Bacillus amyloliquefaciens applied during the hatching phase to control relevant opportunistic pathogens

Abstract

The present invention provides compositions comprising one or two specific isolates 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]BACILLUS AMYLOLIQUEFACIENS APPLIED DURING THE HATCHING PHASE TO CONTROL RELEVANT OPPORTUNISTIC PATHOGENS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63 / 601,979, which was filed on November 22, 2023, the contents of which are incorporated by reference in their entireties. BACKGROUND In commercial poultry operations, eggs are promptly removed from the hen and transferred to a hatchery. As a result, the eggs have limited exposure to the hen’s microbiota and the gastrointestinal tracts of neonatal chicks are instead colonized by the first microorganisms that they encounter in the hatch cabinet. The hatch cabinet provides optimal conditions for microbial proliferation, and opportunistic pathogens flourish in this environment. Formaldehyde fumigation has been utilized to control the microbial bloom that occurs in commercial-scale hatch cabinets for over 100 years. Although this method is effective, formaldehyde is non- selective and acts on both pathogenic and beneficial microorganisms. Furthermore, formaldehyde is a known carcinogen and has been shown to damage the respiratory tract of hatching neonatal chicks, leaving them susceptible to colonization by the predominant microorganism present in the hatch cabinet. Accordingly, there is a need in the art for alternative methods for controlling the growth of pathogenic microorganisms in the hatch cabinet. SUMMARY In a first aspect, the present invention provides compositions comprising a bacterium. The bacterium is selected from: (a) a Bacillus amyloliquefaciens strain designated as MCR002 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B-68465, (b) a Bacillus amyloliquefaciens strain designated as MCR009 and deposited at the NRRL under accession number NRRL B-68466, or (c) a combination of MCR002 and MCR009. The compositions of the present invention are probiotic compositions in that they can be used to introduce beneficial Bacillus bacteria and are antimicrobial compositions in that they can be used to inhibit the growth of deleterious bacteria. 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 subject. These methods comprise administering the composition to the subject. In a third embodiment, the compositions are used to inhibit the growth of a microorganism in a space. These methods comprise spraying the composition into the space. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 and FIG.2 show the effects of Bacillus-based probiotics on Bacillus spp. recovery from the hatching environment. Chicken eggs were challenged with a pathogen mixture (PM), and a ground solid-state fermentate produced using the Bacillus isolate MCR002, the Bacillus isolate MCR009, or a combination thereof was applied to the hatch cabinet via compressed air at four time points during hatching. Bacillus spp. recovery from the hatching environment was assessed at four timepoints during the hatching phase: day 20 of embryogenesis (DOE20) at 8:00 am (~20% pip), DOE20 at 2:00 pm (~50% pip), DOE20 at 5:00 pm (~80% pip), and DOE21 at 7:00 am (~100% hatch). Results are compared to those generated with a non- challenged, non-treated control (NC), a challenged, non-treated control (PM), and a formaldehyde fumigated control (PM + F). This experiment was performed twice. FIG.1 shows the results of the first trial, and FIG.2 shows the results of second trial. FIG.3 and FIG.4 show the effects of Bacillus-based probiotics on cumulative bacterial recovery from the hatching environment. Chicken eggs were challenged with a pathogen mixture (PM), and a ground solid-state fermentate produced using MCR002, MCR009, or a combination thereof was applied to the hatch cabinet as described above. The cumulative recovery of Gram- negative bacteria, Enterococcus spp., and Staphylococcus aureus was assessed at four time points during hatching (i.e., T20, T50, T80, and T100). The graphs show the average recovery across all these bacterial populations and time points for each treatment type (i.e., averages at each time point were combined to reflect “cumulative” recovery across all four time points). Results are compared to those generated with a non-challenged, non-treated control (NC), a challenged, non-treated control (PM), and a formaldehyde fumigated control (PM + F). This experiment was performed twice. FIG.3 shows the results of the first trial, and FIG.4 shows the results of the second trial. DETAILED DESCRIPTION The present invention provides compositions comprising specific isolates of Bacillus amyloliquefaciens. Methods of using these compositions to inhibit the growth of microorganisms are also provided. As is described in the Examples, the present inventors have isolated two stains of Bacillus amyloliquefaciens, referred to herein as MCR002 and MCR009, that exhibit antimicrobial activity against opportunistic pathogens found in the chicken hatching environment. The inventors demonstrate that these Bacillus strains can be used to reduce the load of pathogenic microorganisms in commercial poultry hatch cabinets and to shift enteric colonization in neonatal chicks without negatively affecting early growth performance. Importantly, use of these Bacillus strains could allow commercial hatcheries to move away from the use of formaldehyde fumigation, which would (1) reduce human exposure to