Composition including a metabolite produced by bacillus amyloliquefaciens possessing Anti-bacterial activity

Abstract

The present disclosure provides compositions comprising the cell-free supernatant of a specific isolate of Bacillus amyloliquefaciens. Methods of using these compositions to inhibit the growth of microorganisms are also provided. The methods may be utilized to inhibit the growth of a microorganism on a surface, in a space, or in a subject.

Description

[0001] COMPOSITION INCLUDING A METABOLITE PRODUCED BY BACILLUS

[0002] AM YLOLIQl El A( I ENS POSSESSING ANTI-BACTERIAL ACTIVITY

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 601,973, which was filed on November 22, 2023, the contents of which are incorporated by reference in their entireties.

[0005] BACKGROUND

[0006] Staphylococcus aureus is a Gram-positive, spherically shaped bacterium. It is an omnipresent member of the human microbiome that is frequently found in the nose or on the skin. At the same time, it is an opportunistic pathogen and is one of the leading causes of skin infections, such as abscesses, and life-threatening blood infections, such as sepsis and endocarditis. Livestock can be carriers of S. aureus and can also become infected by it. In dairy cows, this bacterium causes mastitis, and infections in chickens also cause problems. Treatment of S. aureus can be challenging as many strains of this bacterium have become resistant to traditional antibiotics. Dangerous multidrug resistant strains, such as methicillin resistant 5. aureus (MRSA), have become endemic in hospitals worldwide. Despite much research and development, no vaccine for S. aureus has been approved. Accordingly, there is a need in the art for novel methods for controlling S. aureus.

[0007] SUMMARY

[0008] In a first aspect, the present invention provides compositions comprising a cell-free supernatant from a Bacillus amyloliquefaciens bacterium designated MCR009 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B- 68466.

[0009] In a second aspect, the present invention provides methods of using the compositions described herein to inhibit the growth of a microorganism. In a first embodiment, the compositions are used to inhibit the growth of a microorganism on a surface. These methods comprise applying the composition to the surface. In a second embodiment, the compositions are used to inhibit the growth of a microorganism in a space. These methods comprise spraying the composition into the space. In a third embodiment, the compositions are used to inhibit the growth of a microorganism in or on a subject. These methods comprise administering the composition to the subject.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows the results of a pathogen overlay assay that was performed to evaluate the antimicrobial activity of eight Bacillus isolates (i.e., MCR002, MCR005, MCR006, MCR007, MCR009, MCR015, MCR040, and MCR041) against two E. coll isolates (i.e., E. coli isolate 021 and an avian pathogenic E. coli (APEC) isolate). In this assay, IxlO4CFU of E. coli was overlaid over a single Bacillus isolate colony and the zone of inhibition (ZOI) was measured. Notably, E. coli isolate 021 was evaluated against all tested Bacillus isolates, whereas the APEC isolate was only evaluated against MCR002 and MCR009. Data are presented as mean + / - standard error (n=9 plates per candidate per pathogen). Differing superscripts represent significant differences at P < 0.05.

[0012] FIG. 2 shows the results of a pathogen overlay assay that was performed to evaluate the antimicrobial activity of eight Bacillus isolates (i.e., MCR002, MCR005, MCR006, MCR007, MCR009, MCR015, MCR040, and MCR041) against three Staphylococcus aureus isolates (i.e., S. aureus ATCC 25923, S. aureus 004, and S. aureus FS1). In this assay, IxlO4CFU of S. aureus was overlaid over a single Bacillus isolate colony and the ZOI was measured. Data are presented as mean + / - standard error (n=9 plates per candidate per pathogen). Differing superscripts represent significant differences at P < 0.05.

