WO2022118104A1 - Article pour le stockage de bactériophages et procédé associé - Google Patents

Article pour le stockage de bactériophages et procédé associé Download PDF

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Publication number
WO2022118104A1
WO2022118104A1 PCT/IB2021/059585 IB2021059585W WO2022118104A1 WO 2022118104 A1 WO2022118104 A1 WO 2022118104A1 IB 2021059585 W IB2021059585 W IB 2021059585W WO 2022118104 A1 WO2022118104 A1 WO 2022118104A1
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Prior art keywords
nonwoven
phage
article
composition
solid support
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PCT/IB2021/059585
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English (en)
Inventor
Minghua Dai
Raj Rajagopal
Fuming B. Li
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3M Innovative Properties Company
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Publication date
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Publication of WO2022118104A1 publication Critical patent/WO2022118104A1/fr

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    • 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
    • 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
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10051—Methods of production or purification of viral material
    • 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
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10251—Methods of production or purification of viral material

Definitions

  • bacteriophages As objects of research and potential agents for biological control. For example, the use of bacteriophages is an emerging therapy area that is intended to replace or supplement the use of antibiotics.
  • Recombinant bacteriophages offer further selectivity to target bacteria of interest. Effective bacteriophage treatment requires the use of a suitable bacteriophage that is capable of infecting the target bacterial strain.
  • Bacteriophage libraries may be used for screening suitable bacteriophages.
  • Bacteriophages should survive and remain viable during the storage and shipping.
  • the present disclosure provides an article, the article comprising a composition comprising a phage, a gelling agent or thickening agent, and a disaccharide, trisaccharide or a polysaccharide; and a nonwoven; wherein the composition is adsorbed onto the nonwoven.
  • the present disclosure provides a kit, the kit comprising the article of present disclosure; and a solid support; wherein the article is embedded in a solid support.
  • the present disclosure provides a method, the method comprising: immobilizing a composition comprising a phage, a gelling agent, and disaccharide, trisaccharide or a polysaccharide onto a nonwoven; wherein when the phage immobilized onto the nonwoven is stored at room temperature for up to 180 days, at least 60% phages remain viable.
  • phrases inactivation and long term reduction in phage titer upon storage is highly undesirable.
  • the article of the present disclosure can provide a low cost process and improve thermal stability for long term storage of bacteriophages.
  • the present disclosure provides a method of phage viability during storage without the need for refrigeration and allows for phage viability during long distance and ambient temperature shipping.
  • the present disclosure provides an article.
  • the article can include a composition comprising a phage, a gelling agent or thickening agent, and a disaccharide, trisaccharide or a polysaccharide.
  • the article can include a nonwoven.
  • the composition can be adsorbed onto the nonwoven.
  • composition of the present disclosure When the composition of the present disclosure is stored at room temperature for up to 75 days, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% phages remain viable.
  • composition of the present disclosure is stored at room temperature for up to 180 days, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% phages remain viable.
  • composition of the present disclosure when the composition of the present disclosure is stored at room temperature for up to 30 days, up to 75 days, or up to 180 days, all phages remain viable.
  • a bacteriophage may be considered to be viable, if it is capable of infecting prokaryotic cells and subsequently multiplying in them. The viability of bacteriophages may be tested e.g. as described in the examples.
  • bacteriophage and “phage” may be used interchangeably. They may refer to a vims or a recombinant virus that is capable of infecting prokaryotic cells, i.e. bacteria and/or archaea. Bacteriophages may vary in shape and genetic material. For example, a bacteriophage may have a capsid that is icosahedral, octahedral or filamentous. A bacteriophage may also have a head-tail structure, comprising e.g. at) icosahedral capsid (head), a tail and optionally' other parts, such as a collar.
  • the term “a bacteriophage” or “the bacteriophage” may refer to one or more species of bacteriophages, and/or to one or more bacterioplsage particles.
  • the temperature and other conditions during the dtying may vary, although the conditions may be selected such that the viability of the bacteriophage is not reduced or significantly reduced.
  • the drying may work better for some bacteriophages than for others.
  • relatively small phages may tolerate the dry ing better than large ones.
