EP3320108A1 - Phages recombinants et méthodes de détection de la listeria - Google Patents

Phages recombinants et méthodes de détection de la listeria

Info

Publication number
EP3320108A1
EP3320108A1 EP16824938.1A EP16824938A EP3320108A1 EP 3320108 A1 EP3320108 A1 EP 3320108A1 EP 16824938 A EP16824938 A EP 16824938A EP 3320108 A1 EP3320108 A1 EP 3320108A1
Authority
EP
European Patent Office
Prior art keywords
phage
composition
listeria
recombinant
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16824938.1A
Other languages
German (de)
English (en)
Inventor
Michael Sandor Koeris
Michael CAPPILLINO
Edyta Krzymanska-Olejnik
Daniel Robert Brownell
Robert Patrick Shivers
Jayson L. Bowers
Timothy Kuan-Ta Lu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute for Environmental Health Inc
Original Assignee
Sample6 Technologies Inc
SAMPLE6 Tech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sample6 Technologies Inc, SAMPLE6 Tech Inc filed Critical Sample6 Technologies Inc
Publication of EP3320108A1 publication Critical patent/EP3320108A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/00043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This disclosure generally relates to the detection of microbes through the use of codon- optimized recombinant phage.
  • Bacterial contamination and infection is a significant problem to public health and in many other areas. Bacterial food borne diseases pose a significant threat to human health, estimated to cause as many as about 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the US annually.
  • bovine mastitis an infection caused by bacterial cells that results in the inflammation of the bovine breast, reduction in milk yield and a decrease in milk quality. This condition is caused by the bacteria Staphylococcus aureus and Staphylococcus agalactiae. This reduction in milk yields and quality in the western world alone have been suggested to cause annual financial losses of $3.7 billion.
  • bovine tuberculosis (Mycobacterium bovis), a bacteria that causes financial loses worldwide.
  • Mycobacterium bovis a bacteria that causes financial loses worldwide.
  • 12 of a herd of 55 cattle in a small Michigan farm tested positive for bovine tuberculosis.
  • the farm was forced to destroy the entire herd of cattle, along with an entire herd of hogs.
  • Tuberculosis testing in cattle requires the animal to be held for 2 days, and tests are false positive 5 percent of the time. Often entire herds have to be quarantined or destroyed. The annual worldwide financial losses have been estimated at $3 billion.
  • Tuberculosis is a leading cause of death worldwide.
  • One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Every day 25,000 people are infected and 5,000 people die from the disease.
  • multidrug resistant strains of M. tuberculosis are emerging and the reemergence of tuberculosis as a worldwide epidemic has become a real threat.
  • the worldwide annual market for tuberculosis diagnostics has been estimated at $1.8 billion.
  • MRSA is a drug-resistant version of the common Staphylococcus aureus bacteria and is contagious, due to the nature of the MRSA bacterium.
  • the bacteria are highly contagious and spread by touch. Approximately 86% of all infections occur within hospitals, and these infections carry a 20% mortality rate. This bacterium costs an average of $21,000 over the standard costs to treat, and kills approximately 19,000 people in the US annually.
  • Listeria monocytogenes is an intracellular pathogen that can cause invasive disease in humans and animals. Approximately 99% of human listeriosis infections appear to be food borne. While L. monocytogenes has been isolated from a variety of raw and ready-to-eat foods, most human listeriosis infections appear to be caused by consumption of RTE foods that permit postcontamination growth of this pathogen. Listeriosis is estimated to be responsible for about 500 deaths per year in the United States, accounting for 28% of annual deaths attributable to known food-borne patho- gens, second only to deaths due to Salmonella infections.
  • compositions and methods of the present disclosure address these needs.
  • compositions and methods of the disclosure address the long-felt need in the art for compositions and methods of immediate detection of bacterial infection by a non-technical or layperson at the site of potential contamination.
  • composition comprising, consisting essentially of or consisting of at least one recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker.
  • compositions of the disclosure may comprise, consist essentially of or consist of at least two, three, four, five, or six recombinant phages capable of infecting a target microbe, each of said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon- optimized marker.
  • Compositions of the disclosure may comprise, consist essentially of or consist of greater than six recombinant phage capable of infecting a target microbe, each of said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker.
  • the ribosome binding site of each phage may be identical in compositions of the disclosure comprising, consisting essentially of or consisting of one or more recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker.
  • Exemplary ribosome binding sites of the phage of the disclosure may comprise, consist essentially of or consist of SEQ ID NO: 54.
  • Codon-optimized markers of the disclosure may comprise, consist essentially of or consist of a codon-optimized luciferase.
  • Exemplary codon-optimized markers of the disclosure may comprise, consist essentially of or consist of SEQ ID NO: 36 (COP2).
  • codon- optimized markers of the disclosure may comprise, consist essentially of or consist of SEQ ID NO: 37 (COP3) or a UTR7 variant of COP3 (SEQ ID NO: 115).
  • codon-optimized markers of the disclosure may comprise, consist essentially of or consist of SEQ ID NO: 37 (COP3).
  • compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage is selected from the group consisting of LP173, LP80, V18, LP22, LP143, A511, LP101, LP124, LP99, LP48, LP125, P100, and LP40.
  • Compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is LP80, V18, LP22, A511, LP40 or LP124.
  • Compositions of the disclosure may comprise, consist essentially of or consist of LP80, V18, LP22, A51 1, LP40 and LP124.
  • compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is A511.
  • Compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is LP40.
  • Compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is LP124.
  • Compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is LP80.
  • Compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is VI 8.
  • Compositions of the disclosure may comprise, consist essentially of or consist of at least one recombinant phage, wherein the phage is LP22.
  • Target microbes of the disclosure may belong to the genus Listeria.
  • Exemplary target microbes of the disclosure include, but are not limited to, Listeria innocua, Listeria
  • the target microbe is Listeria monocytogenes.
  • Listeria seeligeri Listeria ivanovii, Listeria grayi, Listeria marthii, Listeria rocourti, Listeria welshimeri, Listeria floridensis, Listeria aquatic, Listeria cornellensis, Listeria riparia, Listeria weihenstephanensis, Listeria flieschmannii, Listeria newyorkensis and Listeria grandensis.
  • the target microbe is Listeria monocytogenes.
  • compositions of the disclosure may comprise, consist essentially of, or consist of a recombinant phage of the disclosure and an aqueous solution.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non-target microbe in an
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5 and at least one agent to prevent the decomposition of a marker substrate.
  • Exemplary agents to prevent the decomposition of a marker substrate may comprise, consist essentially of or consist of a compound to prevent the decomposition of luciferin.
  • the compound to prevent the decomposition of luciferin may comprise, consist essentially of or consist of a compound to prevent the decomposition of luciferin.
  • decomposition of luciferin prevents decomposition of luciferin for between 5 and 10 hours. In certain embodiments of the aqueous solutions of the disclosure, the compound to prevent the decomposition of luciferin prevents decomposition of luciferin for less than 5 hours. In certain embodiments of the aqueous solutions of the disclosure, the compound to prevent the
  • decomposition of luciferin prevents decomposition of luciferin for greater than 10 hours.
  • Exemplary agents to prevent decomposition of the luciferin may comprise, consist essentially of or consist of non-ionic detergents, oxygen scavengers and/or emulsifiers.
  • Exemplary agents to prevent decomposition of the luciferin may comprise, consist essentially of or consist of sodium metabi sulfite, sodium thiosulfate, Tween-80, HEPES and/or lecithin.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5 and at least one agent suitable to neutralize a sanitizer present in an environmental sample.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5, at least one agent to prevent the decomposition of a marker substrate and at least one agent suitable to neutralize a sanitizer present in an environmental sample.
  • agents suitable to neutralize a sanitizer may comprise, consist essentially of or consist of sodium metabi sulfite, sodium pyruvate, sodium thiosulfate, Tween-80, HEPES and lecithin.
  • an agent suitable to neutralize a sanitizer may comprise, consist essentially of or consist of sodium pyruvate.
  • an agent suitable to neutralize a sanitizer may comprise, consist essentially of or consist of sodium pyruvate, wherein the sodium pyruvate is 1% or less of the aqueous solution.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; and e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5.
  • Exemplary nutrients include, but are not limited to, a culture medium, alcohol, sugar, sugar derivatives, and combinations thereof.
  • Exemplary nutrients include, but are not limited to, Brain Heart Infusion medium, Tryptic Soy Broth, glucose, glycerol, pyruvate, and combinations thereof.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; and e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5.
  • exemplary selective agents suitable to inhibit growth of a non-target microbe include, but are not limited to, LiCl, acriflavine, nalidixic acid, cycloheximide, and combinations thereof.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; and e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5.
  • the at least one vitamin comprises, consists essentially of or consist of a yeast extract.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; and e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5.
  • Exemplary divalent methods include, but are not limited to, CaCl 2 , MgS04, and combinations thereof.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of: a) at least one nutrient; b) at least one selective agent suitable to inhibit growth of at least one non- target microbe in an environmental sample or an agricultural sample; c) at least one vitamin; d) at least one divalent metal; and e) at least one buffering agent capable of maintaining the composition at pH 7.0-7.5.
  • the at least one buffering agent comprises HEPES buffer.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of Tryptic Soy Broth, LiCl, nalidixic acid, yeast extract, glucose, MgS0 4 , pyruvate, and HEPES.
  • Aqueous solutions of the disclosure may comprise, consist essentially of or consist of Tryptic Soy Broth, LiCl, nalidixic acid, yeast extract, glucose, MgS0 4 , pyruvate, HEPES, Tween-80, lecithin, and potassium phosphate.
  • compositions of the disclosure may comprise, consist essentially of or consist of a recombinant phage of the disclosure, an aqueous solution, and a substrate for luciferase.
  • the substrate for luciferase may comprise, consist essentially of or consist of luciferin.
  • compositions of the disclosure may comprise, consist essentially of or consist of a recombinant phage of the disclosure, an aqueous solution, a substrate for luciferase, and a buffer to facilitate a light reaction.
  • the buffer to facilitate a light reaction may comprise, consist essentially of or consist of at least one agent suitable to neutralize a sanitizer.
  • agents suitable to neutralize a sanitizer may comprise, consist essentially of or consist of sodium pyruvate.
  • sodium pyruvate may comprise 1% or less of the buffer to facilitate a light reaction.
  • the disclosure provides a method of determining the presence or absence of a target microbe in an environmental sample, an agricultural sample or both, comprising: a) contacting an environmental sample, an agricultural sample, or both with a composition of the disclosure to form a test sample; and b) detecting the presence or absence of light in the test sample, thereby determining the presence or absence of a target microbe in an environmental sample or an agricultural sample.
  • Methods of determining the presence or absence of a target microbe in an environmental sample, an agricultural sample or both may further comprise the step of incubating the test sample at a temperature between 30°C and 35°C, inclusive of the endpoints, prior to the detecting step.
  • the test sample is incubated at about 35°C. In certain embodiments, the test sample is incubated at 35°C.
  • Methods of determining the presence or absence of a target microbe in an environmental sample, an agricultural sample or both may further comprise the step of centrifuging the test sample prior to the detecting step.
  • the centrifugation step may be performed at a speed of between 1000 and 14000 rcf.
  • the centrifugation step may be performed at a speed of about 1000 rcf, 3000 rcf, 5000 rcf, 9000 rcf, 14000 rcf or any rcf value in between.
  • the centrifugation step may be performed at a speed of about 9000 rcf.
  • the centrifugation step may be performed at a speed of 9000 rcf.
  • the test sample may have a volume of at least 300 ⁇ at the time the presence or absence of light is detected.
  • the test sample may have a volume of about 600 ⁇ at the time the presence or absence of light is detected.
  • the test sample may have a volume of 600 ⁇ at the time the presence or absence of light is detected.
  • Methods of determining the presence or absence of a target microbe in an environmental sample, an agricultural sample or both may further comprise the step of confirming a positive result of the detecting step.
  • the confirming step may comprise contacting the detected test sample with a confirmation composition, and wherein a decrease in an abundance or intensity of light confirms that the positive result is a true result.
  • exemplary confirmation compositions may comprise, consist essentially of or consist of an organic solvent.
  • the organic solvent may comprise, consist essentially of or consist of acetone or ethanol.
  • the organic solvent may comprise, consist essentially of or consist of ethanol.
  • the organic solvent may comprise, consist essentially of or consist of 70% ethanol.
  • Methods of determining the presence or absence of a target microbe in an environmental sample, an agricultural sample or both may further comprise the step of collecting the environmental sample, the agricultural sample, or both prior to the contacting step.
  • the step of collecting may comprise contacting a sponge to a portion or a surface of the environmental sample and/or the agricultural sample to form a test sponge and subsequently contacting the test sponge to the composition.
  • Exemplary sponges may comprise, consist essentially of or consist of polyurethane.
  • Environmental samples of the disclosure include, but are not limited to, an agricultural production facility, a food production facility, a container, a machine, a processing plant, a storage facility, a health care facility, an educational institution, a loading dock, a cargo hold, a sink, a vehicle, an airport, a customs facility or any portion or surface thereof.
  • Environmental samples of the disclosure may include a health care facility, portion or surface thereof, or sample isolated from a health care facility.
  • exemplary health care facility include, but are not limited to, a clinic, an emergency medical services location, a hospice, a hospital ship, a hospital train, a hospital, a military medical installation, a doctor's office, a long term care facility, respite care facility, or a quarantine station.
  • Environmental samples of the disclosure may include a food production facility, portion or surface thereof, or sample isolated from a food production facility.
  • Exemplary food production facilities include, but are not limited to, a farm, a boat, a food distribution facility, a food processing plant, a food retail location, a home, or a restaurant.
  • Agricultural samples of the disclosure include, but are not limited to, stock feed or food supply.
  • the food supply may be intended for human or non-human (animal) consumption.
  • the food supply may include plant or animal matter.
  • the food supply may be solid or liquid.
  • the food supply comprises, consists essentially of or consists of a dairy product, a fruit product, a grain product, a sweet, a vegetable product, a meat product, or any combination thereof.
  • dairy products include, but are not limited to, milk, butter, yogurt, cheese, ice cream, queso fresco, a derivative thereof or any combination thereof.
  • Exemplary fruit products include, but are not limited to, an apple, orange, banana, berry, lemon, or any combination thereof.
  • Exemplary grain products include, but are not limited to, wheat, rice, oats, barley, bread, pasta, or any combination thereof.
  • Exemplary sweet products include, but are not limited to, candy, soft drinks, cake, pie, or a combination thereof.
  • Exemplary vegetable products include, but are not limited to, spinach, carrots, onions, peppers, avocado, broccoli, or any combination thereof.
  • the vegetable product may comprise, consist essentially of or consist of guacamole.
  • Exemplary meat products include, but are not limited to, chicken, fish, turkey, pork, beef, or any combination thereof. In certain embodiments, the meat product comprises, consists essentially of or consists of whole muscle meat, ground meat, or a combination thereof.
  • the disclosure provides a kit comprising a composition of the disclosure.
  • the kit further comprises a confirmation composition.
  • the confirmation composition may comprise, consist essentially of or consist of an organic solvent.
  • Exemplary organic solvents include, but are not limited to, acetone or ethanol.
  • the organic solvent may comprise, consist essentially of or consist of ethanol.
  • the organic solvent may comprise, consist essentially of or consist of 70% ethanol.
  • the kit may further comprise a polyurethane sponge.
  • the disclosure provides a kit comprising: a first container comprising at least one recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker; a second container comprising an aqueous solution composition comprising Tryptic Soy Broth, LiCl, nalidixic acid, yeast extract, glucose, MgS0 4 , pyruvate, HEPES, Tween-80, lecithin, and potassium phosphate; a third container containing a substrate; and a fourth container containing a buffer to optimize light detection.
  • a first container comprising at least one recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker
  • a second container comprising an aqueous solution composition comprising
  • the disclosure provides a kit comprising: a first container comprising at least one recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker and an aqueous solution composition; a second container comprising an aqueous solution composition comprising Tryptic Soy Broth, LiCl, nalidixic acid, yeast extract, glucose, MgS04, pyruvate, HEPES, Tween-80, lecithin, and potassium phosphate; a third container containing a substrate; a fourth container containing a buffer to optimize light detection; and a fifth container comprising a confirmation composition.
  • a first container comprising at least one recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker
  • the confirmation composition may comprise, consist essentially of or consist of an organic solvent.
  • organic solvents include, but are not limited to, acetone or ethanol.
  • the organic solvent may comprise, consist essentially of or consist of ethanol.
  • the organic solvent may comprise, consist essentially of or consist of 70% ethanol.
  • the kit may further comprise a polyurethane sponge.
  • Kits of the disclosure may contain an aqueous solution that may comprise, consist essentially of or consist of Tryptic Soy Broth, LiCl, nalidixic acid, yeast extract, glucose, MgS0 4 , pyruvate, HEPES, Tween-80, lecithin, and potassium phosphate.
  • Kits of the disclosure may contain buffer to optimize light detection that may comprise, consist essentially of or consist of Tween 80, lecithin, and HK 2 P0 4 at pH 7.4. Kits of the disclosure may contain buffer to optimize light detection that may comprise, consist essentially of or consist of 28% Tween 80, 4% lecithin, and HK 2 P0 4 at pH 7.4.
  • the disclosure provides a method of making a recombinant phage capable of infecting a target microbe, said phage comprising at least a capsid protein sequence, a ribosome binding site, and a codon-optimized marker, comprising (a) inserting into a phage targeting vector (PTV), a nucleic acid sequence encoding the capsid protein sequence, a nucleic acid sequence encoding a ribosome binding site, and a nucleic acid sequence encoding a codon-optimized marker, (b) transforming the PTV of (a) into a phage host cell, and (c) incubating the phage host cell of (b) with a starting phage, thereby generating a recombinant phage capable of infecting a target microbe.
  • PTV phage targeting vector
  • the at least one of the nucleic acid sequence encoding the capsid protein sequence, the nucleic acid sequence encoding a ribosome binding site, and the nucleic acid sequence encoding a codon-optimized marker are a heterologous nucleic acid sequence.
  • the nucleic acid sequence encoding the capsid protein sequence, the nucleic acid sequence encoding a ribosome binding site, and the nucleic acid sequence encoding a codon-optimized marker are each a heterologous nucleic acid sequence.
  • a contiguous nucleic acid molecule comprises the nucleic acid sequence encoding the capsid protein sequence, the nucleic acid sequence encoding a ribosome binding site, and the nucleic acid sequence encoding a codon-optimized marker.
  • Host cells of the methods of the disclosure may be isolated and/or derived from a strain selected from the group consisting of 1816, 1817, 1823, 1825, 1826, 1828, 1832, 1836, 1883, 1886, 1890, 1892, 1893, 1894, 1899, 1900, 1907, 1909, 1912, 1916, 1951, 1962, 1978, 1979, 1981, 1990, 1991, 1992, 1993, 1994, 1995, 2006, 2010, 2011, 2012, 2013, 2067, 2071, 2080, 2081, 2082, 2085, 2087, 2089, 2100, 2101, 2102, 2103, 2104, 2105, 2107, 2108, 2110, 2112, 2134, 2136, 2137, 2138, B4-G7, B5-E10, B6-G7, B7-A10, B7-F6, B9-G4, BG-G10, 085-018-02 1, 085-018-02 2, 085-018-02 3, 088-013 02 SI, 088-013
  • Figure 1 is a map of the insertion site in the A511 : :COP2 engineered phage.
  • Figure 2 is a map of the insertion site in the LP124: :COP2 engineered phage.
  • Figure 3 is a map of the insertion site in the LP40: :COP2 engineered phage.
  • Figure 4 is a graph comparing the luminescence put out (measured in Relative Light Units, RLU) by healthy and sick cells infected with a mixture of A511, LP40 and LP124 engineered phage comprising either nanoluc luciferase or codon optimized COP2 luciferase.
  • Figure 5 is a graph comparing relative performance in terms of luminescence ouput of healthy and sick cells of a mixture of A511, LP40 and LP124 engineered phage comprising either the nanoluc luciferase or codon optimized COP2 luciferase.
  • Figure 6 is a graph comparing the performance in terms of luminescence output of sick cells infected with A511 engineered phage comprising either nanoluc luciferase or codon optimized COP2 luciferase.
  • Figure 7 is a graph comparing relative performance in terms of luminescence output of sick cells of A511 engineered phage comprising either the nanoluc luciferase or codon optimized COP2 luciferase.
  • Figure 8 is a series of graphs that depict the mean light signal detected in Relative Light Units (RLU) per colony forming units (CFU) in samples of various Listeria species (total of 66 species) infected with recombinant codon-optimized phage version 2 (COP2; v.1.0.2; top panel) or with recombinant optimized phage version 3 (COP3 v.1.0.3; bottom panel).
  • RLU Relative Light Units
  • CFU colony forming units
  • Figure 9 is a graph that depicts a comparison of the amounts of mean light signal detected (shown as a percentage) in Listeria samples that were infected with either recombinant codon-optimized phage version 2 (COP2; v.1.0.2) or with recombinant optimized phage version 3 (COP3 v.1.0.3).
  • the y-axis depicts the mean relative signal increase in percentage
  • the x- axis depicts the Listeria strain that utilized.
  • Figure 10 is a graph that depicts optimal phage concentration using codon-optimized, luciferase encoding, recombinant phage.
  • the y-axis depicts RLU
  • the x-axis depicts phage concentration in PFU/mL.
  • Figure 11 is a graph that demonstrating the effect of the addition of various LiCl concentrations to the IX TSB buffer in RLU output following infection of bacteria with luciferase encoding phage and normalized to RLU output obtained from lx BHI buffer. A 3 hour and a 6 hour time point were assayed. The % values are shown on the y-axis.
  • Figure 12 is a graph that summarizes the findings with respect to enzyme activity and infection rate of bacteria exposed to luciferase encoding phage while in the presence of various iterations of the infection buffer formulations and normalized to RLU output obtained from lx BHI buffer. The % values are shown on the y-axis.
