Methods for the diagnosis and treatment of biofilm-related infections

Abstract

To provide methods for the diagnosis and treatment of biofilm-related infections. [Solution] Methods and systems for the rapid diagnosis and treatment of biofilm-related infections in subjects having medical implants are disclosed herein. A reporter cocktail composition is disclosed herein, which can be used for the detection of a target microorganism and for determining the presence of an infection. A therapeutic cocktail composition is also disclosed herein, which can be used for the treatment of subjects diagnosed with a biofilm-related infection. A second aspect of the disclosure is a method for preventing or inhibiting an infection in a subject, comprising the step of applying a cocktail composition containing at least one recombinant bacteriophage to a surgical implant, bandage, or suture.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority and the benefit thereof based on U.S. Provisional Application No. 63 / 039,146, filed on June 15, 2020, which is incorporated herein by reference in its entirety.

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

Background Art

[0003] Background Post - operative infections of implanted devices are a major concern within the medical field. Such infections can be difficult to diagnose and treat. One factor that particularly complicates matters is the formation of biofilms. A biofilm is a layer of bacteria or other microorganisms and can be formed from one or more species of bacteria. Bacteria growing as a biofilm are present within a matrix of extracellular polymeric secretions (EPS) consisting of proteins, polysaccharides, and nucleic acids that enable the biofilm to adhere to the surface of natural materials or medical devices. Bacteria secrete EPS into their environment to establish the functional and structural integrity of the biofilm. Biofilms allow bacteria to share their nutrients and protect the bacteria from harmful agents including antibiotics.

[0004] Biofilms can cause chronic, nosocomial, and medical - device - related infections. Such infections are difficult to treat due to their antibiotic - resistant nature, and thus, the use of antibiotics alone is typically ineffective in treating biofilm - related infections (Khatoon et al., 2018, Heliyon). Biofilm - related infections not only pose difficulties in treatment but also present challenges in establishing an accurate diagnosis based on the speciation / susceptibility of the infection. treatment but also present challenges in establishing an accurate diagnosis based on the speciation / susceptibility of the infection.

[0005] Antibiotics are widely used to treat infectious diseases caused by microorganisms. Some microorganisms are naturally resistant to certain antibiotics, while other microorganisms can acquire such resistance after being treated with antibiotics for some time. Antibiotic resistance can cause undesirable results; microorganisms can still grow in the presence of antibiotics, thus worsening the infectious disease, and ineffective antibiotics can cause serious side effects and in some cases lead to life-threatening situations. As a result, antibiotic resistance can result in higher medical costs, longer hospital stays, and increased mortality.

[0006] Therefore, the detection of microorganisms resistant to specific antibiotics is very important. The ability of healthcare providers to determine whether the microorganisms causing an infectious disease present in the body are resistant to antibiotics is extremely important in the selection of appropriate treatment. Furthermore, the ability to determine the antibiotic resistance of microorganisms from samples with low levels of microorganisms within a short time frame is essential for the successful treatment of infectious diseases before they become severe.

[0007] Current methods for detecting antibiotic resistance often require assays that are time-consuming, technically demanding, and / or lack sufficient sensitivity. Typically, such assays involve immunoassays and molecular-based assays in cultured samples, requiring gel electrophoresis, real-time PCR / multiplexing, and / or multi-locus sequence typing. These tests often take 24–48 hours to complete and / or lack sufficient sensitivity. Currently available methods typically require isolation and / or enrichment of the microorganism by culture prior to detection, thus requiring increased time to results. Therefore, there is a strong interest in rapid and highly sensitive tests to determine whether a target microorganism, e.g., a microorganism causing an infection, is resistant to a particular antibiotic before using that antibiotic. The present invention excels as a rapid test for detecting microorganisms by not requiring isolation of the microorganism prior to detection. This knowledge can assist clinicians in prescribing appropriate antibiotics to control infections in a timely manner and increase the ability to prevent the transmission of serious infections through active monitoring in healthcare settings. [Overview of the project] [Means for solving the problem]

[0008] Bacteriophages have been suggested as an alternative or supplement to antibiotic treatment. Modifying bacteriophages to optimize their enzyme function would be advantageous for effectively treating biofilm-related infections.

[0009] overview Embodiments of the present invention include compositions, methods, and systems for the diagnosis and treatment of microorganism-related infections. The present invention can be embodied in various ways.

[0010] The first aspect of this disclosure is a method for diagnosing and treating a biofilm-related infection in a subject, comprising the steps of (i) providing a biological sample taken from the subject; (ii) diagnosing the subject with a biofilm-related infection by detecting the presence of at least one target microorganism, comprising the steps of (a) contacting at least one aliquot of the biological sample with a certain amount of a diagnostic cocktail composition comprising at least one recombinant bacteriophage; (b) detecting a signal generated after replication of the recombinant bacteriophage, wherein the detection of the signal indicates the presence of the target microorganism in the sample; and (iii) treating the subject diagnosed with a biofilm-related infection, comprising the steps of (a) selecting a therapeutic cocktail composition based on the diagnosis of step (ii); (b) administering a therapeutically effective amount of the therapeutic cocktail composition comprising at least one bacteriophage, wherein the bacteriophage is specific to the detected target microorganism; and (c) administering at least one additional therapeutic agent as needed. In some embodiments, the therapeutic cocktail composition comprises at least one bacteriophage, which is either a recombinant bacteriophage or a wild-type bacteriophage. In some embodiments, the subject has an implant.

[0011] A second aspect of this disclosure is a method for preventing or inhibiting an infection in a subject, comprising the step of applying a cocktail composition comprising at least one recombinant bacteriophage to a surgical implant, bandage, or suture.

[0012] A third aspect of the present disclosure is a surgical implant, bandage, or suture coated with a cocktail composition comprising at least one recombinant bacteriophage.

[0013] Certain specific embodiments of this disclosure utilize methods and constructs described in U.S. Patent Application Publication 2015 / 0218616 and U.S. Patent Application Publication 2019 / 0010534, which are incorporated herein by reference in their entirety. The present invention provides, for example, the following items: (Item 1) A method for diagnosing and treating biofilm-related infections in a subject, (i) A step of providing a biological sample taken from the subject; (ii) (a) A step of contacting at least one aliquot of the biological sample with a certain amount of a reporter cocktail composition containing at least one recombinant bacteriophage; (b) A step of detecting a signal generated after replication of the recombinant bacteriophage, wherein the detection of the signal indicates the presence of the target microorganism in the sample. A step of diagnosing the subject as having a biofilm-related infection by detecting the presence of at least one of the aforementioned target microorganisms, including; and (iii) (a) A step of selecting a therapeutic cocktail composition based on the diagnosis in step (ii); (b) a step of administering a therapeutically effective amount of a therapeutic cocktail composition comprising at least one bacteriophage, wherein the bacteriophage is specific to the microorganism of interest detected; and (c) The step of administering at least one additional therapeutic agent as needed. A process for treating the subject diagnosed with a biofilm-related infection, including Methods that include... (Item 2) The method according to item 1, wherein the biofilm is present on or around the surface of an implant in a subject suspected of having an infection. (Item 3) The method according to item 1, wherein the step of diagnosing the subject includes a step of contacting a plurality of aliquots with a plurality of reporter cocktail compositions. (Item 4) The method according to item 1, wherein the step of diagnosing the target further includes a step of determining the antibiotic resistance of the detected target microorganism. (Item 5) The method according to item 4, wherein the step of determining the antibiotic resistance of the detected target microorganism further includes the step of contacting the biological sample with an antibiotic before contacting the biological sample with the reporter cocktail composition. (Item 6) The method according to item 1, wherein the recombinant bacteriophage in the reporter cocktail composition comprises a genetic construct inserted into a bacteriophage genome, and the genetic construct comprises an indicator gene and an additional late bacteriophage promoter. (Item 7) The method according to item 6, wherein the indicator gene does not encode a fusion protein, and the transcription of the indicator gene is controlled by the additional late bacteriophage promoter. (Item 8) The method according to item 7, wherein the expression of the indicator gene during bacteriophage replication after infection of a host bacterium yields an indicator protein product. (Item 9) The method according to item 8, wherein the indicator gene encodes a luciferase enzyme. (Item 10) The method according to item 1, wherein the bacteriophage in the therapeutic cocktail composition is a recombinant bacteriophage. (Item 11) The method according to item 10, wherein the recombinant bacteriophage in the therapeutic cocktail composition comprises a genetic construct inserted into the bacteriophage genome, and the genetic construct comprises an enzyme. (Item 12) The method according to item 11, wherein the enzyme is glycosidase, amidase, or endopeptidase. (Item 13) The method according to item 1, wherein the target microorganism is Staphylococcus, Klebsiella, Pseudomonas, Cutibacterium acnes, and Shigella. (Item 14) The method according to item 1, wherein at least one type of recombinant bacteriophage is constructed from phage K, MP115, or ISP. (Item 15) The method according to item 1, wherein the biological sample is first incubated under conditions that support growth for an enrichment period of 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours. (Item 16) The method according to item 1, wherein the therapeutic agent is an antibiotic. (Item 17) A method for preventing or inhibiting an infection in a subject, comprising the step of applying a cocktail composition containing at least one recombinant bacteriophage to a surgical implant, bandage, or suture. (Item 18) A surgical implant, bandage, or suture coated with a cocktail composition containing at least one recombinant bacteriophage. [Modes for carrying out the invention]

[0014] Detailed description of the invention Compositions, methods, and systems demonstrating remarkable speed and sensitivity, as well as increased treatment efficacy, for diagnosing bacterial infections are disclosed herein. Diagnosis can be achieved within a shorter timeframe than with currently available methods. This disclosure describes the use of genetically modified infectious agents in assays.

[0015] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention should have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by circumstances, singular terms should include plurals, and plural terms should include singulars. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, as well as the techniques described herein, are well known and commonly used in the art. Known methods and techniques are generally performed, unless otherwise indicated, by common methods well known in the art and as described in the various general and more specific references considered throughout this specification. Enzyme reactions and purification techniques are performed as generally achieved in the art or as specified by the manufacturer, as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is well known and commonly used in the art.

[0016] The following terms should be understood to have the meanings set forth below, unless otherwise indicated:

[0017] In this specification, the terms "a," "an," and "the" may refer to one or more unless otherwise noted.

[0018] The use of the term “or” is used to mean “and / or” unless it is explicitly indicated that only substitutes are being referred to, or that the substitutes are mutually exclusive; however, this disclosure supports the definitions that refer only to substitutes and to “and / or.” As used herein, “another” may mean at least the second or more.

[0019] Throughout this application, the term “approximately” is used to indicate that the value includes inherent error variations in the device or method used to determine the value, or variations present between samples.

[0020] The terms “solid support” or “support” refer to a structure that provides a substrate and / or surface upon which biomolecules can be bound. For example, a solid support may be an assay well (i.e., a microtiter plate or multiwell plate, etc.), or a solid support may be a position on a filter, array, or mobile support such as beads or membranes (e.g., a filter plate, latex particles, paramagnetic particles, or a lateral flow strip).

[0021] The term "binding factor" refers to a molecule that can specifically and selectively bind to a second (i.e., different) target molecule. The interaction may be non-covalent as a result of, for example, hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction may be covalent. The term "soluble binding factor" refers to a binding factor that does not associate (i.e., covalently or non-covalently) with a solid support.

[0022] As used herein, “analyte” means the molecule, compound, or cell being measured. The analyte of interest may interact with binding factors in certain embodiments. As used herein, the term “analyte” may mean the protein or peptide of interest. The analyte may be an agonist, antagonist, or modulator. Alternatively, the analyte may not have any biological effect. Examples of analytes include small molecules, sugars, oligosaccharides, lipids, peptides, peptide mimes, and organic compounds.