  • the composition or phage In the dry state, the composition or phage may be easier to handle, store, transport etc. Its volume may be reduced, and the risk of contamination from the composition or phage may be reduced.
  • bacteriophages for which the present composition or matrix may be suitable, include the order Caudovirales (including the families Myoviridae, Siphoviridae, Podoviridae) and Ligamenvirales (Lipothrixviridae, Rudiviridae); and families Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae. Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and/or Tectiviridae.
  • the composition or matrix, the methods and the uses are not particularly limited to any order, family or species of bacteriophages. However, they may be better suited for some bacteriophages than for others.
  • the exemplary disaccharide, trisaccharide or polysaccharide can include sucrose, lactose, maltose, glucose, fructose, trehalose, mannose, sorbitol, mannitol, pullulan, and combination thereof. These sugars can be used to stabilize phage.
  • the exemplary gelling agent can contain one or more organic cold-water-soluble agents, such as alginate, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxides.
  • organic cold-water-soluble agents such as alginate, carboxymethyl cellulose, tara gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, guar gum, locust bean gum, xanthan gum, polyacrylamide, polyurethane, polyethylene oxides.
  • Combinations of natural and/or synthetic gelling agents are contemplated.
  • Preferred gelling agents include guar gum, xanthan gum, and locust bean gum, these gelling agents being useful individually or, in any embodiment, in combination with one another.
  • gelling agent can include natural gums, starches, pectins, agar-agar, gelatin, agarose,
  • gelling agent can include Polyvinylpyrrolidone (PVP), (Hydroxypropyl)methyl cellulose (HPMC), Poly(D,L-lactide-co-glycolide) (PLGA), Polyvinyl alcohol (PVA), Dextran 35, and Sodium Alginate.
  • PVP Polyvinylpyrrolidone
  • HPMC Hydrophilicity Modulfate
  • PLGA Poly(D,L-lactide-co-glycolide)
  • PVA Polyvinyl alcohol
  • Dextran 35 and Sodium Alginate.
  • a uniform monolayer of a cold-water-soluble hydrogel-forming composition is desired with sufficient surface area exposed for hydration.
  • the powdered cold-water-soluble hydrogelforming composition may further comprise an inducer, and indicator agent, or a combination of these.
  • the thickening agent can be polyethylene glycol (PEG) 6000 or PEG 3000.
  • Nonwoven, for example, nonwoven web, suitable for use in the present disclosure may be made, for example, by conventional air laid, carded, stitch bonded, spunbonded, wet laid, meltspun, and/or melt- blown procedures, more preferably melt-blown, meltspun, and/or spunbonded process.
  • the nonwoven can be a hydrophilic or hydrophobic nonwoven.
  • a nonwoven web can be made by air-laying of fibers.
  • Air-laid nonwoven fiber webs may be prepared using equipment such as, for example, that available as a RANDO WEBBER from Rando Machine Company of Ard, New York.
  • a type of air-laying may be used that is termed gravitylaying, as described, e.g., in U. S. Pat. Application Publication 2011/0247839 to Lalouch.
  • Spunbonded nonwoven fiber webs can be formed according to well-known conventional methods wherein meltspun fibers are deposited on a moving belt where they form a nonwoven continuous fiber web having interfiber bonds. Melt-blown nonwoven fiber webs processes such as described in Van A. Wente, Superfine Thermoplastic Fibers, 48 INDUS. ENGN.
  • the melt-blowing process may further comprise at least one of addition of a plurality of staple fibers to the plurality of discrete, discontinuous, multi-component fibers, or addition of a plurality of particulates to form a composite nonwoven fiber web.
  • Nonwoven fiber webs may be densified and strengthened, for example, by techniques such as crosslapping, stitchbonding, needletacking, hydroentangling, chemical bonding, and/or thermal bonding.
  • Suitable polymeric resins which can be used for making nonwoven fiber webs, for example, include thermoplastic polyolefins (e.g., Polyethylene, Polypropylene, polymethyl pentane); thermoplastic polyesters (e.g., polylactides and polyethylene terephthalate (PET)); and copolyesters; polyvinyl chloride; polystyrene; polycarbonates; styrenic block copolymers (e.g., SIS, SEBS, SBS); elastomeric alloys (e.g., elastomeric thermoplastic acrylate block copolymers such as polymethyl methacrylate-block-poly (butyl acrylate)-block-polymethyl methacrylate; thermoplastic polyurethanes (TPUs); perfluorinated polymers and copolymers, thermoplastic polyamides, and blends of any of the foregoing.