  • Figures 13A and 13B are a pair of graphs depicting the effect of the addition of HEPES buffer to the Formulation- 1 infection buffer.
  • Bacterial cells were exposed, in either Formulation- 1 without HEPES or Formulation- 1 with HEPES, to luciferase encoding phage, followed by assessment of RLU values normalized to RLU output obtained from lx BHI buffer. The % values are shown on the y-axis.
  • Figure 14 is a graph that demonstrates the effects of the addition of Tween-80, or Tween- 80 and lecithin, or the addition of neither of these components to the Listeria growth broth in the presence of increasing concentrations of quaternary ammonium salts.
  • Bacterial cells were exposed to luciferase encoding phage in the following buffers, Listeria growth broth, Listeria growth broth with Tween-80, or to Listeria growth broth with the addition of both Tween-80 and lecithin.
  • the RLU values are shown on the y-axis.
  • Figure 15 is a graph that summarizes the findings with respect to enzyme activity and infection rate of bacteria exposed to luciferase encoding phage while in the presence of various iterations of the infection buffer formulations and normalized to RLU output obtained from lx BHI buffer. The % values are shown on the y-axis.
  • Figure 16 is a graph that depicts the results of a lower limit of detection assay in which Formulation-2 (NIB-12) is used as the infection buffer either alone or with the addition of Tween-80. The bacteria were exposed in the presence of either of these infection buffers to luciferase encoding phage, followed by detection of RLU. RLU values are shown on the y-axis.
  • Figures 17A-17D are a series of graphs and a table that depict the effects of the NIB-14 infection buffer or a base buffer, Letheen, in the presence of various concentrations of the F29 sanitizing solution containing quaternary ammonium compounds.
  • Figure 17A depicts the effects of using either buffer in the presence of various concentrations of F29 on phage infection activity.
  • Figure 17B depicts the effects of using either buffer in the presence of various concentrations of F29 on enzymatic activity.
  • Figure 17C depicts the effects of using either buffer during exposure of the NanoGlo luciferin to various concentrations of the F29 solution.
  • Figure 17D depicts a table with a summary of the results obtained from this series of experiments. When graphs are depicted, the y-axis represents percentage activity compared to no addition of F29.
  • Figures 18A and 18B are a series of graphs that depict the effects of incubating Letheen or NIB-14 over time with the sanitizing solution, Clorox, prior to the downstream effects on the luciferase encoding phage infection assay.
  • Assay time points for this study included 5, 10, 15, 30, 60 and 120 minutes.
  • Three concentrations of Clorox were assessed over this time, including 500ppm, lOOOppm, and 5000ppm.
  • Figure 19 is a graph that depicts the results of a series of experiments in which stressed cells (i.e. cells that were dried for 18 hours on a stainless steel table before further processing) were incubated with various NIB infection buffers, NIB-10, NIB-12 or NIB-14. As a control for the experiments, IX BHI was used. The stressed cells were exposed in each of these buffers to luciferase encoding phage, and subsequently followed by assessing the RLU for each condition. The enzyme activity was also assessed in the presence of each buffer iteration.
  • stressed cells i.e. cells that were dried for 18 hours on a stainless steel table before further processing
  • Figures 20A-20B are a series of graphs that depict lower limit of detection assays (also referred to herein as "LLOD") utilizing L. monocytogenes, incubated in various foods. LLOD assays were performed with J. monocytogenes incubated in 100% (full fat) ice cream and 100% (full fat) milk ( Figure 20 A) or NIB-14 (as a reference), or in Demi-Fraser broth incubated at 30C in with raw ground beef, deli turkey, guacamole and queso fresco (Figure 20B) for 16 hours.
  • LLOD assays were performed with J. monocytogenes incubated in 100% (full fat) ice cream and 100% (full fat) milk ( Figure 20 A) or NIB-14 (as a reference), or in Demi-Fraser broth incubated at 30C in with raw ground beef, deli turkey, guacamole and queso fresco ( Figure 20B) for 16 hours
  • Figures 21A-21D are a series of graphs that depict the time course of L. monocytogenes detection in various food samples.
  • L. monocytogenes was added to the food samples for defined amounts of time, followed by infection with a recombinant, luciferase encoding phage and subsequent detection of the luciferin signal.
  • the food samples used in the assays included turkey ( Figure 21 A), queso fresco (Figure 21B), guacamole (Figure 21C) and beef ( Figure 21D).
  • RLU values are on the y axis.
  • Figure 22A-M is a series of graphs that depict the time course of L. innocua and . monocytogenes detection in various food samples.
  • L. innocua or L. monocytogenes was added to the food samples for defined amounts of time, followed by infection with a recombinant, luciferase encoding phage and subsequent detection of the luciferin signal.
  • the food samples used in the assays included potato salad ( Figure 22A, 22B, 22H, and 221), smoked salmon (Figure 22C, 22D, 22J and 22K), ground turkey (Figure 22E, 22F, 22L and 22M) and sour cream (Figure 22G).
  • RLU values are on the y axis.
  • Figure 23A-B is a series of graphs that depict the time course of L. innocua and monocytogenes detection in either pepperoni or spinach.
  • Figure 24A-C is a series of graphs that depict the time course of detection of L. seeligeri, L. innocua, and L. monocytogenes in turkey, queso, and in guacamole.
  • Figure 25A-D is a series of graphs that depict the detection of L. monocytogenes in various food types when the assay is performed utilizing various dilution of food matrix to incubation buffer.
  • Figure 26 is a sequence alignment of the CPS open reading frame of LP143 (SEQ ID NO: 124), A511 (SEQ ID NO: 125), LPlOl (SEQ ID NO: 126), LP 124 (SEQ ID NO: 127), LP99 (SEQ ID NO: 128), LP48 (SEQ ID NO: 129), LP 125 (SEQ ID NO: 130), PI 00 (SEQ ID NO: 131) and LP40 (SEQ ID NO: 132) phage.
  • Figure 27 is an amino acid alignment of the CPS open reading frame of LP143 (SEQ ID NO: 133), A511 (SEQ ID NO: 134), LPlOl (SEQ ID NO: 135), LP124 (SEQ ID NO: 136), LP99 (SEQ ID NO: 137), LP48 (SEQ ID NO: 138), LP 125 (SEQ ID NO: 139), P100 (SEQ ID NO: 140) and LP40 (SEQ ID NO: 141) phage.
  • Figure 28 is a sequence alignment of recombinant LP143 (SEQ ID NO: 142), A511 (SEQ ID NO: 143), LP101 (SEQ ID NO: 144), LP124 (SEQ ID NO: 145), LP99 (SEQ ID NO: 146), LP48 (SEQ ID NO: 147), LP 125 (SEQ ID NO: 148), and P100 (SEQ ID NO: 149) phage engineered with firefly luciferase.
  • Figure 29 is a sequence alignment of recombinant A511 (SEQ ID NO: 150), LP124 (SEQ ID NO: 151), LP125 (SEQ ID NO: 152), P100 (SEQ ID NO: 153), and LP40 (SEQ ID NO: 154) phage with nanoluc luciferase.
  • Figures 30A and 30B are a series of bar graphs that depict the influence of the volume of Nano-Glo reagent added to the total sample volume ⁇ Listeria lysate) on the resultant relative light units (RLU) detected.
  • Figures 31 A and 31B are a series of bar graphs that depict the effects of varying the volume of sample added to the detection reaction composition while maintaining a constant volume of Nano-Glo reagent in said composition in healthy cells.
  • Figures 32A-32C are a series of bar graphs that depict the effects of varying the volume of sample added to the detection reaction composition while maintaining a constant volume of Nano-Glo reagent in said composition in stressed cells.
  • Figures 33A and 33B are a series of graphs that depict the effect of increasing the incubation temperature from 30°C to either 35°C ( Figure 33A, and 33B), or to 37°C ( Figure 33B).
  • Figure 33A is a bar graph that indicates the ratio of signal detected at 35°C/30°C. A ratio of greater than 1 indicated an increase in signal detected ( Figure 33A).
  • Figure 33B compares the average signal detected following sample incubation at 30°C, 35°C, and 37°C.
  • Figures 34A and 34B are a series of graphs that depict the number of Listeria strains detected at or above RLU cutoff based on the infection cocktail used.
  • Figures 35A and 35B are a series of graphs that depict the effect of the addition of organic solvents ⁇ i.e. acetone, isopropanol, ethanol, and propylene glycol) on the amounts of light signal emitted from a sample.
  • Figure 36 is a graph that indicates the effect of sample volume and the volume of ethanol added to the sample on the light signal emitted from the sample as a means to determine a true positive signal versus a false positive signal.
  • Figure 37 is a graph that depicts the signal to noise observed following incubation of specific healthy Listeria strains (x-axis) with either version 1.0.4 or with version 2.0 of the Listeria detection assay.
  • Figures 38A-38C are a series of graphs that depict the detection of stressed Listeria cells with the use of either vl .0.4 or with v. 2.0 of the Listeria detection assay.
  • Figures 39A and 39B are a series of graphs that depict the detected of Listeria strains from environmental samples with the use of either vl .0.4 or with v. 2.0.
  • Figure 40 depicts the performance of Listeria detection assay v2.0 versus previous iterations of the detection assay.
  • Figures 41A-41C are a series of graphs that depict the effect of substituting individual components of the Listeria detection composition v2.0 with those used in vl .0.4 in the detection of Listeria from a control sample ( Figure 40A) or from environmental samples ( Figures 40A and 40B).
  • Figure 42 is a graph that illustrates the coefficient of variation (CV) for several perameters tested in either a polyurethane (PUR) sponge or a cellulose sponge.
  • Figure 43 is a graph that depicts the average RLU/CFU value across 5 repeats with the standard deviation across the five repeats. Also plotted is the percent coefficient of variation (CV) of the assay.
  • Figure 44 is a graph that depicts the effect of pyruvate or metabisulfite on the activity of NanoLuc enzyme.
  • Figure 45 is a graph that depicts the light signal detected (RLU) in comparison to the amounts of pyruvate added to the NanoGlo buffer in the presence of 0.1% peroxide.
  • Figure 46 is a graph that depicts the effect of the addition of various concentrations of pyruvate in the NanoGlo buffer on the enzymatic activity.
  • Figure 47 is a graph that depicts the neutralization of solid peroxide-based sanitizer on polyurethane sponges by various concentrations of pyruvate in NanoGlo buffer.
  • Figure 48 is a graph that depicts the effect of Quat Neutralizer (28% Tween 80, 4% lecithin, and 3mM KH 2 PO 4 , pH 7.4) on the activity of NanoLuc enzyme.
  • Figure 49 is a graph that depicts the effects of 0.25% sodium pyruvate and 2% Quat Neutralizer in the NanoGlo buffer upon incubation in the presence of varying amounts of sanitizer concentrations.
  • Figures 50A-50C are a series of graphs that depict the LP80 phage's ability to clear Listeria cultures.
  • each point in the graph represents a single strain tested against either LP80 or LP80 RM lysate, where LP80 RM has been produced in a different host than LP80; points in the lower right quandrant produced by the two orthogonal lines in the figure represent strains that are cleared by the alternate host lysate but not by the other host lysate.
  • Figure 50B is a comparison of RLU generated by LP80 produced from either an alternate host (y-axis) or typical host (x-axis); each dot represents one of the 474 Listeria strains tested against the two phage lysates.
  • RM Restriction Modification system
  • an endogenous bacterial DNA modification system for defening against foreign DNA. Strains that show greater RLU output from the alternate host generated phage skew toward the upper left quadrant of the graph.
  • Figure 50C depicts a distribution of the fold signal increase across 474 Listeria strain panel. The average signal increase is roughly 10-fold across the entire panel with a maximum increase of 292-fold.
  • compositions, methods, and kits are presented herein for the detection of target microbes through the use of codon-optimized recombinant phage.
  • This disclosure provides recombinant phage with sequence encoding a codon optimized marker, aqueous solutions that enable robust signal detection following contact with the target microbes in a sample.
  • the compositions and methods of the disclosure provide broad detection coverage of a microbe genus, species or a combination of species.
  • composition and buffer components necessary for robust signal detection following infection by codon optimized phage is dependent on the sampled area.
  • at least one recombinant phage is provided in combination with an aqueous solution, that, together, provide optimal for the detection of microbes in an agricultural facility.
  • a particular challenge of detection of microbes in an agricultural facility is the potential for the presence of trace sanitation solutions that may interfere with signal detection.
  • Another embodiment relates to the use of recombinant phage for the detection of microbes in agricultural products themselves, such as, for example, food stuffs intended for human or animal consumption. Detection of microbes in an agricultural sample presents unique challenges in that components of the agricultural sample may contain substances that interfere with signal detection.
  • the aqueous solutions presented herein are formulated to minimize such interference.
  • compositions presented herein are optimized in order to allow the propagation of microbes from a test sample, infection of the microbes with a recombinant phage that encodes a detectable marker, and the quantification of the amounts of the microbes from the sample by way of detection of the recombinant phage marker.
  • Phage infective engineering is used herein to make recombinant phage.
  • PIE methodology is disclosed in U.S. Patent Application No. 14/226,889, which is hereby incorporated herein in its entirety by reference.
  • This method is sometimes referred to herein as phage infective engineering (PIE).
  • PIE phage infective engineering
  • This method allows insertion of a heterologous nucleic acid sequence into any desired location of a phage genome.
  • the PIE method utilizes a phage targeting vector (PTV) that is transformed into a phage host cell.
  • the PTV comprises a heterologous nucleic acid sequence (such as an open reading frame encoding a marker) for insertion into a phage genome.
  • the heterologous nucleic acid sequence is flanked by upstream and downstream homology regions, which are located adjacent to the desired insertion site.
  • the homology regions in the vector are directly adjacent in a starting phage genome. Such embodiments allow insertion of the heterologous nucleic acid sequence into the phage genome without a loss of endogenous phage sequence.
  • the homology regions in the vector flank a region of the starting phage genome that is not included in the vector.
  • Such embodiments allow insertion of the heterologous nucleic acid sequence into the phage genome while deleting a region of the starting phage genome at the site of insertion.
  • Such embodiments allow, for example, the replacement of an endogenous phage sequence with a replacement sequence.
  • the starting sequence that is deleted and the replacement sequence display sequence homology, such as homology of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher.
  • the upstream homology region, downstream homology region, and heterologous nucleic acid sequence are combined in a vector to make a PTV.
  • a suitable vector is pMK4; however, skilled artisans are aware of many suitable vectors that may be used for this purpose.
  • the plasmid may be isolated in any suitable host, such as E. coli. Upon verification, the plasmid is then transformed into a phage host cell.
  • a cell useful for many Listeria phage is the L. monocytogenes strain EGD-e.
  • the initial recombination was performed by incubating the transformed phage host cell with starting phage.
  • the infection is assayed using any suitable method to identify recombinant phage that comprise the heterologous nucleic acid sequence.
  • PCR is one method that may be used.
  • the heterologous nucleic acid sequence comprises an open reading frame the presence of transcripts encoded by that open reading frame, the presence of the encoded gene product, or functional readouts of the encoded gene product may be screened for in cultures of cells infected with the resultant phage to identify recombinant phage.
  • the disclosure provides recombinant phage comprising a heterologous nucleic acid sequence encoding a codon optimized marker.
  • the phage can be LP40, LP48, LP99, LP101, LP124, LP125, LP143, A511, or P100.
  • the marker can be any detectable marker. In one embodiment the marker is luciferase.
  • codon optimized phages should take into account a variety of factors, including the frequency of codon usage in a host organism, nearest neighbor frequencies, RNA stability, the potential for secondary structure formation, the route of synthesis and the intended future DNA manipulations of that gene.
  • the degeneracy of the genetic code permits the same amino acid sequence to be encoded and translated in many different ways. For example, leucine, serine and arginine are each encoded by six different codons, while valine, proline, threonine, alanine and glycine are each encoded by four different codons.
  • synonymous codons varies from genome to genome among kingdoms and phyla.
  • synonymous codon-choice patterns among mammals are very similar, while evolutionarily distant organisms such as yeast (S. cerevisiae), bacteria (such as E. coli) and insects (such as D. melanogaster) reveal a clearly different pattern of genomic codon use frequencies.
  • codon selection may vary with the species, strain or ribotype of the host to be infected by a particular phage.
  • codon usage may vary with the environment in which the host exists, depending on factors such as temperature, pH, pressure, and other external parameters. Further, codon usage may vary with the state of growth in which the host exists, e.g. depending on rapid division vs. non-division, or within a healthy or an injured cellular state.
  • the preferred codon usage frequencies for a recombinant phage should reflect the codon usages of genes derived from the genome of the intended host organism.
  • a gene can be optimized by replacing codons of the origin species with known preferred codons from a host organism encoding the same amino acid.
  • a host organism is Listeria.
  • software can be utilized which applies an algorithm to a genetic sequence which will codon optimize the sequence for a specific host organism.
  • software from DNA 2.0TM can be used to codon optimize a genetic sequence for a specific host organism.
  • Example algorithms for codon optimization in silico have been described ⁇ see Villalobos et al. BMC Bioinformatics. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. PLoS ONE. 2011 6:el9912.; US8,635,029; US8,401,708; US8, 126,653; US8,005,620; US7,805,252; US7,561,973; US7,561,72.)
  • codon optimization allows for increased expression of phage encoded proteins in a host organism.
  • the host organism is a bacterium.
  • codon optimization allows for increased expression of reporter proteins or polypeptides encoded by a recombinant phage in a host organism.
  • codon optimization of recombinant phage allows for increased expression of a luciferase reported by Listeria.
  • Listeria phages used for recombination may be selected from A511, LP124, and LP40.
  • recombinant phages comprise the entirety of the original phage genome.
  • recombinant phages comprise deletions to the original phage genome and addition of heterologous nucleic acid sequences.
  • recombinant phages comprise added stop codons.
  • recombinant phages comprise added ribosome binding sites.
  • recombinant phages comprise a codon optimized reporter gene.
  • a reporter gene is a sequence encoding luciferase.
  • the luciferase reporter gene is a codon optimized NanoLuc sequence optimized for expression in Listeria.
  • a recombinant phage is an A511 phage comprising added stop codons, an added ribosome binding site, and an added codon optimized NanoLuc sequence.
  • a recombinant phage is a LP124 phage comprising added stop codons, an added ribosome binding site, and an added codon optimized NanoLuc sequence.
  • a recombinant phage is a LP40 phage comprising added stop codons, an added ribosome binding site and added codon optimized NanoLuc sequence.
  • the disclosure provides formulations of an aqueous solution which effectively enable bacteria isolated from a test site to be productively infected by recombinant phage.
  • the aqueous solution is capable of preserving the enzymatic activity used in the phage based detection system.
  • a major difficulty encountered in the detection of bacteria using phage based recombinant markers is the potential interactions between sanitation reagents found in the sample and the test reagent compounds that are used to quantify bacterial presence or absence. This problem is augmented given the propensity of facilities to use amounts of disinfectants in excess of the recommended guidelines presented by the Federal Drug
  • FDA United States Department of Agriculture
  • USD A United States Department of Agriculture
  • CDC Centers for Disease Control
  • Overuse of these sanitization agents may lead to obfuscation of true positive or true negative results due to (i) decreasing enzymatic activity required for the phage- based detection system, (ii) lowering the ability of phage to infect bacteria collected from the test site, or (iii) disrupting the bacterial cells to a degree that they are not detectable.
  • Collection and processing of a an environmental sample from a test site follows a stepwise process that includes: (a) collection of the sample by way of swabbing the surface with a sponge, followed by immediate placement of the sponge into an isolated container; (b) processing the sample begins with the addition of an aqueous solution (infection buffer), and the addition of a marker encoding phage to the sample collecting sponge; (c) incubation of the solution impregnated sponge at an appropriate temperature range; (d) isolation of the liquid from the sponge by way of centrifugation; and (e) detection of a signal in the liquid with an instrument (e.g. luciferase presence with a luminometer).
  • an instrument e.g. luciferase presence with a luminometer
  • the solution added to the sponge is a buffer that contains reagents that minimize the interaction with components of commonly used sanitization solutions that have been found to reduce signal detection ability (e.g. by either reducing phage infection or by reducing enzymatic activity, or by affecting the luciferin substrate).
  • the purposes of the buffer include recovery of the isolated stressed and injured bacteria in order to optimize phage infection and to optimize downstream signal detection.
  • the marker used for signal detection can be any detectable marker.
  • Preferred detection signal systems include luciferase based assays.
  • Bacterial cells collected from the environment present many additional challenges to the downstream processing required for adequate signal detection. Many of these challenges relate to the health of the cells upon collection.
  • the collected cells may be starved, osmotically stressed, and have underlying oxidative stress.
  • Formulations have been developed, and described herein, to overcome these challenges encountered following the collection of the cells. For example, detailed herein, and specifically in the Examples section, are formulations to overcome osmotic stress (e.g. via addition of glycerol), cell starvation (e.g. via addition of nutrients including carbon, nitrogen source, sugars and vitamins), and oxidative stress (e.g. via the addition of vitamins including those contained in yeast extract). Interaction with non-target biologicals also poses a challenge in the downstream signal detection methods. Formulations presented herein have been optimized to overcome non-target biological interactions via the addition of either nalidixic acid and/or lithium salts.
  • a base aqueous solution of the disclosure is Formulation-1 (Table 1).
  • a preferred embodiment of the aqueous solutions of the disclosure is
  • Formulation-2 (also referred to herein as “ ⁇ 3-12”) (Table 3).
  • the addition of 20mM HEPES increases enzyme activity and stability, and increases the buffering capacity against pH extremes (See Example 7).
  • ⁇ 3-14 contains lecithin, Tween-80 and potassium phosphate added to the base components of NIB-12.
  • NIB-14 allows for greater phage infection ability and increased enzymatic activity compared with a base medium (BHI), and also allows for greater neutralization of remnant sanitizer chemicals in comparison to other aqueous solutions tested (See Examples 8 and 11).
  • Table 4 Infection Formulation NIB-14 [00124] Table 5 lists the groups and category of the reagents that are included in the aqueous solutions disclosed herein, and the affect that each of these components has on Listeria detection.
  • compositions and methods of the disclosure are particularly beneficial in comparison to currently used methods of microbial detection in that the present invention, (i) has minimal sample preparation, (ii) is capable of detecting microbes in undiluted or minimally diluted matrix of certain foods resulting in less operator and cross- contamination risk, smaller volumes (less cost) and less waste, (iii) has high sensitivity and specificity, and (iv) has a total time to result of less than 8-10 hours.