a known carcinogen, and (2) prevent damage to the chick tracheal epithelium and thereby mitigate disease caused by opportunistic pathogens. Bacillus compositions: In a first aspect, the present invention provides compositions comprising a bacterium. The bacterium is selected from: (a) a Bacillus amyloliquefaciens strain designated as MCR002 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B-68465, (b) a Bacillus amyloliquefaciens strain designated as MCR009 and deposited at the NRRL under accession number NRRL B-68466, or (c) a combination of MCR002 and MCR009. The compositions of the present invention are probiotic compositions in that they can be used to introduce beneficial Bacillus bacteria and are antimicrobial compositions in that they can be used to inhibit the growth of deleterious bacteria. MCR002 and MCR009 are two strains of Bacillus amyloliquefaciens that the inventors isolated from environmental samples. Specifically, MCR002 was isolated from a sample taken from the surface of a compost pile, and MCR009 was isolated from a manure sample taken from a cattle farm. The inventors selected these Bacillus strains for use in poultry hatch cabinets based on their antimicrobial activity against hatchery-associated opportunistic pathogens in in vitro screening assays. The compositions of the present invention may comprise one or both of these Bacillus strains. The compositions of the present invention were initially designed to be sprayed into commercial poultry hatch cabinets to control the microbial bloom that occurs during hatching. For this application, it is important that the bacteria in the composition are in spore form, as this (1) allows the bacteria to withstand extreme conditions and extends shelf-life, and (2) avoids introducing additional moisture (which promotes bacterial growth) into the hatch cabinet. As used herein, the term “spore” refers to the dormant form of a bacterium. Bacterial spores are small oval or spherical structures that are highly resistant to adverse environmental conditions such as high temperatures, radiation, desiccation, and chemical agents. Thus, in preferred embodiments, the bacterium in the composition comprises or consists of spores. The Bacillus composition that was tested in the Examples comprised a fermentation product produced using MCR002, a fermentation product produced using MCR009, or a combination of both of these fermentation products. Thus, in some embodiments, the bacterium is included in the composition as part of a fermentation product. A “fermentation product” or “fermentate” is a product produced by culturing a microorganism in a fermentation medium, thereby allowing the microorganism to break down nutrients in the fermentation medium into simpler compounds. The “fermentation medium” can be any substance that provides the necessary nutrients for the microorganism to grow. A typical fermentation medium includes a carbon source, a nitrogen source, salts, water, and micronutrients. A fermentation medium may be a solid, liquid, or semi-solid substance. “Solid-state fermentation” (SSF) is a process in which a microorganism is grown on a solid fermentation medium in the absence of free liquid. The fermentation medium used in this process is referred to as a “solid-state fermentation (SSF) medium”, and the product produced by this process is referred to as a “solid-state fermentation (SSF) product”. In the Examples, the inventors produced SSF products by inoculating a SSF medium with either MCR002 or MCR009 and culturing the inoculated SSF medium. As used herein, “inoculating” refers to a process in which a microorganism is introduced into a medium. Thus, in some embodiments, the fermentation product is an SSF product. As is detailed in Table 4, the SSF medium utilized by the inventors comprises wheat bran, rice hulls, and a dry mix. The dry mix provides essential nutrients, i.e., substances that must be obtained through the diet. It includes trace minerals, yeast extract, and a starch. Thus, in some embodiments, the SSF medium used to produce the SSF product comprises one or more of these ingredients. 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 Examples, the inventors cultured Bacillus-inoculated SSF medium for 18 hours at 37°C to produce their SSF products. Thus, in some embodiments, inoculated medium is cultured for 17-19 hours at 34-40°C to produce the fermentation product. In the Examples, the fermentation products were dried to induce bacterial sporulation, eliminate vegetative bacterial cells, prevent contamination, and allow the fermentation product to be powderized. Specifically, the inventors dried their fermentation product for 3 days at 65°C in a drying oven. Thus, in some embodiments, the composition is dried. 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. Additionally, to allow it to be sprayed into a hatch cabinet, the inventors ground their dried fermentation product into a fine powder. Thus, in some embodiments, the composition is a powder. As used herein, the term “powder” refers to a bulk solid composed of many fine, dry particles. In the Examples, the inventors used a coffee grinder to powderize their dried fermentation product. However, any means of powderizing a substance may be utilized, including grinding, crushing, and disintegration. For efficient spray application (i.e., to avoid clumping and unequal distribution), the particle size of the powder should be 400 microns or smaller. The bacterium may be included in the composition at any concentration that is appropriate for the intended application. For example, the concentration of the bacterium may be at least 1x107CFU per gram of composition, at least 1x108CFU per gram of composition, at least 1x109CFU per gram of composition, at least 1x1010CFU per gram of composition, at least 1x1011CFU per gram of composition, at least 1x1012CFU per gram of composition, or higher. 