Bacillus subtilis subspecies

Reference to a Deposit of Biological Material

This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference.

Reference to sequence listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

Index to sequence listing:

SEQ ID NO: 1 is the DNA sequence of the gyrB gene from DSM 29870 (obtained as described in Example 3).

SEQ ID NO: 2 is the partial amino acid sequence deduced from the gyrB gene of SEQ ID NO: 1 from DSM 29870.

SEQ ID NO: 3 is the DNA sequence of the rpoB gene from DSM 29870 (obtained as described in Example 4).

SEQ ID NO: 4 is the partial amino acid sequence deduced from the rpoB gene of SEQ ID NO: 3 from DSM 29870.

SEQ ID NO: 5 is the DNA sequence of the gyrA gene (published genome sequence of Bacillus subtilis subsp. Spizizenii CP002905).

SEQ ID NO: 6 is the partial amino acid sequence deduced from the gyrA gene of SEQ ID NO: 5.

SEQ ID NO: 7 is the DNA sequence of the gyrA gene from DSM 29870.

SEQ ID NO: 8 is the amino acid sequence of gyrA gene product of SEQ ID NO: 7 from DSM 29870.

SEQ ID NO: 9 is the 16S rDNA of DSM 29870.

SEQ ID NO: 10 to SEQ ID NO: 31 are PCR and sequencing primers.

SEQ ID NO: 32 is DNA sequence of GyrB gene from DSM 29870 (obtained from PCR product).

SEQ ID NO: 33 is the partial amino acid sequence deduced from the gyrB gene of SEQ ID NO: 32 from DSM 29870.

SEQ ID NO: 34 is DNA sequence of rpoB gene from DSM 29870 (obtained from PCR product).

SEQ ID NO: 35 is the partial amino acid sequence deduced from the rpoB gene of SEQ ID NO: 34 from DSM 29870. BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to novel subspecies of Bacillus subtilis. The novel subspecies can improve health and performance of production animals. In one embodiment the Bacillus subtilis subspecies has activity against Clostridium perfringens and/or E. coli. In a preferred embodiment the Bacillus subtilis subspecies has a high compatibility with monensin such as being compatible with at least 2.4 μg/ml monensin as determined in Example 12. The invention further relates to compositions comprising one or more strains of the Bacillus subtilis subspecies and to use of the strain(s) of the Bacillus subtilis subspecies in an animal feed.

Background of the Invention

Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes (oxygen reliant), or facultative anaerobes (having the ability to be aerobic or anaerobic). Ubiquitous in nature, Bacillus includes both free-living (non-parasitic) and parasitic pathogenic species. Under stressful environmental conditions, the bacteria can produce oval endospores that are not true spores but which the bacteria can reduce themselves to and remain in a dormant state for very long periods.

Clostridium perfringens (C. perfringens) is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium of the genus Clostridium. Infections due to C. perfringens show evidence of tissue necrosis, bacteremia, emphysematous cholecystitis, and gas gangrene, which is also known as clostridial myonecrosis. C. perfringens can also result in polymicrobial anaerobic infections. The incidence of Clostridium perfringens-asso ated necrotic enteritis in poultry has increased in countries that stopped using antibiotic growth promoters. Necrotic enteritis is an enterotoxemic disease that results in significant economic losses in the poultry industry.

There is a need for development of tools and strategies for prevention and control of C. perfringens in mono-gastric animals such as poultry. Whilst the vaccination of animals has been suggested, there are challenges associated with vaccinating thousands of animals. Thus discovering a solution which could be administered as an additive in an animal feed would be advantageous.

It is an object of the invention to provide solutions which prevents and/or controls C. perfringens in poultry by use of an animal feed comprising one or more one or more bacteria with activity against Clostridium perfringens.

A challenge of delivering Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore it is necessary to determine the compatibility of strains with commonly-used feed antibiotics such as monensin in order to identify any potential conflicts with use as a direct fed microbial. The present invention relates in one embodiment to a Bacillus subtilis subspecies with high compatibility with monensin. Description of the Related Art

WO 2010/033714 describes a method for enhancing the health of an animal comprising administering to the animal a composition comprising Bacillus subtilis QST713.

US 4,919,936 describes a method for increasing the weight gain in animals comprising feeding an animal a probiotic comprising Bacillus subtilis C-3102.

Knap et al. describes that Bacillus licheniformis has an effect on necrotic enteritis in broiler chickens (Knap et al., 2010, "Bacillus licheniformis Prevents Necrotic Enteritis in Broiler Chickens", Avian Diseases 54(2): 931-935).

Summary of the Invention

The present invention relates to a novel subspecies of Bacillus subtilis. It has been surprisingly found that the addition of direct fed microbes (DFM) from these Bacillus subtilis subspecies to animal feed can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in mono-gastric animal such as pigs and/or poultry.