  • compositions and methods of the disclosure incorporate the use of marker encoding phage, infection buffer/media, and a quantification of the amounts of phage marker present following phage infection of a sample in order to identify microbial presence in food samples.
  • Preferred embodiments of the compositions and methods of the disclosure enable the detection of microbes in various food sources, including fatty foods, such as for example, whole milk, ice cream, queso fresco, and guacamole; salty foods, such as for example, deli turkey; and other foods, such as for example, beef.
  • microbial target species for the current invention include species of Listeria.
  • compositions and methods of the disclosure are capable of detecting target microbes in an undiluted food matrix. These properties contribute to the minimal sample preparation steps and associated rapid processing associated with the use of the present methods and compositions.
  • the use of the recombinant phage containing the codon-optimized marker sequence in the compositions and methods of the disclosure enables the rapid detection of extremely low numbers of microbes (e.g. Listeria monocytogenes in various foods, see Example 13), and the detection of microbes in lower limit of detection assays (also referred to herein as "LLOD”) of down to 1 CFU in certain foods (see Example 14).
  • LLOD lower limit of detection assays
  • the phage LP40, LP48, LP99, LP101, LP124, LP125, LP143, and A511 were selected for engineering.
  • the examples describe making recombinant versions of the phage LP40, LP48, LP99, LP101, LP124, LP125, LP143, A511, and PlOO, comprising a heterologous nucleic acid sequence encoding a marker.
  • those phage are useful, for example, to detect target bacteria, as further disclosed throughout this application.
  • this disclosure provides recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker.
  • the recombinant phage comprises a genome comprising a region of at least 1 kb that comprises substantial homology to a region of at least 1 kb of the genome of at least one phage selected from LP40, LP48, LP99, LP101, LP124, LP125, LP143, A511, and P100.
  • the region of homology comprises at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, or more.
  • the region of homology is the entire genome of the recombinant Listeria phage.
  • the substantial homology is nucleotide sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% across the region of homology.
  • This disclosure provides the amino acid sequences of the cps gene of the phage LP40 (SEQ ID NO: 6), LP48 (SEQ ID NO: 8), LP99 (SEQ ID NO: 10), LP101 (SEQ ID NO: 12), LP 124 (SEQ ID NO: 14), LP 125 (SEQ ID NO: 16), LP 143 (SEQ ID NO: 18), A511 (SEQ ID NO: 20), and P100 (SEQ ID NO: 22).
  • this disclosure provides recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker, wherein the recombinant Listeria phage comprises a nucleic acid sequence that encodes a protein selected from SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, and 22, and muteins thereof.
  • This disclosure also provides the nucleotide sequences of the open reading frames of the cps gene of the phage LP40 (SEQ ID NO: 5), LP48 (SEQ ID NO: 7), LP99 (SEQ ID NO: 9), LP101 (SEQ ID NO: 11), LP124 (SEQ ID NO: 13), LP125 (SEQ ID NO: 15), LP143 (SEQ ID NO: 17), A511 (SEQ ID NO: 19), and PI 00 (SEQ ID NO: 21).
  • this disclosure provides recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker, wherein the recombinant Listeria phage comprises a nucleic acid sequence selected from SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17, 19, and 21, and nucleic acid sequences comprising substantial homology thereto.
  • the recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker comprises a screenable marker.
  • the marker is a luciferase.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2.
  • the luciferase is encoded by a nucleic acid sequence comprising SEQ ID NO: 1 or a nucleic acid sequence comprising substantial homology to SEQ ID NO: 1 capable of encoding a luciferase that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to SEQ ID NO: 2.
  • the recombinant Listeria phage is selected from LP48: :ffluc, LP99: :ffluc, LP101 : :ffluc, LP124: :ffluc, LP125: :ffluc, LP143 : :ffluc, A511 : :ffluc, P100: :ffluc, LP48: :COP2, LP48::COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP2, LP48: :COP
  • the recombinant Listeria phage is selected from phage comprising genomes comprising substantial homology to at least one phage selected from LP48: :ffluc, LP99: :ffluc, LP101 : :ffluc,
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%) or at least 99% identical to SEQ ID NO: 4.
  • the luciferase is encoded by a nucleic acid sequence comprising SEQ ID NO: 3 or a nucleic acid sequence comprising substantial homology to SEQ ID NO: 3 capable of encoding a luciferase that is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to SEQ ID NO: 4.
  • the recombinant Listeria phage is selected from LP040: :nluc, LP124: :nluc, LP125: :nluc, A511 : :nluc, P100: :nluc, LP040: :COP2, LP124: : COP2, LP125: : COP2, A511 : : COP2, P100: : COP2, LP040: :COP3, LP124: : COP3, LP125: : COP3, A511 : : COP3, P100: : COP3.
  • the recombinant Listeria phage is selected from phage comprising genomes comprising substantial homology to at least one phage selected from LP040: :nluc, LP124: :nluc, LP125: :nluc, A511 ::nluc, P100: :nluc, LP040: :COP2, LP124: : COP2, LP125: : COP2, A511 : : COP2, P100: : COP2, LP040: :COP3, LP124: : COP3, LP125: : COP3, A511 : : COP3, P100: : COP3.
  • the heterologous nucleic acid sequence encoding a marker is operatively linked in the recombinant phage genome to at least one regulatory element that is also heterologous to the phage genome.
  • expression of the heterologous nucleic acid sequence encoding a marker in target bacteria is controlled exclusively by regulatory elements that are heterologous to the phage genome.
  • the heterologous nucleic acid sequence encoding a marker is operatively linked in the recombinant phage genome to at least one regulatory element that is endogenous to the phage genome.
  • the heterologous nucleic acid sequence encoding a marker is operatively linked to the endogenous regulatory element by virtue of the location in the starting phage genome where the heterologous nucleic acid sequence encoding a marker is placed.
  • expression of the heterologous nucleic acid sequence encoding a marker in target bacteria is controlled exclusively by regulatory elements that are endogenous to the phage genome.
  • expression of the heterologous nucleic acid sequence encoding a marker in target bacteria is controlled in part by regulatory elements that are endogenous to the phage genome and in part by regulatory elements that are
  • the recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker comprises more than one heterologous nucleic acid sequence encoding a marker.
  • the recombinant phage comprises multiple copies of the same nucleic acid sequence encoding a marker (i.e., copy encodes the same marker).
  • the recombinant phage comprises copies of more than one type of nucleic acid sequence encoding a marker (i.e., at least two copies encode different markers).
  • the more than one copy are positioned at adjacent locations in the recombinant phage genome. In other embodiments at least one (up to all) of the more than one copy are located at non-adjacent locations in the recombinant phage genome.
  • the length of the heterologous nucleic acid sequence is at least 100 bases, at least 200 based, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, at least 1.0 kilobase (kb), at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, at least 1.7 kb, at least 1.8 kb, at least 1.9 kb, at least 2.0 kb, at least 2.1 kb, at least 2.2 kb, at least 2.3 kb, at least 2.4 kb, at least 2.5 kb, at least 2.6 kb, at least 2.7 kb, at least 2.8 kb, at least 2.9 kb, at least 3.0 kb, at least 3.1 kb, at least 3.2 kb, at
  • the length of the heterologous nucleic acid sequence is 500 bases or less, 1,0 kb or less, 1.5 kb or less, 2.0 kb or less, 2.5 kb or less, 3.0 kb or less, 3.5 kb or less, 4.0 kb or less, 4.5 kb or less, 5.0 kb or less, 5.5 kb or less, 6.0 kb or less, 6.5 kb or less, 7.0 kb or less, 7.5 kb or less, 8.0 kb or less, 8.5 kb or less, 9.0 kb or less, 9.5 kb or less, or 10.0 kb or less.
  • the heterologous nucleic acid sequence comprises a length that is less than the maximum length of heterologous nucleic acid sequence that can be packaged into a phage particle encoded by the phage genome and comprising the phage genome.
  • the length of the heterologous nucleic acid sequence is from 100 to 500 bases, from 200 to 1,000 bases, from 500 to 1,000 bases, from 500 to 1,500 bases, from 1 kb to 2 kb, from 1.5 kb to 2.5 kb, from 2.0 kb to 3.0 kb, from 2.5 kb to 3.5 kb, from 3.0 kb to 4.0 kb, from 3.5 kb to 4.5 kb, from 4.0 kb to 5.0 kb, from 4.5 kb to 5.5 kb, from 5.0 kb to 6.0 kb, from 5.5 kb to 6.5 kb, from 6.0 kb to 7.0 kb, from 6.5 kb to 7.5 kb, from 7.0 kb to 8.0 kb, from 7.5 kb to 8.5 kb, from 8.0 kb to 9.0 kb, from 8.5 kb to 9.5 kb, or from 9.0 kb
  • the ratio of the length of the heterologous nucleic acid sequence to the total length of the genome of the recombinant phage is at least 0.05, at least 0.10, at least 0.15, at least 0.20, or at least 0.25. In some embodiments the ratio of the length of the genome of the recombinant phage to the length of the genome of the corresponding starting phage is at least 1.05, at least 1.10, at least 1.15, at least 1.20, or at least 1.25.
  • the heterologous nucleic acid sequence is inserted into the starting phage genome with no loss of endogenous starting phage genome sequence. In some embodiments the inserted heterologous nucleic acid sequence replaces endogenous starting phage genome sequence. In some such embodiments the heterologous nucleic acid sequence replaces an amount of endogenous genomic sequence that is less than the length of the heterologous nucleic acid sequence. Thus, in such embodiments the length of the recombinant phage genome is longer than the length of the starting phage genome. In some such
  • heterologous nucleic acid sequence replaces an amount of endogenous genomic sequence that is greater than the length of the heterologous nucleic acid sequence.
  • the length of the recombinant phage genome is shorter than the length of the starting phage genome.
  • the heterologous nucleic acid sequence replaces an amount of endogenous genomic sequence that is equal to the length of the heterologous nucleic acid sequence.
  • the protein or polypeptide encoded by a heterologous open reading frame is modified to reduce cleavage by proteases present in phage host cells.
  • computational algorithms can be used to identify known protease cleavage sites and the sequence of the open reading frame may be modified using conservative substitutions to remove these sites.
  • directed mutagenesis is used to evolve the open reading frame sequence to encode a product that has an increased resistance to at least one protease present in a phage host cell or in the culture of a phage host cell.
  • This disclosure also provides isolated nucleic acids obtainable from a recombinant phage of this disclosure.
  • the isolated nucleic acid is an isolated genome of a recombinant phage of this disclosure.
  • the isolated nucleic acid comprises a fragment of less than the total genome of recombinant phage of this disclosure, the fragment comprising at least 10%, at least 20%>, at least 30%>, at least 40%>, at least 50%>, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the genome of the recombinant phage.
  • the isolated nucleic acid comprises a fragment of less than the total genome of recombinant phage of this disclosure, the fragment comprising at least 20 bp, at least 50 bp, at least lOObp, at least 500 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, or at least 5 kb of the phage genome.
  • the isolated nucleic acid comprises a fragment that is homologous to a fragment disclosed in this paragraph.
  • the recombinant phage of this disclosure may be used to detect the presence of bacteria. Detection of target bacteria is based on the ability of the recombinant phage to bind to target bacteria, transfer of the phage genome into the target bacteria, and express the
  • heterologous nucleic acid sequence encoding a marker by the bacteria is based on the range of bacterial types that support expression of the marker following exposure to the phage.
  • the range of bacterial types that support expression of the marker following exposure to the phage is referred to herein as the "host range” of the phage.
  • the set of bacterial types that make up the host range of the phage is sometimes referred to herein as "target bacteria" for the phage.
  • the methods comprise exposing a candidate type of bacteria to a phage in a liquid culture.
  • the ability of the phage to cause clearing of the culture, which reflects infection and lysis of bacteria in the culture by the phage, is an indication that the bacteria in the culture are target bacteria of the phage.
  • this method is surprisingly more accurate in assessing the true phage host range for a phage than prior art plate-based plaque assays.
  • the "host range" of a phage or the "target bacteria" of a phage are defined based on a set of bacteria that a phage can clear in a liquid culture-based assay.
  • the liquid culture method is an improvement over prior methods and is very useful for many purposes, it does embody all aspects of methods of using a recombinant phage to detect target bacteria. Such methods rely on the ability of the recombinant phage to bind to target bacteria, transfer of the phage genome into the target bacteria, and expression of the heterologous nucleic acid sequence encoding a marker by the bacteria. Accordingly, even if a phage is unable to lyse a liquid culture of a particular bacterial cell type the phage may nonetheless be able to bind to the bacteria type, transfer the phage genome into the target bacteria, and thus cause expression of a heterologous nucleic acid sequence encoding a marker by the bacteria. Indeed, as demonstrated by the examples, assays that detect the presence of the marker in a type of bacteria following exposure to a recombinant phage are in some
  • the "host range" of a phage or the "target bacteria" of a phage are defined by a process that comprises 1) providing a recombinant phage comprising a heterologous nucleic acid sequence encoding a marker; 2) exposing a sample to the phage; and 3) assaying for the presence of the marker in the exposed sample.
  • This type of assay is sometimes referred to herein generally as a "marker host range assay.”
  • assaying for the presence of the marker in the exposed sample is by a method comprising detection of an mRNA.
  • assaying for the presence of the marker in the exposed sample is by a method comprising direct detection of marker protein, such as using an antibody.
  • assaying for the presence of the marker in the exposed sample is by a method comprising functional detection of marker protein. For example, if the marker protein is a luciferase the exposed sample may be exposed to luciferin and production of light may be assayed. This method may be adapted to any type of marker disclosed herein and skilled artisans are aware that many variations on the detection method of the marker may be used.
  • Certain variables may modify the host range of phage under certain conditions.
  • bacteriophage is capable of recognizing. Furthermore, it includes the chemical and physical composition of the environment that the bacteriophage and the bacterium experience when the phage contacts a bacterium. Environmental factors of the solution such as but not limited to pH, osmolarity, temperature, rheological properties and others all may impact the ability of a bacteriophage to infect a bacterium.
  • the step of exposing a sample of bacteria to a phage in the liquid clearing host-range assay and the marker host range assay may be conducted under defined conditions.
  • the defined conditions may comprise at least one of: a defined time duration, a defined temperature, and the presence of at least one of a) at least one compound selected from carbohydrates and related compounds, b) at least compound selected from nitrogen containing compounds, c) at least compound selected from nucleic acids and related compounds, d) at least compound selected from lipid, e) at least one inorganic compound, and f) at least one organic compound.
  • the carbohydrates and related compounds are selected from sugars such as glucose, mannose, and maltose.
  • the carbohydrates and related compounds are selected from carboxy sugars that are degraded by the pentose phosphate pathway, which may but need not generate more moles of NADPH per mole consumed as compared to glucose.
  • the carbohydrates and related compounds are selected from compounds feeding into central metabolism, such as but not limited to a ketoglutarate, D-malic acid, or pyruvic acid.
  • the carbohydrates and related compounds are selected from glycerol and other carbohydrate (or other) osmoprotectants that may but need not provide osmotic support to cells that exist in a potentially weakened or damaged state in the environment.
  • glycerol functions as a volume excluder that increases the efficiency of phage infection.
  • the carbohydrates and related compounds are selected from sugars such as glucose, mannose, and maltose.
  • carboxy sugars that are degraded by the pentose phosphate pathway, which may but need not
  • carbohydrates and related compounds are selected from sugar alcohols, such as aminoethanol.
  • the nitrogen containing compounds are selected from ammonium, other amino acid building blocks, and free amino acids.
  • the free amino acid may be any genome encoded standard amino acid or any non-standard amino acid.
  • the amino acid is selected from glutamic acid and glutamine.
  • the amino acid is selected from branched chain amino acids.
  • the nitrogen containing compounds are selected from degradation products of branched amino acids such as propionic acid.
  • nucleic acids and related compounds are selected from nucleotides, nucleosides, deoxynucleotides, and deoxynucleosides. In some embodiments the nucleic acids and related compounds are selected from metabolites of the nucleotide generation pathways such as inosine.
  • the lipid compounds are selected from fatty acids and related compounds. Tween 20, 40, and 80 are converted to fatty acids upon ester hydrolysis and can also be used. In some embodiments the lipid compounds are selected from lecithin and related compounds.
  • the inorganic compounds are selected from salts, such as for example thiosulfate.
  • the organic compounds are selected from aliphatics, aromatics, heterocyclics, and non-biogenic polymers.
  • the at least one compound is selected from:
  • Another approach to modify the host range detected in a host range assay is to pretreat bacteria before exposing the bacterial samples to the phage. This allows for a decoupling of steps designed to modify the state of a bacterial cell (and possibly its susceptibility to phage infection) from conditions used for the infection itself. For example the metabolic rate may be increased during a pre-incubation step, which in turn may increase at least one of the replicative, transcriptive, and translative functions that influence clearing or production of a marker following infection of a bacterial cell by a phage. Furthermore, it is possible that such an incubation period also changes the surface receptor expression, or changes the composition of the cell wall of the bacterium, which may also modify whether a phage can productively infect the bacteria.
  • samples of bacteria are incubated in metabolic stimulation conditions before exposure to the phage for the phage host range assay.
  • exposure of the cells to metabolic stimulation conditions stimulates cell division in the cells.
  • exposure of the cells to metabolic stimulation conditions does not stimulate cell division in the cells.
  • exposure of the cells to metabolic stimulation conditions stimulates at least one of the replicative, transcriptive, and translative functions that influence clearing or production of a marker following infection of a bacterial cell by a phage.
  • metabolic stimulation conditions are conditions that promote development of a microorganism metabolic state in which the microorganism is permissive to infection and maintenance of a phage life cycle and/or infection followed by expression of a marker gene produce encoded by a heterologous nucleic acid sequence in the genome of the phage.
  • the microorganism prior to exposure to the metabolic stimulation conditions is not permissive to infection and maintenance of a phage life cycle.
  • the microorganism prior to exposure to the metabolic stimulation conditions is in a metabolic state that reduces its susceptibility to infection and maintenance of a phage life cycle compared to a comparable microorganism grown under log phase conditions.
  • metabolic stimulation conditions comprise at least one of a permissive temperature, pH, Po 2 , and nutrient combination.
  • the target microbe undergoes at least one cell division under metabolic stimulation conditions.
  • the target microbe does not undergo at least one cell division under metabolic stimulation conditions.
  • the sample is exposed to metabolic stimulation conditions before the sample is contacted with a phage. In some such embodiments the sample is then removed from metabolic stimulation conditions prior to contacting with a phage while in other embodiments the sample is maintained under metabolic stimulation conditions when contacted by a phage. In some embodiments the sample is exposed to a first set of metabolic stimulation conditions for a first period of time and then transferred to a second set of metabolic stimulation conditions. In some embodiments the recombinant phage is exposed to the sample while the sample is maintained under the second set of metabolic stimulation conditions.
  • the sample is exposed to metabolic stimulation conditions for from 5 minutes to 24 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 5 minutes to 6 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 10 minutes to 6 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 20 minutes to 6 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 30 minutes to 6 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 1 to 6 hours before the sample is contacted by a phage.
  • the sample is exposed to metabolic stimulation conditions for from 2 to 6 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 2 to 12 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 3 to 12 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 6 to 12 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for from 12 to 24 hours before the sample is contacted by a phage. In some embodiments the sample is exposed to metabolic stimulation conditions for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 1.5 hours, or at least 2 hours.
  • the conditions used for the host range analysis are also used for methods of detecting target bacteria using the phage when those phage are used to detect target bacteria in other contexts (i.e., when testing environmental samples).
  • the recombinant phage are useful to detect target microbes.
  • This disclosure provides exemplary recombinant phage and methods of making further recombinant phage.
  • This disclosure also defines the target bacteria of certain disclosed recombinant phage and provides methods of identifying the target bacteria of any phage, including any recombinant phage.
  • this disclosure enables methods of detecting target microbes using recombinant phage.
  • enabling a detailed characterization of the target bacteria of the recombinant phage this disclosure in certain embodiments provides useful methods not available in the prior art.
  • the methods are broadly applicable and in view of the teachings of this disclosure skilled artisans will understand how to apply the methods to detect any type of archaea and/or bacteria.
  • the archaea is a Euryarcheota.
  • the archaea is a Crenarcheota.
  • the bacteria is a member of a phyla selected from Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus,
  • Dictyoglomi Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Synergistets, Tenericutes,
  • Thermodesulfobacteria Thermotogae.
  • the bacteria is at least one
  • the bacteria is at least one Proteobacteria selected from Acidobacillus, Aeromonas, Burkholderia, Neisseria, Shewanella, Citrobacter, Enterobacter, Erwinia, Escherichia, Klebsiella, Kluyvera, Morganella, Shigella, Yersinia, Coxiella, Rickettsia, Legionella, Avibacterium, Haemophilus, Pasteurella, Acinetobacter, Moraxella, Pseudomonas, Vibrio, Xanthomonas.
  • the bacteria is at least one Tenericutes selected from Mycoplasma, Spiroplasma, and Ureaplasma.
  • Common bacterial contaminates of food that are detected using the phage and methods disclosed herein include, without limitation, E. coli (including without limitation pathogenic E. coli, E. coli 0157:H7, Shiga-toxin producing E. coli, E. coli 026, E. coli 111, E. coli 0103, E. coli 0121, E. coli 045 and E. coli 0145), coliform bacteria (which include without limitation, Citrobacter, Enter obacter, Hafiiia, Klebsiella, Serratia), Shigella, Listeria,
  • Clostridium including Clostridium botulinum and Clostridium perfringens
  • Vibrio including Vibrio cholera and Vibrio vulnificus
  • Enter obacteriacae Staphylococcus (including
  • Staphylococcus aureus and Staphylococcus epidermis Bacillus (including Bacillus cereus), Campylobacter (including Campylobacter jejuni), Pseudomonas, Streptococcus, Acinetobacter, Klebsiella, Campylobacter, and Yersinia.
  • the methods comprise providing a sample; exposing the sample to at least a first type of recombinant phage capable of infecting at least a first set of target bacteria, comprising a heterologous nucleic acid sequence encoding at least a first marker and assay for the at least one first marker in the exposed sample.