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 (e.g., microcrystalline cellulose). Common propellants that can be used to spray the compositions into a space include, for example, compressed gas, propane, isobutane, and butane. 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. Methods for inhibiting the growth of microorganisms: In a second aspect, the present invention provides methods of using the compositions described herein to inhibit the growth of a microorganism. A “microorganism” is a microscopic organism. Microorganisms include bacteria, viruses, and fungi. The most problematic microorganisms that thrive in the poultry hatching environment are pathogenic bacteria, i.e., bacteria that can cause disease. Thus, in preferred embodiments, the microorganism is a pathogenic bacterium. Examples of pathogenic bacteria that are commonly found in the poultry hatching environment include Escherichia coli (e.g., avian pathogenic Escherichia coli), Salmonella enterica, Citrobacter spp., Enterobacter spp., Klebsiella spp., Staphylococcus aureus, Staphylococcus chromogenes, Psuedomonas aeruginosa, and Enterococcus spp.. In the Examples, the inventors assessed the effects of their Bacillus compositions on the presence of Gram-negative bacteria, Enterococcus spp., and Staphylococcus aureus in the hatch cabinet and in the gastrointestinal tracts of chicks following hatch. Thus, in some embodiments, the microorganism inhibited by the methods is a Gram-negative bacterium, an Enterococcus spp., or Staphylococcus aureus. 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), subject (e.g., of the same species, sex, age), or space (e.g., of the same size, having the same temperature, humidity, and contents) 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 (OD600). 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. 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. 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. The treated surface may be a surface found in a household, an industrial setting, or a commercial setting. In the Examples the surface is a hatchery surface where eggs are incubated and are allowed to hatch. 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. In a second embodiment, the compositions are used to inhibit the growth of a microorganism in a subject. These methods comprise administering the composition to the subject, i.e., as a probiotic treatment. 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. “Administering” refers to the introduction of a substance into 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. In a third embodiment, the compositions are used to inhibit the growth of a microorganism in a space. These methods comprise spraying the composition into the space. 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. The treated space may be a room in a household, business, or public area. In some embodiments, the space is a space where livestock are born or raised, such as a barn, shelter, maternity pen, farrowing house, or hatch cabinet. The compositions of the present invention were designed to be sprayed into commercial poultry hatch cabinets to control the microbial bloom that occurs during hatching. Thus, in preferred 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 fumigation has been utilized to control the microbial bloom in commercial poultry hatch cabinets. In this method, formaldehyde is typically applied via drip application of 6 mL of formalin (18.5% formaldehyde solution) every 3 hours. Fumigation is typically initiated immediately after eggs have been transferred into the hatch cabinet (i.e., at 18 days of embryogenesis) and is typically ceased about 12 hours prior to the removal of chicks from the hatch cabinet. While this method is effective for controlling microbial growth, formaldehyde is a carcinogen and is known to damage the chick respiratory system, making chicks more susceptible to disease. The methods of the present invention were designed to be used in hatch cabinets as an alternative to formaldehyde fumigation. Thus, in some embodiments in which the compositions are sprayed into a hatch cabinet, the methods allow the hatch cabinet to be treated with a reduced amount of formaldehyde as compared to conventional practices. In preferred embodiments, the hatch cabinet is not treated with formaldehyde. In the Examples, the inventors demonstrate that administration of their Bacillus compositions into a hatch cabinet shifts the composition of microorganisms in the hatch cabinet. Specifically, they show that this treatment increases the amount of Bacillus spp. and reduces the amounts of pathogenic bacteria in the hatch cabinet. Thus, in some embodiments, the methods increase the amount of Bacillus spp. bacteria in the hatch cabinet as compared to an untreated control hatch cabinet and / or decrease the amounts of pathogenic bacteria in the hatch cabinet as compared to an untreated control hatch cabinet. As used herein, an “untreated control hatch cabinet” is a hatch cabinet that is comparable to the hatch cabinet treated with the Bacillus composition (e.g., same size, shape, etc.) and operated under comparable