[0023] The terms “indicator moiety,” “detectable biomolecule,” “reporter,” or “indicator protein product” refer to molecules that can be measured in a quantitative assay. For example, the indicator moiety may include an enzyme that can be used to convert a substrate into a measurable product. The indicator moiety may be an enzyme (e.g., luciferase) that catalyzes a reaction that produces bioluminescent emission. Alternatively, the indicator moiety may be a radioisotope that can be quantified. Alternatively, the indicator moiety may be a fluorophore. Alternatively, other detectable molecules may be used.

[0024] As used herein, “bacteriophage” or “phage” includes one or more types of bacterial viruses. In this disclosure, the terms “bacteriophage” and “phage” refer to viruses that can invade living bacteria, fungi, mycoplasmas, protozoa, yeasts, and other microscopic living organisms, and that use such organisms to replicate themselves, including viruses such as mycobacteriophages (e.g., with respect to TB and paraTB), mycophages (e.g., with respect to fungi), mycoplasma phages, and any other terms. Here, “microscopic” means having a maximum dimension of 1 millimeter or less. Bacteriophages are viruses that have evolved in nature to use bacteria as a means of replication. Phages do this by attaching the phage itself to a bacterium, injecting its DNA (or RNA) into the bacterium, and inducing the bacterium to replicate the phage hundreds or even thousands of times. This is also known as phage amplification.

[0025] As used herein, “late gene region” refers to a region of the viral genome that is transcribed late in the viral life cycle. Typical late gene regions include the most abundantly expressed genes (e.g., structural proteins assembled into the bacteriophage particle). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, in phage T7, the late genes (synonymous with class III) are transcribed from, for example, 8 minutes after infection until lysis, while class I genes (e.g., RNA polymerase) are transcribed early, between 4 and 8 minutes, and class II genes are transcribed between 6 and 15 minutes, so the timing of II and III genes overlaps. A late promoter is a promoter that is naturally located and active in such late gene regions.

[0026] As used herein, “culturing for enrichment” means conventional culturing (e.g., incubation in a medium favorable to microbial growth) and should not be confused with other possible uses of the term “enrichment” (e.g., enrichment by taking out the liquid components of a sample and concentrating the microorganisms contained therein) or other forms of enrichment that do not involve conventional promotion of microbial growth. Culturing for enrichment over a period of time may be used in some embodiments of the methods described herein.

[0027] As used herein, “recombinant” means otherwise found. This term refers to genetic (i.e., nucleic acid) modification, typically performed in a laboratory setting, to combine genetic material that does not exist otherwise. This term is used herein as interchangeable with the term "modified."

[0028] As used herein, "RLU" refers to the relative luminescence measured by a luminometer (e.g., GLOMAX® 96) or a similar light-detecting instrument. For example, the detection of a reaction between luciferase and a suitable substrate (e.g., NANOLUC® and NANO-GLO®) is often reported by the detected RLU.

[0029] As used herein, “time to result” refers to the total time from the start of sample incubation to the generation of results. Time to result does not include any confirmation test time. Data collection may be performed at any time after the results have been generated.

[0030] sample Each of the embodiments of the methods and systems of this disclosure can enable rapid and highly sensitive diagnosis and treatment of biofilm-related infections. For example, the methods of this disclosure can be performed with excellent results in a shortened time period.

[0031] The microorganisms of interest detectable by this disclosure include, but are not limited to, bacterial cells present in biological samples. In some embodiments, the biological sample may be debridenated tissue, blood, serum, plasma, mucosal lymphoid tissue, articular fluid, pleural fluid, saliva, and urine. In some embodiments, irrigation is used to collect the biological sample. Irrigation is a flow of solution (e.g., saline solution) through an open wound or implanted orthotic device. Thus, in some embodiments, the biological sample may be a wound cleanser or orthotic cleanser.

[0032] The sample may be a liquid, solid, or semi-solid. The sample may be a swab of a solid surface (e.g., a medical implant). In other embodiments, the sample may be taken from the biological fluid surrounding a medical implant. Medical implants include, but are not limited to, central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthroprostheses, fracture fixation devices, inflatable penile implants, breast implants, cochlear implants, intraocular lenses, and dental implants.

[0033] In some embodiments, a sample may be used directly in the detection method of the present disclosure without preparation, concentration, dilution, purification, or isolation. For example, liquid samples, including but not limited to biological fluids, may be assayed directly. A sample may be diluted or suspended in a solution, including but not limited to a buffer solution or bacterial culture medium. A sample that is solid or semi-solid may be suspended in a liquid by chopping, mixing, or macerating the solid in the liquid. The sample should be maintained within a pH range that facilitates bacteriophage adhesion to host bacterial cells. The sample may also contain Na + Mg 2+ and Ca 2+ The sample should contain appropriate concentrations of divalent and monovalent cations, including but not limited to these. Preferably, the sample is maintained at a temperature that preserves the viability of any pathogenic cells contained within the sample.

[0034] In some embodiments of the detection assay, the sample is maintained at a temperature that preserves the viability of any pathogen cells present in the sample. For example, it is preferable to maintain the sample at a temperature that facilitates bacteriophage attachment during the process of bacteriophage attachment to bacterial cells. It is also preferable to maintain the sample at a temperature that promotes bacteriophage replication and host lysis during the process of bacteriophage replication within infected bacterial cells or lysis of such infected cells. Such temperatures are at least about 25 degrees Celsius (°C), more preferably about 45°C or less, and most preferably about 37°C.

[0035] The assay can include a variety of suitable control samples. For example, control samples that do not contain bacteriophages, or control samples that contain bacteriophages but do not contain bacteria, can be assayed as controls for background signal levels.

[0036] Indicator Recombinant Bacteriophage As described in more detail herein, the compositions, methods, and systems of this disclosure may include infectious agents for use in the diagnosis of biofilm-related infections. In certain embodiments, this disclosure may include a composition comprising a recombinant indicator bacteriophage, the bacteriophage genome being genetically modified to include an indicator or reporter gene.

[0037] Recombinant indicator bacteriophages may include a genetic construct containing a reporter or indicator gene. In certain embodiments of recombinant indicator bacteriophages, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during bacteriophage replication after infection with a host bacterium results in a soluble indicator protein product. In some examples, the genetic construct may further include an exogenous promoter. In certain embodiments, the genetic construct 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 contain a gene for a major capsid protein.

[0038] Some embodiments involve designing (and, if necessary, preparing) a sequence for homologous recombination downstream of the major capsid protein gene. Other embodiments involve designing (and, if necessary, preparing) a sequence for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequence includes a codon-optimized reporter gene after an untranslated region. The untranslated region may include a late-stage phage gene promoter and a ribosome entry site.

[0039] In some embodiments of recombinant indicator phages, an additional exogenous late promoter (a class III promoter, e.g., derived from phage K, T7, or T4) has high affinity for the RNA polymerase of the same native phage (e.g., phage K, T7, or T4, respectively) that transcribes the genes for the structural proteins assembled into the phage particle. These proteins are the most abundant proteins produced by the phage, as each phage particle contains dozens or hundreds of copies of these molecules. The use of a viral late promoter can ensure optimally high levels of expression of the indicator protein product. The use of a late viral promoter that is specific to or active in the native wild-type phage from which the indicator phage originates (e.g., a phage K, T4, or T7 late promoter in a system based on phage K, T4, or T7) can further ensure optimal enzyme expression. The use of standard bacterial (non-viral / non-phage) promoters can be detrimental to expression in some cases, because such promoters are often downregulated during phage infection (as the phage prioritizes bacterial resources for phage protein production). Therefore, in some embodiments, the phage is preferably engineered to encode an indicator protein product and be expressed at high levels.

[0040] In some embodiments, recombinant indicator phages are constructed from bacteriophages specific to biofilm-forming bacterial species. Bacterial cells detectable by this disclosure include, but are not limited to, all species of Staphylococcus, including S. aureus, Salmonella spp., Pseudomonas spp., Streptococcus spp., all strains of Escherichia coli, Listeria, including, but not limited to L. monocytogenes, Campylobacter spp., Bacillus spp., Bordetella pertussis, Campylobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens, Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Shigella sonnei, and Streptococcus spp.

[0041] Additional microorganisms whose antibiotic resistance can be detected using the claimed method and system include Abiotrophia adiacens and Acinetobacter. baumanii, Actinomycetaceae, Bacteroides, Cytophaga and Flexibacter phylum, Bacteroides fragilis, Bordetella pertussis, Bordetella spp., Campylobacter jejuni and E. coli, Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Candida spp., Chlamydia pneumoniae, Chlamydia trachomatis, Clostridium spp., Corynebacterium spp., Cronobacter spp., Crypococcus neoformans, Cryptococcus spp., Cryptosporidium parvum, Entamoeba spp., Enterobacteriaceae group, Enterococcus casseliflavus-flavescens-gallinarum group, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus spp., Escherichia coli and Shigella spp. group, Gemella spp., Giardia spp., Haemophilus influenzae, Klebsiella oxytoca、Klebsiella pneumoniae、Legionella pneumophila、Legionella spp.、Leishmania spp., Mycobacteriaceae, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Pseudomonads group, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus spp. .

[0042] In certain embodiments, the indicator bacteriophage is derived from a phage specific to Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, Streptococcus viridans, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, or Pseudomonas aeruginosa. In some embodiments, the indicator phage is derived from a bacteriophage that is highly specific to the particular pathogenic microorganism of interest.

[0043] As described herein, such phages can replicate inside bacteria to produce hundreds of progeny phages. Detection of indicator genes inserted into phages can be used as a measure of bacteria in a sample. S. aureus phages include, but are not limited to, phages K, SA1, SA2, SA3, SA11, SA77, SA187, Twort, NCTC9857, Ph5, Ph9, Ph10, Ph12, Ph13, U4, U14, U16, and U46. Well-studied E. coli phages include T1, T2, T3, T4, T5, T7, and Lambda; other E. coli phages available in ATCC collections include, for example, phiX174, S13, Ox6, MS2, phiV1, fd, PR772, and ZIK1. Alternatively, native phages can be isolated from various environmental sources. Sources for phage isolation can be selected based on the location where the target microorganism is expected to be found.

[0044] As described above regarding the compositions of the present invention, the phage is derived from T7, T4, T4-like, phage K, MP131, MP115, MP112, MP506, MP87, ISP, 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 with the phages disclosed above. In some aspects, the present invention includes recombinant phages comprising an indicator gene inserted into the late gene region of the phage. In some embodiments, the phage belongs to the genus Tequatrovirus or Kayvirus. In one embodiment, the recombinant phage is derived from phage K, ISP, or MP115. In certain embodiments, the recombinant phage is highly specific to a particular bacterium. For example, in a particular embodiment, the recombinant phage is highly specific to MRSA. In a particular embodiment, the recombinant phage can distinguish MRSA from at least 100 other types of bacteria.

[0045] In some embodiments, the selected wild-type bacteriophage is derived from a phage of the order Caudovirales. Caudovirales is an order of bacteriophages with a tail containing a double-stranded DNA (dsDNA) genome. Each virion of the order Caudovirales has an icosahedral head containing the viral genome and a flexible tail. The order Caudovirales includes five families of bacteriophages: Myoviridae (long contractile tail), Siphoviridae (long non-contractile tail), Podoviridae (short non-contractile tail), Ackermannviridae, and Herelleviridae. The term myovirus may be used to describe any bacteriophage having an icosahedral head and a long contractile tail, encompassing bacteriophages that fall within both the families Myoviridae and Herelleviridae.