Thus the invention relates to a Bacillus subtilis subspecies or one or more Bacillus strain(s) comprising one or more of the features selected from the group consisting of:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2;

ii) a rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4;

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 and/or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8; and/

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%. In a preferred embodiment the Bacillus subtilis subspecies has a high compatibility with monensin such as being compatible with at least 2.4 μg/ml monensin as determined in Example 12.

The present invention also relates to compositions, such as animal feed or animal feed additives, which comprise one or more Bacillus strains of the invention; methods of feeding an animal with the composition of the invention and methods of treating a Clostridium perfringens infection or for treating necrotic enteritis in an animal with the composition of the invention. Use of the composition according to the invention to improve the performance of an animal, such as improving the feed conversion rate, improving the body weight gain and/or improving the feed efficiency and/or improving the health is also part of this invention. The invention further relates to a method for preventing and/or controlling C. perfringens infections and/or necrotic enteritis in a mono-gastric animal such as swine or poultry by use of an animal feed, wherein the animal feed comprises the strain having the deposit accession number DSM 29870, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.

In an embodiment, the invention relates to an animal feed that is fed to a mono- gastric animal such as poultry or swine; comprising the strain having the deposit accession number DSM 29870, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.

In a preferred embodiment the invention relates to an animal feed composition such as an animal feed additive comprising a Bacillus strain wherein:

i. the Bacillus strain has the deposit accession number DSM 29870; a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof, and ii. the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.

In a further aspect, the invention relates to a method for preventing and/or controlling C. perfringens infections and/or necrotic enteritis in an animal such as pigs or poultry, comprising the steps of:

(a) feeding an animal such as a pig feed or a poultry feed; and

(b) administering to an animal such as pigs or poultry one or more Bacillus strains or spores of said strains wherein the strain has the deposit accession number DSM 29870; a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli, and wherein step (a) occurs before, after, or simultaneously with step (b).

In an embodiment, the method relates to an animal feed that is fed to a mono-gastric animal such as pigs and/or poultry. The poultry can, e.g., be chickens and/or broilers and/or layers.

In an embodiment, the animal feed composition comprises one of more additional bacteria; is fed to a mono-gastric animal such as pigs and/or poultry; and further comprises concentrate and/or one or more enzymes and/or one or more additional microbes and/or one or more vitamins and/or one or more minerals and/or one or more amino acids and/or one or more other feed ingredients.

Figure legends

Figure 1 : Core genome phylogram of Bacillus species. Note that B. amyloliquefaciens subspecies amyloliquefaciens and plantarum separate with a bootstrap support of 1 (100%). Figure 2: Rectangular phylogram with midpoint rooting of the members of the Bacillus species. All the draft and completed genomes of Bacillus subtilis that were available on NCBI on 29-12-2014 were included in the analysis along with representatives from other species. Numbers indicate bootstrap support (1 = 100%). Scale bar on top left indicates an evolutionary distance of 0.1 amino acid substitutions per position. See the table for species descriptions.

Figure 3: Rectangular phylogram of members of Bacillus genus. This is a zoomed in view of Figure 2 to show the relationship of 052BCU (alias strain DSM 29870) to its nearest neighbors.

Definitions

In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in context of the disclosure.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

ANI (Average nuclear identity): DNA-DNA hybridization (DDH) has been used traditionally for phylogenetic definition of a species. Based on DDH, strains with greater than 70% relatedness would be considered to belong to the same species [Wayne et al., 1987, Report of the Ad-Hoc-Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacterid 37: 463-464]. With the availability of cheap and affordable whole genome sequencing technologies as well as the increased availability of public genomes, whole genome-based species estimation methods are becoming popular as they offer elegant in silico solutions as alternatives DDH methods. ANI is a distance based approach to delineate species based on pair-wise comparisons of genome sequences [Goris et al., 2007, "DNA- DNA hybridization values and their relationship to whole-genome sequence similarities", Int. J. Syst. Evol. Microbiol. 57: 81-91]. Goris et al. used 28 different strains to conclude that 95% ANI is similar to the aforementioned 70% DDH cutoff value and can be used for species delineation. ANI has been evaluated in multiple labs and has become the gold standard for species delineation [cf. Kim et al., 2014, "Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes", Int. J. Syst. Evol. Micr. 64: 346-351 ; Richter et al., 2009, "Shifting the genomic gold standard for the prokaryotic species definition", P Natl Acad Sci USA 106: 19126-19131 ; and Chan et al., 2012, "Defining bacterial species in the genomic era: insights from the genus Acinetobacter", Bmc. Microbiol. 12)]. An evaluation of AN I for designation for a strain of Bacillus amyloliquefaciens is presented here as an example. AN I calculations were obtained for pair-wise comparisons of B. amyloliquefaciens strain FZB42 to members of B. amyloliquefaciens subspecies plantarum, B. amyloliquefaciens subspecies amyloliquefaciens, Bacillus atrophaeus and Bacillus subtilis. The AN I calculator on the Kostas lab website (enve- omics.ce. gatech.edu/ani/newjob) was used. Comparisons with B. subtilis strain 160 and B. atrophaeus strain C89 give AN I less than 90% indicating that AN I can be successfully used to distinguish between these species (Table A). Table A - Pairwise AN I of B. amyloliquefaciens FZB42 compared to other bacterial genomes (in percentage).