conditions (e.g., same temperature, humidity, etc.) but is not treated with a Bacillus composition described herein. The microorganisms present in a hatch cabinet can be assessed by enumerating or sequencing the bacteria present in hatch cabinet samples. Examples of suitable hatch cabinet samples include samples collected using air sampling equipment, open agar plates that were exposed to the hatch cabinet environment for some time (e.g., 1-5 minutes), hatch basket swabs, and fluff / dander samples. The terms “fluff” and “dander” are used interchangeably herein to refer to any down or small particulate matter than may have accumulated in the hatch cabinet environment during the hatching phase. Development of a healthy gut microbiome is a crucial for the health and performance of livestock animals. In the Examples, the inventors demonstrate that administration of their Bacillus compositions into a hatch cabinet shifts the composition of microorganisms in the gastrointestinal tracts of chicks hatched therein. Specifically, they show that this treatment increases the amount of Bacillus spp. and decreases the amounts of pathogenic bacteria in the gastrointestinal tracts of chicks. Thus, in some embodiments, chicks hatched in the hatch cabinet have an increased amount of Bacillus spp. bacteria in their gastrointestinal tract at hatch as compared to control chicks hatched in an untreated control hatch cabinet and / or decreased amounts of pathogenic bacteria in their gastrointestinal tract at hatch as compared to control chicks hatched in an untreated control hatch cabinet. As used herein, the term “control chick” refers to a comparable chick (e.g., of the same breed, sex, and age) that was hatched in an untreated control hatch cabinet. The microorganisms present in the gastrointestinal tract of a chick can be assessed by collecting chick droppings or gastrointestinal tract samples and enumerating the bacteria present therein on various culture media. Alternatively, the microorganisms present in the gastrointestinal tract of a chick can be assessed via metagenomic sequencing. In the Examples, the inventors demonstrate that hatching chicks in a Bacillus-treated hatch cabinet does not negatively affect their growth performance at 14 days post-hatch. Growth performance may be assessed in terms of body weight gain, i.e., the difference between an animal’s final weight and initial weight. Thus, in some embodiments, the body weight gain of chicks hatched in the hatch cabinet is not significantly lower than the body weight gain of control chicks hatched in an untreated control hatch cabinet. In the disclosed methods, the compositions may be applied to the surface, administered to the subject, or sprayed into the space 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 sprayed their Bacillus compositions into hatch cabinets at four timepoints during chick hatching that correlate with chick hatching milestones, i.e., 20% pip, 50% pip, 80% pip, and 100% hatch. Hatching begins around day 20 of embryogenesis when the chick cracks the eggshell, forming a “pipped” egg, and ends on day 21 of embryogenesis when the chick has left the egg. The term “percent pip” refers to the percentage of eggs in a hatch cabinet that have been cracked and the term “percent hatch” refers to the percentage of eggs in a hatch cabinet from which chicks have fully emerged. Thus, in some embodiments, the composition is sprayed into a hatch cabinet at one or more of the hatching milestones listed above. The compositions may be applied, administered, or sprayed using any carrier that is suitable for the intended application. In the Examples, the Bacillus compositions were dried and powderized and were sprayed using compressed air to spray them into a hatch cabinet. Thus, in some embodiments, the composition is sprayed via compressed air (i.e., air that has been compressed to a pressure higher than atmospheric pressure). The methods of the present invention can be used in combination with other methods for controlling microorganisms. In some embodiments, the methods further comprise applying, administering, or spraying at least one additional composition to the surface, subject, or space. The additional composition may be a probiotic composition that is used to introduce beneficial microorganisms, an antimicrobial composition used to inhibit the growth of deleterious microorganisms, or both. The additional composition may comprise one or more living microorganisms or may comprise a cell-free supernatant produced using a microorganism. The additional composition may be applied, administered, or sprayed using the same method used for the Bacillus composition or using a different method. For example, to control a microorganism in a poultry hatching environment, the additional composition may be sprayed into the hatch cabinet, applied to the surface of eggs, injected into eggs, or administered directly to chicks after they have hatched. 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. 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. 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. 