[0046] Furthermore, phage genes that appear non-essential may possess unrecognized functions. For example, seemingly non-essential genes may have crucial functions in increasing burst size, such as minor cutting, fitting, or trimming functions in assembly. Therefore, deleting genes to insert indicators may be detrimental. Most phages can package DNA several percent larger than their native genome. This suggests that smaller indicator genes may be a more appropriate choice for modifying bacteriophages, particularly those with smaller genomes. OpLuc and NANOLUC® proteins are only about 20 kDa (approximately 500-600 bp to encode), while FLuc is about 62 kDa (approximately 1,700 bp to encode). Moreover, reporter genes should not be endogenously expressed by bacteria (i.e., not part of the bacterial genome), should generate a high signal-to-background ratio, and should be readily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, NANOLUC®, combined with Promega's NANO-GLO®, an imidazopyrazinon substrate (furimazine), can provide a robust signal with low background.

[0047] In some indicator phage embodiments, the indicator gene may be inserted into the untranslated region to avoid disruption of the functional gene while leaving the wild-type phage gene intact, which may result in a better fit when infecting non-laboratory bacterial strains. Furthermore, including stop codons in all three reading frames may help increase expression by reducing read-through (also known as leaky expression). This strategy may also eliminate the possibility of low-level production of fusion proteins that appear as background signals (e.g., luciferase) that cannot be isolated from the phage.

[0048] An indicator gene can express various biomolecules. The indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. In alternative embodiments, and as described in more detail herein, the luciferase is one of the following: Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as NANOLUC®.

[0049] Accordingly, in some embodiments, the present invention includes a genetically modified bacteriophage containing a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-natural indicator gene is under the control of a late promoter. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like a viral capsid protein, but is also not arrested, like an endogenous bacterial gene or even an early viral gene.

[0050] Genetic modifications to infectious agents may include insertions, deletions, or substitutions of small fragments of nucleic acids, substantial parts of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acids contain non-natural sequences. Non-natural indicator genes may be inserted into the bacteriophage genome so as to be under the control of the bacteriophage promoter. Therefore, in some embodiments, the non-natural indicator gene is not part of the fusion protein. That is, in some embodiments, the genetic modification may be configured such that the indicator protein product does not contain polypeptides of the wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present invention encompasses a method for detecting a bacterium of interest, the method comprising the step of incubating a test sample with such recombinant bacteriophage.

[0051] In some embodiments, the expression of an indicator gene in progeny bacteriophages after infection with a host bacterium results in the production of a free soluble protein product. In some embodiments, the non-native indicator gene is not contiguous with the gene encoding the phage structural protein and therefore does not produce a fusion protein. Unlike systems that use a fusion of the indicator protein product to a capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., soluble luciferase). In some embodiments, the indicator or reporter ideally does not contain a bacteriophage structure; that is, the indicator or reporter is not attached to the phage structure. Therefore, the gene of the indicator or reporter is not fused with other genes in the recombinant phage genome. This can significantly increase the sensitivity of the assay (down to a single bacterium), simplify the assay, and allow the assay to be completed in two hours or less, in some embodiments, in contrast to the several hours required due to the additional purification steps necessitated by constructs producing detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to constraints on protein folding, which can alter, for example, the conformation of the enzyme active site or access to the substrate. If the concentration is 1,000 bacterial cells per 1 mL of sample, for example, less than 4 hours may be sufficient for the assay.

[0052] Furthermore, fusion proteins, by definition, limit the number of sites that attach to the protein subunits in the bacteriophage. For example, the use of a commercially available system designed to function as a platform for fusion proteins results in approximately 415 copies of fusion sites, corresponding to approximately 415 copies of the 10B capsid protein in each T7 bacteriophage particle. Without this constraint, it can be expected that infected bacteria will express more than a few additional copies of indicator protein products (e.g., luciferase) than can fit in the bacteriophage. Moreover, larger fusion proteins, such as capsid-luciferase fusions, can inhibit the assembly of bacteriophage particles, thus producing fewer bacteriophage offspring. Therefore, soluble, non-fusion indicator gene products may be preferable.

[0053] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may be light-emitting and / or 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 can function as indicator protein products. In some embodiments, firefly luciferase is the indicator protein product. In some embodiments, Oplophorus luciferase is the indicator portion. In some embodiments, NANOLUC® is the indicator protein product. Other manipulated luciferases or other enzymes that produce a detectable signal may also be suitable indicator portions.

[0054] In some embodiments, the use of soluble indicator protein products eliminates the need to remove stock phages contaminating lysates of infected sample cells. The fusion protein system ensures that any bacteriophage used to infect sample cells possesses the attached indicator protein product and becomes indistinguishable from daughter bacteriophages that similarly contain the indicator protein product. Since the detection of sample bacteria relies on the detection of newly created (de novo synthesized) indicator protein products, the use of fusion constructs requires an additional step to separate older (stock phage) indicators from newly synthesized indicators. This can be achieved by washing infected cells multiple times prior to the completion of the bacteriophage life cycle, inactivating excess stock phages after infection by physical or chemical means, and / or chemically modifying stock bacteriophages with binding sites (e.g., biotin) that can be bound and / or separated thereafter (e.g., by streptavidin-coated Sepharose beads). However, even if all these attempts are made to remove them, if high concentrations of stock phages are used to ensure infection of a small number of sample cells, the stock phages may remain and create background signals that can obscure the detection of signals from infected cell progeny phages.

[0055] In contrast, since stock phage compositions do not contain any indicator protein products, purification of stock phages from the final lysate is unnecessary due to the soluble indicator protein products expressed in some embodiments of the present invention. Therefore, any indicator protein products present after infection (which indicate the presence of infected bacteria(s)) must be generated de novo. To take advantage of this benefit, phage production and preparation may include the purification of phages from any free indicator protein products produced during the production of recombinant bacteriophages in bacterial cultures. Some embodiments of phages according to the present invention can be purified using standard bacteriophage purification techniques such as sucrose density gradient centrifugation, cesium chloride isodense density gradient centrifugation, HPLC, size exclusion chromatography, and dialysis or derivative techniques (e.g., Amicon brand concentrators - Millipore, Inc.). Stock phage particles can be separated from contaminating luciferase proteins produced after phage proliferation in the bacterial host using cesium chloride isodense ultracentrifugation as part of the preparation of recombinant phages of this disclosure. In this way, the recombinant bacteriophage of the present invention substantially contains no luciferase produced during bacterial production. Removal of residual luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with the test sample.

[0056] In some embodiments of modified recombinant bacteriophages, the late promoter (class III promoter) has high affinity for the RNA polymerase of the same bacteriophage, which transcribes the genes for structural proteins that are assembled into bacteriophage particles. These proteins are the most abundant proteins produced by the phage, as each bacteriophage particle contains dozens or hundreds of copies of these molecules. The use of a viral late promoter can ensure optimally high levels of expression of the luciferase indicator protein product. The use of a late viral promoter that is specific to or active in the original wild-type bacteriophage from which the indicator phage originates can further ensure optimal expression of the indicator protein product. For example, an indicator phage specific to MRSA may include a consensus late gene promoter derived from the S. aureus phage ISP. The use of standard bacterial (non-viral / non-bacteriophage) promoters can be detrimental to expression in some cases because such promoters are often downregulated during bacteriophage infection (because bacteriophages prioritize bacterial resources for phage protein production). Therefore, in some embodiments, phages are manipulated to preferably encode and express at high levels a soluble (free) indicator portion, using genomic arrangements that do not limit expression to the number of subunits of the phage structural components.

[0057] The compositions of this disclosure 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 capable of encoding and expressing the same or different indicator proteins. In some embodiments, the cocktail of indicator bacteriophages may include at least two different types of recombinant bacteriophages.

[0058] Therapeutically effective bacteriophages As described in more detail herein, the compositions, methods, and systems of this disclosure may include infectious agents for use in the diagnosis and treatment of biofilm-related infections. In certain embodiments, this disclosure includes a therapeutically effective bacteriophage. In some embodiments, the therapeutically effective bacteriophage is a wild-type bacteriophage. In other embodiments, the therapeutically effective bacteriophage is a recombinant bacteriophage, and the bacteriophage genome is genetically modified to include a genetic construct containing a gene encoding an enzyme.

[0059] In certain embodiments, the gene does not encode a fusion protein. For example, in certain embodiments, the expression of an enzyme during bacteriophage replication after infection with a host bacterium results in the production of a free enzyme. In some examples, the genetic construct may further include an exogenous promoter. In certain embodiments, the genetic construct may be inserted into the late gene region of the bacteriophage. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. The late gene region may be a class III gene region and may contain the gene for the major capsid protein.

[0060] In some embodiments of the modified phage, an additional exogenous late promoter (a class III promoter, e.g., derived from phage K, T7, or T4) has high affinity for the RNA polymerase of the same native phage (e.g., phage K, T7, or T4, respectively) that transcribes the genes for structural proteins assembled into the phage particle. These proteins are the most abundant proteins produced by the phage, as each phage particle contains dozens or hundreds of copies of these molecules. The use of a viral late promoter can ensure optimally high levels of enzyme expression. The use of a late viral promoter that is specific to or active in the original wild-type phage from which the therapeutic phage is derived (e.g., a phage K, T4, or T7 late promoter in a system based on phage K, T4, or T7) can further ensure optimal enzyme expression. The use of standard bacterial (non-viral / non-phage) promoters can be detrimental to expression in some cases because such promoters are often downregulated during phage infection (because the phage prioritizes bacterial resources for phage protein production). Therefore, in some embodiments, the phage is preferably engineered to encode an enzyme and be expressed at high levels.

[0061] Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix, allowing bacteria to adhere to inactive (e.g., implantable medical devices) or living surfaces. Furthermore, biofilms have been shown to increase the chances of infection in a target and to be resistant to both antibiotics and phagocytic cells. Bacteriophages are known to produce enzymes that can disrupt the extracellular matrix, and thus bacteria within biofilms can be targeted.

[0062] Bacteriophages are known to naturally produce enzymes that can disrupt the biofilm matrix. In some cases, the bacteriophage genome contains genes encoding soluble enzymes intended to penetrate the cell wall. Such enzymes can hydrolyze the bacterial cell wall, thus allowing the phage to escape the cell. The composition of the extracellular matrix surrounding biofilms is similar to that of the bacterial cell wall; therefore, increasing the expression of bacteriophage enzymes may be advantageous for treating biofilm-related infections. In some embodiments, bacteriophages are modified to increase the levels of enzymes produced (e.g., lysin and endolysin) or to enable the production of different enzymes. Furthermore, some bacteriophages (e.g., T4) have additional enzymes present in the bacteriophage tail that further aid in bacterial cell wall penetration. However, in the natural infection process, such enzymes are shielded until the tail is reconstituted during the infection cycle.

[0063] In some embodiments, bacteriophages are modified to enable the production of enzymes specific to the microorganism of interest. In some embodiments, bacteriophages are genetically engineered to contain pathogenicity-enhancing factors. In some embodiments, the gene encoding the enzyme is inserted into the bacteriophage genome. After infection of bacterial cells, the inserted gene encoding the enzyme is produced at high levels and released from the lysed bacterial cells into the extracellular matrix of the biofilm. In certain embodiments, the enzyme is glycosidase, amidase, or endopeptidase. Glycosidase, amidase, and endopeptidase are the main enzymes produced by phages for cell lysis or DNA injection through the cell wall. For example, in some embodiments, a recombinant bacteriophage for therapeutic purposes may be specific to Staphylococcus infection. It contains dispersin B (DspB), a glycoside hydrolase enzyme produced by Actinobacillus actinomycetemcomitans that hydrolyzes β-1,6-N-acetyl-D-glucosamine. Staphylococcus-specific phages can be used to treat biofilm-related Staphylococcus infections.