  • the first type of recombinant phage comprises a heterologous nucleic acid sequence; a codon optimized at least first markers.
  • detection of the first marker in the sample indicates the presence of bacteria of the first set of target bacteria in the sample.
  • the methods comprise providing a sample; exposing the sample to a first type of phage capable of infecting a first set of target bacteria and comprising a heterologous nucleic acid sequence encoding a first marker; exposing the sample to a second type of phage capable of infecting a second set of target bacteria and comprising a heterologous nucleic acid sequence encoding a second marker; and assaying for the presence of the first marker and the second marker in the exposed sample.
  • detection of the first marker in the sample indicates the presence of bacteria of the first set of target bacteria in the sample.
  • detection of the second marker in the sample indicates the presence of bacteria of the second set of target bacteria in the sample.
  • the first marker and the second marker are the same, and detection of the marker in the sample indicates the presence of bacteria of at least one of the first set of target bacteria and the second set of target bacteria in the sample.
  • the first set of target bacteria and the second set of target bacteria independently comprise at least two species of a single genus of bacteria. In some embodiments, the first set of target bacteria and the second set of target bacteria independently comprise at least three species of a single genus of bacteria. In some embodiments, the first set of target bacteria and the second set of target bacteria independently comprise at least four species of a single genus of bacteria. In some embodiments, the single genus of bacteria is Listeria. In some embodiments, the first set of target bacteria and the second set of target bacteria comprise at least one species of bacteria in common. In some embodiments, the first set of target bacteria and the second set of target bacteria comprise at least two species of bacteria in common.
  • the first set of target bacteria and the second set of target bacteria comprise at least three species of bacteria in common. In some embodiments, the first set of target bacteria and the second set of target bacteria comprise at least four species of bacteria in common.
  • the species of Listeria are selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii, Listeria marthii, Listeria rocourti and Listeria welshimeri. In some embodiments, the species of Listeria are selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri.
  • the target bacteria comprise at least one sig B allelotype of Listeria innocua selected from 11, 22, 37, and 56. In some embodiments, the target bacteria comprise at least four allelotypes of Listeria innocua. In some embodiments, the at least four allelotypes of Listeria innocua are 11, 22, 37, and 56.
  • the target bacteria comprise at least one ribotype of Listeria monocytogenes selected from DUP-10142, DUP-1030A, DUP-1030B, DUP-1038B, DUP-1039A, DUP-1039B, DUP-1039C, DUP-1042A, DUP-1042B, DUP-1042C, DUP-1043A, DUP-1044A, DUP-1044B, DUP-1044E, DUP-1045B, DUP-1052A, DUP-1053A, DUP-1062A, and DUP-1062D.
  • the target bacteria comprise at least nineteen ribotypes of Listeria monocytogenes.
  • the at least nineteen ribotypes of Listeria monocytogenes are DUP-10142, DUP-1030A, DUP-1030B, DUP-1038B, DUP-1039A, DUP- 1039B, DUP-1039C, DUP-1042A, DUP-1042B, DUP-1042C, DUP-1043A, DUP-1044A, DUP- 1044B, DUP-1044E, DUP-1045B, DUP-1052A, DUP-1053A, DUP-1062A, and DUP-1062D.
  • the target bacteria comprise at least one sig B allelotype of Listeria seeligeri selected from 3, 20, 24, and 35. In some embodiments, the target bacteria comprise at least four allelotypes of Listeria seeligeri. In some embodiments, the at least four allelotypes of Listeria seeligeri are 3, 20, 24, and 35.
  • the target bacteria comprise at least one sig B allelotype of Listeria welshimeri selected from 15, 27, 32, and 89. In some embodiments, the target bacteria comprise at least four allelotypes of Listeria welshimeri. In some embodiments, the at least four allelotypes of Listeria welshimeri are 15, 27, 32, and 89.
  • the first set of target bacteria are all members of the same genus.
  • the second set of target bacteria are all members of the same genus.
  • all of the target bacteria are Listeria.
  • the target bacteria do not include at least one of Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterococcus durans, Enterococcus faceium, Enterococcus hirae, Kocuria varians, Kurthia gibsonii, Kurthia zopfii, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus equi, Streptococcus galloyticus, Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactus, Lactobacillus fermentum, Micrococcus lutue
  • the target bacteria do not include Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterococcus durans, Enterococcus faceium,
  • Streptococcus equi Streptococcus galloyticus, Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactus, Lactobacillus fermentum, Micrococcus lutues, Pseudomonas protogens, Pseudomonas florescens, Aeromonas sp, Serratia liquefaciens, Serratia proteamaculans, Serratia liquefaciens, Bacillaceae bacterium, Serratia proteamaculans, Pseudomonas florescens, Pseudomonas poae, Pseudomonas sp, Pseudomonas fragi, Providencia alcalifaciens, Serratia sp, Serratia grime sii, Hafnia sp., Serratia proteamaculans, Pseudomonas flores
  • the methods further comprise exposing the sample to a third type of phage capable of infecting a third set of target bacteria and comprising a
  • the methods further comprise exposing the sample to a fourth type of phage capable of infecting a fourth set of target bacteria and comprising a heterologous nucleic acid sequence encoding a fourth marker. In some embodiments, the methods further comprise exposing the sample to a fifth type of phage capable of infecting a fifth set of target bacteria and comprising a heterologous nucleic acid sequence encoding a fifth marker. In some embodiments, the methods further comprise exposing the sample to a sixth type of phage capable of infecting a sixth set of target bacteria and comprising a heterologous nucleic acid sequence encoding a sixth marker. In some
  • the methods further comprise exposing the sample to a seventh type of phage capable of infecting a seventh set of target bacteria and comprising a heterologous nucleic acid sequence encoding a seventh marker. In some embodiments, the methods further comprise exposing the sample to an eighth type of phage capable of infecting an eighth set of target bacteria and comprising a heterologous nucleic acid sequence encoding an eighth marker. In some embodiments, the methods further comprise exposing the sample to a ninth type of phage capable of infecting a ninth set of target bacteria and comprising a heterologous nucleic acid sequence encoding a ninth marker.
  • the methods further comprise exposing the sample to ten or more types of phage capable of infecting ten or more sets of target bacteria and comprising a heterologous nucleic acid sequences encoding ten or more markers.
  • all of the three or more markers are different.
  • all of the three or more markers are the same.
  • two, three, four, five, six, seven, eight, or nine of the markers are the same.
  • At least one type of phage used in the method is selected from A511, P100, LP40, LP48, LP99, LP101, LP124, LP125, and LP143, and derivatives thereof.
  • every type of phage used in the method is selected from A511, P100, LP40, LP48, LP99, LP101, LP124, LP125, and LP143, and derivatives thereof.
  • the first marker is a screenable marker.
  • the first marker is a luciferase.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 2.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 4.
  • the phage is selected from LP48: :ffluc, LP99: :ffluc, LP101 : :ffluc, LP124: :ffiuc, LP125: :ffluc, LP143 : :ffiuc, A511 : :ffluc, P100: :ffluc, LP48: : COP2, LP99: : COP2, LP101 : : COP2, LP 124: : COP2, LP 125: : COP2, LP 143 : COP2, A511 : : COP2, P100: : COP2, LP48: : COP3, LP99: : COP3, LP101 : : COP3, LP124: : COP3, LP125: : COP3, LP143 : : COP3, A511 : : COP3, and PlOO: : COP3.
  • the phage is selected from LP40: :nluc, LP124: :nluc, LP125: :nluc, A511 : :nluc, P100: :nluc, LP40: :COP2,
  • the sample is an environmental sample.
  • the sample is an agricultural sample. In some embodiments, the sample is an agricultural sample.
  • the agricultural sample is stock feed or food supply.
  • the food supply is for human or non-human consumption.
  • the food supply is a plant or an animal.
  • the agricultural sample in the composition is selected from a dairy product, a fruit product, a grain product, a sweet, a vegetable product, and a meat product.
  • the dairy product includes foods derived from milk products comprising milk, butter, yogurt, cheese, ice cream and queso fresco.
  • the fruit product comprises apple, oranges, bananas, berries and lemons.
  • the grain product comprises wheat, rice, oats, barley, bread and pasta.
  • the sweet product comprises candy, soft drinks, cake, and pie.
  • the vegetable product comprises spinach, carrots, onions, peppers, avocado and broccoli.
  • the vegetable product is guacamole.
  • the meat product comprises chicken, fish, turkey, pork and beef.
  • the meat product further comprises deli meats and ground meets, as well as deli turkey and ground beef.
  • the food sample in the composition is selected from a dairy product, a fruit product, a grain product, a sweet, a vegetable product, and a meat product.
  • the dairy product includes foods derived from milk products comprising milk, butter, yogurt, cheese, ice cream and queso fresco.
  • the fruit product comprises apple, oranges, bananas, berries and lemons.
  • the grain product comprises wheat, rice, oats, barley, bread and pasta.
  • the sweet product comprises candy, soft drinks, cake, and pie.
  • the vegetable product comprises spinach, carrots, onions, peppers, avocado and broccoli.
  • the vegetable product is guacamole.
  • the meat product comprises chicken, fish, turkey, pork and beef.
  • the meat product further comprises deli meats and ground meets, as well as deli turkey and ground beef.
  • the marker is detected in the sample, indicating the presence of bacteria of the first set of target bacteria in the sample.
  • the target microbe of the method is selected from the group consisting oicoliform bacteria, Escherichia, Shigella, Listeria, Clostridium, Vibrio, Enter obacteriacae, Staphylococcus, Bacillus, Campylobacter, Pseudomonas, Streptococcus, Acinetobacter, Klebsiella, Cronobacter, Mycobacterium, Campylobacter ; and Yersinia.
  • the target microbe is E. coli.
  • the target microbe is Listeria selected from the group consisting of Listeria innocua, Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii, Listeria grayi, Listeria marthii, Listeria rocourti, Listeria welshimeri, Listeria floridensis, Listeria aquatic, Listeria yakmannii, Listeria weihenstephanensis, Listeria cornellensis, Listeria riparia, Listeria newyorkensis and Listeria grandensis.
  • the second marker is a screenable marker.
  • the second marker is a luciferase.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 2.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 4.
  • the second type of phage is selected from LP48::ffluc, LP99: :ffluc, LP101 : :ffluc, LP124: :ffluc, LP125: :ffluc, LP143 : :ffluc, A511 : :ffluc, P100: :ffluc, LP48: :COP2, LP99: :COP2, LP101 : :COP2, LP124: :COP2, LP125: :COP2, LP143 : :COP2, A511 : :COP2, P100: :COP2, LP48: :COP3, LP99: :COP3, LP101 ::COP3, LP124: :COP3,
  • the second type of phage is selected from LP40: :nluc, LP124: :nluc, LP125: :nluc, A511 : :nluc, P100: :nluc, LP40: :COP2, LP124::COP2, LP125: :COP2, A511 : :COP2, P100: :COP2, LP40: :COP3,
  • the method comprises exposing the sample to the first type of phage and the second type of phage at the same time.
  • the sample is an environmental sample.
  • the first marker is detected in or on the sample, or in situ, indicating the presence of bacteria of the first set of target bacteria in or on the sample, or in situ.
  • the second marker is detected in the sample, indicating the presence of bacteria of the second set of target bacteria in the sample.
  • the first marker and the second marker are the same, and the marker is detected in or on the sample, or in situ, indicating the presence of bacteria of at least one of the first set of target bacteria and the second set of target bacteria in or on the sample, or in the in situ location.
  • the sample is exposed to metabolic stimulation conditions before it is exposed to the phage.
  • the methods further comprise incubating the sample under metabolic stimulation conditions for a period of time before exposing the sample to the phage capable of infecting target bacteria.
  • the methods comprise providing a sample; exposing the sample to at least one recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker, the recombinant Listeria phage selected from recombinant LP40 and derivatives thereof, recombinant LP48 and derivatives thereof, recombinant LP99 and derivatives thereof, recombinant LP101 and derivatives thereof, recombinant LP 124 and derivatives thereof, recombinant LP 125 and derivatives thereof, and recombinant LP 143 and derivatives thereof; and assaying for the presence of the marker in the exposed sample.
  • the methods further comprise exposing the sample to at least one recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker, the recombinant Listeria phage selected from recombinant A511 and recombinant PI 00.
  • detection of the marker in the sample indicates the presence of Listeria in the sample.
  • target bacteria of the recombinant Listeria phage comprise at least one species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii, Listeria marthii, Listeria rocourti, and Listeria welshimeri.
  • detection of the marker in the sample indicates the presence of the at least one species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii, Listeria marthii, Listeria rocourti, and Listeria welshimeri in the sample.
  • target bacteria of the Listeria phage comprise at least one species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri.
  • detection of the marker in the sample indicates the presence of the at least one species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri in the sample.
  • target bacteria of the Listeria phage comprise at least one sig B allelotype of Listeria innocua selected from 11, 22, 37, and 56, and detection of the marker in the sample indicates the presence of at least one sig B allelotype of Listeria innocua selected from 11, 22, 37, and 56.
  • the at least one Listeria phage is capable of infecting Listeria innocua sig B allelotypes 11, 22, 37, and 56.
  • target bacteria of the Listeria phage comprise at least one ribotype of Listeria monocytogenes selected from DUP-10142, DUP-1030A, DUP-1030B, DUP- 1038B, DUP-1039A, DUP-1039B, DUP-1039C, DUP-1042A, DUP-1042B, DUP-1042C, DUP- 1043A, DUP-1044A, DUP-1044B, DUP-1044E, DUP-1045B, DUP-1052A, DUP-1053A, DUP- 1062 A, and DUP-1062D; and detection of the marker in the sample indicates the presence of at least one ribotype of Listeria monocytogenes selected from DUP-10142, DUP- 103 OA, DUP- 1030B, DUP-1038B, DUP-1039A, DUP-1039B, DUP-1039C, DUP-1042A, DUP-1042B, DUP- 1042C, DUP-1043A, DUP-1044
  • target bacteria of the Listeria phage comprise Listeria monocytogenes ribotypes DUP-10142, DUP-1030A, DUP-1030B, DUP-1038B, DUP-1039A, DUP-1039B, DUP-1039C, DUP-1042A, DUP-1042B, DUP-1042C, DUP- 1043 A, DUP-1044A, DUP-1044B, DUP-1044E, DUP-1045B, DUP-1052A, DUP- 1053 A, DUP-1062A, and DUP-1062D.
  • target bacteria of the Listeria phage comprise at least one sig B allelotype of Listeria seeligeri selected from 3, 20, 24, and 35, and detection of the marker in the sample indicates the presence of at least one sig B allelotype of Listeria seeligeri selected from 3, 20, 24, and 35.
  • target bacteria of the Listeria phage comprise Listeria seeligeri sig B allelotypes 3, 20, 24, and 35.
  • target bacteria of the Listeria phage comprise at least one sig B allelotype of Listeria welshimeri selected from 15, 27, 32, and 89, and detection of the marker in the sample indicates the presence of at least one sig B allelotype of Listeria welshimeri selected from 15, 27, 32, and 89.
  • target bacteria of the Listeria phage comprise Listeria welshimeri sig B allelotypes 15, 27, 32, and 89.
  • the target bacteria comprise at least two species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri. In some embodiments, the target bacteria comprise at least three species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri. In some embodiments, the target bacteria comprise at least four species of Listeria selected from Listeria innocua, Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii, Listeria marthii, Listeria rocourti, and Listeria welshimeri.
  • the target bacteria do not include at least one of Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterococcus durans, Enterococcus faceium, Enterococcus hirae, Kocuria varians, Kurthia gibsonii, Kurthia zopfii, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus equi, Streptococcus galloyticus, Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactus, Lactobacillus fermentum, Micrococcus lutues, Pseudomonas protogens, Pseudomonas florescens, Aeromonas sp, Serratia liquefaciens, Serratia proteamaculans, Serratia lique
  • proteamaculans Pseudomonas florescens, Pseudomonas poae, Pseudomonas sp, Pseudomonas jragi, Providencia alcalifaciens, Serratia sp, Serratia grime sii, Hafnia sp., Serratia
  • the target bacteria do not include Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterococcus durans, Enterococcus faceium,
  • Streptococcus equi Streptococcus galloyticus, Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactus, Lactobacillus fermentum, Micrococcus lutues, Pseudomonas protogens, Pseudomonas florescens, Aeromonas sp, Serratia liquefaciens, Serratia proteamaculans, Serratia liquefaciens, Bacillaceae bacterium, Serratia proteamaculans, Pseudomonas florescens, Pseudomonas poae, Pseudomonas sp, Pseudomonas fragi, Providencia alcalifaciens, Serratia sp, Serratia grime sii, Hafnia sp., Serratia proteamaculans, Pseudomonas flores
  • the sample is exposed to the phage for a period of time before assaying for the presence of a marker in the exposed sample is conducted.
  • the period of time is from 1 minute to 24 hours, from 5 minutes to 12 hours, from 5 minutes to 6 hours, from 5 minutes to 3 hours, from 5 minutes to 2 hours, from 5 minutes to 1 hour, from 5 minutes to 50 minutes, from 5 minutes to 40 minutes, from 5 minutes to 30 minutes, from 5 minutes to 20 minutes, or from 5 minutes to 10 minutes.
  • the period of time is from 1 to 2 hours, from 1 to 4 hours, or from 2 to 4 hours. In some
  • the period of time is for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 1 hour.
  • any phage and/or parts of phage in the exposed sample are substantially removed before the assaying for the presence of a marker in the exposed sample is conducted.
  • the methods further comprise comparing a detected level of marker in a test sample to at least one of a positive control and a negative control.
  • the positive and/or negative control may be used to calibrate the assay including for the purpose of defining a positive result and/or a negative result.
  • the methods of assaying phage host range allow, in certain embodiments, for the characterization of the host range of phage— and thus definition of target bacteria for phage— at a resolution not previously provided.
  • One use of the methods and of phage characterized by the methods is to identify useful combinations of phage that may be used together in a system to detect target bacteria.
  • such systems provide phage separately and the phage are then mixed before or during an assay.
  • such systems comprise useful mixtures of phage, such as phage provided in a buffer for use in an assay.
  • compositions comprising useful combinations of phage are also, necessarily, produced during the assay in several embodiments. Accordingly, this disclosure also provides compositions that comprise phage. [00201] In some embodiments the composition comprises: at least one recombinant
  • Listeria phage comprising a heterologous nucleic acid sequence encoding a marker, the recombinant Listeria phage selected from recombinant A511 and derivatives thereof, recombinant PI 00 and derivatives thereof, recombinant LP40 and derivatives thereof, recombinant LP44 and derivatives thereof, recombinant LP48 and derivatives thereof, recombinant LP99 and derivatives thereof, recombinant LP101 and derivatives thereof, recombinant LP 124 and derivatives thereof, recombinant LP 125 and derivatives thereof, and recombinant LP 143 and derivatives thereof and at least one non-phage component selected from Table 5 and/or from at least one of a) at least one compound selected from carbohydrates and related compounds, b) at least compound selected from nitrogen containing compounds, c) at least compound selected from nucleic acids and related compounds, d) at least compound selected from lipid, e) at least one inorgan
  • the composition comprises at least one of 1,2-Propanediol, 2-Aminoethanol, Glucuronamide, Tyramine, b-Phenylethylamine, L-Aspartic Acid, L-Proline, D-Alanine, D- Serine, L-Glutamic Acid, L-Asparagine, D-Aspartic Acid, L-Glutamine, Gly-Asp, D-Threonine, Gly-Glu, L-Serine, L-Threonine, L-Alanine, Ala-Gly, Gly-Pro, L-Arabinose, N-Acetyl-D- Glucosamine, D-Galactose, D-Trehalose, D-Mannose, Dulcitol, D-Sorbitol, Glycerol, L-Fucose, D,L-a-Glycerol, Phosphate, D-Xylose, D-Mannitol, D-Glu
  • recombinant LP48 and derivatives thereof recombinant LP99 and derivatives thereof, recombinant LP101 and derivatives thereof, recombinant LP 124 and derivatives thereof, recombinant LP 125 and derivatives thereof, and recombinant LP 143 and derivatives thereof.
  • the systems or compositions comprise at least three, four, five, six, seven, eight, nine, or more recombinant Listeria phage, selected from recombinant LP040 and derivatives thereof, recombinant LP048 and derivatives thereof, recombinant LP99 and derivatives thereof, recombinant LP101 and derivatives thereof, recombinant LP 124 and derivatives thereof, recombinant LP 125 and derivatives thereof, and recombinant LP 143 and derivatives thereof.
  • the system and or composition comprising at least one recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker is provided in the form of an article of manufacture.
  • Such an article of manufacture is useful, for example, as a means to provide the at least one recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker in combination with other components that can be used together to perform an assay to detect target bacteria.
  • the article of manufacture comprises at least one container comprising the at least one recombinant Listeria phage comprising a heterologous nucleic acid sequence encoding a marker.
  • the article of manufacture comprises at least one container comprising at least two recombinant Listeria phage selected from recombinant LP40 and derivatives thereof, recombinant LP48 and derivatives thereof, recombinant LP99 and derivatives thereof, recombinant LP101 and derivatives thereof, recombinant LP 124 and derivatives thereof, recombinant LP 125 and derivatives thereof, and recombinant LP 143 and derivatives thereof.
  • the systems or compositions comprise at least three, four, five, six, seven, eight, nine, or more recombinant Listeria phage, selected from recombinant LP40 and derivatives thereof, recombinant LP48 and derivatives thereof, recombinant LP99 and derivatives thereof, recombinant LP101 and derivatives thereof, recombinant LP 124 and derivatives thereof, recombinant LP 125 and derivatives thereof, and recombinant LP 143 and derivatives thereof.
  • the article of manufacture comprises more than one phage all of the phage are provided in separate containers. In other embodiments two or more of the phage are provided in combination in a single container.
  • the article of manufacture comprises at least one container comprising at least one recombinant phage selected from A511, PI 10, LP40, LP48, LP99, LP107, LP124, LP125 and LP143, and derivatives thereof.
  • the phage comprises a heterologous nucleic acid sequence encoding a first marker.
  • the first marker is a screenable marker.
  • the first marker is a luciferase.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 2.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 4.
  • the luciferase is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO: 41.
  • the first type of phage is selected from LP48: :ffluc, LP99: :ffluc, LP101 : :ffluc, LP124: :ffluc, LP125: :ffluc, LP143 : :ffluc, A511 : :ffluc, P100: :ffluc, LP48: :COP2, LP99: :COP2, LP101 : :COP2, LP124: :COP2, LP125: :COP2, LP143 : :COP2, A511 : :COP2, and P100: :COP2, LP48: :COP3, LP99: :COP3, LP101 : :COP3, LP124: :COP3, LP125: :COP3, LP143 : :COP3, A51 1 : :COP3, and P100: :COP3 and derivatives of those phage.
  • the first type of phage is selected from LP48: :f
  • the first type of phage is selected from A511 : : COP2, LP 124 : : COP2, LP40 : : COP2, LP 125 : : COP2, A511 : : COP3 ,
  • the article of manufacture further comprises an aqueous solution including one or more reagents from Table 5 and/or at least one non-phage component selected from at least one of a) at least one compound selected from carbohydrates and related compounds, b) at least compound selected from nitrogen containing compounds, c) at least compound selected from nucleic acids and related compounds, d) at least compound selected from lipid, e) at least one inorganic compound, and f) at least one organic compound.
