (iv) obtaining a second biological sample from the subject, the second biological sample being of the same type as the first biological sample; (v) using the indicator phage to detect the specific microorganism or category of microorganism in the second biological sample; and (vi) determining a decreased, increased, or steady-state level of the particular microorganism or category of microorganisms in the subject based on the amounts detected in the first and second biological samples; A method comprising:

Claims

[Claim 1] The invention described in the specification.

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