[0064] In other embodiments, bacteriophages are modified to enhance the production of naturally occurring enzymes. For example, such enzymes may be recombinatively inserted into the phage genome to create a fusion protein, which can then be soluble and spread to the virion surface or from the infected bacterium into a biofilm. This can be done by homologous recombination cloning, CRISPR-based cloning, or any other method generally known in the art.

[0065] Methods for using bacteriophages for the diagnosis and treatment of biofilm-related infections As noted herein, in certain embodiments, the present invention may include a method for using infectious particles to detect microorganisms. The method of the present invention can be embodied in a variety of ways.

[0066] In some embodiments, diagnostic recombinant bacteriophages can determine the bacterial strain(s) present in a biofilm-associated infection. Detection of the bacterial strain(s) present in the biofilm is crucial for determining appropriate treatment of the infection.

[0067] In one embodiment, the present invention includes a method for diagnosing and treating a biofilm-related infection in an implanted subject, comprising: (i) providing a biological sample taken from an implanted subject; (ii) diagnosing the subject with a biofilm-related infection by detecting the presence of at least one target microorganism, comprising (a) contacting at least one aliquot of the biological sample with a certain amount of a reporter cocktail composition containing at least one recombinant bacteriophage; (b) detecting a signal generated after replication of the recombinant bacteriophage, wherein the detection of the signal indicates the presence of the target microorganism in the sample; and (iii) selecting a therapeutic cocktail composition based on the diagnosis of (a) step (ii); administering a therapeutically effective amount of the therapeutic cocktail composition containing at least one bacteriophage, wherein the bacteriophage is specific to the detected target microorganism; and (c) administering at least one additional therapeutic agent as needed, comprising treating the subject diagnosed with a biofilm-related infection.

[0068] In a particular embodiment, the step of diagnosing a subject with a biofilm-associated infection includes the step of detecting at least one microorganism of interest. In a particular embodiment, a method for detecting at least one microorganism of interest in a sample includes the steps of: incubating the sample with a bacteriophage that infects the bacteriophage of interest, wherein the bacteriophage comprises a genetic construct, and the genetic construct comprises an indicator gene, such that the expression of an indicator gene during bacteriophage replication after infection with the bacteriophage of interest results in the production of 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. In a particular embodiment, the genetic construct further comprises an additional exogenous promoter.

[0069] In some embodiments, the assay can be made to utilize a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments using the recombinant bacteriophage (i.e., indicator bacteriophage) of the present invention can be made to accommodate sample types, sample sizes, and assay formats, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 1 Rapid detection of specific bacterial strains can be enabled with a total assay time of less than 2, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, or 26.0 hours. For example, the amount of time required may be slightly shorter or longer depending on the bacteriophage strain and the bacterial strain to be detected in the assay, the type and size of the sample to be examined, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target bacterial contaminants. For example, detection of Gram-negative strains (e.g., E. coli, Klebsiella, Shigella) can be completed in total assay times of less than 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 hours without detection of antibiotic resistance, or in total assay times of less than 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 hours with detection of antibiotic resistance. Detection of Gram-positive strains can be completed in total assay times of less than 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 hours without detection of antibiotic resistance, or in total assay times of less than 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5 hours with detection of antibiotic resistance.

[0070] Bacteriophages (e.g., phage K, ISP, MP115) can be engineered to express soluble luciferase during phage replication. Luciferase expression is driven by a viral capsid promoter (e.g., bacteriophage Pecentumvirus or T4 late promoter) to achieve high expression. Since stock phages are prepared to be luciferase-free, the luciferase detected in the assay should originate from the replication of progeny phages during bacterial cell infection. Therefore, it is generally not necessary to isolate the parent phage from the progeny phage.

[0071] In some embodiments, bacterial enrichment of the sample is not required prior to testing. In some embodiments, the sample may be enriched prior to testing by incubation under growth-promoting conditions. In such embodiments, the enrichment period may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or longer, depending on the sample type and size.

[0072] In some embodiments, the indicator bacteriophage comprises a detectable indicator protein product, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by the amplified signal produced by the indicator protein product. Thus, the method may include the step of detecting an indicator protein product produced during phage replication, and the detection of the indicator indicates the presence of the bacterium of interest in the sample.

[0073] In some embodiments, the present invention may include a method for detecting a target bacterium in a sample, comprising the steps of: incubating the sample with a recombinant bacteriophage that infects the target bacterium, wherein the recombinant bacteriophage includes an indicator gene inserted into the late gene region of the bacteriophage such that the expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in the production of a soluble indicator protein product; and detecting the indicator protein product, wherein the positive detection of the indicator protein product indicates the presence of the target bacterium in the sample. In some embodiments, the amount of indicator protein product detected corresponds to the amount of the target bacterium present in the sample. In some embodiments, the positive detection of a particular target bacterium is used to diagnose a subject with a biofilm-related infection.

[0074] As described in more detail herein, the compositions, methods, and systems of this disclosure can utilize a parent indicator bacteriophage at a range of concentrations to infect bacteria present in a sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly locate, bind to, and infect target bacteria present in very small numbers in the sample, such as 10 cells. In some embodiments, the phage concentration may be sufficient to locate, bind to, and infect target bacteria in less than one hour. In other embodiments, these events may occur in less than two, three, or four hours after the addition of the indicator phage to the sample. For example, in a particular embodiment, the bacteriophage concentration for the incubation step may be 1 × 10⁻⁶. 5 Exceeding PFU / mL, 1 × 10 6 exceeding PFU / mL or 1 × 10⁻¹⁰ 7 exceeding PFU / mL or 1 × 10⁻¹⁰ 8 Exceeding PFU / mL

[0075] In certain embodiments, the recombinant stock phage composition may be purified to be free from any residual indicator proteins that may be produced after the production of the phage stock. Thus, in certain embodiments, the recombinant bacteriophage may be purified using a sucrose gradient or cesium chloride isodensity gradient centrifugation prior to incubation with the sample. If the infectious agent is a bacteriophage, this purification may have the added benefit of removing bacteriophages that do not possess DNA (i.e., empty phages or "ghosts").

[0076] In some embodiments of the method of the present invention, the microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or more target microorganisms may be applied directly to an assay vessel (e.g., a spin column, a microtiter well, or a filter), and the assay is performed in that assay vessel. Various embodiments of such assays are disclosed herein.

[0077] In some embodiments, at least one aliquot of a biological sample is contacted with a certain amount of an indicator bacteriophage cocktail composition. In a particular example, the indicator cocktail composition contains at least one recombinant bacteriophage specific to a particular bacterium of interest. In other embodiments, the indicator cocktail composition contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten types of recombinant bacteriophages specific to a particular bacterium of interest. In a particular embodiment, the step of diagnosing a subject with a biofilm-associated infection further includes the step of contacting multiple aliquots of a biological sample with multiple indicator cocktail compositions. In some examples, each indicator cocktail composition is specific to a different microorganism of interest. For example, a first aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to Enterococcus faecalis, and a second aliquot may be contacted with Staphylococcus A third aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to *Aureus*, a fourth aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to *Staphylococcus epidermidis*, a fifth aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to *Streptococcus viridans*, a sixth aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to *Escherichia coli*, a seventh aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to *Proteus mirabilis*, and an eighth aliquot may be contacted with a recombinant bacteriophage cocktail composition specific to *Pseudomonas aeruginosa*. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aliquots of a biological sample are brought into contact with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different reporter cocktail compositions.

[0078] Aliquotes of the test sample may be directly distributed into the wells of a multiwell plate, an indicator phage may be added, and after a sufficient period for infection, lysis buffer may be added along with a substrate for the indicator portion (e.g., a luciferase substrate for a luciferase indicator) to assay for the detection of the indicator signal. Some embodiments of the method may be performed in a filter plate or a 96-well plate. Some embodiments of the method may be performed with or without concentration of the sample before infection with the indicator phage.

[0079] For example, in many embodiments, multi-well plates are used to perform the assay described above. The choice of plate (or any other container in which the detection step may be performed) can affect the detection step. For example, some plates may contain a colored or white background, which can affect the detection of light emission. Generally, white plates have higher sensitivity but also produce a higher background signal. Other colors of plates may produce a lower background signal but may have slightly lower sensitivity. Furthermore, one reason for the background signal is light leakage from one well to another adjacent well. Some plates have white wells, while the rest of the plate is black. This allows for a high signal within the wells but prevents light leakage from well to well, and thus can reduce the background. Therefore, the choice of plate or other assay container can affect the sensitivity and background signal for the assay described above.

[0080] The methods of this disclosure may include various other steps to increase sensitivity. For example, as described in more detail herein, the methods may include a step of washing the captured and infected bacteria after the addition of the bacteriophage, but before incubation, to remove any excess bacteriophage and / or luciferase or other reporter protein that may be introduced into the bacteriophage preparation.

[0081] In some embodiments, the detection of the target microorganism can be completed without the need to culture the sample as a way to increase the microbial population. For example, in certain embodiments, the total time required for detection is 28.0 hours, 27.0 hours, 26.0 hours, 25.0 hours, 24.0 hours, 23.0 hours, 22.0 hours, 21.0 hours, 20.0 hours, 19.0 hours, 18.0 hours, 17.0 hours, 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, less than 1.5 hours, or less than 1.0 hour. Minimizing the time to results is of significant importance in diagnostic testing.

[0082] In contrast to assays known in the art, the methods of this disclosure can detect individual microorganisms. Thus, in certain embodiments, the method can detect approximately 10 cells of a microorganism present in a sample. For example, in certain embodiments, the recombinant indicator bacteriophage is highly specific to Staphylococcus spp., E. coli strains, Shigella spp., Klebsiella spp., or Pseudomonas spp. In certain embodiments, the recombinant indicator bacteriophage can distinguish the target bacterium in the presence of other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a specific type in a sample. In certain embodiments, the recombinant indicator bacteriophage can detect approximately 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.

[0083] Therefore, aspects of this disclosure provide a method for detecting microorganisms in a test sample using indicator protein products. In some embodiments, if the microorganism of interest is a bacterium, the indicator protein product may be related to an infectious agent such as an indicator bacteriophage. The indicator protein product can react with a substrate to emit a detectable signal or an endogenous signal (e.g., a bioluminescent protein). In some embodiments, the detection sensitivity can reveal the presence of approximately 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, even a single cell of the microorganism of interest can produce a detectable signal. In some embodiments, the bacteriophage is phage K, ISP, or MP115. In certain embodiments, recombinant Staphylococcus spp.-specific bacteriophages are highly specific to Staphylococcus spp.

[0084] In some embodiments, indicator protein products encoded by recombinant indicator bacteriophages may be detectable during or after bacteriophage replication. Many different types of detectable biomolecules suitable for use as indicator moieties are known in the art and many are commercially available. In some embodiments, the indicator phage comprises an enzyme that functions as an 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 be able to emit light and / or be detectable by a change in color of an added substrate. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes can function as indicator moieties. 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 manipulated luciferases or other enzymes that produce a detectable signal could also be suitable indicator portions.

[0085] Therefore, in some embodiments, the recombinant indicator bacteriophage of the composition, method, or system is prepared from a wild-type bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an endogenous signal, such as a fluorescent protein (e.g., green fluorescent protein or others). The indicator may be able to emit light and / or be detectable by a change in color. In some embodiments, the indicator gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to produce a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is Oplophorus luciferase, firefly luciferase, sea urchin luciferase, External Gaussia luciferase, Lucia luciferase, or NANOLUC®, Rluc8. It is one of the manipulated luciferases, such as 6-535 or Orange Nano-lantern.