  • aqueous solution including one or more reagents from Table 5 and/or at least one non-phage component selected from at least one of a) at least one compound selected from carbohydrates and related compounds, b) at least compound selected from nitrogen containing compounds, c) at least compound selected from nucleic acids and related compounds, d) at least compound selected from lipid, e) at least one inorganic compound, and f) at least one organic compound.
  • the article of manufacture comprises a container comprising a solution comprising at least one of 1,2-Propanediol, 2-Aminoethanol, Glucuronamide, Tyramine, b- Phenylethylamine, L-Aspartic Acid, L-Proline, D-Alanine, D-Serine, L-Glutamic Acid, L- Asparagine, D-Aspartic Acid, L-Glutamine, Gly-Asp, D-Threonine, Gly-Glu, L-Serine, L- Threonine, L-Alanine, Ala-Gly, Gly-Pro, L-Arabinose, N-Acetyl-D-Glucosamine, D-Galactose, D-Trehalose, D-Mannose, Dulcitol, D-Sorbitol, Glycerol, L-Fucose, D,L-a-Glycerol, Phosphate, D-Xy
  • At least one recombinant Listeria phage present in the article of manufacture is present in the aqueous solution comprising at least one non-phage component.
  • the phage and solution are provided separately and may, for example, be combined by a user.
  • the article of manufacture includes a substrate for a light reaction, or other required component for the marker to operate.
  • the substrate is luciferin.
  • the article of manufacture includes an additional aqueous solution that is optimized for a light reaction, or that provides conditions that are optimal for detection of a marker.
  • the article of manufacture is a kit.
  • the kit may further comprise instructions for performing one or more of the assays described herein. Definitions
  • sequence database entries e.g., UniProt/SwissProt or GENBANK records
  • sequence database entries e.g., UniProt/SwissProt or GENBANK records
  • information on the internet including sequence database entries, is updated from time to time and that, for example, the reference number used to refer to a particular sequence can change.
  • reference is made to a public database of sequence information or other information on the internet it is understood that such changes can occur and particular embodiments of information on the internet can come and go. Because the skilled artisan can find equivalent information by searching on the internet, a reference to an internet web page address or a sequence database entry evidences the availability and public dissemination of the information in question.
  • the term "comprising" as used herein is synonymous with "including" or
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • in vivo refers to events that occur within an organism
  • an assay that occurs at least in part in vivo within a microbe may nonetheless occur in vitro if parts of the assay occur outside of the microbe in culture, for example.
  • isolated refers to a substance or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%), about 90%), or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%), or more than about 99% pure. As used herein, a substance is "pure” if it is substantially free of other components.
  • peptide refers to a short polypeptide, e.g., one that typically contains less than about 50 amino acids and more typically less than about 30 amino acids.
  • the term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.
  • polypeptide encompasses both naturally-occurring and non-naturally occurring proteins, and fragments, mutants, derivatives and analogs thereof.
  • a polypeptide may be monomelic or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities. For the avoidance of doubt, a "polypeptide" may be any length greater two amino acids.
  • isolated protein or "isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds).
  • polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • isolated does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from a cell in which it was synthesized.
  • polypeptide fragment refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide, such as a naturally occurring protein.
  • the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, or at least 12, 14, 16 or 18 amino acids long, or at least 20 amino acids long, or at least 25, 30, 35, 40 or 45, amino acids, or at least 50 or 60 amino acids long, or at least 70 amino acids long.
  • fusion protein refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements that can be from two or more different proteins.
  • a fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, or at least 20 or 30 amino acids, or at least 40, 50 or 60 amino acids, or at least 75, 100 or 125 amino acids.
  • the heterologous polypeptide included within the fusion protein is usually at least 6 amino acids in length, or at least 8 amino acids in length, or at least 15, 20, or 25 amino acids in length.
  • Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein ("GFP") chromophore- containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
  • GFP green fluorescent protein
  • a protein has "homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
  • a protein has homology to a second protein if the two proteins have similar amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.)
  • homology between two regions of amino acid sequence is interpreted as implying similarity in function.
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the
  • GCG Genetics Computer Group
  • Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.
  • An exemplary algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3 :266-272 (1993); Madden et al., Meth. Enzymol. 266: 131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • Exemplary parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max.
  • polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, or at least about 20 residues, or at least about 24 residues, or at least about 28 residues, or more than about 35 residues.
  • searching a database containing sequences from a large number of different organisms it may be useful to compare amino acid sequences.
  • Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183 :63-98 (1990). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.
  • polymeric molecules e.g., a polypeptide sequence or nucleic acid sequence
  • polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%) or at least 99% identical.
  • polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%>, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar.
  • the term "homologous" necessarily refers to a comparison between at least two sequences
  • nucleotide sequences or amino acid sequences are considered to be homologous if the polypeptides they encode are at least about 50%) identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%), at least 97%, at least 98% or at least 99% identical for at least one stretch of at least about 20 amino acids.
  • homologous nucleotide sequences are
  • nucleotide sequences characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Both the identity and the approximate spacing of these amino acids relative to one another must be considered for nucleotide sequences to be considered homologous. In some embodiments of nucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.
  • two protein sequences are considered to be homologous if the proteins are at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%), at least 98% or at least 99% identical for at least one stretch of at least about 20 amino acids.
  • a "modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence to a reference polypeptide sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the reference polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the
  • radioactive isotopes such as I, P, S, and H
  • ligands that bind to labeled antiligands e.g., antibodies
  • fluorophores e.g., fluorophores
  • chemiluminescent agents e.g., enzymes
  • antiligands that can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation.
  • Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002).
  • polypeptide mutant refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide, such as a native or wild-type protein.
  • a mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini.
  • a mutein may have the same or a different biological activity compared to the reference protein.
  • a mutein has, for example, at least 70% overall sequence homology to its counterpart reference polypeptide or protein. In some embodiments, a mutein has at least 75%, at least 80%, at least 85%, or at least 90% overall sequence homology to the wild-type protein or polypeptide. In other embodiments, a mutein exhibits at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% at least 99.5%, at least 99.9% sequence identity, or 98%, or 99%, or 99.5% or 99.9% overall sequence identity.
  • recombinant refers to a biomolecule, e.g., a gene or protein, that (1) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (2) is operatively linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature.
  • recombinant refers to a biomolecule that does not occur in nature.
  • the term “recombinant” can be used in reference to cloned DNA isolates, chemically
  • a protein synthesized by a microorganism is recombinant, for example, if it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.
  • a phage is "recombinant” if it comprises a recombinant biomolecule.
  • a phage is “recombinant” if it comprises a recombinant biomolecule that does not occur in nature.
  • a phage is recombinant if the genome of the phage comprises a recombinant nucleic acid sequence.
  • nucleic acid refers to a polymeric form of nucleotides of at least 10 bases in length.
  • the term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both.
  • the nucleic acid can be in any topological conformation.
  • the nucleic acid can be single-stranded, double-stranded, triple- stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.
  • the nucleic acid also referred to as polynucleotides
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carb
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" nucleic acids.
  • RNA, DNA or a mixed polymer is one created outside of a cell, for example one synthesized chemically.
  • nucleic acid fragment refers to a nucleic acid sequence that has a deletion, e.g., a 5 '-terminal or 3 '-terminal deletion compared to a full-length reference nucleotide sequence.
  • the nucleic acid fragment is a contiguous sequence in which the nucleotide sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence.
  • fragments are at least 10, 15, 20, or 25 nucleotides long, or at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides long.
  • a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence.
  • such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed "recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself
  • a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it.
  • a nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention.
  • a "recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
  • a "recombinant phage genome” is a phage genome that contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention and does not occur in nature.
  • the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.
  • the term "degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
  • sequence identity refers to the residues in the two sequences, which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32, and even more typically at least about 36 or more nucleotides.
  • polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183 :63-98 (1990).
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.
  • sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
  • nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions.
  • Stringent hybridization conditions and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
  • “stringent hybridization” is performed at about 25°C below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions.
  • “Stringent washing” is performed at temperatures about 5°C lower than the Tm for the specific DNA hybrid under a particular set of conditions.
  • the Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65°C for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65 °C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.
  • an "expression control sequence” refers to polynucleotide sequences that affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences that control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences is intended to encompass, at a minimum, any component whose presence is essential for expression, and can also encompass an additional component whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • operatively linked or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
  • a "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of vector is a
  • Plasmid which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
  • Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below).
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply “expression vectors").
  • recombinant host cell (or simply “recombinant cell” or “host cell”), as used herein, is intended to refer to a cell into which a recombinant nucleic acid such as a recombinant vector has been introduced.
  • the word "cell” is replaced by a name specifying a type of cell.
  • a “recombinant microorganism” is a recombinant host cell that is a microorganism host cell. It should be understood that such terms are intended to refer not only to the particular subject cell, but to the progeny of such a cell.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
  • bacteria refers to a virus that infects bacteria.
  • archaeophage refers to a virus that infects archaea.
  • phage is used to refer to both types of viruses but in certain instances as indicated by the context may also be used as shorthand to refer to a bacteriophage or archaeophage specifically.
  • Bacteriophage and archaeophage are obligate intracellular parasites that multiply inside bacteria/archaea by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria).
  • viruses that infect bacteria.
  • different bacteriophages and archaeophages may contain different materials, they all contain nucleic acid and protein, and can under certain circumstances be encapsulated in a lipid membrane.
  • the nucleic acid may be either DNA or RNA but not both and it can exist in various forms.
  • heterologous nucleic acid sequence is any sequence placed at a location in the genome where it does not normally occur.
  • a heterologous nucleic acid sequence may comprise a sequence that does not naturally occur in a particular bacteria/archaea and/or phage or it may comprise only sequences naturally found in the bacteria/archaea and/or phage, but placed at a non-normally occurring location in the genome.
  • the heterologous nucleic acid sequence is not a natural phage sequence; in some embodiments it is a natural phage sequence, albeit from a different phage; while in still other embodiments it is a sequence that occurs naturally in the genome of the starting phage but is then moved to another site where it does not naturally occur, rendering it a heterologous sequence at that new site.
  • starting phage or “starting phage genome” is a phage isolated from a natural or human made environment that has not been modified by genetic engineering, or the genome of such a phage.
  • a "recombinant phage” or “recombinant phage genome” is a phage that comprises a genome that has been genetically modified by insertion of a heterologous nucleic acid sequence into the phage, or the genome of the phage.
  • a “recombinant phage” or “recombinant phage genome” is a phage that does not occur in nature, i.e., does not comprise a genome that occurs in nature.
  • the genome of a starting phage is modified by recombinant DNA technology to introduce a heterologous nucleic acid sequence into the genome at a defined site.
  • the heterologous sequence is introduced with no corresponding loss of endogenous phage genomic nucleotides.
  • the heterologous sequence is inserted between Nl and N2.
  • the heterologous sequence is flanked by nucleotides Nl and N2.
  • the heterologous sequence is inserted and endogenous nucleotides are removed or replaced with the exogenous sequence.
  • the exogenous sequence is inserted in place of some or all of the endogenous sequence which is removed.
  • endogenous sequences are removed from a position in the phage genome distant from the site(s) of insertion of exogenous sequences.
  • a "phage host cell” is a cell that can be infected by a phage to yield progeny phage particles.
  • “Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with coding sequences of interest to control expression of the coding sequences of interest, as well as expression control sequences that act in trans or at a distance to control expression of the coding sequence.
  • a "coding sequence” or “open reading frame” is a sequence of nucleotides that encodes a polypeptide or protein. The termini of the coding sequence are a start codon and a stop codon.
  • expression control sequence refers to polynucleotide sequences which affect the expression of coding sequences to which they are operatively linked.
  • Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • a "phage genome” includes naturally occurring phage genomes and derivatives thereof. Generally (though not necessarily), the derivatives possess the ability to propagate in the same hosts as the parent. In some embodiments the only difference between a naturally occurring phage genome and a derivative phage genome is at least one of a deletion and an addition of nucleotides from at least one end of the phage genome if the genome is linear or at least one point in the genome if the genome is circular.
  • target microbe includes bacteria, however, this term may also include other unicellular pathogens that cause infection in animals and/or humans. Preferred target microbes are bacteria.
  • target bacteria are bacteria that can be infected by a phage to yield a detectable output or signal.
  • a detectable output includes cell lysis.
  • lysis of bacterial cells by a phage indicates that the bacterial cells are "target bacteria" of that phage.
  • Another example of a detectable output is expression of a marker following infection of a bacterial cell by a phage. Suitable markers include RNAs and polypeptides.
  • a "marker” includes selectable and/or screenable markers.
  • a "selectable marker” is a marker that confers upon cells that possess the marker the ability to grow in the presence or absence of an agent that inhibits or stimulates, respectively, growth of similar cells that do not express the marker. Such cells can also be said to have a "selectable phenotype" by virtue of their expression of the selectable marker.
  • the ampicillin resistance gene confers the ability to grow in the presence of ampicillin on cells, which possess and express the gene.
  • genes that confer resistance to chloramphenicol, kanamycin, and tetracycline include URA3, TRP and LEU, which allow growth in the absence of said uracil, tryptophan and leucine, respectively.
  • a "screenable marker” is a detectable label that that can be used as a basis to identify cells that express the marker. Such cells can also be said to have a "screenable phenotype" by virtue of their expression of the screenable marker.
  • selectable markers may also function as screenable markers in so far as the gene product of the selectable marker may be used as a basis to identify cells that express the marker independently of the function of the gene product to confer selectability on cells that express it.
  • Any molecule that can be differentially detected and encoded by the recombinant phage can serve as a screenable marker.
  • a screenable marker can be a nucleic acid molecule or a portion thereof, such as an RNA or a DNA molecule that is single or double stranded.
  • a screenable marker can be a protein or a portion thereof.
  • Suitable protein markers include enzymes that catalyze formation of a detectable reaction product.
  • An example is a chemiluminescent protein such as luciferase or variations, such as luxAB, and ⁇ -galactosidase.
  • Another example is the
  • Proteins used to generate a luminescent signal fall into two broad categories: those that generate light directly (luciferases and related proteins) and those that are used to generate light indirectly as part of a chemical cascade (horseradish peroxidase).
  • the most common bioluminescent proteins used in biological research are aequorin and luciferase.
  • the former protein is derived from the jellyfish Aequorea victoria and can be used to determine calcium concentrations in solution.
  • the luciferase family of proteins has been adapted for a broad range of experimental purposes. Luciferases from firefly and Renilla are the most commonly used in biological research.
  • proteins have also been genetically separated into two distinct functional domains that will generate light only when the proteins are closely co-localized. A variety of emission spectrum-shifted mutant derivatives of both of these proteins have been generated over the past decade. These have been used for multi-color imaging and co- localization within a living cell.
  • the other groups of proteins used to generate chemiluminescent signal are peroxidases and phosphatases. Peroxidases generate peroxide that oxidizes luminol in a reaction that generates light. The most widely used of these is horseradish peroxidase (HRP), which has been used extensively for detection in western blots and ELISAs.
  • HRP horseradish peroxidase
  • a second group of proteins that have been employed in a similar fashion are alkaline phosphatases, which remove a phosphate from a substrate molecule, destabilizing it and initiating a cascade that results in the emission of light.
  • Fluorescent proteins include but are not limited to blue/UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3 A, mTurquoise, monomelic Midoriishi-Cyan, TagCFP, and mTFPl), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP,
  • blue/UV fluorescent proteins for example, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, and T-Sapphire
  • cyan fluorescent proteins for example, ECFP, Cerulean, SCFP3 A, mTurquoise, monomelic Midoriishi-Cyan, TagCFP, and mTFPl
  • green fluorescent proteins for example, EGFP, Emerald, Superfolder GFP,
  • Monomeric Azami Green, TagGFP2, mUKG, and mWasabi yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, ⁇ , mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed- Tandem, mKate2, mNeptune, and NirFP), near-IR fluorescent proteins (for example,
  • TagRFP657, IFP1.4, and iRFP long stokes-shift proteins (for example, mKeima Red, LSS- mKatel, and LSS-mKate2), photoactivatible fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), and photoswitchable fluorescent proteins (for example, Dronpa).
  • long stokes-shift proteins for example, mKeima Red, LSS- mKatel, and LSS-mKate2
  • photoactivatible fluorescent proteins for example, PA-GFP, PAmCherryl, and PATagRFP
  • photoconvertible fluorescent proteins for example, Kaede (green), Ka
  • Suitable markers include epitopes.
  • a protein comprising an epitope that can be detected with an antibody or other binding molecule is an example of a screenable marker.
  • An antibody that recognizes the epitope can be directly linked to a signal generating moiety (such as by covalent attachment of a chemiluminescent or fluorescent protein) or it can be detected using at least one additional binding reagent such as a secondary antibody, directly linked to a signal generating moiety, for example.
  • the epitope is not present in the proteins of the phage or the target microorganism so detection of the epitope in a sample indicates that the protein comprising the epitope was produced by the microorganism following infection by the recombinant phage comprising a gene encoding the protein
  • the marker may be a purification tag in the context of a protein that is naturally present in the target microorganism or the phage.
  • the tag e.g., a 6-His tag
  • the tag can be used to purify the heterologous protein from other bacterial or phage proteins and the purified protein can then be detected, for example using an antibody.
  • an "environmental sample” is a sample obtained from any setting other than a laboratory cell culture setting.
  • an environmental sample is obtained from a setting that comprises at least one of a) a temperature that does not support maximum growth and/or metabolism of bacterial cells, b) a nutrient profile that does not support maximum growth and/or metabolism of bacterial cells, and c) bacterial cells that are not target bacteria for a phage used in an assay.
  • some or all of the bacteria present in an environmental sample are not in a metabolically active state.
  • environmental samples may be obtained from industrial plants, food processing plants, veterinary sources, food, livestock, medical settings and surfaces, schools, assisted living centers, cruise ships, other confined quarters and homes.
  • the surface may be of any material.
  • the surface can be metal, glass, wood, brick, concrete, tile, rug and the like.
  • the surface can also be on an agricultural product.
  • the sample can also be found inside of an agricultural produce.
  • the "environmental sample" can be in situ, in other words, the assay can be performed at the site itself, rather than removed from the site. Alternatively, the
  • “environmental sample” can be removed for assay from a collection point, as through the use of an absorbent material, such as a cotton swab to physically collect the sample.
  • agricultural refers to cultivated or wild plants, animals, and fungi. The term also refers to stock feed or food supply. "Food supply” encompasses food for either human or non-human animal consumption. Accordingly, an “agricultural sample” refers to a sample from of, within, or on the exterior of a plant, animal and fungi.