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. EXAMPLES In the following example, the inventors describe the isolation of two stains of Bacillus amyloliquefaciens, referred to herein as MCR002 and MCR009, that exhibit antimicrobial activity against opportunistic pathogens found in the chicken hatching environment. They demonstrate that these Bacillus strains can be used to reduce the load of pathogenic microorganisms in commercial hatch cabinets and to shift enteric colonization in neonatal chicks without negatively affecting early performance. Results: Environments with a presumably high microbial load (i.e., compost, poultry litter, and a cattle farm) were sampled for Bacillus species.52 Bacillus species were isolated.43 of these isolates were evaluated for antimicrobial activity against the hatchery-associated opportunistic pathogens Escherichia coli 021 and Staphylococcus aureus 004 in a preliminary overlay assay screen. Isolates that showed inhibition against the two tested pathogens were selected for further evaluation in a second agar overlay assay screen followed by a cell-free supernatant (CFS) assay screen. Two Bacillus isolates, referred to as MCR002 and MCR009, were selected as the top performing isolates based the number of pathogens they inhibited and the degree of inhibition they exhibited in these assays. MCR002 and MCR009 were isolated from samples collected from different source locations. Specifically, MCR002 was isolated from a sample taken from the surface of a compost pile, and MCR009 was isolated from a manure sample taken from a cattle farm. Both MCR002 and MCR009 were identified as Bacillus amyloliquefaciens using an API kit. The ability of MCR002 and MCR009 to control the microbial bloom that occurs during the hatching phase in commercial poultry hatch cabinets was tested using an in vivo challenge model (Graham et al., 2022). In these experiments, MCR002 and MCR009 were individually grown via solid-state fermentation (SSF), and the resulting SSF products were ground into fine powders.100 µL of challenge material, which consisted of a mixture of pathogenic microorganisms found in commercial poultry hatch cabinets, was applied on day 19 of embryogenesis (DOE19) to the air cell end of the egg. Following application of the challenge material, the SSF product from one or both Bacillus isolates was applied to hatching cabinets via compressed air at four timepoints: immediately post challenge, at 50% hatch, at 80% hatch, and at 100% hatch. For comparison, formaldehyde was applied every four hours starting immediately post challenge and ending 12 hours prior to hatch pull for a total of 10 applications. Thus, treatment groups included: a MCR002 only group, a MCR009 only group, a MCR002 / MCR009 combination group, and a formaldehyde group. Control groups included both a non-challenged, non-treated control (NC), and a challenged, non-treated control (PM). The microbial load in the hatch cabinet environment was measured using an open agar plate exposed to the hatching environment for one or five minutes at four timepoints: at 20% hatch, at 50% hatch, at 80% hatch, and just before hatch pull (100% hatch). A variety of selective, semi-selective, and non-selective media were used to identify and differentiate between the recovered microorganisms. Additionally, bacteria present in day-of-hatch whole gut gastrointestinal tract (GIT) samples and fluff collected immediately post hatch pull were enumerated using the various media. This in vivo challenge experiment was performed twice, and the results of the two trials of this experiment are discussed below. Application of the pathogen mix (PM) to the eggshell at DOE19 increased total Gram- negative bacteria, Enterococcus spp., and Staphylococcus aureus circulating in the hatch cabinet environment during the hatching phase (FIG.3, FIG.4, Table 1, Table 2). As expected, formaldehyde fumigation reduced the total microbial load in the hatching environment in both trials (FIGs.1-4). Also, across both trials, spray application of a Bacillus-based probiotic (i.e., MCR002 fermentate and / or MCR009 fermentate) during late embryogenesis increased Bacillus species recovery from the hatching environment at all time points and recovery from fluff and gastrointestinal tract (GIT) samples collected at hatch (FIG.1, FIG.2, Table 1, Table 2). Further, application of a Bacillus-based probiotic appeared to displace the pathogen load in the hatch cabinet, GIT samples, and fluff samples similarly to formaldehyde fumigation (FIG.3, FIG.4, Table 1, Table 2). However, the benefits associated with this treatment were more prominent in the first trial (FIG.3, Table 1) as compared to second trial (FIG.4, Table 2). The results of this experiment also suggest that shifting the composition of the microbial load in the hatch cabinet during late embryogenesis altered enteric colonization at hatch. This is indicated by significant differences in bacterial recovery from the gut of chicks that hatched in the formaldehyde-treated and Bacillus-treated cabinets as compared to the challenged control group (Table 1, Table 2). Specifically, recovery of Gram-negative bacteria was significantly lower in the Bacillus-treated groups as compared to the challenged control group and was comparable to the formaldehyde-treated group. Furthermore, there were no significant effects on growth performance (i.e., body weight gain) associated with challenge or hatch cabinet treatment (Table 3). Thus, MCR002 and MCR009 do not negatively impact performance under these conditions. Table 1. Bacterial recovery from gastrointestinal tract and fluff samples collected at hatch in trial 1. Data are expressed as mean Log10CFU / g ± standard error (SE). GIT1Total aerobic Gram- Enterococcus Staphylococc (Log10 CFU / g) teria negat us bac2ive bacteria spp. aureus NC 0.22 ± 0.22c0d0.29 ± 0.29a0aPM 0c5.86 ± 0.47a2.62 ± 1.03b0.46 ± 0.46aPM + F 0.67 ± 0.35c1.75 ± 0.68bc3.26 ± 0.87a0aPM + MCR002 2.72 ± 0.39b2.13 ± 0.75bc1.47 ± 0.80ab0.47 ± 0.47aPM + MCR009 3.81 ± 0.25a0.44 ± 0.44cd3.25 ± 0.80a0.28 ± 0.28aPM + Combo 3.93 ± 0.15a3.02 ± 0.77b2.47 ± 0.78a0aFluff Total aerobic Gram- Enterococcus Staphylococcus bacteria negative ba spp. aureus cteria NC 3.96 ± 0.14d0c2.47 ± 0.14b0cPM 6.52 ± 0.11c5.75 ± 0.12a2.46 ± 1.23a5.04 ± 0.07bPM + F 3.80 ± 0.10d2.47 ± 1.23b0c0cPM + MCR002 8.33 ± 0.20b5.63 ± 0.19a0c1.23 ± 1.23cPM + MCR009 8.37 ± 0.33ab0c0c4.11 ± 0.06bPM + Combo 8.90 ± 0.10a6.10 ± 0.10a0c6.85 ± 0.15a1Treatment abbreviations: negative control (NC); pathogen mix (PM); pathogen mix + formaldehyde (PM + F); pathogen mix + MCR002 (PM + MCR002); pathogen mix + MCR009 (PM + MCR009); and pathogen mix + MCR002 + MCR009 (PM + Combo).2A portion of the homogenized GIT sample was pasteurized at 70°C for 10 minutes to quantify Bacillus spp. recovery from the GIT.a,b,cIndicates significant difference between treatments by row (P<0.05). Means separated using Student’s t test. Table 2. Bacterial recovery from gastrointestinal tract and fluff samples collected at hatch in trial 2. Data are expressed as mean Log10CFU / g ± standard error (SE). 1 GIT Total aerobic 2 Gram-negative Staphylococcus 10g) bacteria b Enterococcus spp. (Log CFU / acteria aureus NC0b d b a 0 0 0 PM0b a ab a 7.74 ± 0.51 2.32 ± 0.98 0.37 ± 0.37 PM + F0.31 ± 0.31b cd b a 1.49 ± 0.76 0.22 ± 0.22 0 PM + MCR0023.53 ± 0.49a bc ab a 3.80 ± 1.22 1.78 ± 0.93 0.33 ± 0.33 PM + MCR0094.01 ± 0.15a d a a 0.64 ± 0.64 4.06 ± 1.23 0 PM + Combo3.76 ± 0.18a b a a 4.29 ± 1.19 4.11 ± 1.19 0 Fluff Total aerobic Gram-negative g10 CFU / g) bacteria bacteria Enteroco Staphylococcus (Lo ccus spp. aureus NC4.33 ± 0.09b d d 0 0 PM4.46 ± 0.16b b b c 6.34 ± 0.11 6.34 ± 0.12 6.45 ± 0.20 PM + F1.85 ± 0.83c d c d 0 0 0.78 ± 0.78 PM + MCR0029.31 ± 0.19a c c bc 5.51 ± 0.07 0 6.95 ± 0.11 PM + MCR0098.65 ±0.17a b b ab 6.48 ± 0.38 6.36 ± 0.21 7.60 ± 0.18 PM + Combo9.68 ±0.24a a a a 8.00 ± 0.31 7.65 ± 0.09 8.20 ± 0.251Treatment abbreviations: negative control (NC); pathogen mix (PM); pathogen mix + formaldehyde (PM + F); pathogen mix + MCR002 (PM + MCR002); pathogen mix + MCR009 (PM + MCR009); and pathogen mix + MCR002 + MCR009 (PM + Combo).2A portion of the homogenized GIT sample was pasteurized at 70°C for 10 minutes to quantify Bacillus spp. recovery from the GIT.a,b,cIndicates significant difference between treatments by row (P<0.05). Means separated using Student’s t test. Table 3. Impact of treatment on body weight gain (BWG) in trial 2. Data are expressed as mean BWG (g) ± standard error (SE). Treatment10-7 day 7-14 day 0-14 day BWG (g) BWG (g) BWG (g) NC 89.84 ± 6.98 210.22 ± 4.08 348.62 ± 4.52 PM 86.95 ± 2.67 211.96 ± 6.71 349.02 ± 8.91 PM + F 87.09 ± 2.83 217.48 ± 7.15 350.51 ± 9.56 PM + MCR002 88.76 ± 4.22 218.23 ± 11.90 351.86 ± 15.00 PM + MCR009 90.31 ± 1.89 210.61 ± 10.41 348.75 ± 11.82 PM + Combo 93.34 ± 2.30 203.23 ± 11.85 342.25 ± 12.061Treatment abbreviations: negative control (NC); pathogen mix (PM); pathogen mix + formaldehyde (PM + F); pathogen mix + MCR002 (PM + MCR002); pathogen mix + MCR009 (PM + MCR009); and pathogen mix + MCR002 + MCR009 (PM + Combo). Materials and Methods: Bacterial isolation Samples from several environments, including compost, poultry litter, and a cattle farm, were collected for isolation of Bacillus species. These environments were selected due to their high microbial loads, which encourages the evolution of microbes with mechanisms that provide competitive advantages. Samples were desiccated in a drying oven to remove moisture and to promote sporulation of Bacillus species present in the samples. A portion of each desiccated sample was pasteurized via water bath after dilution and was then plated on Tryptic Soy Agar (TSA) via a spread plate technique. After incubation, individual colonies were selected based on colony morphology and were transferred to a second TSA plate via isolation streak. From this initial isolation streak, two more subsequent isolation streaks were performed to further isolate and confirm purity. From the third round of isolation, individual colonies were selected from plates that contained a single colony morphology and were transferred to a culture tube containing Tryptic Soy Broth (TSB). After incubation, purity was evaluated via isolation streak to confirm that the cultures remained pure. Aliquots were stored at -80°C and were utilized for in vitro screening assays as needed. Agar overlay assay The in vitro antimicrobial activity of Bacillus spp. candidates was evaluated using an agar overlay assay. Opportunistic bacterial pathogens were overlayed over a single Bacillus sp. colony and the zone of inhibition surrounding the Bacillus colony was measured after overnight aerobic incubation at 37°C. For this assay, 10 µL of an overnight culture (~108CFU / mL) of each Bacillus candidate was pipetted in the center of a TSA plate (n=3 replicate plates / Bacillus candidate / pathogen) and 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 were incubated aerobically at 37°C for another 15 hours. Specifically, 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 between 50-60°C before being inoculated with the pathogen of interest. Based on previous studies, the short duration of exposure to these temperatures does not affect pathogen viability. To seed TSSA overlay media, 100 µL of each pathogen was added to the TSSA tubes and mixed gently before application. For E. coli, 30 µL of 25 mg / mL novobiocin (Sigma Catalog # N1628) was added to the TSSA