[0086] Indicator detection may include detection of light emission. In some embodiments, an indicator protein product (e.g., luciferase) is reacted with a substrate to generate a detectable signal. Signal detection can generally be achieved by any machine or device known in the art. In some embodiments, the signal may be detected using an In Vivo Imaging System (IVIS). The IVIS measures light emission by total flux using a CCD camera or CMOS sensor. Total flux = radiance (photons / second). Average radiance is photons / second / cm². 2It is measured as steradians. In other embodiments, signal detection can be achieved by a luminometer, spectrophotometer, CCD camera, or CMOS camera capable of detecting color changes and other light emissions. In some embodiments, the signal is measured as absolute RLU. In further embodiments, a high signal-to-background ratio (e.g., >2.0, >2.5, or >3.0) is required for a single cell or a small number of cells to be reliably detected.

[0087] In some embodiments, the indicator phage is genetically engineered to include a gene for an enzyme (e.g., luciferase) that is produced only during infection by a bacterium specifically recognized and infecting the phage. In some embodiments, the indicator portion is expressed later in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused with a phage structural protein that limits its copy number.

[0088] Accordingly, in some embodiments utilizing indicator phages, the present invention encompasses a method for detecting a target microorganism, the method comprising the steps of: capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phages; giving time for infection and replication to generate progeny phages and express a soluble indicator portion; and detecting the progeny phages, preferably the indicator, wherein the detection of the indicator indicates the presence of the bacterium in the sample.

[0089] For example, in some embodiments, the bacteria in the test sample may 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 some embodiments, infectious agents (e.g., indicator phages) are added in a minimum volume to the sample directly captured in the filter. In some embodiments, the microorganisms captured on the filter or plate surface are then washed once or multiple times to remove excess unbound infectious agents. In some embodiments, a culture medium (e.g., Luria-Bertani (LB) broth, buffered peptone water (BP)) is used. W) or trypsin soy broth or tryptone soy broth (TSB), BrainHeart Infusion (BHI)) further incubation Adding the indicator phage over an incubation period may enable replication of bacterial cells and phages, as well as high-level expression of the gene encoding the indicator portion. However, a remarkable aspect of some embodiments of the assay is that the incubation step with the indicator phage only needs to be long enough for a single phage life cycle. A single replication cycle of the indicator phage may be sufficient to facilitate highly sensitive and rapid detection according to some embodiments of the present invention.

[0090] In some embodiments, aliquots of a test sample containing bacteria may be applied to a spin column, and after infection with recombinant bacteriophages and optional washing to remove any excess bacteriophages, the amount of soluble indicator detected is proportional to the amount of bacteriophages produced by the infected bacteria.

[0091] The soluble indicator (e.g., luciferase) released into the surrounding liquid during the lysis of the above-mentioned bacteria can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter, the filtrate is collected in a new container for an assay (e.g., in a luminometer), and then a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

[0092] In various embodiments, the parental phage can be purified before being used for incubation with the test sample, so that the purified parental indicator phage does not contain the detectable indicator itself. Late (class III) gene expression occurs in the later stages of the viral life cycle. In some embodiments of the present invention, the parental phage can be purified to eliminate any existing indicator proteins (e.g., luciferase). In some embodiments, the expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. Therefore, in many embodiments, it is not necessary to separate the parent from the progeny phage prior to the detection step. In some embodiments, the microorganism is a bacterium, and the indicator phage is a bacteriophage. In some embodiments, the indicator protein product is freed soluble luciferase released upon lysis of the host microorganism.

[0093] The assay can be performed in various ways. In one embodiment, the sample is added to at least one well in a 96-well plate, incubated with the phage, lysed, incubated with the substrate, and then read. In another embodiment, the sample is added to a 96-well filter plate before incubation with the phage, the plate is centrifuged, and the culture medium is added to the bacteria collected on the filter. In yet another embodiment, the sample is captured in at least one well of a 96-well plate using an antibody before incubation with the phage, washed with culture medium to remove excess cells.

[0094] In some embodiments, bacterial lysis may occur before or during the detection step. Experiments suggest that, in some embodiments, infected, unlyzed cells may be detectable after the addition of the luciferase substrate. Presumably, without complete cell lysis, luciferase can leave the cells and / or the luciferase substrate can enter the cells. For example, in some embodiments, the luciferase substrate is cell-permeable (e.g., flimazine). Thus, lysis is required for detection in embodiments utilizing a spin filter system, where only the luciferase released into the lysate (not the luciferase still inside the intact bacteria) is analyzed in the luminometer. However, lysis is not required for detection in embodiments utilizing a filter plate or 96-well plate containing the sample in solution or suspension, where the original plate filled with intact and lysed cells is directly assayed in the luminometer.

[0095] In some embodiments, the reaction between the substrate and the indicator moiety (e.g., luciferase) can last for 60 minutes or longer, and detection at various time points may be desirable to optimize sensitivity. For example, in embodiments using a 96-well filter plate as a solid support and luciferase as an indicator, luminescence meter readings can be obtained initially and at 10 or 15 minute intervals until the reaction is complete.

[0096] Surprisingly, high concentrations of phage utilized for infection of test samples have been successful in achieving detection of very small numbers of target microorganisms within a very short time frame. Incubation of the test sample with the phage, in some embodiments, only needs to be long enough for a single phage life cycle. In some embodiments, the bacteriophage concentration for this incubation step is 1.0×10 6 、2.0×10 6 、3.0×10 6 、5.0×10 6 、6.0×10 6 、7.0×10 6 、8.0×10 6 、9.0×10 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 greater than 1.0×10 8 PFU / mL.

[0097] The success with such high concentrations of phages is surprising, as many phages have previously been associated with "lysis from without," which kills target cells and thereby prevents the generation of useful signals from initial phage assays. In addition to removing any phage-related contaminating luciferases, this clean-up can also remove ghost particles (particles that have lost DNA), so the clean-up of the prepared phage stock described herein (e.g., clean-up by sucrose gradient or cesium chloride isodensity gradient ultracentrifugation) can help mitigate this problem. Ghost particles can lyse bacterial cells by "lysis from without," prematurely killing the cells and thereby preventing the generation of indicator signals. Electron microscopy demonstrates that crude phage lysates (i.e., before cesium chloride clean-up) can have more than 50% ghost particles. Such ghost particles can contribute to the premature death of microorganisms via the action of many phage particles puncturing the cell membrane. Therefore, ghost particles may have contributed to the previous problem where high PFU concentrations were reported to be harmful. Furthermore, very clean phage preps allow the assay to be performed without a washing step, which in turn allows the assay to be performed without an initial enrichment step. Some embodiments include an initial enrichment step, and in some embodiments, this enrichment step allows for a shorter enrichment incubation time.

[0098] Some embodiments of the testing method may further include confirmatory assays. Typically, various assays for confirming initial results at a later stage are known in the art. For example, the sample can be cultured (e.g., by selective chromogenic plating), PCR can be used to confirm the presence of microbial DNA, or other confirmatory assays can be used to confirm the initial results.

[0099] In certain embodiments, the method of the present disclosure combines the use of a binder (e.g., an antibody) with detection using an infectious agent to purify and / or concentrate a target microorganism, such as Staphylococcus spp., from a sample. For example, in certain embodiments, the present invention is a method for detecting a target microorganism in a sample, comprising the steps of capturing the microorganism from the sample on a prior support using a capture antibody specific to the target microorganism, such as Staphylococcus spp.; and the sample , a step of incubating with a recombinant bacteriophage that infects Staphylococcus spp., wherein the recombinant bacteriophage contains an indicator gene inserted into the late gene region of the bacteriophage such that the expression of the indicator gene during bacteriophage replication after infection of the host bacterium yields a soluble indicator protein product; and a step of detecting the indicator protein product, wherein positive detection of the indicator protein product yields Staphylococcus The method includes a step that indicates the presence of spp. in the sample.

[0100] In some embodiments, synthetic phages are designed to optimize desirable traits for use in pathogen detection assays. In some embodiments, bioinformatics and prior analysis of genetic modifications are used to optimize desirable traits. For example, in some embodiments, the gene encoding the phage tail protein may be optimized to recognize and bind to a specific species of bacteria. In other embodiments, the gene encoding the phage tail protein may be optimized to recognize and bind to an entire genus of bacteria or a specific group of species within a genus. In this way, the phage may be optimized to detect a broader or narrower group of pathogens. In some embodiments, synthetic phages may be designed to improve the expression of indicator genes. Moreover and / or alternatively, in some examples, synthetic phages may be designed to improve detection by increasing the phage burst size.

[0101] In some embodiments, phage stability may be optimized to improve shelf life. For example, enzyme solubility may be increased to enhance subsequent phage stability. In addition, and / or alternatively, the thermal stability of the phage may be optimized. Thermally stable phages better preserve functional activity during storage, thereby improving shelf life. Therefore, in some embodiments, thermal stability and / or pH tolerance may be optimized.

[0102] In some embodiments, genetically modified or synthetically derived phages include a detectable indicator. In some embodiments, the indicator is luciferase. In some embodiments, the phage genome includes an indicator gene (e.g., a luciferase gene or another gene encoding a detectable indicator).

[0103] In some embodiments, the detection of recombinant bacteriophages is used to diagnose the presence of biofilm-related infections and to identify specific strains that cause biofilms. The diagnosis can then be used to select appropriate treatment.

[0104] In certain embodiments, the therapeutic cocktail composition is selected based on the determined diagnosis. For example, if Staphylococcus spp. is detected in the diagnosis of interest, a therapeutic cocktail composition containing a recombinant bacteriophage specific to Staphylococcus spp. is selected. In other embodiments, a broad-spectrum therapeutic cocktail composition is selected to treat multiple potential infections.

[0105] In some embodiments, the therapeutic cocktail composition comprises at least one type of bacteriophage. In other embodiments, the therapeutic cocktail composition comprises at least two, three, four, five, six, seven, eight, nine, or ten types of bacteriophages. These bacteriophages may be specific to the same species of bacteria or to different species of bacteria. In some embodiments, the bacteriophage is a wild-type bacteriophage. In other embodiments, the bacteriophage is a recombinant bacteriophage.

[0106] Biofilm-associated infections are difficult to treat because typical antimicrobial agents (e.g., antibiotics) cannot break down the biofilm. As an alternative or complement to antibiotic treatment, genetically modified bacteriophages can be used to express enzymes that can hydrolyze bacterial cells. Such phages can infect bacterial cells in the biofilm, replicate to produce progeny phages, and also produce enzymes that can break down the biofilm. As the infection progresses, the progeny bacteriophages continue to infect other bacterial cells, which then release enzymes into the environment, thereby removing the biofilm.

[0107] In some embodiments, a therapeutically effective dose of the therapeutic cocktail composition is administered to a subject diagnosed with a biofilm-related infection. In some embodiments, the therapeutic cocktail composition is administered intravenously (e.g., to treat prosthetic heart valve infection). In other embodiments, the therapeutic cocktail composition is administered topically (e.g., to treat prosthetic joint infection). In some embodiments, repeated doses of the therapeutic cocktail composition are administered. The frequency of doses may vary based on the severity of the infection, the specific phage, and the route of administration. For example, the therapeutic cocktail composition may be administered every 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours. The therapeutically effective dose of the cocktail composition also varies depending on which phage is used. In some embodiments, a therapeutically effective dose of the therapeutic cocktail composition is 1.0 × 10⁻⁶ 6 , 1.0 × 10 7 , 1.0 × 10 8 , 1.0 × 10 9 , 1.0 × 10 10 , 1.0 × 10 11 , 1.0 × 10 12 It contains at least one therapeutic phage having a concentration exceeding [a certain value].