  • thermoplastic polyolefins e.g., Polyethylene, Polypropylene
  • the nonwoven can be an uncharged or charged nonwoven.
  • Electret nonwoven webs which is an electrostatically charged nonwoven fibrous webs, can also be suitable for use in the present invention.
  • the nonwoven fiber webs as described in the above sessions can be further charged by the charging processes described in U.S. Pat. 30,782 (van Turnhout), U.S. Pat. No. 4,215,682 (Davis et al.), U.S. Pat. No. 5,401,446 (Wadsworth et al.), & U.S. Pat. No. 5,496,507 (Angadjivand) to make electret nonwoven fiber webs.
  • the nonwoven comprises polyethylene terephthalate (PET) fibers, PET ⁇ rayon fibers, PET ⁇ cellulose fibers, polyethylene ⁇ polyester bicomponent fibers, polyester ⁇ cellulose fibers, or polypropylene fibers.
  • PET polyethylene terephthalate
  • PET ⁇ rayon fibers PET ⁇ cellulose fibers
  • polyethylene ⁇ polyester bicomponent fibers PET ⁇ cellulose fibers
  • polyester ⁇ cellulose fibers or polypropylene fibers.
  • the article of the present disclosure can provide materials which can evenly absorb phage on the nonwoven so that the composition is evenly distributed on the nonwoven.
  • the article of the present disclosure can prevent the deactivation of phage, especially at high temperatures and extend the shelf-life of phage for phage or reporter-phage based assay.
  • the dried phages adsorbed onto the nonwoven can remain stable and evenly distributed on nonwoven materials.
  • the article of the present disclosure can provide a low cost process and improve thermal stability for long term storage of bacteriophages. Thus, the method of phage viability without the need for refrigeration storage allows for phage viability during storage and long distance and ambient temperature shipping.
  • the solid support for example a receptacle, may have a recess, or a plurality of recesses, for receiving and/or containing the composition.
  • the solid support may simply have an area on which the composition may be arranged.
  • the composition or matrix may be impregnated in the solid support, for example to a gauze.
  • Multiwell plates such as 6-, I2-. 24-, 48-, 96-, 384-, and 1536-weU plates, or strips of tubes such as 8 strip may be commercially available and may be well suited as the solid support.
  • Such multiwell plates or strips of tubes may be made of various materials, such as plastic, for example polystyrene.
  • the solid support for example a receptacle, may have a recess, or a plurality of recesses, for receiving and/or containing the composition ⁇ ) or article and subsequently for receiving and/or containing a prokaryotic ceil suspension and/or a plurality of different prokaryotic ceils suspensions.
  • the prokaryotic cells suspension(s) may be added directly to the solid support and to the composition(s) or article, such that the bacteriophage! s) in the composition! s) or article may infect the prokary otic cells.
  • the arrangement may be closable and optionally also sealable so as to prevent spreading and/or contamination by the bacteriophage(s).
  • a multiwell plate or strips of lubes may be closable and optionally sealable so as to retain the bacteriophage(s) in the well(s) of the multiwell plate or strips of tubes and to prevent it from contaminating other wells of the multiwell plate or strips of tubes (crosscontamination).
  • the arrangement may further comprise a cover for the solid support.
  • multiwell plates or strips of tubes may be provided w ith a cover or a lid; such covers or lids may be suitably shaped so as to fit the edge of the rvell(s), for example to prevent cross-contamination.
  • receptacles such as tubes, bottles, vessels, or bioreactors may be provided with e.g. a cover, a lid, a cork, a plug, a cap, or other suitable means for closing the receptacle.
  • solid supports and materials for the solid support may be contemplated.
  • Various synthetic polymers, bio compounds, microbead, cellulose-based material and natural polymers may be suitable. Examples of suitable materials may be e.g. the support materials described in WO 2013/072563 (p, 25, 1.12-p, 26, 1.19), which is herein incorporated in its entirety.