to slow Bacillus growth so that E. coli could sufficiently grow. Once 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 the TSSA containing the pathogen sufficiently covered the Bacillus colony on the TSA plate without damaging the physical integrity of the colony itself. Plates were incubated for 15 hours and then 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). Solid-state fermentation A solid-state fermentation (SSF) medium consisting of wheat bran, rice hulls, and a dry mix that supplies essential nutrients (Table 4) was utilized to generate test material for the Bacillus isolates MCR002 and MCR009. To obtain the necessary amount of inoculum for each Bacillus strain, 47 mL of TSB was inoculated with a 1 mL bacterial stock aliquot and incubated for 18 hours at 37°C. The ingredients for the SSF growth medium (Table 4) were combined in a metal pan and sealed with a lid. The SSF growth medium was autoclaved, allowed to cool overnight, and inoculated with the 48 mL inoculum. The inoculated SSF growth medium was incubated at 37°C for 5 days. After incubation, the SSF product was dried at 65°C for 3 days in a drying oven. This temperature and time had been previously determined to be sufficient to induce sporulation, eliminate vegetative cells, prevent contamination, and remove nearly all moisture from the material. The dried SSF product was then processed three times through a commercial coffee grinder to produce a fine powder. The powdered product was stored at room temperature in a sealed container. The concentration (CFU / g) of final product was determined by conducting 10-fold serial dilutions in sterile saline and drop plating 10 µL volumes of each dilution onto TSA plates. Plates were incubated aerobically at 37°C and enumerated after overnight incubation. Table 4. Solid-state fermentation (SSF) medium Ingredient Amount Units Dry mix 19.48 g Water 720.00 mL Wheat bran 336.00 g Rice hulls 153.60 g KOH / NaOH 6.72 g Product application The SSF product described above was spray-applied into a hatching cabinet four times during the hatching phase. For each application, 1 g of SSF product, which included MCR002 or MCR009 at a concentration of 1x1010CFU / g, was loaded into an applicator fixed to the side of a GQF1550 hatching cabinet. A time activated actuator connected to an air compressor supplied a 10 second burst of air at 80 PSI into the applicator, which forced the SSF product into the hatching cabinet. The applicators were fixed in a position where, when the SSF product was applied, it entered the hatch cabinet in front of the air circulating fan which immediately dispersed the product throughout the hatching cabinet. The SSF product was administered at four hatch milestones: 20% pip, 50% pip, 80% pip, and 100% hatch. Microbial load assessment The hatch cabinet environment was sampled at four timepoints: day 20 of embryogenesis (DOE20) at 8:00 am (~20% hatch), DOE20 at 2:00 pm (~50% hatch), DOE20 at 5:00 pm (~80% hatch), and DOE21 at 7:00 am (~100% hatch). These time points were selected to assess the microbial load in the hatch cabinet environment at early, mid, late, and final timepoints during the hatching phase. The open-agar plate method (Berrang et al., 1995; Kim et al., 2010; Graham et al., 2018) was used to enumerate bacteria. For recovery of Bacillus spp. specifically, three tryptic soy agar (TSA) plates (with the lids removed) were placed open side up on the top tray of the hatchers (G.Q.F.1550 Digital Cabinet Egg Incubator) using a modified sample port, as previously described (Graham et al., 2021). The plates were exposed to the hatch cabinet environment for 1 minute and were then incubated at 37°C for 18 hours to enumerate Bacillus spp.. References: Berrang, M., N. Cox, and J. Bailey.1995. Measuring air-borne microbial contamination of broiler hatching cabinets. J. of Appl. Poult. Res.4:83–87. Graham, B.D., Selby, C.M., Forga, A.J., Coles, M.E., Beer, L.C., Graham, L.E., Teague, K.D., Tellez-Isaias, G., Hargis, B.M. and Vuong, C.N., 2022. Development of an environmental contamination model to simulate the microbial bloom that occurs in commercial hatch cabinets. Poultry Science, 101(6), p.101890. Graham, L., K. Teague, J. Latorre, Y. Yang, M. Baxter, B. Mahaffey, X. Hernandez- Velasco, L. Bielke, B. Hargis, and G. Tellez. 2018. Use of probiotics as an alternative to formaldehyde fumigation in commercial broiler chicken hatch cabinets. J. of Appl. Poult. Res. 27:371–379. Graham, B., C. Selby, L. Graham, K. Teague, G. Tellez-Isaias, B. Hargis, and C. Vuong. 2021. Development of a wild-type Escherichia coli environmental bloom model to evaluate alternatives to formaldehyde fumigation in broiler chicken hatch cabinets. Poult. Sci.100:100975. Kim, J. H., and K. S. Kim. 2010. Hatchery hygiene evaluation by microbiological examination of hatchery samples. Poult. Sci.89:1389–98. DEPOSIT INFORMATION A deposit of the Board of Trustees of the University of Arkansas proprietary Bacillus amyloliquefaciens strain designated as MCR002 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 of the strain, 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-68465. 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. A deposit of the Board of Trustees of the University of Arkansas proprietary Bacillus amyloliquefaciens strain designated as MCR009 disclosed above and recited in the appended claims has been made with the 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 of the strain, 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