[0013] FIG. 3 shows the results of a cell-free supernatant (CFS) assay that was performed to evaluate the antimicrobial activity of thirteen Bacillus isolates (i.e., MCR002, MCR005, MCR006, MCR007, MCR009, MCR014, MCR015, MCR021, MCR028, MCR031, MCR036, MCR039, and MCR040) against one S. aureus isolate (i.e., S. aureus isolate 004). In this assay, a liquid culture medium was inoculated with a single Bacillus isolate followed by S. aureus 004. Then, S. aureus growth was evaluated by measuring absorbance and percent inhibition was calculated relative to a S. aureus-ov y positive control. Data are presented as mean ± standard error (n=12 for MCR002, MCR005, MCR007, MCR014, MCR015, MCR031, MCR039, and MCR040; n=24 for MCR021 and MCR036; and n=36 for MCR006, MCR009, and MCR028). Negative values represent a reduction in absorbance as compared to the positive control. FIG. 4 shows the results of a CFS assay that was performed to evaluate the antimicrobial activity of MCR009 CFS that had been boiled for 5 minutes, frozen at -80°C and then thawed, or fdtered using either a 5 kDa fdter or 10 kDa filter against S. aureus isolate 004. In this assay, a liquid culture medium was inoculated with the boiled, frozen, or fdtered CFS followed by S. aureus 004. Then S. aureus 004 growth was evaluated by measuring absorbance and percent inhibition was calculated relative to a S. aureus-onVy positive control. Data are presented as mean ± standard error of the mean (n=12 replicate wells per condition). Note: 20% v / v Bacillus sp. CFS was utilized in this assay.

[0014] DETAILED DESCRIPTION

[0015] The present disclosure provides compositions comprising the cell-free supernatant of a specific isolate of Bacillus amyloliquefaciens . Methods of using these compositions to inhibit the growth of microorganisms are also provided.

[0016] As is described in the Examples, to identify new and useful bacteria, the present inventors isolated Bacillus spp. from samples collected from complex microbial environments. The antimicrobial activity of the Bacillus isolates was tested in vitro using a pathogen overlay assay and a cell-free supernatant (CFS) assay. In both assays, one Bacillus amyloliquefaciens isolate, referred to herein as MCR009, exhibited strong antimicrobial activity against Staphylococcus aureus. These data indicate that MCR009 secretes one or more antimicrobial agents that inhibit the growth of this opportunistic pathogen. Thus, compositions comprising the cell-free supernatant of MCR009, or the antimicrobial agent(s) present therein, offer a novel means for controlling the growth of microorganisms.

[0017] Compositions:

[0018] In a first aspect, the present invention provides compositions comprising a cell-free supernatant from a Bacillus amyloliquefaciens bacterium designated MCR009 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B- 68466.

[0019] The bacterium used to produce the compositions of the present invention is a Bacillus amyloliquefaciens isolate referred to herein as MCR009. MCR009 was isolated from a manure sample taken from a cattle farm. A “cell-free supernatant” is a liquid in which a bacterium was grown and then removed. Cell-free supernatant is produced by: (a) inoculating a medium with the bacterium, (b) culturing the inoculated medium, and (c) removing the bacterium from the medium. Cell-free supernatant comprises residual nutrients from the medium as well as substances (e.g., metabolites, growth factors, and extracellular matrix proteins) that were secreted by the bacterium prior to its removal.

[0020] As used herein, “inoculating” refers to a process in which a microorganism is introduced into a medium. Bacillus bacteria can exist in both a vegetative state and as a spore. Bacteria in the vegetative state are actively growing, reproducing, and secreting substances, whereas spores are dormant. Thus, in preferred embodiments, the MCR009 bacterium used to produce cell-free supernatant is inoculated into medium in a vegetative state to ensure that the supernatant comprises MCR009-secreted substances.

[0021] A “medium” or “culture medium” is a substance that provides the necessary nutrients (i.e., amino acids, carbohydrates, vitamins, minerals), growth factors, and / or hormones for a cell to grow. A medium may be a solid, liquid, or semi-solid substance. However, the medium used with the present invention (i.e., to produce a cell-free supernatant) should be a liquid. The medium used with the present invention may be any nutrient broth in which the MCR009 bacterium can grow. In the Examples, the inventors produced cell-free supernatant by culturing MCR009 in Tryptic Soy Broth (TSB). Thus, in some embodiments, the medium is TSB.