  • the present disclosure also provides a method.
  • the method may include immobilizing a composition comprising a phage, a gelling agent, and disaccharide, trisaccharide or a polysaccharide onto a nonwoven.
  • the composition immobilized on the nonwoven can be stored at room temperature for up to 180 days, with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% phages remain viable.
  • the composition immobilized on the nonwoven can be stored at room temperature for up to 75 days, with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% phages remain viable.
  • the composition immobilized on the nonwoven can be stored at room temperature for up to 30 days, with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% phages remain viable.
  • immobilizing the phage onto the nonwoven comprises drying a phage containing solution onto the nonwoven.
  • the drying may be done e.g. by air-drying, i.e. allowing die composition or phage containing solution to air dry.
  • the temperature and other conditions during the drying may vary, although the conditions may be selected such that the viability of the bacteriophage is not reduced or significantly reduced.
  • the drying may be done at room temperature, but lower or higher temperatures may also be used, depending e.g. on the bacteriophage.
  • Tire time period required for foe drying may depend e.g. on foe consistency of foe composition or matrix, its volume, the conditions etc.
  • foe phage containing solution can be air-dried at room temperature.
  • the method can include filtering the phage containing solution through the nonwoven before drying.
  • the method can include evenly distributing the phage on the nonwoven.
  • the method can include lyophilizing the phage on the nonwoven.
  • the method can further include detecting bacteria using the phage on the nonwoven.
  • the method can further include treating a wound infection using the phage on the nonwoven.
  • the method can further include using the immobilized phage for detection of microorganisms.
  • the microorganisms can be foodbome pathogens, such as, Salmonella, E. coli 0157, Cronobacter, Listeria, Listeria monocytogenes, Shiga-toxin producing E. coli (STEC) Campylobacter, Shigella, Bacillus, Staphylococcus, and others.
  • the phage is a reporter phage.
  • the reporter phage can be used in a detection assay for viable bacterial species in food matrices or water.
  • reporter phages have been engineered to include genes that produce a bio luminescence detection signal when used with the appropriate substrate in a phage infection assay of viable bacterial cells.
  • reporter phages have been engineered for luciferase and beta-galactosidase expression.
  • Reporter phages have also been engineered for fluorescence detection in a phage infection assay of viable bacterial cells.
  • phages have been engineered for green fluorescent protein expression.
  • the luc, lux AB. or luxCD ABE genes can be incorporated in an engineered reporter phage to provide a reporter phage that expresses luciferase on bacterial infection.
  • the lacZ gene can be incorporated in an engineered reporter phage to provide a reporter phage that expresses a beta-galactosidase enzyme on bacterial infection.
  • the gfp gene can be incorporated in an engineered reporter phage to provide a reporter phage that expresses the green fluorescent protein on bacterial infection.
  • Reporter phage can be genetically engineered to express the luceriferase NanoLuc.
  • NanoLuc luciferase is derived from the deep-sea shrimp Oplophorus gracilirostris.
  • the preparation of reporter phages engineered to express NanoLuc luciferase for E. coli detection has been described by Hinkley, et. al., Analyst, 2018, 143, pages 4974-4080; Zhang, et. al., Nature Scientific Reports, 2016, 6, 33235 (published online September 14, 2016 ; doi: 10.1038/srep33235); and Oda, et. al., Applied and Environmental Microbiology, 2004, 70(1), pages 527-534.
  • a luciferase reporter phage (LRP) for Listeria monocytogenes has been described by Loessner, et. al., Applied and Environmental Microbiology, 1996, 70(1), pages 1133- 1144.
  • a bioluminescent reporter phage for Salmonella thyphimurium has been described by Kim, et. al., Analytical Chemistry, 2014, 86(12), pages 5858-64; and in Korean patent KR101617166.
  • the phage immobilized on a nonwoven substrate is a reporter phage.
  • the phage immobilized on a nonwoven is a reporter phage engineered to include a gene that expresses an enzyme for a bioluminescent reaction.
  • the phage immobilized on a nonwoven is a reporter phage engineered to include a gene that produces a fluorescent signal.
  • the phage immobilized on a nonwoven is a reporter phage engineered to include a gene that expresses luciferase.