CLAIMS We claim:

1. A composition comprising a bacterium, wherein the bacterium is a Bacillus amyloliquefaciens strain designated as MCR002 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B-68465, a Bacillus amyloliquefaciens strain designated as MCR009 and deposited at the NRRL under accession number NRRL B-68466, or a combination thereof.

2. The composition claim 1, wherein the bacterium comprises or consists of spores.

3. The composition of claim 1 or 2, wherein the bacterium is included in the composition as part of a fermentation product.

4. The compositions of claim 3, wherein the fermentation product is a solid-state fermentation (SSF) product.

5. The composition of claim 4, wherein the SSF product was produced by: a) inoculating a SSF medium with the bacterium; and b) culturing the inoculated SSF medium to produce the SSF product.

6. The composition of claim 5, wherein the SSF medium comprises wheat bran, rice hulls, and essential nutrients.

7. The composition of claim 5 or 6, wherein the inoculated SSF medium is cultured for 17-19 hours at 34-40°C in step (b).

8. The composition of any one of the preceding claims, wherein the composition is dried.

9. The composition of claim 8, wherein the composition is a powder.

10. The composition of claim 8 or 9, wherein the concentration of the bacterium is at least 1x107CFU per gram of composition.

11. The composition of claim 10, wherein the concentration of the bacterium is at least 1x1010CFU per gram of composition.

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

13. A method for inhibiting the growth of a microorganism on a surface by applying the composition of any one of claims 1-12 to the surface.

14. The method of claim 13, wherein the surface is a surface in a healthcare facility, a surface of a food product, or a surface of a plant.

15. A method for inhibiting the growth of a microorganism in a subject by administering the composition of any one of claims 1-12 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. A method for inhibiting the growth of a microorganism in a space by spraying the composition of any one of claims 1-12 into the space.

19. The method of claim 18, wherein the composition is sprayed via compressed air.

20. The method of claim 18 or 19, wherein the space is a poultry hatch cabinet.

21. The method of claim 20, wherein the hatch cabinet is not treated with formaldehyde.

22. The method of claim 20 or 21, wherein the composition is sprayed during chick hatching.

23. The method of claim 22, wherein the composition is sprayed at 20% pip, 50% pip, 80% pip, and / or 100% hatch.

24. The method of any one of claims 20-23, wherein the method: a) increases the amount of Bacillus spp. bacteria in the hatch cabinet as compared to an untreated control hatch cabinet; b) decreases the amount of pathogenic bacteria in the hatch cabinet as compared to an untreated control hatch cabinet; or c) both (a) and (b).

25. The method of any one of claims 20-24, wherein chicks hatched in the hatch cabinet have: a) an increased amount of Bacillus spp. bacteria in their gastrointestinal tract at hatch as compared to control chicks hatched in an untreated control hatch cabinet; b) a decreased amount of pathogenic bacteria in their gastrointestinal tract at hatch as compared to control chicks hatched in an untreated control hatch cabinet; or c) both (a) and (b).

26. The method of any one of claims 20-25, wherein the body weight gain of chicks hatched in the hatch cabinet is not significantly lower than the body weight gain of control chicks hatched in an untreated control hatch cabinet.

27. The method of any one of claims 13-26, wherein the microorganism is a pathogenic bacterium.

28. The method of claim 27, wherein the pathogenic bacterium is a Gram-negative bacterium, an Enterococcus spp., or Staphylococcus aureus.

29. The method of any one of claims 13-28, wherein the composition is applied to the surface, administered to the subject, or sprayed into the space multiple times.

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