  • Recombinant phage were designed for increased expression of the luciferase reporter. Phages selected for recombination were A511, LP124, and LP40. The capsid (CPS) nucleotide sequences for A511 (SEQ ID NO: 19), LP124 (SEQ ID NO: 13) and LP40 (SEQ ID NO: 5) are provided herewith.
  • the NanoLuc luciferase reporter was selected for recombination by phage optimization. Phage optimization was performed using DNA 2.0TM software. The software uses an algorithm described Villalobos et al. (see Villalobos et al. BMC Bioinformatics. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. PLoS ONE. 2011 6:el9912.) to replace synonymous codons with those preferred by a host organism, in this case listeria.
  • the codon optimized Nanoluc (COP2; SEQ ID NO: 36) was inserted into the phage CPS open reading frame following stop codons and a ribosome binding site (SEQ ID NO: 54) using methods as described in Example 19 herein.
  • Primers used in engineering listeria phage include pMAK upf (SEQ ID NO: 55), dbono380 (SEQ ID NO: 56), S0472 (SEQ ID NO: 57), S0473 (SEQ ID NO: 58), S0474 (SEQ ID NO: 59) and dbono 382 (SEQ ID NO: 62) oligos.
  • a sequence map of the insertion site for A511 : :COP2 recombinant phage (SEQ ID NO: 39) recombinant phage is shown in FIG. 2 indicating the location of insertion of the COP2 reporter, ribosome binding site and the flanking sequence following CPS.
  • a sequence map of the insertion site for LP124: :COP2 recombinant phage (SEQ ID NO: 39) is shown in FIG. 2 indicating the location of insertion of the COP2 reporter, ribosome binding site and the flanking sequence following CPS.
  • a sequence map of the insertion site for LP40: :COP2 recombinant phage (SEQ ID NO: 40) is shown in FIG. 3 indicating the location of insertion of the COP2 reporter, ribosome binding site and the flanking sequence following CPS.
  • Recombinant phage comprising the native NanoLuc luciferase was compared to recombinant phage comprising the codon optimized COP2 luciferase.
  • Comparisons utilized a mixture of recombinant phage.
  • the COP2 mixture comprising the A511 :COP2, LP124: :COP2, and LP40: :COP2 phages. These experiments were done using a cocktail of A511 : :COP2, LP124: :COP2, and LP40: :COP2.
  • the final concentration of each phage is 1.5e7 pfu/ml, the final concentration of the mixture is 4.5e7 pfu/ml.
  • the mixture of NanoLuc phages comprised phages selected from Example 19. Protocols for the comparison assay are as follows.
  • Healthy cells 5ml overnight culture (18-24h in 0.5X BHI) diluted 1 :4 into
  • Healthy cells in this case, refer to an overnight culture that has reached stationary phase being back-diluted to re-enter log phase.
  • Sick cells 250 ⁇ 1 of a CDW 1554 overnight culture diluted to ⁇ le7 CFU/ml in BHI+1% glucose was spread on a 4"x4" square on a stainless steel table. Cells were allowed to dry overnight (18-24h). Cells were recovered using a cotton swab moistened with Letheen Broth, and placed in a conical tube containing 2ml of Letheen Broth. Cells were allowed to recover for 30 minutes at 30°C. Cells were diluted in 0.5X BHI to the point where ⁇ should contain -100 CFU. ⁇ was spiked onto each sponge. The model mimics a factory condition where cells are surviving on a steel surface that may or may not have food contact. Sick cells are less metabolically active and produce less light upon phage infection than their healthy counterparts.
  • the mixture of phages comprising codon optimized luciferase shows a 2.6 fold increase in relative light units (RLU) per colony forming units (CFU) over recombinant phage encoding basic NanoLuc when infecting healthy Listeria cells.
  • RLU relative light units
  • CFU colony forming units
  • the codon optimized COP2 encoding phage mixture shows a 4.1 fold increase in RLU/CFU over regular NanoLuc encoding phage mixtures (see FIG. 4).
  • the codon optimized COP2 encoding phage When normalized as an indication of performance, the codon optimized COP2 encoding phage perform at 264% of their NanoLuc encoding counterparts in healthy cells (see Table 7 and FIG. 5). In sick cells, the COP2 encoding phage perform at 409% of their NanoLuc encoding counterparts (see Table 12 and FIG. 5). This significant enhancement in the output of light from the recombinant phage encoding codon optimized COP2 improves the detectable limits of Listeria contamination.
  • NanoLuc was used to create three new engineered phages: A511 : :VIP178, LP124: :VIP178, and LP40: :VIP178 (also referred to herein as A511 : :COP3, LP124: :COP3 and LP40::COP3).
  • the phages were engineered using the primers described herein.
  • the primers pMAK upf (see SEQ ID NO:55) and DBONO380 (see SEQ ID NO:56) were used to amplify the upstream homology fragments for each phage.
  • the VIP 178 fragment was amplified using the SO670 (see SEQ ID NO:64) and S0671 (see SEQ ID NO:65).
  • the downstream homology fragments were amplified using the primers S0672 (see SEQ ID NO: 66) and DBON0382 (see SEQ ID NO: 60).
  • Signal intensity levels of COP2 and COP3 phages were assessed as detailed below in Example 3.
  • Validation Plates (342 strains across Listeria species), Omni-Tray with 0.5x BHI agar, Deep-Well 96-well plate (Axygen), Clear flat-bottom 96-well plate (Evergreen),White flat-bottom 96-well plate (Greiner Bio-One), Plate-sealing film (breathable), 15mL conical tubes, Letheen Broth, 0.5x BHI, NIB-14, Nano-Glo, Substrate, Nano-Glo Buffer, 200 ⁇ L ⁇ multichannel pipette, 1000 ⁇ multichannel pipette, 20 ⁇ . multichannel pipette, 96-pin replicator tool (frogger).
  • GM plate is not a full plate— use the empty wells to act as negative controls for the assay
  • COP3 target panel consists of strains producing between 100 RLU/CFU and
  • Table 10 Full Listing of Listeria Strains Assessed in COP2 and COP3 Signal Intensity Assay
  • NP#1839 were infected at various concentrations of phage.
  • the infected samples were incubated for 6 hours at 30°C.
  • the luciferase signal was detected using the Glomax 96 luminometer.
  • the resultant signal intensities for the various concentrations of phage used was plotted on a graph for comparison of signal.
  • the data from these experiments indicate that the concentration for maximum signal is approximately between 1.5X10 6 to 1.8X10 6 pfu/mL. See Figure 10. All assays were performed minimally in triplicate.
  • BHI and TSB media were further titrated to assess whether there was an increase in RLU at different concentrations of base media.
  • the data indicate that stressed cells recovered best in IX TSB medium. Additional benefits of the IX TSB medium include that the TSB does not contain animal byproducts, it contains more nutrients, and there is better consistency of the product among different lots tested.
  • Example 7 Selection of Media Additives— selective agents, neutralizers, and nutrients
  • Antifungal agents were also added to the base medium and tested in order to determine whether there is a decrease in RLU activity, either as a result of loss of enzyme activity or because of reduced infection ability.
  • the anti-fungal agents tested included cycloheximide solution in DMSO. Neither cycloheximide nor DMSO resulted in a decrease in the infection rate or in the enzymatic activity.
  • a comparison of enzyme activity and infection ability with various media formulations indicated that a preferred embodiment of the formulation includes the base Formulation- 1, with the addition of 0.08% MgS04 and 0.1% pyruvate (also referred herein as Formulation-IA). (See Figure 27).
  • additives that are geared towards reducing the interference from quaternary ammonium salts found in various sanitizing solutions including among others: “Sani-Step,” “Sani-Save,” “Boost-FT,” “Quorum Clear V,” “Whisper V,” “Sparkle QF-BH,” “F29”.
  • Particularly good candidates that have the capability of reducing interference of quaternary ammonium salts are Tween-80 and lecithin.
  • NIB-14 This formulation is herein referred to as NIB-14, the full
  • the NIB-14 formulation included the use of neutralizers (e.g.Tween-80 and lecithin) meant to play a role in reducing the effects of remnant amounts of sanitizers in a sample. As such, NIB-14 was shown to allow for high amounts of infection and subsequent signal stability. (See Example 8). Subsequent assays, meant to ascertain the levels of protection provided by the NIB-14 formulation towards various kinds remnant sanitizer samples were performed.
  • neutralizers e.g.Tween-80 and lecithin
  • the 1893 cell-type was used at a density of 900 cells per well BHI basal medium, and further incubated with serially diluted (into either NIB-14 or Letheen buffer) sanitizing chemicals for 30 minutes. Following the 30 minute incubation period, the luciferase marker containing phage was added in BHI medium, and the samples were further allowed to incubate for an additional 3 hours at 30 C. The enzyme was then detected by the addition of the NanoGlo luciferin substrate and luminescence measurement.
  • the sanitizer chemicals were added to the enzyme only in either NIB-14 or in Letheen for 3 hours at 30 °C.
  • Another variant of this assay involved the direct incubation of the sterilization chemical with the NanoGlo luciferin substrate in the absence of either cells, phage or enzyme.
  • the sanitizer chemicals were added at various time intervals ⁇ e.g. 5min to 120min) directly to the NIB-14 or to the Letheen, followed by the addition of cells and phage and incubated for 3 hours at 30 C.
  • the assays indicate that both the infection ability and the enzymatic activity are protected by the use of the NIB-14 medium in comparison to the Letheen medium. (See Figures 17A-17D).
  • NIB-14 Another sanitizer used to assess the protective ability of NIB-14 was Quorum Clear.
  • the Quorum Clear sanitizer contains quaternary ammonium salts.
  • the recommended usage concentration for Quorum Clear is a 3% solution.
  • Addition of the NIB-14 medium was able to preserve up to 50% phage infection ability and enzyme activity at concentrations of Quorum Clear of up to 2.0% and 3.0%>, respectively.
  • NIB-14 was also able to provide protection to the NanoGlo substrate during exposures to Quorum Clear.
  • NIB-14 Another commonly used sanitizer component that was tested in the microbial detection system assays was hydrogen peroxide (H 2 0 2 ).
  • H 2 0 2 hydrogen peroxide
  • the protective ability of NIB-14 was determined in situations where various concentrations of the sanitizing component were added either during the phage infection step, the enzymatic activity step, or to the NanoGlo substrate.
  • the data indicate that NIB-14 provides protection to peroxide presence in comparison to Letheen. (See Figure 17A, 17B and 17D).
  • the data do not indicate protection by NIB-14 to the NanoGlo substrate. (See Figure 17C).
  • there are no deleterious effects to the NanoGlo substrate during exposure to peroxide at concentrations recommended for sanitizing i.e. 500ppm).
  • Boost FT was another sanitizer used in the microbial detection system assays to determine the protective effects of NIB-14 in the microbial detection assays.
  • Boost FT contains a combination of quaternary ammonium salts, peroxide (H 2 O 2 ), and EDTA at elevated pH.
  • the recommended concentration of use for Boost FT is 0.7% concentration of the active ingredient.
  • Addition of the NIB-14 medium was able to preserve up to 50% phage infection ability and enzyme activity at concentrations of Boost FT of up to 0.07%) and greater than 0.7%, respectively.
  • the increase noted in enzymatic activity may largely be due to oxidization of the NanoGlo substrate. The need for additional neutralization of peroxides was demonstrated.
  • a direct comparison of infection and enzymatic activity using stressed cells infected with recombinant luciferase encoding phage was performed utilizing NIB buffers. These tests measured the direct performance of NIB-10*, NIB-12, and NIB-14 with regard to enhancing either enzymatic activity or infection rate.
  • the base buffer, IX BHI was used as a comparison buffer for these tests.
  • stressed cell assays bacterial cells were purposefully stressed by way of drying for 18 hours on a stainless steel table followed by downstream processing. The effect of the buffers was assessed either during the infection stage or during the enzymatic processing stage. Subsequently, the RLU activity was recorded for each of the buffer conditions.
  • NIB-12 had the greatest beneficial effect during the infection step, as indicated by the highest RLU values among all of the buffers tested.
  • the second most beneficial buffer in enhancing the infection step was NIB-14. This buffer offers instead higher neutralization power against residual sanitizers. The influence of the buffers was not as pronounced during the enzymatic activity phase; however, of the NIB buffers tested, NIB-12, supported the most enzymatic activity, followed by the similar enzymatic activity rates of both NIB-10 and NIB-14 as determined by RLU output. (See Figure 19).
  • *NIB-10 is composed of: IX BHI, 0.5% LiCl, 0.002% nalidixic acid, 0.2% yeast extract, 2mM CaC12, 40 mM HEPES, pH 7.4, ImM sodium metabisulfite, 0.1% sodium thiosulfate, 0.5% Tween-80, and 0.1% lecithin.
  • Example 13 Lower Limit of Detection Assays in the Detection of Microbes in Food
  • LLOD Lower limit of detection assays
  • 25 mL of 100% (full fat) whole milk, 25 mL of NIB-14 infection buffer (see Table 4), and 4.5X10 7 pfu/mL recombinant marker encoding phage were used.
  • the recombinant phage had luciferase as the recombinant marker.
  • the results of the LLOD assays revealed that within 2 hours of the addition of L. monocytogenes to the food sample there was detectable signal in the assays, wherein up to 50 cells in a 50 mL sample was detectable. (See Figure 20A).
  • 25 g of food matrix was spiked with either 2 or 20 CFU and then incubated with 75 mL NIB-14. Following the incubation, a sample of the NIB-14 liquid was incubated with the recombinant phage for 3 hours.
  • a time course to detection assay was also performed using three species of Listeria, L. seeligeri, L. innocua, and L. monocytogenes incubated in turkey, queso, and in guacamole. ⁇ See Figure 24A-C). The data indicate that the detection of the various species of Listeria is dependent on food matrix type ⁇ e.g. shorter time to detection in Turkey) and the Listeria species.
  • a bacterial strain panel comprising a diverse combination of Listeria species and subspecies was selected for characterization of Listeria phages.
  • the panel comprises strains that have been isolated from various geographic and environmental niches including food processing plants and food retail locations. Special consideration was given to obtain bacterial strains from food processing environments with sufficient geographic separation to maximize natural variation within the bacterial strain panel.
  • L. monocytogenes strains from each of the most common nbotypes represented from isolates from food and human disease were selected for the collection. These populations are largely overlapping and have a strong correlation in prevalence and, therefore, represent the strains most useful to identify in food processing plants. When looking at breadth of coverage of L.
  • monocytogenes strains based on nbotypes isolated in human disease and food processing plants, the panel as constructed represents -86% and 91% coverage, respectively.
  • the purpose for selecting 10 strains of each L. monocytogenes ribotype was to allow for the identification of natural variation within a group to ensure a reasonably complete coverage of the L. monocytogenes species.
  • the Listeria bacterial strain panel was used to determine the host range for a particular bacteriophage. To do this a culture of each Listeria strain to be tested was started in 5 ml of LBL1 and grown overnight at 30C in an orbital shaker and allowed to grow for 16 hours. For each bacterial host strain 30 ⁇ of the 16-hour culture was mixed with 270 ⁇ of fresh LBL1 medium. To each cell dilution, 4 ml of LBL1 soft agar was added and overlay ed onto LBL1 agar in 100 mm petri dish. The soft agar overlay was allowed to cool and solidify at room temperature.
  • a reference strain (FSL F6- 367 for A511 and P100) was treated in a similar manner to the host range isolates.
  • a 10- fold dilution series of the bacteriophage in LBL1 medium was prepared from 10 "1 to 10 "8 . 5 ⁇ 1 of each dilution of the bacteriophage was spotted onto the soft agar overlay and the liquid was allowed to adsorb and then the plate was incubated at 30C for 16 hours. After incubation the plaques present at each dilution series were counted and compared to the reference strain to provide an efficiency of plaquing for each host range isolate.
  • the host range was represented as a percentage of the titer observed on the experimental host compared to the reference strain.
  • Bacterial strains that showed a plaquing efficiency greater than 10% (Table 19, dark gray shading) of the reference strain were considered to be within the host range.
  • Bacterial strains that showed a plaquing efficiency less than 10% but greater than .01% (Table 19, light gray shade) of the reference strain were considered to be weakly susceptible to the phage.
  • Bacterial strains that showed a plaquing efficiency less than .01 % (Table 19, unshaded) of the reference strain were considered to be outside of the host range for a phage.
  • a phenomenon that was seen for many of the bacterial strains tested was what has been described in the literature and art as "extracellular killing" (ECK) (Table 19, black), see e.g. Shaw et al. (J Immunol Methods. 1983;56(l):75-83).
  • a strain was defined as demonstrating ECK for a particular phage when at high phage concentration completely cleared the lawn, however, subsequent dilutions did not produce clearing.
  • the prevalence of the extra-cellular killing (ECK) phenomenon exhibited by both A511 and PI 00 in the plate-based host range method demonstrates that the plate based is not as useful as it could be for determining the host range for either phage.
  • the liquid-based host range assay is an end point assay where the ability of a phage to infect a particular bacterial isolate is determined by comparing the optical density of a culture with or without bacteriophage.
  • BHI Heart Infusion
  • agar plates and single colonies were inoculated in 1 ml BHI liquid in a 2-ml 96-deep well dish, covered with a sterile breathable sterile membrane and grown at 30C for 16 hours.
  • Each of the 16-hour cultures from the 96-well plates were diluted 1 : 10,000 in 198 ⁇ of LBL1 in a 300 ⁇ flat-bottom optical 96-well plate and then either 1 X 10 5 pfu of the bacteriophage or an equivalent volume of LBL1 was added to each well of the 96-well plate. This concentration of bacteriophage and bacterial cell dilutions was to approximate a multiplicity of infection (MOI) of 1 in each well.
  • MOI multiplicity of infection
  • the plates were incubated at 26C with shaking at 50 rpm for 16-hours. Plates were placed in a 96-well plate reader (Biotek Eon Microplate Reader) and agitated for 3 seconds with orbital shaking to resuspend cells that had settled out of culture. After the agitation, the optical density of each well was measure at 600 nm (OD600) wavelength light. The ratio of OD600 of the bacterial isolate in the presence of bacteriophage to the uninfected bacterial isolate culture was used as a metric to determine the efficiency of infection for a bacterial strain. A bacterial strain with a ratio of less than or equal to 0.4 (Table 2, dark gray shade) was considered to be sensitive to infection by the bacteriophage.
  • the liquid-based host range assay identified 192 and 153 bacterial strains sensitive to A511 and PI 00, respectively, of the 272 strains in the bacterial strain panel (Table 19). This data shows that A511 is capable of infecting approximately 70% and P100 is capable of infecting approximately 58% of the host range panel.
  • the plate-based host range method identified 62 and 120 bacterial strains that demonstrated a plaquing-efficiency for A511 and PI 00, respectively. Of the strains identified in the plate-based host range methods, only 8 A511 -sensitive bacterial strains and 3 P100-sensitive bacterial strains did not show clearance in the liquid-based clearance assay.
  • the liquid-based assay is an endpoint assay and represents a kinetic interaction between bacteriophage infection and bacterial cell growth
  • certain bacterial strains with increased cell growth rates may be able to saturate a culture even though the strain is susceptible to infection and this may explain the reason why a small number of strains identified in the plaque-based assay were not identified in the liquid assay.
  • the additional strains identified by the liquid-based host range assay were due to the ability to collect data on strains that demonstrated an ECK phenotype in the plate-based host range assay.
  • the large number of strains that demonstrated this phenotype created a large amount of unknown information regarding the host range for A511 and PI 00.
  • the liquid-based assay eliminated the ECK phenomenon, one of the large drawbacks of the plate-based host range method. Two factors contributed to the lack of ECK.
  • First the concentration of phages used in the liquid-based assay is a set
  • the delocalized concentration of bacteriophage within the liquid infection and the low MOI decreases the number of interactions between the bacterial cells and bacteriophage. The limited interaction decreases the possibility of non-productive encounters and lowers super-infection, or infection by multiple
  • bacteriophages of a cell By eliminating ECK, the sensitivity for measuring susceptibility of a particular bacterial cell to a bacteriophage was increased substantially and provided a more accurate representation of the host range of a bacteriophage across the Listeria species.
  • the liquid-based host range assay showed substantial advances over the prior method of using a plate-based system for determining host range of a bacteriophage. Previous literature did not report the ability of growing these bacteriophages in a format other than a plate-based method.
  • the liquid format is also useful because the speed with which the liquid-based host range assay can be performed increases the speed of determining the host range of a bacteriophage from 7-10 days for the panel as it was assembled to several hours of hands on labor. Additionally, the high-throughput nature of the scoring of host susceptibility allowed for multiple bacteriophage host ranges to be determined concurrently, a possibility that did not exist previously. The ability to process multiple bacteriophages concurrently allowed for a more direct comparison of
  • the liquid-based host range assay allowed for a more accurate representation of the functional determination of a potential bacteriophage in a predicted product compared to a plate-based host range assay. The combination of the increased speed, ability for more direct comparison and ability to assess functionality of a bacteriophage in a more direct method to the final product makes the liquid-based host range assay significantly more useful than the plate-based host range method in most contexts.
  • the efficacy of a cocktail of a PI 00 and A511 bacteriophage can be determined by the ability of each of the bacteriophages to infect a particular strain.
  • Infections of the host panel with a cocktail of PI 00 and A511 show the additive host range expected from the extrapolation of the individual host ranges. Based on observations regarding the bacteriophage concentration required for optimum luciferase production during the course of infection, the concentration of bacteriophage added was maintained at a constant total phage concentration of 1X10 7 whether a single bacteriophage or multiple phage cocktail was used for infections.
  • the cocktail of A511 and PI 00 shows coverage of 74% of the panel constructed, while the individual bacteriophages show 70% and 55% coverage, respectively.
  • the seven bacteriophages selected in addition to A511 and P100 were LP44, LP40, LP48, LP99, LP101, LP124, LP125, and LP143. No individual phage assayed covers more than 78% of the Listeria host strain panel. In combination, the bacteriophages cover approximately 92% of the host strain panel as assayed by liquid- based host range assay (Tables 21-23). This combinatorial approach allows for the construction of a bacteriophage cocktail that provides the necessary coverage of the Listeria species to provide a reliable determination of the presence of Listeria in environmental sample collection.
  • PIE Phage Infective Engineering
  • the coding sequence (SEQ ID NO: 1) for the firefly luciferase (SEQ ID NO: 2) or the coding sequence (SEQ ID NO: 3) for the nanoluc luciferase (SEQ ID NO: 4) was inserted at this location.
  • the PIE method uses Phage Targeting Vectors PTVs which include the luciferase gene sequence flanked by -1KB of phage sequence directly upstream and downstream of the desired insertion site (referred to as an upstream homology region (UHR) and downstream homology region (DHR)).
  • UHR upstream homology region
  • DHR downstream homology region
  • Each of these inserts was created using PCR primers that would amplify the desired amplicon, while adding 20bp of homology to facilitate assembly.
  • Plasmids were assembled using the GeneArt Seamless Assembly Kit (Life Technologies).
  • the 3 inserts (UHR, luc, DHR) were assembled into the gram positive/gram negative shuttle vector pMK4, which was restriction-digested with Smal and Pstl (NEB).
  • A511 phage genome sequence is available in Genbank (NC 009811).
  • A511 phage may be obtained from ATCC (PTA-4608TM).
  • the PIE method was used to insert the firefly luciferase gene (SEQ ID NO: 1) directly after the stop codon of the cps gene of A511, between bases 46,695 and 46,696 of the genomic sequence. No sequence was deleted from the phage genome.
  • a 16bp sequence containing a ribosome-binding site (GAGGAGGTAAATATAT) (SEQ ID NO: 67) was placed before the start (ATG) of the firefly luciferase gene.