[0022] As used herein, “culturing” refers to a process in which microorganisms are grown in an artificial environment. Microorganisms are typically cultured in a vessel (e.g., a dish, flask, plate, or tube) comprising a medium. Other factors, such as the concentration of gases (e.g., CO2, O2), pH, osmotic pressure, and temperature may be manipulated in the culture environment. In the Examples, the inventors produced cell-free supernatant by culturing media inoculated with MCR009 for 15 hours at 37°C. Thus, in some embodiments, inoculated medium is cultured for 14-16 hours at 34-40°C. This incubation period allowed the bacterium in the culture to reach a concentration between IxlO7and IxlO9CFU / g. Thus, in some embodiments, the inoculated medium is cultured until the bacterium reaches a concentration between IxlO7and IxlO9CFU / g.

[0023] Bacterial cells may be removed from a medium via centrifugation and / or filtration. “Centrifugation” is a method of separating substances of different densities in a solution by spinning the solution around an axis at high speed. Centrifuging a vessel containing bacteria at an appropriate speed will cause the bacteria to be compacted into a pellet at the bottom of the vessel. Suitable centrifuge conditions for pelleting bacteria are known in the art and include spin speeds ranging from about 1,000 g to 12,000 g and times ranging from about 1 minute to 1 hour. For example, the inventors pelleted bacteria by centrifuging media at 3000 x g for 30 minutes. “Filtration” is a method of separating substances of different sizes by passing them through a porous material, i.e., a fdter. Substances that are larger than the pore size of fdter are retained by the filter whereas substances that are smaller than the pore size pass through the filter. Suitable filter pore sizes for removing bacteria range from about 0.2 m to about 0.45 pm. In the Examples, the inventors removed bacteria from media by pelleting the bacteria, decanting the supernatant, and then sterile filtering the supernatant twice using a 0.2 pm syringe filter. Thus, in some embodiments, bacterial cells are removed from a culture medium via a combination of multiple centrifugation steps and / or filtration steps.

[0024] In the Examples, the inventors demonstrate that the antimicrobial activity of the MCR009 cell-free supernatant against Staphylococcus aureus was dramatically reduced when molecules that are larger than 5 kDa were filtered out of the cell-free supernatant, suggesting that the agent responsible for antimicrobial activity of the cell-free supernatant is larger than 5 kDa. Thus, in some embodiments, the cell-free supernatant only comprises molecules that are larger than 5 kDa.

[0025] In some embodiments, the compositions are concentrated by drying or removing liquid from the composition. Drying can be performed, for example, to allow the composition to be sprayed into a space. As used herein, a “dried” composition is a composition from which some amount of water has been removed. Drying can be accomplished using any method known in the art including, without limitation, heat drying, dielectric drying, freeze drying (lyophilization), supercritical drying, and air drying.

[0026] The compositions of the present invention may comprise additional reagents. In some embodiments, the compositions further comprise a carrier. As used herein, the term “carrier” refers to a diluent, excipient, or propellant. In embodiments in which the composition is designed to be administered to a subject, the carrier may be a pharmaceutically acceptable carrier, i.e., a carrier that is not deleterious to a recipient to which it is administered. Examples of pharmaceutically acceptable carriers include diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizing agents (e.g., glycerol, polyethylene glycerol), emulsifiers, liposomes, nanoparticles, and adjuvants. In embodiments in which the composition is dried, the carrier may be a dry substance, such as a powder. Common propellants that can be used to spray the compositions into a space include, for example, compressed gas, propane, isobutane, and butane.

[0027] The compositions may further include additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking substances or tonicity modifiers (e.g., lactose, mannitol). Components of the compositions may be covalently attached to polymers (e.g., polyethylene glycol), complexed with metal ions, or incorporated into or onto particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels) or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. The compositions may also be formulated in lipophilic depots (e.g., fatty acids, waxes, oils) for controlled or sustained release.

[0028] Methods for inhibiting the growth of microorganisms:

[0029] In a second aspect, the present invention provides methods of using the compositions described herein to inhibit the growth of a microorganism.