  • the reporter phage is a luciferase reporter phage (RP).
  • the engineered phage is a T7 phage engineered to express luciferase (T7-luc) or NanoLuc luciferase (T7-nluc).
  • the reporter phage is T7-nl (reporter phage with accession number MH651797) or T7-nlc (reporter phage with accession number MH651798).
  • a method for detecting a microorganism comprising a reporter phage immobilized on a nonwoven and an enzyme substrate.
  • the reporter phage is engineered to include a gene that expresses a luciferase and the enzyme substrate is a substrate for luciferase.
  • the enzyme substrate is luciferin.
  • the microorganism is a foodbome pathogen.
  • the microorganism is selected from Salmonella, E. coli, Cronobacter, and Listeria.
  • a method for detecting E. coli in a water sample comprising a reporter phage immobilized on a nonwoven and an enzyme substrate.
  • the reporter phage is engineered to include a gene that expresses a luciferase and the enzyme substrate is a substrate for luciferase.
  • the enzyme substrate is luciferin.
  • Soft LB Agar was prepared by adding tryptone (10 g), yeast extract (5 g), sodium chloride (5 g), magnesium chloride (1 g), and agar (7.5 g) to deionized water (1 L). The resulting product was autoclaved (121 °C for 15 minutes), separated into 50 mL aliquots, and stored as a solid at room temperature. Prior to use, the soft agar was melted using a microwave oven.
  • PEGYNaCl phage precipitating solution was prepared by adding PEG 8000 at a concentration of 20% (weight/volume) to a solution of NaCl (2.5 M) in deionized water. The solution was autoclaved (121 °C for 15 minutes) and stored in a sealed bottle at room temperature.
  • Pseudomonas aeruginosa was obtained from ATCC (Manassas, VA).
  • E.coli strain BL21 was obtained from New England Biolabs (Ipswich, MA).
  • Escherichia coli bacteriophage T7 (ATCC BAA-1025-B2) and Pseudomonas aeruginosa bacteriophage 2 (ATCC 14203-B1) were obtained from ATCC (Manassas, VA).
  • SM Buffer and Modified SM Buffer Stock solutions of NaCl (IM), Tris-HCl (IM), and magnesium sulfate (IM) were prepared using deionized water. The reagents were combined to provide a solution of NaCl (200 mM), Tris-HCl (10 mM, pH 7.4), and magnesium sulfate (1 mM). Gelatin (100 micrograms/mL) was added to prepare the SM buffer.
  • Modified SM buffer was prepared by adding sucrose, PEG 6000 and trehalose to the SM buffer so that the final concentrations were sucrose (0.5 mM), PEG 6000 (0.1 M), and trehalose (0.5 mM).
  • Nonwoven A was a drylaid, resin bonded PET ⁇ rayon fiber nonwoven sheet (basis weight of 42 gsm (grams per square meter)) obtained as Unfit 125 nonwoven from Midwest Filtration LLC (Cincinnati, OH).
  • Nonwoven B was a spunbonded ⁇ thermobonded polyethylene ⁇ polyester (PE ⁇ PET) bicomponent fiber nonwoven sheet (basis weight of 58 gsm) obtained as Unitherm 170 HK nonwoven from Midwest Filtration LLC (Cincinnati, OH).
  • PE ⁇ PET spunbonded ⁇ thermobonded polyethylene ⁇ polyester
  • Nonwoven C was a wetlaid, resin bonded polyester ⁇ cellulose fiber nonwoven sheet (basis weight of 46 gsm) obtained as Uniblend 135 nonwoven from Midwest Filtration LLC.
  • Nonwoven D was a wetlaid PET ⁇ cellulose fiber nonwoven sheet (basis weight of 50.9 gsm) obtained as Hydroguard 150 HEM nonwoven from Hanes Company (Conover, NC).
  • Nonwoven E was a spunbonded PET fiber nonwoven sheet (basis weight of 37 gsm) obtained as REEMAY 2100 nonwoven from Talas (Brooklyn, NY).
  • Nonwoven F was a melt-blown polypropylene fiber nonwoven sheet (basis weight of 55 gsm, effective fiber diameter of 8 micrometers, solidity of 6.3%, and thickness of 1 mm).