[0108] In additional embodiments, at least one additional therapeutic agent is administered. In some embodiments, the additional therapeutic agent is an antibiotic. Non-limiting examples of antibiotics that may be used in the present invention include aminoglycosides, carbasephalosporins, carbapenems, cephalosporins, glycopeptides, macrolides, monobactams, penicillins, beta-lactam antibiotics, quinolones, bacitracin, sulfonamides, tetracyclines, streptogramin, chloramphenicol, clindamycin and lincosamides, cephamycin, lincomycin, daptomycin, oxazolidinone and glycopeptide antibiotics.

[0109] In another aspect, the disclosure includes a method for preventing or inhibiting an infection in a subject, comprising the step of applying a cocktail composition comprising at least one recombinant bacteriophage to a surgical implant, bandage, or suture. In certain examples, the cocktail composition comprises a therapeutic recombinant bacteriophage. In further embodiments, the cocktail composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 types of recombinant bacteriophages. The recombinant bacteriophages constituting the cocktail composition may be specific to the same or different species of bacteria.

[0110] In yet another aspect, the disclosure includes surgical implants, bandages, or sutures coated with a cocktail composition comprising at least one recombinant bacteriophage. In certain examples, the cocktail composition comprises a therapeutic recombinant bacteriophage. In further embodiments, the cocktail composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 types of recombinant bacteriophages. The recombinant bacteriophages comprising the cocktail composition may be specific to the same or different species of bacteria.

[0111] Determination of antibiotic resistance In some aspects, the present invention includes methods for detecting antibiotic resistance in microorganisms. In some embodiments, the present disclosure provides a method for detecting antibiotic-resistant microorganisms in a sample, comprising the steps of (a) contacting the sample with an antibiotic; (b) contacting the sample with an infectious agent, wherein the infectious agent comprises an indicator gene, is specific to the microorganism, and the indicator gene encodes an indicator protein product; and (c) detecting a signal produced by the indicator protein product, wherein the detection of the signal is used to determine antibiotic resistance.

[0112] The method may use an infectious agent to detect the microorganism of interest. For example, in a particular embodiment, the microorganism of interest is a bacterium and the infectious agent is a phage. The antibiotic referred to in this application may be any agent that is bacteriostatic (can inhibit the growth of microorganisms) or bactericidal (can kill microorganisms). Thus, in a particular embodiment, the method may include detecting the resistance of the microorganism of interest in a sample to an antibiotic by contacting the sample with the antibiotic and incubating the antibiotic-contacted sample with an infectious agent that infects the microorganism of interest. This is separate from an assay that detects the presence of a gene (e.g., PCR) or protein (e.g., antibody) that can confer antibiotic resistance, but does not test its functionality. Thus, this assay enables phenotypic detection, as opposed to genotypic detection.

[0113] In certain embodiments, the method may include the detection of functional resistance genes to antibiotics in a microorganism of interest in a sample. PCR enables the detection of antibiotic resistance genes; however, PCR cannot distinguish between bacteria with functional antibiotic resistance genes and those with non-functional antibiotic resistance genes, thus leading to false-positive detection of antibiotic-resistant bacteria. The method embodied herein can positively detect bacteria with functional antibiotic resistance genes without positively detecting bacteria with non-functional antibiotic resistance genes. The method disclosed herein enables the detection of functional resistance to antibiotics even if the resistance mechanism is not a single gene / protein or mutation. Therefore, the method does not rely on knowledge of the resistance-mediating gene (PCR) or protein (antibody).

[0114] In certain embodiments, the infectious agent includes an indicator gene capable of expressing an indicator protein product. In some embodiments, the method may include a step of detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the microorganism of interest in the sample and that the microorganism is resistant to antibiotics. In some examples, the microorganism of interest is not isolated from the sample prior to testing for antibiotic resistance. In certain embodiments, the sample is an uncultured or unenriched sample. In some cases, the method for detecting antibiotic resistance can be completed within 5 hours. In some embodiments, the method includes treatment with a lysis buffer to lyse the microorganism infected with the infectious agent prior to detection of the indicator portion.

[0115] In another aspect of the present invention, the present invention provides a method for determining an effective dose of an antibiotic in killing a microorganism, comprising: (a) incubating one or more antibiotic solutions separately with one or more samples containing a microorganism, wherein the concentrations of the one or more antibiotic solutions are different and define a range; (b) incubating the microorganism in one or more of the 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 one or more of the samples, wherein the detection of an indicator protein product in one or more of the samples indicates that the concentration of the antibiotic solution used to treat one or more of the samples is ineffective, and the absence of detection of the indicator protein indicates that the antibiotic is effective, thereby determining an effective dose of the antibiotic.

[0116] The methods disclosed herein can be used to detect whether a microorganism of interest is susceptible to or resistant to an antibiotic. Certain antibiotics may be specific to the type of microorganism they kill or inhibit; antibiotics kill or inhibit the growth of microorganisms that are susceptible to the antibiotic, while neither killing nor inhibiting the growth of microorganisms that are resistant to the antibiotic. In some cases, a previously susceptible microbial strain may become resistant. Microbial resistance to antibiotics can be mediated by a number of different mechanisms. For example, some antibiotics interfere with cell wall synthesis in microorganisms; resistance to such antibiotics can be mediated by altering the antibiotic's target, i.e., cell wall proteins. In some cases, bacteria create resistance to antibiotics by producing compounds that can inactivate the antibiotic before it reaches the bacteria. For example, some bacteria cleave the beta-lactam of penicillin and / or carbapenems, thus producing beta-lactamases that can inactivate these antibiotics. In some cases, antibiotics are removed from cells by specific pumps before they reach their target. One example is the RND transporter. In some cases, certain antibiotics act by binding to ribosomal RNA (rRNA), inhibiting protein biosynthesis in microorganisms. Microorganisms resistant to such antibiotics may contain mutated rRNAs that have reduced binding ability to the antibiotic but function essentially normally within the ribosome. In other cases, bacteria possess genes that can confer resistance. For example, some MRSA strains possess the mecA gene. The mecA gene product is an alternative transpeptidase that has low affinity for the cyclic structure of certain antibiotics that typically bind to transpeptidases required for bacterial cell wall formation. Therefore, antibiotics containing beta-lactams cannot inhibit cell wall synthesis in such bacteria.Some bacteria possess non-functional antibiotic resistance genes, likely due to genetic or regulatory mutations. These can be mistakenly detected as antibiotic resistance by conventional nucleic acid methods such as PCR, but are not detected by functional methods such as antibiotic plating or culture, or by the method described here.

[0117] Non-limiting examples of antibiotics that may be used in the present invention include aminoglycosides, carbasephalosporins, carbapenems, cephalosporins, glycopeptides, macrolides, monobactams, penicillins, beta-lactam antibiotics, quinolones, bacitracin, sulfonamides, tetracyclines, streptogramin, chloramphenicol, clindamycin and lincosamides, cephamycin, lincomycin, daptomycin, oxazolidinone and glycopeptide antibiotics.

[0118] As noted herein, in certain embodiments, the present invention may include a method for using infectious particles to detect microbial resistance to antibiotics, or, to put it another way, to detect the effectiveness of antibiotics against microorganisms. In another embodiment, the present invention may include a method for selecting an antibiotic for the treatment of an infection. Furthermore, the method may include a method for detecting antibiotic-resistant bacteria in a sample. The methods of the present invention may be embodied in a variety of ways.

[0119] The method may include, as described above, the steps of bringing a sample containing microorganisms into contact with an antibiotic and an infectious agent. 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, comprising: (a) incubating one or more antibiotic solutions separately with one or more samples containing microorganisms, wherein the concentrations of the one or more antibiotic solutions are different and define a range; (b) incubating the microorganisms in 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 one or more samples, wherein the detection of an indicator protein product in one or more of the samples indicates that the concentration of the antibiotic solution used to treat one or more of the samples is ineffective, and the absence of detection of the indicator protein indicates that the antibiotic is effective, thereby determining an effective dose of the antibiotic.

[0120] In other embodiments, the antibiotic and infectious agent are added sequentially; for example, the sample is in contact with the antibiotic before the sample is in contact with the infectious agent. In certain embodiments, the method may include a step of incubating the sample with the antibiotic over a period prior to the step of contacting the sample with the infectious agent. The incubation time may vary depending on the properties of the antibiotic and 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, or less than 30 minutes. The incubation time for infectious agents and microorganisms may also vary depending on the life cycle of the particular infectious agent, and in some cases, the incubation time is less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 45 minutes, or less than 30 minutes. Microorganisms resistant to antibiotics may survive and reproduce, and their specific infectious agents will replicate, leading to the production of indicator protein products (e.g., luciferase); conversely, microorganisms sensitive to antibiotics are killed, and therefore their infectious agents do not replicate. Furthermore, bacteriostatic antibiotics do not kill bacteria; however, they do halt bacterial growth and / or enrichment. In some cases, bacteriostatic antibiotics may interfere with bacterial protein synthesis, and it is expected that bacteriophages will not produce indicator molecules (e.g., luciferase). The infectious agent in this method includes an indicator moiety, the amount of which corresponds to the amount of microorganisms present in the sample treated with the antibiotic. Therefore, positive detection of the indicator moiety indicates that the microorganism is resistant to the antibiotic.

[0121] 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 to 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 with a recombinant phage specific to S. aureus for a period of time. In samples containing antibiotic-resistant S. aureus, detection of an indicator protein produced by the recombinant phage will be positive. In samples containing antibiotic-susceptible S. aureus, detection of the indicator protein will be negative. In some embodiments, the method for detecting antibiotic resistance can be used to select an effective therapeutic agent to which the pathogenic bacteria are susceptible.

[0122] In a particular embodiment, the total time required for detection is 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, less than 1.5 hours, or less than 1.0 hour. The total time required for detection depends on the target bacterium, the type of phage, and the antibiotic being tested.

[0123] If necessary, the method further includes the step of lysing the microorganism before detecting the indicator portion. Cells can be lysed using any solution that does not affect the activity of luciferase. In some cases, the lysis buffer may contain nonionic detergents, chelating agents, enzymes, or proprietary combinations of various salts and agents. 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 after the addition of luciferase substrates. Perhaps, without complete cell lysis, luciferase can exit the cell and / or luciferase substrates can enter the cell. For example, in some embodiments, luciferase substrates are cell-permeable (e.g., flimazine). Thus, lysis is required for detection in embodiments utilizing a spin filter system, where only the luciferase released into the lysate (not the luciferase remaining inside the intact bacteria) is analyzed in the luminometer. However, in embodiments utilizing filter plates or 96-well plates with phage-infected samples in solution or suspension, as described later, intact and lysed cells can be directly assayed in a luminometer, lysis may not be necessary for detection. Therefore, in some embodiments, methods for detecting antibiotic resistance do not involve the step of lysing microorganisms.