[0030] A “microorganism” is a microscopic organism. Microorganisms include bacteria, viruses, and fungi. In the Examples, the inventors tested the antimicrobial activity of their compositions against several hatchery and poultry derived isolates of Staphylococcus aureus and Escherichia coli. Thus, in some embodiments, the microorganism is a strain of Staphylococcus aureus or Escherichia coli. In specific embodiments, the microorganism is a Staphylococcus aureus isolate selected from S. aureus ATCC 25923, S. aureus 004, and S. aureus FS1. Compositions comprising MCR009 cell-free supernatant were shown to have antimicrobial activity against S'. aureus 004. Thus, in preferred embodiments, the microorganism is S. aureus 004, which was isolated from a poultry hatchery.

[0031] In a first embodiment, the compositions are used to inhibit the growth of a microorganism on a surface. These methods comprise applying the composition to the surface. The compositions may be applied to the surface via spraying, fogging, misting, wiping, pouring, or a combination of these methods. Application tools such as wipes, mops, towels, sprayers, sponges, and scrubbers may be utilized. In these embodiments, the methods may further comprise pre-cleaning the surface (e.g., using soap and water) to remove any visible grime before the composition is applied.

[0032] The treated surface may be a surface found in a household, an industrial setting, or a commercial setting. Staphylococcus aureus is a leading cause of hospital-acquired infections. Thus, in some embodiments, the surface is in a healthcare facility (e.g., a clinic, hospital, nursing home, hospice, birth center, dentist office, etc.). Examples of surfaces found in healthcare facilities include, but are not limited to, bed rails, beside tabletops, chairs, call bells, door handles, light switches, sink taps, solution dispensers, blood pressure cuffs, crutches, IV poles, bedpans, computers, counters, pens, clipboards, and thermometers. Further, Staphylococcus aureus produces toxins that can cause food poisoning. Thus, in some embodiments, the surface is a surface of a food product or a food contact surface. Examples of food contact surfaces include, but are not limited to, food packaging, knives, blades, cutting boards, countertops, conveyor belts, and food processing machinery. Additionally, Staphylococcus aureus can cause disease in plants. Thus, in some embodiments, the surface is a surface of a plant. As used herein, the term “plant” includes whole plants and any portion of a plant including, without limitation, an embryo, pollen, ovule, flower, glume, panicle, root, root tip, tuber, anther, pistil, leaf, stem, seed, fruit, pod, calli, clump, cell, protoplast, germplasm, asexual propagate, or tissue culture.

[0033] In a second embodiment, the compositions are used to inhibit the growth of a microorganism in a space. These methods comprise spraying the composition into the space.

[0034] As used herein, the term “spraying” refers to the process of distributing a powder, liquid, or fine mist into a space. Spaying may be accomplished using a sprayer device and / or a propellant.

[0035] The treated space may be a room in a household, business, or public area. Livestock can be carriers of Staphylococcus aureus and can also be infected by it. Thus, in some embodiments, the space is a space where livestock are bom or raised, such as a barn, shelter, maternity pen, farrowing house, or hatch cabinet. In the Examples, the antimicrobial activity of the compositions was demonstrated against hatchery and poultry derived pathogens. Thus, in some embodiments, the space is a poultry hatch cabinet. A “hatch cabinet” or “egg incubator” is a machine that creates ideal conditions (i.e., temperature, humidity) for an egg to incubate and hatch; it mimics the conditions created by a broody hen. These conditions are also ideal for microbial proliferation and produce microbial blooms. Traditionally, formaldehyde has been used to control such microbial blooms. However, formaldehyde is a carcinogen and is known to damage the chick respiratory system, making chicks more susceptible to disease. The methods disclosed herein may be used as an alternative means to control the growth of microorganisms in the hatch cabinet environment and may allow the hatch cabinet to be treated with a reduced amount of formaldehyde as compared to conventional practices.

[0036] In a third embodiment, the compositions are used to inhibit the growth of a microorganism in or on a subject. These methods comprise administering the composition to the subject.