FZB42 shows >95% identify with all members of B. amyloliquefaciens subspecies plantarum, confirming its species designation. However, FZB42 comparisons to DSM7 and other amyloliquefaciens subspecies show an AN I of 94-94.5%, which is less than the acceptable species definition cutoff of 95%. Reciprocal comparison of DSM7 to other genomes also shows > 95% AN I within the subspecies amyloliquefaciens isolates and 94- 95% AN I with subspecies plantarum isolates (Table B). Table B - Pairwise ANI of B. amyloliquefaciens DSM7 compared to other bacterial genomes (in percentage).

Borriss et al. also observed that the DDH between FZB42 and DSM7 varied from 63.7-71.2% which are ranges below the DDH threshold of 70% and is one of the many reasons why they were classified into separate subspecies [Borriss et al., 201 1 , "Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7(T) and FZB42(T): a proposal for Bacillus amyloliquefaciens subsp amyloliquefaciens subsp nov and Bacillus amyloliquefaciens subsp plantarum subsp nov based on complete genome sequence comparisons", Int. J. Syst. Evol. Micr. 61 : 1786-1801].

Chan et al. recommended complementing AN I with core genome phylogenetics [Chan et al., 2012, "Defining bacterial species in the genomic era: insights from the genus Acinetobacter", Bmc. Microbiol. 12: 302], where common elements (generally coding sequences or their translations) can be used to derive phylogenetic inferences. To test this, Hyatt et al. [Hyatt et al., 2010 "Prodigal: prokaryotic gene recognition and translation initiation site identification", BMC Bioinformatics 1 1 : 1 19] was used for gene calling and PhyloPhlan [Segata et al., 2013, "PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes", Nat. Commun. 4: 2304] was used to generate a core genome phylogenetic tree. Dendroscope 3 [Huson et al. , 2012, "Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks", Syst. Biol. 61 : 1061-1067] was used to visualize the tree as a rectangular phylogram with midpoint rooting along with bootstrap support (cf. Figure 1). It is clear that all of the B. amyloliquefaciens strains cluster together in one distinct branch; and the amyloliquefaciens and plantarum subspecies separate into two subclades.

Based on the data presented here, the following species definition could be applied to B. amyloliquefaciens: "Bacillus amyloliquefaciens" shall mean a monophyletic group of strains that reside in either amyloliquefaciens or plantarum subspecies branches of a core genome phylogenetic tree and whose genomes exhibit at least 95% pairwise average- nucleotide identity (ANI) [Goris et al., 2007, "DNA-DNA hybridization values and their relationship to whole-genome sequence similarities", Int J Syst Evol Microbiol 57: 81-91] when compared to members of the same subspecies and greater than 94% ANI when compared to members of the other subspecies. Alternative definition: "Bacillus amyloliquefaciens" shall mean a monophyletic group of strains whose genomes exhibit at least 95% pairwise average-nucleotide identity (ANI) to type strain DSM7 if belonging to subspecies amyloliquefaciens or at least 95% pairwise ANI to type strain FZB42 if belonging to subspecies plantarum, as inferred by core genome phylogenetics.

Animal feed: The term "animal feed" refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a mono-gastric animal comprises concentrates as well as vitamins, minerals, enzymes, amino acids and/or other feed ingredients (such as in a premix). The animal feed may further comprise forage.

Antimicrobial activity against Clostridium perfringens: The term "Antimicrobial activity against Clostridium perfringens" means that the growth of Clostridium perfringens is inhibited and/or that some or all of the Clostridium perfringens are killed. This can be determined by the assay described in Example 6.

Blend: the term "blend" means more than one of the bacterial strains described herein. Body Weight Gain: The Body Weight Gain of an animal is the increase of body weight of the animal over a specified time period.

Composition: The term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be mixed with an animal feed(s) and referred to as a "mash feed."

Concentrates: The term "concentrates" means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

Control C. perfringens infections and/or necrotic enteritis: The term "control C. perfringens infections and/or necrotic enteritis" means a method and/or composition that partly or completely inhibits C. perfringens infections and/or necrotic enteritis in an animal. Accordingly, the term "control C. perfringens infections and/or necrotic enteritis" means the C. perfringens infections and/or the necrotic enteritis is reduced or completely eliminated or prevented.