[0124] A surprising aspect of the assay embodiments is that the step of incubating microorganisms in the sample with an infectious agent only requires that the infectious agent, e.g., a phage, be long enough for a single life cycle. The amplification power using phages was previously thought to require more time for the phage to replicate over several cycles. A single replication of an indicator phage may be sufficient to facilitate highly sensitive and rapid detection according to some embodiments of the present invention. Another surprising aspect of the assay embodiments is that high concentrations of phages (i.e., high MOI) used to infect the test sample have successfully achieved the detection of a small number of antibiotic-resistant target microorganisms treated with antibiotics. Factors including phage burst size can influence the number of phage life cycles, and therefore the amount of time required for detection. A phage with a large burst size (approximately 100 PFU) may require only one cycle for detection, while a phage with a smaller burst size (e.g., 10 PFU) may require multiple phage cycles for detection. In some embodiments, the incubation of the test sample and phage is required to be only long enough for a single phage life cycle. In other embodiments, the incubation of the test sample and phage spans a period of time exceeding a single life cycle. The phage concentration for the incubation step varies depending on the type of phage used. In some embodiments, the phage concentration for this incubation step is 1.0 × 10⁻⁶. 6 , 2.0×10 6 , 3.0×10 6 , 5.0×10 6 , 6.0×10 6 , 7.0×10 6 , 8.0×10 6 , 9.0×10 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 , 1.7×107 , 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 The levels exceed PFU / mL. The success of such high concentrations of phages is surprising, as such a large number of phages have previously been associated with "non-infectious lysis," which immediately kills target cells, thereby preventing the generation of useful signals from early phage assays. In addition to removing any phage-related contaminating luciferase, purification can also remove ghost particles (particles that have lost DNA), so the purification of phage stocks described herein can help mitigate this problem (e.g., purification by sucrose gradient cesium chloride isodensity gradient ultracentrifugation). Ghost particles can lyse bacterial cells via "non-infectious lysis," prematurely killing the cells and thereby preventing the generation of indicator signals. Electron microscopy demonstrates that crude recombinant phage lysates (i.e., before cesium chloride purification) can contain more than 50% ghost particles. Such ghost particles can contribute to the premature death of microorganisms via the action of many phage particles puncturing the cell membrane. Therefore, ghost particles may have contributed to the previous problem where high PFU concentrations were reported to be harmful.

[0125] Any of the indicator portions described in this disclosure may be used to detect the viability of a microbial organism after antibiotic treatment, thereby detecting antibiotic resistance. In some embodiments, indicator portions related to infectious agents may be detectable during or after replication of the infectious agent. For example, as described above, in some cases the indicator portion may be a protein that emits an endogenous signal, such as a fluorescent protein (e.g., green fluorescent protein or others). The indicator may be detectable by producing light and / or by a change in color. In some embodiments, an indicator (e.g., luciferase) can be detected using a luminometer. However, other machines or devices may also be used. For example, a spectrophotometer, a CCD camera or a CMOS camera can detect changes in color and other light emissions.

[0126] In some embodiments, the exposure of the sample to the antibiotic can be continued for 5 minutes or longer, and detection at various time points may be desirable for optimal sensitivity. For example, aliquots of a primary sample treated with the antibiotic may be taken at different time intervals (e.g., 5 minutes, 10 minutes, or 15 minutes). Samples obtained from the varying time intervals can then be infected with phages, and the indicator portion is measured after the addition of a substrate.

[0127] In some embodiments, signal detection is used to determine antibiotic resistance. In some embodiments, the signal generated by the sample is compared to an experimentally determined value. In further embodiments, the experimentally determined value is the signal generated by a control sample. In some embodiments, the background threshold is determined using a control without microorganisms. In some embodiments, a control without phages or antibiotics, or other control samples, may also be used to determine an appropriate threshold. In some embodiments, the experimentally determined value is the mean background signal + mean background The background threshold is calculated from 1 to 3 times the standard deviation of the mean background signal, or higher. In some embodiments, the background threshold may be calculated from the mean background signal plus twice the standard deviation of the mean background signal. In other embodiments, the background threshold may be calculated from the mean background signal multiplied by a multiple (e.g., 2 or 3). Detection of a sample signal exceeding the background threshold indicates the presence of one or more antibiotic-resistant microorganisms in the sample. For example, the mean background signal may be 250 RLU. The threshold background value may be calculated by multiplying the mean background signal (e.g., 250) by 3 to obtain a value of 750 RLU. A sample containing bacteria with a signal value exceeding 750 RLU is determined to be positive for the presence of antibiotic-resistant bacteria.

[0128] Instead, the value determined by the experiment is the signal generated by the control sample. The assay can include a variety of suitable control samples. For example, a sample that does not contain a microorganism-specific infectious agent, or a sample that contains an infectious agent but no 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 the infectious agent.

[0129] In some embodiments, the sample signal is compared to a control signal to determine whether antibiotic-resistant microorganisms are present in the sample. Unchanged detection of the signal compared to a control sample that has been in contact with an infectious agent but not with an antibiotic indicates that the microorganism is resistant to the antibiotic, while reduced detection of the indicator portion compared to a control sample that has been in contact with an infectious agent but not with an antibiotic indicates that the microorganism is susceptible to the antibiotic. Unchanged detection means that the signal detected from the sample treated with the antibiotic and infectious agent is at least 80%, at least 90%, or at least 95% of the signal from the control sample that has not been treated with the antibiotic. Reduced detection means that the signal detected from the sample treated with the antibiotic and infectious agent is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or at least 30% of the signal from the control sample that has not been treated with the antibiotic.

[0130] If necessary, the sample containing the microorganism of interest is an uncultured sample. If necessary, the infectious agent is a phage and includes an indicator gene inserted into the late gene region of the phage such that the expression of the indicator gene during phage replication after infection of the host bacterium results in a soluble indicator protein product. As described above, each feature of the composition used in the method can also be utilized in a method for detecting antibiotic resistance of the microorganism of interest. In some embodiments, the transcription of the indicator gene is controlled by an additional bacteriophage late promoter.

[0131] Methods for determining an effective dose of an antibiotic for killing a microorganism are also provided herein. In some embodiments, the antibiotic is effective in killing Staphylococcus species. For example, the antibiotic may be cefoxitin, which is effective against most methicillin-sensitive S. aureus (MSSA). Typically, one or more antibiotic solutions are prepared at different concentrations, such that the different concentrations of the solutions define a range. 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, for example, 1:5 to 1:30 or 1:10 to 1:20. In some cases, the minimum concentration of one or more antibiotic solutions is at least 1 μg / mL, for example, 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 one or more antibiotic solutions is incubated with a single aliquot of the sample containing the target microorganism. In some cases, a microorganism-specific infectious agent (e.g., a bacteriophage) is added simultaneously with the antibiotic solution. In other cases, the sample aliquots are incubated with the antibiotic solution for a period prior to the addition of the infectious agent. Indicator protein products can be detected; a positive detection indicates that the antibiotic solution is ineffective, and a negative detection indicates that the antibiotic solution is effective. The concentration of the antibiotic solution is expected to correlate with the effective clinical dose.Accordingly, in some embodiments, a method for determining an effective dose of an antibiotic in killing a target microorganism includes the steps of: incubating each of one or more antibiotic solutions separately with the target microorganism in a sample, wherein the concentrations of the one or more antibiotic solutions are different and define a range; incubating the microorganism in one or more samples with an infectious agent including an indicator portion; and detecting an indicator protein product of the infectious agent in one or more samples, wherein positive detection of the indicator protein product in one or more of the one or more samples indicates that the concentration of the antibiotic solution used to treat one or more of the one or more samples is ineffective, and the absence of detection of the indicator protein indicates that the antibiotic is effective, thereby determining an effective dose of the antibiotic.

[0132] In some embodiments, the method allows for the determination of categorical assignments for antibiotic resistance. For example, the categorical assignments of antibiotics (e.g., susceptible, intermediate, and resistant) can be determined using the method disclosed herein. Susceptible antibiotics are those that may inhibit pathogenic microorganisms but do not guarantee this; these may be a suitable choice for treatment. Intermediate antibiotics are those that are effective at higher doses or more frequent doses, or that may only be effective at specific body sites where the antibiotic penetrates to achieve an appropriate concentration. Resistant antibiotics are those that are ineffective in inhibiting the growth of organisms in laboratory testing; these may not be a suitable choice for treatment. In some embodiments, two or more antibiotic solutions are tested, and the concentration ratio of the least concentrated to the most concentrated solution in one or more antibiotic solutions ranges from 1:2 to 1:50, for example, 1:5 to 1:30 or 1:10 to 1:20. In some cases, the minimum concentration of one or more antibiotic solutions is at least 1 μg / mL, for example, 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.

[0133] In some embodiments, the present invention includes a method for detecting antibiotic-resistant microorganisms in the presence of antibiotic-sensitive microorganisms. In certain specific examples, the detection of antibiotic-resistant bacteria can be used to prevent the transmission of infections in a healthcare setting. In some embodiments, patients in a healthcare setting may be monitored for colonization of antibiotic-resistant bacteria. Preventive measures can then be taken to prevent the transmission of antibiotic-resistant bacteria.

[0134] In some embodiments of methods for detecting antibiotic-resistant microorganisms, a sample may contain both antibiotic-resistant and antibiotic-sensitive bacteria. For example, a 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 produce a signal above the threshold, but MRSA present in the sample can produce 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).

[0135] In contrast to many assays known in the art, the detection of antibiotic resistance in microorganisms can be achieved without prior isolation. Many methods require patient samples to be pre-cultured on agar plates to purify / isolate individual bacterial colonies. The increased sensitivity of the methods disclosed herein is partly due to the ability of numerous specific infectious agents, e.g., phages, to bind to a single microorganism. After phage infection and replication, the target microorganism can be detected via indicator protein products produced during phage replication.

[0136] Therefore, in certain embodiments, the method can detect antibiotic resistance of microorganisms in a sample containing ≤10 cells of microorganisms (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 microorganisms). In certain embodiments, antibiotic resistance can be detected using recombinant phages by detecting a single bacterium of a specific type in a sample treated with an antibiotic. In certain embodiments, recombinant phages detect the presence of approximately 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 exposed to an antibiotic.

[0137] The sensitivity of the methods for detecting antibiotic resistance disclosed herein may be further increased by washing the captured and infected microorganisms prior to incubation with the antibiotic. Isolation of the target bacteria may be required if the antibiotic being evaluated is known to be degraded by other bacterial species. For example, penicillin resistance is difficult to assess without purification because other bacteria present in the clinical sample may degrade the antibiotic (beta-lactamase secretion), leading to false positives. Furthermore, the captured microorganisms can be washed after incubation with the antibiotic and infectious agent, prior to the addition of lysis buffer and substrate. Such an additional washing step helps remove excess parental phages and / or luciferase or other reporter proteins that may contaminate the phage preparation. Therefore, in some embodiments, the method for detecting antibiotic resistance may include a step of washing the captured and infected microorganisms after the addition of the phage, but before incubation.

[0138] In many embodiments, multi-well plates are used to perform the assay. The choice of plate (or any other container in which detection can be performed) can affect the detection process. For example, some plates may have a colored or white background, which can affect the detection of light emission. Generally speaking, white plates have higher sensitivity but also produce a higher background signal. Plates of other colors may produce a lower background signal but also have slightly lower sensitivity. Furthermore, one reason for the background signal is the leakage of light from one well to another adjacent well. There are some plates that have white wells but the rest of the plate is black. This allows for a high signal inside the wells but prevents light leakage between wells and thus reduces the background. Thus, the choice of plate or other assay container can affect the sensitivity and background signal of the assay.

[0139] Therefore, several embodiments of the present invention address the need by using infectious factor-based methods to amplify a detectable signal, thereby indicating whether or not a microorganism is resistant to antibiotics. The present invention enables users to detect antibiotic resistance in microorganisms present in unpurified and unisolated samples. In certain embodiments, as few as a single bacterium can be detected. This principle, based on the specific recognition of microbial surface receptors, allows for the amplification of indicator signals from one or more cells. For example, even a single microbial cell can be exposed to multiple phages, and then the indicator signal is amplified to detect a single microorganism by allowing high levels of expression of encoded indicator gene products during phage amplification and replication. The present invention is excellent as a rapid test for microbial detection because it does not require isolation of the microorganism prior to detection. In some embodiments, detection is possible within one or two replication cycles of the phage or virus.

[0140] In additional embodiments, the Disclosure includes a system (e.g., a computer system, an automated system, or a kit) comprising components for carrying out the methods disclosed herein and / or for using the modified infectious agents described herein.