[0037] “Administering” refers to the introduction of a substance into or onto a subject's body. Methods of administration are well known in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraoral administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent.

[0038] The “subject” to which the methods are applied may an animal of any species. For example, in some embodiments, the subject is a livestock animal, such as a cow, horse, pig, rabbit, goat, sheep, chicken, turkey, duck, or goose. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected of having an infection caused by the microorganism. Staphylococcus aureus is the main causative agent of mastitis in cattle, sheep, goats, and horses. It also causes dermatitis in sheep and goats, botryomycosis in pigs and horses, and suppurative infections in cats and dogs. In humans, it can cause a variety of infections, including skin infections, such as abscesses, and life-threatening bloodstream infections, such as sepsis and endocarditis. Thus, in some embodiments, the methods are used to treat a Staphylococcus aureus infection in the subject.

[0039] In the methods of the present invention, the compositions may be applied to the surface, sprayed into the space, or administered to the subject once or multiple times. For example, the compositions may be applied, sprayed, or administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In the Examples, the inventors demonstrated that subjecting MCR009 cell-free supernatant to extreme temperatures does not reduce its antimicrobial activity against S. aureus 004. Specifically, the inventors showed that boiling MCR009 cell-free supernatant for five minutes or freezing it at -80°C (and then thawing it before use) did not reduce its antimicrobial activity. Thus, in some embodiments, the ability of the composition to inhibit the growth of the microorganism is maintained after the composition has been boiled or frozen.

[0040] Inhibited growth of a microorganism may be detected as reduced growth as compared to an untreated control. As used herein, an “untreated control” is a comparable surface (e.g., of the same material and size, in the same environment), space (e.g., of the same size, having the same temperature, humidity, and contents), or subject (e.g., of the same species, sex, age) that was not treated with a composition described herein. The most common way to assess microbial growth is to measure of the optical density of a solution comprising the microorganism at 600 nm (ODeoo). Other microbial growth assays, such as sequencing-based assays, agar overlay assays, cell-free supernatant inhibition assays, and assays that measure microbial metabolism may also be utilized to detect growth inhibition. Notably, in the Examples, the inventors demonstrate that treatment with MCR009 cell-free supernatant results in 100% inhibition of S. aureus 004 using a cell-free supernatant inhibition assay.

[0041] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0042] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0043] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or descriptions found in the cited references.

[0044] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0045] EXAMPLES In the following example, the inventors describe the isolation and characterization of the Bacillus amyloliquefaciens isolate MCR009.

[0046] Results:

[0047] Bacterial isolation

[0048] Bacteria live in complex polymicrobial communities and are constantly competing for resources. Thus, environments with high microbial loads promote the evolution of bacteria with unique mechanisms for acquiring nutrients. Accordingly, to identify novel Bacillus species with interesting properties, samples were collected from several such environments, including compost, poultry litter, and a cattle farm. The samples were desiccated in a drying oven to remove moisture and to promote sporulation of Bacillus sp. present in the sample. Then, individual bacteria were purified via isolation streaking.

[0049] Overlay assay

[0050] A series of overlay assays were performed to evaluate the antimicrobial activity of Bacillus isolates against select hatchery and poultry derived pathogens. In these assays, a bacterial pathogen is overlaid over a single Bacillus isolate colony and the zone of inhibition (ZOI) surrounding the Bacillus isolate colony is measured after incubation. The results of these assays are shown for eight Bacillus isolates (i.e., MCR002, MCR005, MCR006, MCR007, MCR009, MCR015, MCR040, and MCR041) in FIG. 1 and FIG. 2.

[0051] FIG. 1 shows the results of a pathogen overlay assay against two Escherichia coli isolates, i.e., E. coli isolate 021 and an avian pathogenic E. coli (APEC) isolate. In this assay, Bacillus isolate MCR002 produced a ZOI of more than 2 mm for both evaluated E. coli isolates, which was similar to or larger than the ZOI produced by the other tested Bacillus isolates. MCR009 did not produce a ZOI when evaluated against E. coli isolate 021, but it did produce a ZOI of nearly 1 mm when evaluated against the APEC isolate.