  • E. coli bacteriophage T7 and P. aeruginosa bacteriophage 2 were separately purified according to the following procedure.
  • the appropriate bacterial strain P. aeruginosa (ATCC 39327 ) or E.coli strain BL21
  • LB Luria-Bertani
  • the corresponding lyophilized phage powder was added to the bacterial culture (50 mL) and the mixture was incubated at 37 °C for 3 hours or until bacterial lysis.
  • the mixture was then centrifuged (6,000 x g) for 15 minutes and the supernatant was fdtered through a 0.45 micron filter.
  • the resulting filtrate was serially diluted (10-fold dilutions) and the dilution samples were assayed for vims titer. Each dilution sample (200 microliters) was added to 400 microliters of corresponding bacterial culture and the mixture was incubated at room temperature for 15 minutes. To each mixture, 3 mL of melted soft agar was added and the resulting mixture was poured onto a prepared LB agar plate. Each LB agar plate was incubated at 37 °C for 3 hours and the appearance of plaques indicated the presence of phages. The plaques in each plate were counted and the PFU (Plaque Forming Units) of the phage solution was calculated.
  • PFU Protein Forming Units
  • the phage filtrate was centrifuged (5000 rpm for 10 minutes) to pellet bacterial cells.
  • the supernatant solution was transferred to a new tube and then mixed with the PEG ⁇ NaCl phage precipitating solution (20% by volume) and held at 4 °C for 2 hours to precipitate the phage.
  • the resulting phage precipitate was recovered by centrifuging the sample (5000 rpm for 30 minutes), removing the supernatant, and then adding 2 mL of SM buffer.
  • the amplified phage solution was stored at 4 °C.
  • each nonwoven sheet (10 cm x 10 cm section) was placed flat in a separate 10 cm x 10 cm NUNC square bioassay dish (product #240835, Thermo Fisher Scientific, Waltham, MA).
  • Phage solution (10 mL selected from either E. coli bacteriophage T7 or P. aeruginosa bacteriophage 2 in modified SM buffer) was added to each bioassay dish. Each dish was then placed on an orbital shaker and shaken at 75 rpm for 1 hour. The resulting nonwoven sheets wetted with phage solution were hung from hooks and air-dried at room temperature for 48 hours to provide nonwoven sheets with adsorbed phage.
  • Nonwoven F a section of the nonwoven sheet (12 cm diameter) was punched and placed on top of the filter component of a 0.45 micrometer sterile bottle top filter unit (Coming Inc., Coming, NY). Phage solution in modified SM buffer (100 mL selected from either E. coli bacteriophage T7 or P. aeruginosa bacteriophage 2) was vacuum filtered through the nonwoven sheet and the filtrate was refiltered through the nonwoven. The resulting nonwoven sheet wetted with phage solution was hung from a hook and air-dried at room temperature for 48 hours to provide Nonwoven Sheet F with adsorbed phage.
  • modified SM buffer 100 mL selected from either E. coli bacteriophage T7 or P. aeruginosa bacteriophage 2
  • Viability determination of the phages adsorbed on the nonwoven sheets prepared in Example 1 was conducted using a zone inhibition assay. Determinations of phage viability were made at time points of 30 days, 75 days, and 180 days after sample preparation. During the study, the prepared nonwoven sheets were individually wrapped in aluminum foil, placed in a sealed plastic bag and stored at room temperature. At each timepoint, three discs (5 mm in diameter) were punched from different sections of each nonwoven sheet.
  • Bacterial culture 200 microliters, about 10 8 CFU/mL
  • a sterile tube was mixed with 5 mL of soft LB agar and then poured onto an LB agar plate. The plate was swirled to produce a uniform top layer.
  • the 3 nonwoven discs were transferred using sterile forceps to the surface of solidified agar.
  • Discs containing E. coli bacteriophage T7 were assayed using E. coli strain BL21 and discs containing P. aeruginosa bacteriophage 2 were assayed using P. aeruginosa (ATCC 39327). Plates containing nonwoven discs with E.