  • the infection was assayed on the following day. First, the lysate was mixed with chloroform to kill any remaining cells, and to destroy the background luciferase made by the PTV. The phage is
  • chloroform-resistant which is a common trait in bacteriophages.
  • 4% v/v CHC13 was added to the lysate, vortexed, spun down, and the supernatant was recovered.
  • a test infection was done, adding a 1 : 10 dilution of an overnight culture of EGD-e was mixed with the recombinant lysate (90 ⁇ 1 cell dilution, ⁇ phage lysate).
  • a control infection was set up without cells. The infections were incubated statically at 30°C for 3hr, then assayed for luminescence on the Glomax 20/20.
  • the initial recombinant lysate was titered. 20 1- ml lysates were set up each with le6, le5, and le4 pfu/ml of the recombinant lysate: 1ml EGD-e @ OD 0.02, leX phages; O/N, 30C, 50rpm. On the following day, the CHC13 treatment was done, as described above, for each lysate. The lysates were used to set up infections as above. Each lysate was assayed on the Glomax 20/20 (20 ⁇ 1 infection, ⁇ Reagent for FF, 20 ⁇ 1 infection, 20 ⁇ 1 NanoGlo for nluc).
  • PI 00 may be obtained from ATCC (PTA-4383TM).
  • the luciferase insertion site for P100 was also downstream of the same cps gene.
  • the location of the firefly luciferase insertion in P100 is between base 13,196 and 13, 197 of the PI 00 genomic sequence.
  • PI 00 was engineered in the same manner as A511 with the following exceptions: the "PI 00 DUR" fragment was amplified using the primers "pMAK dnf ' and "pMAK dnr PI 00". The single recombinant plaque was identified by picking the plaque into ⁇ SM buffer. ⁇ of this soakate was mixed with 50 ⁇ 1 of luciferin and luminescence was seen on the luminometer. This method of identifying positives was utilized in subsequent recombinant phage isolation.
  • phages were engineered using the firefly luciferase gene and the methods described for A511 : :ffluc: LP48, LP124, LP125, LP99, LP101, LP143.
  • phages were engineered using the NanoLuc gene: A511, PI 00, LP40, LP 124 and LP 125.
  • the PTV for A511 : :nluc was constructed by amplifying the following PCR fragments : Using an A511 lysate as the template, the UHR fragment was generated using oligos pMAK upf and DBON0356; the DHR fragment was amplified using oligos DBON0359 and pMAK dnr. Using the Promega plasmid p Ll .1 as a template, the NanoLuc fragment was amplified using oligos DBON0357 and DBON0358. The assembly and subsequent PIE methods were similar to those described.
  • the PTVs for LP124, LP125, and LP40 were constructed in the same way as A51 1 : :nluc, with the following changes.
  • the DHR fragment amplified was shorter to allow for more efficient assembly of the plasmid, using oligos DBON0359 and
  • DBON0382 Also, the insertion site was modified by adding two additional stop codons (TAATAA) directly downstream of the cps gene of these phages. These 6 bases were added by creating additional primers DBON0379 and DBONO380.
  • the UHR fragments for these phages were amplified using oligos pMAK upf and DBONO380.
  • the NanoLuc fragments were amplified using oligos DBON0379 and DBON0358.
  • DBON0361 TGGCTCTACATGCTTAGGGTTCC (SEQ ID NO: 75)
  • AAAACGTTCATAGCAACTAATAATAAGAGGAGGTAAATATATATGGTCTTCAC ACTCGAAGATTT (SEQ ID NO: 80)
  • 96-well microtiter plates were used to grow the PIE lysates at a 200 ⁇ 1 volume.
  • the initial step was making 96 lysates at le6 pfu/lysate (5e6 pfu/ml), 96 at le5, and 96 at le4.
  • the NanoLuc phages it was found that the recombination efficiency of the recombinant lysate was significantly higher, and that dilutions down to leO pfu/lysate could be used.
  • These lysates were made by incubating at 30°C, shaking at 50rpm overnight.
  • the lysates were assayed using the appropriate luciferase assay system (ff or nanoglo). Instead of using the lysates to infect fresh cells, it was found that the background signal of the lysate itself was an indication of the presence of recombinant phage.
  • DBON0398 TGCTATATTATAGGAACATGGGAA (SEQ ID NO: 82).
  • the gene was sequenced using oligos DBON0273, DBON0398, and pMAK upr.
  • DBON0273 TGCTTACATGCCAGTAGGGGT (SEQ ID NO: 83);
  • nanoluc phages were sequenced using oligos:
  • DBON0361 TGGCTCTACATGCTTAGGGTTCC (SEQ ID NO: 75);
  • DBONO360 CCTCTAGCTCAAATTAACGCATCTGT (SEQ ID NO: 74);
  • DBON0362 GTATGAAGGTCTGAGCGGCG (SEQ ID NO: 84) and
  • DBON0363 GATCTGGCCCATTTGGTCGC (SEQ ID NO: 85).
  • DBON0274 CGCATAGAACTGCCTGCGTC (SEQ ID NO: 86);
  • DBON0151 CACCCCAACATCTTCGACGC (SEQ ID NO: 87);
  • DBON0152 GCGCAACTGCAACTCCGATA (SEQ ID NO: 88)
  • LP125::nluc SEQ ID NO: 32
  • A511 : nluc (SEQ ID NO: 33);
  • LP40 :nluc (SEQ ID NO: 35).
  • the insertion site regions of the phages comprising an inserted firefly luciferase coding sequence are aligned in Figure 28.
  • the insertion site regions of the phages comprising an inserted firefly luciferase coding sequence contain the following parts as indicated in Table 14.
  • the insertion site regions of the phages comprising an inserted nanoluc luciferase coding sequence are aligned in Figure 29.
  • the insertion site regions of the phages comprising an inserted nanoluc luciferase coding sequence contain the following parts as indicated in Table 15.
  • the cps gene sequences are aligned in Figure 26 and the protein sequences in Figure 27.
  • the cps genes of the engineered phage display a relatively high degree of homology.
  • recombinant phage To allow for the concentration of signal produced by the infection of listeria by recombinant phages, alternate versions of recombinant phage were produced that included a HIS tag.
  • the 6xHIS tag (SEQ ID NO: 89) is a commonly used affinity tag for concentrating and purifying recombinant proteins.
  • HIS tags are commonly placed at the N-terminus or C-terminus of a protein, as it is often unknown a priori which location is optimal. Depending on the structure of the protein being tagged, as well as interactions with substrates, the tag sequence can interfere with, inhibit, or enhance enzyme function. For this reason phages were engineered with the HIS tag at either the N- or C-terminus.
  • a spacer sequence comprising a small number of amino acid residues is place between the HIS tag and the gene being tagged. The size, charge, and other characteristics of this spacer can effect interactions with the enzyme, substrate, or HIS-binding beads/resins/antibodies. For this reason 2 different spacer were used between the HIS tag and the Nanoluc protein.
  • the HIS-tagged nanoluc versions of A511, LP 124, and LP40 were constructed using the same methods as the untagged phages.
  • the HIS tag and spacer were introduced during PTV construction by adding sequence to the oligos used to amplify the various DNA fragments.
  • the oligos used in constructing the PTVs for A511, LP124 and LP40 are common to all 3 phages.
  • NLUC fragment DBON0379 and DBONO400
  • NLUC fragment DBON0379 and DBONO402
  • DHR fragment DBONO401 and DBON0382
  • DHR fragment DBON0359 and DBON0382
  • NLUC fragment DBONO404 and DBON0358
  • DHR fragment DBON0359 and DBON0382
  • CAGCAGTCTTCACACTCGAAGATTTCG (SEQ ID NO: 94)
  • HIS tag amino acid sequence HHHHHH (SEQ ID NO: 89)
  • HIS tag DNA sequence CATCATCACCATCACCAT (SEQ ID NO: 95)
  • C-terminal HIS with long spacer amino acid sequence AAGGGHHHHHH
  • GCAGCAGGTGGAGGTCATCATCACCATCACCAT (SEQ ID NO: 97)
  • N-terminal HIS with long spacer amino acid sequence HHHHHHGGGAA (SEQ ID NO: 100)
  • N-terminal HIS with long spacer DNA sequence [00499] N-terminal HIS with long spacer DNA sequence:
  • N-terminal HIS with short spacer DNA sequence [00501] N-terminal HIS with short spacer DNA sequence:
  • Engineered phages were tested in the liquid-based host range assay and compared to non-modified bacteriophages.
  • the engineered bacteriophages did not show a change in their host range compared to the non-modified wild-type versions (Tables 24-25).
  • a two phage cocktail would consist of 5X10 4 pfu of each of the two component phages).
  • the combination of bacteriophages in a cocktail did not cause a loss of host range and provided the expected additive effects of the host range of the individual bacteriophages (Tables 24-25).
  • the additive effect of the bacteriophages was independent of the genomic modifications as neither the engineered Firefly luciferase and Nanoluc luciferase expressing bacteriophages had an altered liquid- based host range compared to the unengineered bacteriophages.
  • Example 21 Comparison of Liquid-Based Host Range Versus Marker-Based Host Range
  • the ability of a bacteriophage to clear an actively growing culture is determined by a number of factors including the rate of growth of a particular strain and the rate of bacteriophage replication, in addition to the ability of the bacteriophage to infect a specific strain. Therefore, the output of culture clearance measure used in the liquid culture method disclosed herein is potentially more restrictive than the host range that could be determined by exposing bacterial strains to an recombinant phage comprising a heterologous nucleic acid sequence encoding a marker and assaying for marker production.
  • a marker is luciferase.
  • the host range was determined for phage LP124:nluc by both the liquid-based host range assay and by an infection based luciferase detection assay.
  • the Listeria host panel strain collection was struck out on Brain Heart Infusion (BHI) agar plates and single colonies were inoculated in 1 ml BHI liquid in a 2-ml 96-deep well dish, covered with a sterile breathable sterile membrane and grown at 30C for 16 hours. Each of the 16-hour cultures from the 96-well plates were diluted 1 : 10,000 in 198 ⁇ of BHI.
  • 12.5 ⁇ of the culture dilution were mixed added to 12.5 ⁇ of LP124:nluc at a
  • Table 5 shows the host range determined by the two methods.
  • a strain was considered to be within host range for the clearance assay if the ratio of infected culture OD600 to the uninfected culture OD600 was less than 0.4.
  • For the luciferase detection- based host range assay strains were stratified in three categories, high RLU strains (Table 26, dark gray shading,), medium RLU strains (Table 26, light gray shading), and low RLU strains (Table 26, unshaded). Based on the performance of the assay a strain was considered to be within the host range of the bacteriophage if the RLU measurement was greater than 10,000 Random Light Units (RLU) (Table 26, light gray shading).
  • luciferase activity cut-off was used because it characterizes a useful level of sensitivity in bacterial assays. Based on these criteria the liquid-based host range clearance, LP124 shows a broad host range by clearing 50.5% (140 of 276) of the Listeria strains tested. By the luciferase detection assay, 78.2% (216 of 276) of the Listeria strains tested showed high RLU levels.
  • bacteriophage life-cycle can be separated into discrete steps, a bacteriophage is capable to produce phage encoded proteins, in this case luciferase, without clearance of the culture or producing additional bacteriophage. While additional strains that produce luciferase without producing bacteriophage would not fall within the classical definition of host range for a bacteriophage, the strains do meet inclusion in the host range definition for the purpose of this disclosure because the host range that matters in methods of detecting target bacteria using a phage comprising a heterologous nucleic acid sequence encoding a marker is the types of bacteria that support marker production. This increased host-range observed when using the engineered bacteriophage is an advantageous byproduct of the engineering process and could not be determined a priori for the Listeria host panel.
  • the cultures were diluted to a concentration of 10 5 cfu/ml and tested for both the production of luciferase in the presence of the bacteriophage cocktail and inhibition of Listeria infection by the bacteriophage cocktail as above. None of the bacterial species, consisting of both Gram-positive and Gram-negative bacteria, showed any luciferase production in the presence of the bacteriophage (Table 28). Additionally, incubation of Listeria in the presence of the collected samples failed to show any decrease in the production of luciferase,
  • the increased host range observed by the RLU-based luciferase detection assay compared to the liquid-based host range assay identified a novel method for distinguishing differences between the host range of bacteriophages. Additionally, the RLU-based luciferase detection assay as a means to assess phage host range allows for a highly accurate assessment of the target bacteria identified by an engineered bacteriophage under conditions similar to those of methods of detecting target bacteria. One way this information may be used is to identify useful combinations of phage that can be combined to make a combination of phage having a useful cumulative host range.
  • bacteriophage cocktail a RLU-based luciferase detection assay was compared between A511 : :nluc and LP124: :nluc for a portion of the Listeria host range panel.
  • LP124: :nluc had a larger RLU-based host range (detects 77 of 96 strains, 80.2%) compared to
  • A511 :nluc (detects 37 of 96 strains, 38.5%) (Table 29). Moreover, LP124:nluc produces greater than 100-times higher RLU values compared to A511 :nluc in 73 of 96 strains (76%)). This increased RLU output from LP124:nluc infections predicts that a
  • bacteriophage cocktail that contains both A511 and LP124:nluc would have greater sensitivity and accuracy over a A511 :nluc alone.
  • LP124: :nluc would increase the levels of RLU produced in the presence of A511 and PI 00 the RLU values were compared between samples infected with both a two-phage cocktail (A511 : :nluc/P100: :nluc) and a three-phage cocktail (A511 : :nluc/P100: :nluc/LP124: :nluc). To test this, 1 ml of complex environmental samples grown in UVM medium were pelleted by centrifugation.
  • the supernatant was removed and the cells were resuspended in 100 ⁇ of either the two-phage or three-phage cocktail at a total bacteriophage concentration of 1X10 7 and incubated at 30C.
  • RLU levels were measured by using Promega NanoGlo reagent and the Promega 20/20 luminometer.
  • the environmental samples showed higher levels of RLU in the presence of the three-phage cocktail than the two-phage cocktail (Table 30). This increase in the RLU output of the infection demonstrates a clear advantage from having LP124: :nluc present over P100::nluc and A511 ::nluc alone.
  • the bacteriophage cocktail was able to clear the bacterial culture in 25 of 100 strains (25%). This decreased level of clearance is due to a greater growth rate for the environmentally isolated strains compared to common lab isolates tested in the Listeria host range panel.
  • the RLU based host range assay identified 75 of 100 strains (75%) (Table 31). These environmental samples represented complex microbiological communities and had multiple Listeria isolates per environmental sample. The presence of multiple strains of Listeria within these microbiological communities improves the sensitivity of the assay. In this example the environmental samples were collected using sponges and the sponges were incubated for up to 24h with media, after which an aliquot was removed and assayed for the presence or absence of the bacterial population to be detected. Based on the ability of the
  • the bacteriophage cocktail to identify individual Listeria strains identified from the same environmental samples it would have been predicted that the bacteriophage cocktail of A511 and LP124:nluc would be able to detect 48 of 57 (84.2%) Listeria positive sponges.
  • the environmental sponge was incubated in a growth medium and a sample of the enriched sample is tested using the assay the bacteriophage cocktail containing A511 and LP124:nluc was able to detect 49 of 57 (85.9%) Listeria-positive sponges.
  • This increased sensitivity demonstrates that the presence of multiple Listeria strains, including those out of host range for the bacteriophage cocktail, does not diminish the sensitivity of the assay to detect Listeria strains that are sensitive to the bacteriophage cocktail.
  • the phage detection composition was further optimized to promote sensitivity, specificity and usability.
  • the optimized Listeria detection composition is herein referred to as "Listeria detection composition v2.0.”
  • the methodology and the components of the detection composition were modified to achieve the 2.0 version which is superior to previous iterations of the Listeria detection composition in terms of sensitivity, specificity and ease of use.
  • T e Listeria detection composition v2.0 was generated through modification of buffer cocktails, incubation temperature, detection volume, centrifugation speed, and through the use of a different sponge material for sampling. See Table 33.
  • the examples that follow demonstrate the increased benefits provided by each component of the Listeria detection composition v2.0.
  • the Listeria composition v2.0 provides approximately a 5-fold increase over a previous iteration (vl .0.4) on control strains.
  • the v2.0 provides increased host range coverage and light output.
  • the "reagent” refers to the Nano-Glo substrate. Prior iterations of the Listeria detection composition used a 1 : 1 ratio of sample volume to reagent volume. For these experiments the
  • Figures 30A and 30B indicate that the use of a 2: 1 ratio of sample volume to Nano-Glo reagent does not have a negative impact on the resultant RLU signal detected. These data indicate that it is possible to double the sample volume removed from the sampling bag without increasing the amounts of additional Nano-Glo to acquire detectable signal.
  • Another iteration of experiments meant to assess the impact on varying the volume of the detection reaction included varying the volume of the sample added to the detection reaction composition while maintaining a constant volume of the Nano-Glo reagent added to the reaction.
  • the following protocol was followed: 1) spike 100 cells onto sponge, 2) infect with 6mL of phage cocktail (v 1.0.1 phage, vl .0.3 or 1.0.4), 3) incubate for 6 hours at 30°C, 4) remove 300 ⁇ ., 600 ⁇ ., or 900 ⁇ . of sample from bag, 5) add 300 ⁇ . of Nano-Glo reagent to each sample, and 6) detect the emitted light.
  • the cells were allowed to dry overnight, 3) the spots were swabbed with 3M Letheen Sponge Stick, 4) the sample was incubated for 2 hours at 25°C, 5) the sample was infected with 6mL COP2 at the concentration of 4.5E7PFU/mL (referred to as FCF), 6) the sample was subsequently incubated at 30°C, 7) 300 ⁇ ., 600 ⁇ ., or 900 ⁇ . of the sample was removed, 8) 300 ⁇ . of Nano-Glo reageant was added to each sample, and 9) the emitted light was detected.
  • FCF 6mL COP2 at the concentration of 4.5E7PFU/mL
  • Nano-Glo luciferin/detection buffer prepared according to the manufacterer' s instructions and the luminescence was measured on a Promega GloMax96 luminometer using the built-in "SteadyGlo" protocol (1 second integration, or equivalent to detect emitted light signal.
  • LP80 UTR7 also refered to herein as LP80H4
  • LP80H4 optimized phage to the phage cocktail
  • Table 34 is a complete list of the phage components in the v2.0 detection composition.
  • the detection composition v2.0 phage cocktail was maintained at a constant 1E7 pfu/mL, while the LP80 phage was added to the v2.0 cocktail at 1E5, 5E5 or 1E6 pfu/mL.
  • LP80 is used as an additional phage for inclusion in the v2.0 cocktail because it is able to infect unique Listeria strains.
  • the data from these experiments indicate that the addition of LP80 at 1E5 pfu/mL does not harm the aggregate signal, while adding the benefit of the detection of a greater amount of Listeria strains. See Figure 35B and Table 35.
  • Table 35 Amount of Listeria strains detected at or above cutoff threshold with v2.0 cocktail, and v2.0 cocktail with the addition of LP80 at various concentrations.
  • the currently used sponge for the detection assays is a cellulose sponge.
  • Some of the disadvantages of using a cellulose sponge include supply chain vulnerability, potential for residual quaternary ammonium compounds from the manufacture of the cellulose sponge, batch-to-batch inconsistency in the production of the sponges, and the possibility that the sponges can have extraneous materials from the manufacturing process such as, for example, heavy metals, chloro-organic compounds and residual sulfur from the manufacturing process.
  • polyurethane sponges were tested in the detection protocol.
  • the benefits of polyurethane sponges include that these sponges are non-toxic, there is a greater amount of batch-to-batch consistency in the manufacturing of the polyurethane sponges, the sponges are routinely used in medical devices, they have good water absorbency and retention, and the sponges are strong, characterized by the polyurethane sponge's high tensile strength.
  • One of the objectives is to replace the cellulose sponges with polyurethane sponges as a sample collection device, and to assess whether there is any improvement in the consistency in liquid retention in the polyurethane sponges in comparison to the cellulose sponges.
  • sanitizers used in the food industry fall into several categories: chlorine-based sanitizers (bleach, chlorine dioxide), peroxyacid compounds +/- organic acids, organic acids (ascorbic acid, citric and lactic acid, caprylic acid), quaternary ammonium compounds, iodophors, surfactants and detergens. Despite the neutralization in the previous S6
  • the S6 Infection buffer is composed of: lx Tryptic soy broth, 0.25% lithium chloride, 20mg/L nalidixic acid, 0.5% yeast extract, 0.25% glucose, 0.08% magnesium sulfate, 0.1% sodium pyruvate, 20mM Hepes (pH7.4), 0.22% lecithin, 1.5% Tween 80, and 0.02% potassium phosphate.
  • Various compounds were assayed to develop a more robust neutralization of residual sanitizers in the samples.
  • Three potential neutralizers were of interest: sodium metabi sulfite, sodium pyruvate and catalase. Of these three, sodium metabisulfite and sodium pyruvate were tested in the assays.
  • centrifugation speed was assessed with regards to the resultant light emitted signal obtained. Previous iterations of the detection protocol relied on centrifugation speeds of 14 k rpm. Various kinds of centrifugation speeds (expressed as relative centrifugal force-rcf) were assessed such as 1,000 rcf, 2,000 rcf, 5,000 rcf, 9,000 rcf or 14,000 rcf, including a no spin condition. The data obtained from these various centrifugation speeds indicate that centrifugation speed of 9,000 rcf resulted in the greatest amounts of light emitted signal and greatest reduction in false positives.
  • sanitizers and enzyme were diluted in NIB-14 to generate an approximate 10 3 dilution in 300 ⁇ .
  • 300 ⁇ . of Nano-Glo was added to 600 ⁇ . of each sample and detected
  • 300 ⁇ . of the organic solvent was added to the sample and immediately re-read.
  • the analysis for these experiments was a comparison of the increase or the decrease in signal detected following incubation with various organic solvents.
  • the solvents tested in these studies included acetone,
  • isopropanol, ethanol, and propylene glycol isopropanol, ethanol, and propylene glycol.
  • sample for example 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 and ⁇ . can be added to the sample.
  • CDW 1554 control
  • P1836 v2.0 Target 1
  • NP1883 v2.0 Target 2
  • centrifugation speed (maximum centrifugation speed used was 13.4K RPM), v2.0 drop 600 ⁇ . (300 ⁇ . used).
  • the results of the component substitution experiments are presented in Figures 41 A-41C.
  • Example 26 LP80 alternate host characterization
  • LP80:COP3 bacteriophage were used to establish a host range for said bacteriophage.
  • a liquid-based host range analysis was performed as described in Examples 20 and 21.
  • the tested Listeria strains comprised 96 strains that are difficult to target. See Table 36 below.
  • the LP80 lysate generated on a typical host or an alternate host was compared.
  • the data are depicted in Figures 50A-50C. The data indicate the ability of LP80 to clear Listeria cultures by the standard liquid host range clearance.
  • Example 27 Supplemental Methodology
  • S0818 (SEQ ID 105): ATATTTACCTCCTCTTATTAttaggcaggtaaagtaattg
  • S0649 (SEQ ID 106): CGCATTTTAGCCTAAtaaagactaagcccagcttc
  • SO650 (SEQ ID 107): acgacggccagtgaattcccttacctgctggcacgtct
  • S0648 (SEQ ID 109): tgggcttagtctttaTTAGGCTAAAATGCGCTCGC
  • Template for the COP3 (SEQ ID 115) was an A511 phage engineered with COP3 (UTR 7 Variant). Lowercase letters are the UTR7 sequence, Uppercase are the COP3 sequence.
  • S0646 (SEQ ID 116): tatTTATTAttaggcaggtaaagtaattgtaacagaagaagc [00575]
  • S0647 (SEQ ID 117): actttacctgcctaaTAATAAataattttgattaacttt
  • the tested Listeria strains comprised 96 strains that are difficult to target.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une composition et des méthodes de détection d'un ou de plusieurs microbes cibles. Les compositions de l'invention comprennent au moins un phage recombinant capable d'infecter un microbe cible, ledit phage comprenant au moins une séquence de protéine de capside, un site de fixation du ribosome et un marqueur à codons optimisés (ou optimisation de codons). Les compositions de l'invention peuvent, en outre, comprendre une solution aqueuse qui améliore la capacité de détection de l'expression du marqueur suite à une infection du microbe cible par le phage. Dans certains modes de réalisation, le microbe cible peut être la Listeria.
EP16824938.1A 2015-07-10 2016-07-08 Phages recombinants et méthodes de détection de la listeria Withdrawn EP3320108A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201562191229P 2015-07-10 2015-07-10
US201562191867P 2015-07-13 2015-07-13
PCT/US2016/041529 WO2017011309A1 (fr) 2015-07-10 2016-07-08 Phages recombinants et méthodes de détection de la listeria