[0052] FIG. 2 shows the results of a pathogen overlay assay against three Staphylococcus aureus isolates, i.e., S. aureus ATCC 25923, . aureus 004, and S. aureus FS1. In this assay, Bacillus isolate MCR009 produced the largest ZOI against the 5. aureus isolates with one exception: for S. aureus 004, there was no significant difference between the ZOIs produced by MCR009 and MCR002. In 70.8% of the Bacillus isolate-5. aureus combinations, MCR009 resulted in a significantly larger ZOI. Cell-free supernatant assay

[0053] Additionally, a cell-free supernatant (CFS) assay was performed to evaluate the antimicrobial activity of the CFS of Bacillus isolates against S. aureus isolate 004. The results of this assay for thirteen Bacillus isolates (i.e., MCR002, MCR005, MCR006, MCR007, MCR009, MCR014, MCR015, MCR021, MCR028, MCR031, MCR036, MCR039, and MCR040) are shown in FIG. 3. In this assay, Bacillus isolate MCR009 was the only isolate that showed near complete inhibition of S. aureus growth.

[0054] To provide clues regarding the identity of the unknown antimicrobial agent produced by MCR009, the antimicrobial activity of MCR009 CFS that had been boiled, frozen, or filtered was tested against S. aureus 004 using the CFS assay. Specifically, MCR009 CFS that had been (a) boiled for 5 minutes, (b) frozen at -80°C and then thawed, or (c) filtered using either a 5 kDa filter (i.e., a filter that removes substances larger than 5 kDa) or a 10 kDa filter (i.e., a filter that removes substances larger than 10 kDa) was tested. The results of these assays are shown in FIG. 4. These results reveal that boiling or freezing MCR009 CFS does not reduce its ability to inhibit the growth of S. aureus 004, whereas removing substances larger than 5 kDa from MCR009 CFS dramatically reduces this ability.

[0055] Conclusions: Bacillus isolate MCR009, which was isolated from a cattle farm, was identified as a strain of Bacillus amyloliquefaciens via API, i.e., a kit that identifies bacteria based on their ability to ferment various carbohydrates. As is demonstrated by the data described above, MCR009 caused modest inhibition of avian pathogenic E. coli (APEC) and substantial inhibition of three S. aureus isolates (i.e., ATCC 25923, 004, and FS1) in a pathogen overlay assay. Further, it completely inhibited the growth of one poultry hatchery-associated S. aureus isolate (i.e., 004) in a CFS assay. These data suggest that MCR009 secretes an antimicrobial agent that controls the growth of S. aureus. This antimicrobial agent may prove to be useful for various applications, including applications in both the poultry industry and human medicine.

[0056] Accordingly, in future work, the antimicrobial activity of MCR009 CFS will be tested against Staphylococcus sp. isolated from various sources, including multidrug resistant strains. Additionally, efforts will be made to characterize and ultimately identify the antimicrobial agent(s) secreted by MCR009. Identification of an antimicrobial agent produced by MCR009 may allow the agent to be produced synthetically.

[0057] Materials and Methods:

[0058] Isolation streaking

[0059] A portion of each desiccated sample was diluted, plated on Tryptic Soy Agar (TSA) via the spread plate technique, and pasteurized via water bath. After incubation, individual colonies were selected based on colony morphology and transferred to a second TSA plate via isolation streak. From this initial isolation streak, two more subsequent isolation streaks were performed to further purify each bacterium. From the third isolation streak, individual colonies were selected from plates that exhibited a single colony morphology and were transferred to a culture tube containing Tryptic Soy Broth (TSB). After incubation, purity was evaluated via another isolation streak to confirm that the cultures remained pure. Aliquots of each bacterium were stored at -80°C and were utilized for in vitro testing as needed.