[0141] System and Kit of the Present Invention In some embodiments, the Disclosure includes a system (e.g., an automated system or kit) comprising components for carrying out the methods disclosed herein. In some embodiments, an indicator phage is included in the system or kit according to the present invention. The methods described herein may also utilize such an indicator phage system or kit. Given the minimum amounts of reagents and materials required to carry out the methods, some embodiments described herein are particularly suited to automation and / or kits. In certain embodiments, each component of the kit may include a self-contained unit that can be delivered from a first site to a second site.

[0142] In some embodiments, the disclosure includes a system or kit for the rapid detection of a microorganism of interest in a sample. In certain embodiments, the system or kit may include components for incubating a sample with a recombinant bacteriophage specific to the microorganism of interest, wherein the recombinant bacteriophage comprises a genetic construct, and the genetic construct comprises a gene encoding an indicator protein product; and components for detecting the indicator protein product. Some systems further include components for capturing the microorganism of interest on a solid support.

[0143] In other embodiments, the Disclosure includes a method, system, or kit for the rapid detection of a microorganism of interest in a sample, comprising a recombinant bacteriophage component specific to the microorganism of interest, wherein the recombinant bacteriophage comprises a genetic construct, the genetic construct comprising a gene encoding an indicator protein product; and a component for detecting the indicator protein product. In certain embodiments, the recombinant bacteriophage is highly specific to a particular bacterium. In certain embodiments, the recombinant bacteriophage can distinguish the bacterium of interest in the presence of more than 100 other types of bacteria. In certain embodiments, the system or kit detects a single bacterium of a specific type in a sample. In certain embodiments, the system or kit detects approximately 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.

[0144] In certain embodiments, the system and / or kit may further include components for washing the captured microbial sample. Alternatively, the system and / or kit may further include components for determining the amount of an indicator protein product, wherein the amount of the detected indicator portion 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.

[0145] In some systems and / or kits, the same components can be used for multiple processes. In some systems and / or kits, the processes are automated by computer input or controlled by a user, and / or a liquid-handling robot performs at least one of the processes.

[0146] Therefore, in certain embodiments, the present invention may include a system or kit for the rapid detection of a target microorganism in a sample, comprising: a component for incubating the sample with a recombinant bacteriophage specific to the target microorganism, wherein the recombinant bacteriophage comprises a gene encoding an indicator protein product; a component for capturing the microorganism from the sample onto a solid support; a component for washing the captured microbial sample to remove unbound infectious agents; and a component for detecting the indicator protein product. In some embodiments, the same component (e.g., a filter component) may be used for the capture and / or incubation and / or washing steps. In some embodiments, further comprising a component for determining the amount of the target microorganism in the sample, wherein the amount of indicator protein product 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 for the rapid detection of microorganisms. In some embodiments, the microorganism is a bacterium, The infectious agent is a bacteriophage. In computerized systems, the system can be fully automated, semi-automated, or directed by a user (or a combination of these) via a computer.

[0147] In one embodiment, the present disclosure includes a system or kit comprising components for detecting a microorganism of interest, comprising components for infecting at least one microorganism with a plurality of recombinant bacteriophages; components for lysing at least one infected microorganism; and components for detecting a soluble indicator protein product encoded and expressed by the recombinant bacteriophage, wherein the detection of a soluble protein product of an infectious agent indicates the presence of the microorganism in the sample.

[0148] In some embodiments, the present disclosure includes a system or kit comprising components for treating biofilm-related infections.

[0149] These systems and kits of this disclosure include various components. As used herein, the term “component” is broadly defined and includes any suitable apparatus or collection of suitable apparatus for carrying out the methods described herein. The components do not need to be integrally connected or installed with respect to one another in any particular way. The present invention includes any suitable arrangement of the components with respect to one another. For example, the components do not need to be in the same space. However, in some embodiments, the components are connected to each other in an integral unit. In some embodiments, the same component may perform multiple functions.

[0150] Computer systems and computer-readable media The above system may be embodied in the form of a computer system, as described in any of the current technologies or their components. Typical examples of computer systems include general-purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices that can implement the processes constituting the methods of this technology.

[0151] The computer system may include a computer, input devices, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further include storage devices. The storage devices may be hard disk drives or removable storage devices (e.g., floppy disk drives, optical disk drives, etc.). The storage devices may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit enables the computer to connect to other databases and the Internet through an I / O interface. The communication unit enables transfers to and from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that enables the computer system to connect to databases and networks (e.g., LAN, MAN, WAN, and the Internet). The above computer system can therefore facilitate user input through input devices that are accessible to the system via an I / O interface.

[0152] A computing device typically includes an operating system that provides executable program instructions for the general management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., a hard disk, random-access memory, read-only memory, etc.) that stores instructions causing the computing device to perform its intended functions when executed by a server processor. Suitable implementations of the operating system and the general functionality of the computing device are known or commercially available and can be readily implemented by those skilled in the art, particularly in view of the disclosure herein.

[0153] The above computer system executes a set of instructions stored in one or more memory elements in order to process input data. The memory elements may also hold data or other information as described. The memory elements may be in the form of information sources present in the processing machine or physical memory elements.

[0154] The environment may include various data stores and other memory and storage media as considered above. These may reside in various locations (e.g., in storage media local to (and / or located within) one or more of the computers, or remotely from any or all of the computers across a network). In a particular group of embodiments, information may reside in a storage area network ("SAN") as is familiar to those skilled in the art. Similarly, any essential files for performing functions attributable to the computers, servers, or other network devices may be stored locally and / or remotely, where appropriate. Where the system includes computing devices, each such device may include hardware elements that can be electrically connected via a bus, such as, for example, at least one central processing unit (CPU), at least one input device (e.g., mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., display device, printer, or speaker). Such a system may also include one or more storage devices (e.g., disk drives, optical storage devices, and solid-state storage devices (e.g., random-access memory ("RAM") or read-only memory ("ROM")), as well as removable media devices, memory cards, flash cards, etc.).

[0155] Such devices may also include a computer-readable storage medium reader, a communication device (e.g., a modem, a network card (wireless or wired), an infrared communication device, etc.), and working memory as described above. The computer-readable storage medium reader may be connected to or configured to receive computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for storing, transmitting, and retrieving computer-readable information temporarily and / or more permanently. The system and various devices also typically include many software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs (e.g., a client application or a web browser). It should be recognized that alternative embodiments may have many variations from those described above. For example, customized hardware may also be used, and / or certain elements may be implemented in hardware, software (including portable software (e.g., applets)), or both. Furthermore, connections to other computing devices (e.g., network input / output devices) may be used.

[0156] Non-temporary storage media and computer-readable media for containing code or a portion of code may include any suitable media known or used in the art (including, but not limited to, volatile and non-volatile, removable and non-removable media implemented in any method or technique for storing and / or transmitting information such as computer-readable instructions, data structures, program modules, or other data), such as RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital multipurpose disc (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other media that may be used to store desired information and can be accessed by the system devices. Based on the above disclosure and the teachings provided herein, a person skilled in the art will recognize other ways and / or methods for implementing the various embodiments described above.

[0157] Computer-readable media may include, but are not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, SRAMs, DRAMs, associative memory ("CAM"), DDRs, flash memory (e.g., NAND flash or NOR flash), ASICs, configured processors, optical storage media, magnetic tapes or other magnetic storage media, or any other media from which a computer processor can read instructions. In one embodiment, the computing device may include a single type of computer-readable media (e.g., random access memory (RAM)). In other embodiments, the computing device may include two or more types of computer-readable media (e.g., random access memory (RAM), disk drives, and caches). The computing device may be in communication with one or more external computer-readable media (e.g., external hard disk drives, or external DVD or Blu-ray drives).

[0158] As discussed above, the above embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. These instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language (e.g., C, C++, C#, Visual Basic, Java®, Python, Perl, JavaScript®, and ActionScript (Adobe Systems, Mountain View, Calif.)). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and state machines. Such processors may include programmable electronic devices (e.g., PLCs), programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memory (PROMs), and electronically programmable read-only memory (EPROMs or EEPROMs). or other similar devices may further include.

[0159] The computing device described above includes a network interface. In some embodiments, the network interface is configured to communicate via a wired or wireless communication link. For example, the network interface may enable communication over a network via Ethernet®, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth®, infrared, etc. As another example, the network interface may enable communication over a network (e.g., CDMA, GSM®, UMTS, or other cellular communication network). In some embodiments, the network interface may enable communication with another device via, for example, Universal Serial Bus (USB), 1394 Point-to-point connectivity may be enabled via FireWire®, serial or parallel connections, or similar interfaces. Some embodiments of a suitable computing device may include two or more network interfaces for communication over one or more networks. In some embodiments, the computing device may include a data store in addition to or instead of network interfaces.

[0160] Some embodiments of a suitable computing device may include, or be in communication with, numerous external or internal devices (e.g., a mouse, CD-ROM, DVD, keyboard, display, audio speakers, one or more microphones, or any other input or output devices). For example, the computing device may be in communication with various user interface devices and displays. The display may use any suitable technology (including, but not limited to, LCD, LED, CRT, etc.).

[0161] The set of instructions for execution by the computer system described above may include a variety of commands that instruct a processing machine to perform a specific task (e.g., a step constituting the method of this technique). The set of instructions may be in the form of a software program. Furthermore, the software may be in the form of a collection of separate programs, a program module with a larger program, or part of a program module, as in this technique. The software may also include modular programming in the form of object-oriented programming. The processing of input data by the processing machine may be in response to user commands, the results of previous processing, or requests created by another processing machine.

[0162] While the present invention has been disclosed with reference to certain embodiments, many modifications, changes, and alterations to the embodiments described are possible without departing from the scope and spirit of the invention, as defined in the appended claims. Thus, the present invention is not intended to be limited to the embodiments described above, but has the entire scope as defined by the following claims and their equivalents. [Examples]

[0163] The following embodiments are included to provide guidance to those skilled in the art for carrying out typical embodiments of the subject matter of this disclosure. In light of the general state of the art and the present disclosure, those skilled in the art will recognize that the following embodiments are intended to be merely illustrative and that numerous variations, modifications, and alterations may be made without departing from the scope of the subject matter of this disclosure.

[0164] (Example 1) Staphylococcus aureus biofilm and irrigation washing inspection protocol Overnight cultures of S. aureus were diluted to 200 μL of either TSB (100% tryptone soy broth), TSBg (66% TSB + 0.2% glucose), or TSB-HS (90% TSB + 10% human serum). The initial inoculum was a 200-fold dilution of the overnight culture and was prepared in a 96-well plate. The plate was covered and statically incubated at 37°C for at least 16 hours. Non-biofilm plankton cells were removed by discarding the medium and gently washing with 200 μL of saline. Saline irrigation washing was performed by force-pipeting 200 μL of saline onto the biofilm. This direct washing was expected to mechanically release portions of the adhesive biofilm. 150 μL of each saline irrigation washing sample was transferred to separate 96-well plates containing dried concentrated BHI (Brain Heart Infusion). The final concentration of BHI in each well was 1 × (37 g / L). To evaluate residual adhesive biomass, 150 μL of BHI was added to each well containing biofilm after irrigation washing. All samples (residual biofilm + saline irrigation washing) were covered and statically incubated at 37°C for 4 hours to facilitate enrichment. After enrichment, 10 μL of recombinant phage cocktail was added to each well and mixed by pipetting. The plate was covered again and statically incubated at 37°C for 2 hours. After infection, 65 μL of a detection master mix containing NANO-GLO® buffer, NANO-GLO® substrate, and sea urchin lysis buffer was added to each well and mixed again by pipetting. After a 3-minute waiting period, the samples were read on a GLOMAX® luminometer using a 1-second integration. The data are presented in Table 1 as relative luminous units (RLU). [Table 1]

Claims

[Claim 1] The invention described in the specification.

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