  • coli bacteriophage T7 phage were incubated for 3 hours at 37 °C. Plates containing nonwoven discs with P. aeruginosa bacteriophage 2 were incubated overnight at 37 °C. The plates were inspected by visual examination for the formation of cleared zones (plaques) of no bacterial growth around the perimeter of a disc. Discs in which a cleared zone was observed were determined to have viable phages. Discs in which a cleared zone was not observed were determined to not have viable phages. The results are reported in Tables 1-2.
  • a 5 cm x 5 cm section of nonwoven material (selected from Nonwovens A-F) is placed in a 50 mL lyophilization bottle.
  • a solution of modified SM buffer (1500 micro liters) and 500 microliters of a phage stock solution (5 x 10 7 PFU/mL) is prepared. The solution is soaked into the nonwoven material.
  • the lyophilization bottle is closed with a cap and the nonwoven sample is lyophilized using a VIRTIS Benchtop K Series freeze dryer (SP Industries, Warminster, PA). The sample holding shelves are cooled to 5 °C and the sample is precooled at -80 °C.
  • the shelves are cooled to -30 °C (at 1 °C/minute) and maintained at -30 °C for 90 minutes.
  • the complete solidification of the nonwoven sample occurs within 90 minutes at -30 °C.
  • Primary drying of the sample continues at -30 °C for 12 hours at 100 millitorr. Following the primary drying step, the temperature is increased from -30 °C to 25 °C (at 0.1 °C/minute) maintaining vacuum of 100 millitorr.
  • the nonwoven sample is removed from the freeze dryer, tightly sealed and stored at either 4, 25, 37, or 42 °C. Viability Testing of the sample is conducted as described in Example 2.
  • T7-luc, T7-nluc, T7-nl, or T7-nlc luciferase reporter phage
  • Kinetic bioluminescent measurements are taken using a luminescence plate reader (SpectraMax M5 Microplate Reader, Molecular Devices, LLC.). The presence or absence of S. Typhimurium in each well is determined by adjusting the measured luminescence signals (RLU) based on background.
  • RLU measured luminescence signals
  • Kinetic bioluminescent measurements are taken using a luminescence plate reader (SpectraMax M5 Microplate Reader, Molecular Devices, LLC). The presence or absence of /.. monocytogenes in each well is determined by adjusting the measured luminescence signals (RLU) based on background.
  • RLU measured luminescence signals
  • /.. monocytogenes cultures are grown in Demi-Fraser broth by shaking at 37 °C and 220 rpm for 16 hours. Cultures of /.. monocytogenes are enumerated to determine CFU/mL (colony forming units per mL). Twenty -five gram of a food sample such deli meat is weighed into a sterile filter bag (WHIRL-PAK Homogenizer Blender Filter Bag (710 mL), Nasco, Fort Atkins, WI) and 225 mL of Demi-Fraser broth is added. The sample is homogenized and is spiked with about 1- 5 CFU (low concentration) or 10 CFU (high concentration) and mixed.
  • a control sample (uninoculated) is similarly prepared.
  • the samples are incubated at 37 °C for 24-30 hours.
  • aliquots (1 mL) from each sample are added wells in a 96-well plate as a single sample per well.
  • Each well in the plate contains a nonwoven disc with reporter phage A51 1 ::luxAB adsorbed on the nonwoven.
  • the plate is incubated at 37 °C for 1-2 hours and then 100 microliters of luciferase substrate is added to each well.
  • Bioluminescent measurements are taken using a luminescence plate reader (SpectraMax M5 Microplate Reader, Molecular Devices, LLC). The presence or absence of L .

Abstract

L'invention concerne un article, un kit et un procédé. L'article comprend une composition comprenant un phage, un agent gélifiant ou un agent épaississant, et un disaccharide, un trisaccharide ou un polysaccharide ; et un non-tissé ; la composition étant adsorbée sur le non-tissé. Le kit comprend l'article de la présente invention ; et un support solide ; l'article étant incorporé dans un support solide. Le procédé comprend l'immobilisation d'une composition comprenant un phage, un agent gélifiant, et un disaccharide, un trisaccharide ou un polysaccharide sur un non-tissé ; lorsque le phage immobilisé sur le non-tissé est stocké à température ambiante pendant jusqu'à 180 jours, au moins 60 % de phages restent viables.
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