[0060] Overlay assay

[0061] The in vitro antimicrobial activity of 43 Bacillus spp. isolates was evaluated using an overlay assay. In this assay, an opportunistic bacterial pathogen is overlayed over a single Bacillus sp. colony and the zone of inhibition surrounding the Bacillus colony is measured following incubation. Specifically, 10 pL of an overnight culture (~108CFU / mL) of each Bacillus candidate was pipetted in the center of a Tryptic Soy Agar (TSA) plate (n=3 replicate plates / Bacillus candidate / pathogen) and the plates were incubated aerobically at 37°C for 15 hours. Then, individual Bacillus sp. colonies were overlaid with Tryptic Soy Soft Agar (TSSA) containing a single pathogen at ~106CFU / mL and incubated aerobically at 37°C for another 15 hours.

[0062] The TSSA was prepared and added to borosilicate glass tubes in 5 mL increments and stored at 4°C. Immediately prior to conducting the overlay assay, TSSA was liquified via autoclaving for 5 minutes and placed in a hot water bath held at 80°C to prevent solidification of the agar prior to pathogen inoculation. As needed, TSSA tubes were removed from the hot water bath and allowed to cool to 50-60°C before being inoculated with the pathogen of interest. Based on previous studies, this short duration exposure to these temperatures does not affect pathogen viability. To seed the TSSA overlay media, 100 pL of each pathogen was added to the TSSA tubes and was mixed gently before application. For E. coli. 30 pL of 25 mg / mL novobiocin (Sigma Catalog # N1628) was added to the TSSA to slow Bacillus growth so that E. coll could grow sufficiently. Once the TSSA was inoculated, it was carefully poured onto the edge of the agar surface and the plate was gently moved in a circular motion on a flat surface to ensure that inoculated TSSA sufficiently covered the Bacillus colony on the TSA plate without damaging the physical integrity of the colony itself.

[0063] The zone of inhibition (ZOI) was measured using a dial caliper. Specifically, both the diameter of the Bacillus colony and the diameter of the zone of clearing (which includes the diameter of the Bacillus colony) were measured, and the ZOI was calculated by subtracting the diameter of the Bacillus colony from the diameter of the zone of clearing (ZOI = diameter of clearing - colony diameter). Cell-free supernatant assay

[0064] Additionally, the in vitro antimicrobial activity of Bacillus spp. isolates was evaluated against S. aureus 004 using a cell-free supernatant (CFS) assay (Fernandez et al., 2019) that was modified in several ways. Specifically, growth temperatures and incubation durations were modified to align with pre-determined optimal growth conditions for the evaluated Bacillus spp. and pathogens. Further, preliminary tests indicated that a single filtration step did not adequately sterilize the Bacillus spp. supernatants, so a second filtration step was utilized. The inventors also performed similar assays using two additional bacteria (E. coli 021, E. faecalis).

[0065] To perform this assay, a 1 mL frozen aliquot of each Bacillus sp. was thawed and added to 9 mL of Tryptic Soy Broth (TSB) and incubated for 15 hours at 37°C. The cultures were centrifuged at 3000 x g for 30 minutes at 4°C. The supernatant was decanted and sterile filtered 2x using a 0.2 pm syringe filter, replacing the filter between each filtration. The CFS assay was conducted in a flat bottom 96-well plate. Wells containing only 300 pL sterile TSB were used as negative controls, and wells containing 297 pL TSB and 3 pL of 104CFU / mL (~103CFU / well) S. aureus 004 (1% v / v) were used as positive controls. The remaining wells were loaded with TSB that was first inoculated with fresh sterile-filtered CFS from a single Bacillus sp. culture at 10% (30uL) or 20% (60uL) v / v and then inoculated at 1% v / v with IxlO4CFU / mL S', aureus 004. Plates were incubated in aerobic conditions for 15 hours at 37°C while being agitated gently on a shaker.

[0066] Post-incubation, pathogen growth was evaluated by measuring absorbance at 450 nm (Synergy HT, multimode microplate reader, BioTek Instruments, Inc., VT). Percent inhibition was determined by calculating the corrected absorbance for each treated well (i.e., by subtracting the absorbance of the negative control wells), dividing the corrected absorbance of the CFS- treated wells by the corrected absorbance of the positive control wells, inverting by subtracting from one, and multiplying by 100, as detailed in the equations below. After calculating the percent inhibition for each CFS well, the average for each treatment was calculated. Negative values represent a reduction in absorbance as compared to the positive control.