Publications (1)

Publication Number Publication Date
EP3320108A1 true EP3320108A1 (fr) 2018-05-16

Family

ID=57757729

Family Applications (1)

Application Number Title Priority Date Filing Date
EP16824938.1A Withdrawn EP3320108A1 (fr) 2015-07-10 2016-07-08 Phages recombinants et méthodes de détection de la listeria

Country Status (2)

Country Link
EP (1) EP3320108A1 (fr)
WO (1) WO2017011309A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2022218190A1 (en) * 2021-02-04 2023-09-21 Laboratory Corporation Of America Holdings Methods and systems for the rapid detection of listeria using infectious agents

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090081715A1 (en) * 2007-09-07 2009-03-26 Cobalt Technologies, Inc., A Delaware Corporation Engineered Light-Emitting Reporter Genes
WO2012159774A1 (fr) * 2011-05-26 2012-11-29 Dsm Ip Assets B.V. Nouveau bactériophage p825n de listeria et ses utilisations
JP2016517687A (ja) * 2013-03-27 2016-06-20 サンプル テクノロジーズ，インコーポレイティド 組換えファージ及び細菌検出方法
AU2014281406A1 (en) * 2013-06-19 2016-01-21 Sample6 Technologies, Inc. Phage-based bacterial detection assay

Also Published As

Publication number Publication date
WO2017011309A1 (fr) 2017-01-19

Similar Documents

Publication Publication Date Title
US10273460B2 (en) Recombinant phage for listeria detection
US9828625B2 (en) Phage-based bacterial detection assay
Carstens et al. A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions
US20180274004A1 (en) Recombinant phage and methods of detecting listeria
Bergholz et al. Omics approaches in food safety: fulfilling the promise?
Mouftah et al. Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 C among hyper-aerotolerant generalist Campylobacter jejuni
Bollback et al. Parallel genetic evolution within and between bacteriophage species of varying degrees of divergence
Gill et al. Bacteriological analysis of wheat flour associated with an outbreak of Shiga toxin-producing Escherichia coli O121
Harrand et al. Assembly and characterization of a pathogen strain collection for produce safety applications: pre-growth conditions have a larger effect on peroxyacetic acid tolerance than strain diversity
JP2021521824A5 (fr)
Xu et al. Whole-genome resequencing of Bacillus cereus and expression of genes functioning in sodium chloride stress
Cherrad et al. New insights from short and long reads sequencing to explore cytochrome b variants in Plasmopara viticola populations collected from vineyards and related to resistance to complex III inhibitors
US10039795B2 (en) Codon optimized recombinant phage and methods of using same
WO2014049938A1 (fr) Milieu de culture pour bactéries responsables d'intoxications alimentaires, et méthode de détection de bactéries responsables d'intoxications alimentaires
El Baaboua et al. A review of current knowledge and gaps about campylobacter methods: from culture to characterization
EP3320108A1 (fr) Phages recombinants et méthodes de détection de la listeria
Hodges et al. Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I: 4,[5], 12: i:-isolated from Canadian Atlantic coast shellfish
Azinheiro et al. Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon
Martinez‐Malaxetxebarria et al. Regulation of Energy Metabolism by the E xtra c ytoplasmic function (ECF) σ factors of Arcobacter butzleri
Srinivasa et al. Tracing Foodborne Pathogens Using Molecular-Based Approaches
Calvez et al. Relative expression of genes involved in the resistance/sensitivity of Enterococcus faecalis JH2-2 to recombinant divercin RV41
Hwang et al. A Robust Genome-Wide Association Study Uncovers Signature Genetic Alterations among Pseudomonas aeruginosa Cystic Fibrosis Isolates
Petersen Rapid determination of viable but non-culturable Campylobacter jejuni in agri-food products by loop-mediated isothermal amplification coupling propidium monoazide treatment
Choudhary et al. A TIR-SAVED effector mediates antiviral immunity via a conserved host signal
Tanasuica et al. Prevalence and Genotypes of Shiga Toxin (Verotoxin)-Producing Escherichia coli in Romanian Food

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20180209

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INSTITUTE FOR ENVIRONMENTAL HEALTH, INC.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20180822