[0067] - Cabs - corrected absorbance value

[0068] Cabs = abs - NCabs - abs - raw absorbance value

[0069] - NCabs - raw negative control absorbance value

[0070] % inhibition = (Cabsl / CabsPC)-l)*100 - Cabsl - corrected absorbance value of

[0071] CFS-treated well

[0072] CabsPC - corrected absorbance value of positive control well

[0073] References:

[0074] Fernandez, S. M., M. Cretenet, and M. Bernardeau. 2019. In vitro inhibition of avian pathogenic Enterococcus cecorum isolates by probiotic Bacillus strains. Poultry Science 98:2338- 2364.

[0075] DEPOSIT INFORMATION

[0076] A deposit of the University of Arkansas Division of Agriculture proprietary Bacillus amyloliquefaciens strain designated as MCR009, which is disclosed above and recited in the appended claims, has been made with the Agricultural Research Service Culture Collection (NRRL), 1815 N. University Street, Peoria, IL 61604, and has been accepted under the terms of the Budapest Treaty. The date of deposit was November 6, 2024. The deposit comprises agar plates, which were found viable on November 13, 2024. All restrictions will be irrevocably removed upon granting of a patent, and the deposit is intended to meet all the requirements of 37 C.F.R. §§1.801-1.809. The NRRL accession number is NRRL B-68466. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.

Claims

CLAIMSWhat is claimed:

1. A composition comprising a cell-free supernatant from a Bacillus amyloliquefaciens bacterium designated MCR009 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B-68466.

2. The composition of claim 1, wherein the cell-free supernatant was produced by: a) inoculating a medium with the bacterium; b) culturing the inoculated medium; and c) removing the bacterium from the medium to produce the cell-free supernatant.

3. The composition of claim 2, wherein the medium is Tryptic Soy Broth (TSB).

4. The composition of claim 2 or 3, wherein the bacterium is in a vegetative state in step (a).

5. The composition of any one of claims 2-4, wherein the inoculated medium is cultured until the bacterium reaches a concentration between IxlO7and IxlO9CFU / g in step (b).

6. The composition of any one of claims 2-5, wherein the inoculated medium is cultured for 14- 16 hours at 34-40°C in step (b).

7. The composition of any one of claims 2-6, wherein the bacterium is removed from the medium via centrifugation and / or fdtration in step (c).

8. The composition of any one of the preceding claims, wherein the cell-free supernatant only comprises molecules that are larger than 5 kDa.

9. The composition of any one of the preceding claims, further comprising a carrier.

10. A method for inhibiting the growth of a microorganism on a surface by applying the composition of any one of claims 1-9 to the surface.

11. The method of claim 10, wherein the surface is in a healthcare facility.

12. The method of claim 10, wherein the surface is a surface of a food product or a plant.

13. A method for inhibiting the growth of a microorganism in a space by spraying the composition of any one of claims 1-9 into the space.

14. The method of claim 13, wherein the space is a poultry hatch cabinet.

15. A method for inhibiting the growth of a microorganism in a subject by administering the composition of any one of claims 1-9 to the subject.

16. The method of claim 15, wherein the subject is a human, a cow, or a chicken.

17. The method of claim 15 or 16, wherein the subject has or is suspected of having an infection caused by the microorganism.

18. The method of any one of claims 10-17, wherein the composition is applied to the surface, sprayed into the space, or administered to the subject multiple times.

19. The method of any one of claims 10-18, wherein the microorganism is Staphylococcus aureus.

20. The method of claim 19, wherein the microorganism is a Staphylococcus aureus isolate selected from S. aureus ATCC 25923, S. aureus 004, and S. aureus FS1.

21. The method of any one of claims 10-20, wherein the ability of the composition to inhibit the growth of the microorganism is maintained after the composition has been boiled or frozen.

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