Direct Fed Microbial: The term "direct fed microbial" means live micro-organisms including spores which, when administered in adequate amounts, confer a benefit, such as improved digestion or health, on the host.

European Production Efficacy Factor (EPEF): The European Production Efficacy Factor is a way of comparing the live-bird performance of flocks. This single-figure facilitates comparison of performance within and among farms and can be used to assess environmental, climatic and managemental variables. The EPEF is calculated as [(liveability (%) x Liveweight (kg)) / (Age at depletion (days) x FCR)] x 100, wherein livability is the percentage of birds alive at slaughter, Liveweight is the average weight of the birds at slaughter, age of depletion is the age of the birds at slaughter and FCR is the feed conversion ratio at slaughter.

Effective amount/concentration/dosage: The terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the digestion or yield of an animal or to control C. perfringens infections and/or necrotic enteritis. The actual effective dosage in absolute numbers depends on factors including: the state of health of the animal in question, other ingredients present. The "effective amount", "effective concentration", or "effective dosage" of the bacterial strains may be determined by routine assays known to those skilled in the art. FCR (Feed Conversion Rate): FCR is a measure of an animal's efficiency in converting feed mass into increases of the desired output. Animals raised for meat - such as swine, poultry and fish - the output is the mass gained by the animal. Specifically FCR is the mass of the food eaten divided by the output, all over a specified period.

Feeding an animal: the terms "feeding an animal" or "fed to an animal" means that the composition of the present invention is administered orally to the animal one or more times in an effective amount. The oral administration is typically repeated, e.g., one or more times daily over a specified time period such as several days, one week, several weeks, one months or several months. Accordingly, the term "feeding" or "fed" means any type of oral administration such as administration via an animal feed or via drinking water.

Forage: The term "forage" as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.

Isolated: The term "isolated" means that the one or more bacterial strains described herein are in a form or environment which does not occur in nature, that is, the one or more bacterial strains are at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

Non-hemolytic: Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify bacterial strains into hemolytic and non-hemolytic strains. In this context hemolysis is defined as described in EFSA Journal 201 1 ; 9(1 1): 2445, using Bacillus subtilis 168 (BGSC-1A1 , Bacillus Genetic Stock Center) as a negative control. A Bacillus strain can be classified as non-hemolytic using the assay described in Example 8.

Pellet: The terms "pellet" and/or "pelleting" refer to solid rounded, spherical and/or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the terms "extrusion" or "extruding" are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure. Poultry: The term "poultry" means domesticated birds kept by humans for the eggs they produce and/or their meat and/or their feathers. Poultry includes broilers and layers. Poultry include members of the superorder Galloanserae (fowl), especially the order Galliformes (which includes chickens, Guineafowls, quails and turkeys) and the family Anatidae, in order Anseriformes, commonly known as "waterfowl" and including domestic ducks and domestic geese. Poultry also includes other birds that are killed for their meat, such as the young of pigeons. Examples of poultry include chickens (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.

Prevent C. perfringens infections and/or necrotic enteritis: The term "prevent C. perfringens infections and/or necrotic enteritis" means a method and/or composition that prevents development of a C. perfringens infection and/or necrotic enteritis in an animal.

Roughage: The term "roughage" means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).

Sensitive to antibiotics: The term "sensitive to antibiotics" means the phenotypic property of a bacterial strain, that growth of said bacterial strain is inhibited under conditions where the bacterial strain would otherwise grow. In this context sensitivity to antibiotics is tested after the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). The S. aureus ATCC 29213 is used as reference strain, which means that it should be included in the test, and that the results are only valid if S. aureus ATCC 29213 show results in compliance with the breakpoints of the CLSI guideline (see example Table 5.5) (M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014). A strain of Bacillus is considered sensitive if the growth is detected at or below the breakpoint concentration of the appropriate antibiotic specified in EFSA Journal 2012; 10(6): 2740.

Silage: The term "silage" means fermented, high-moisture stored fodder which can be fed to ruminants (cud-chewing animals such as cattle and sheep) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called ensilage, ensiling or silaging, and is usually made from grass or cereal crops (e.g., maize, sorghum, oats, rye, timothy etc forage grass plants),) or legume crops like clovers/trefoils, alfalfa, vetches, using the entire green plant (not just the grain). Silage can be made from many field crops, and special terms may be used depending on type (oatlage for oats, haylage for alfalfa). Silage is made either by placing cut green vegetation in a silo, by piling it in a large heap covered with plastic sheet, or by wrapping large bales in plastic film.

Spore: The terms "spore" and "endospore" are interchangeable and have their normal meaning which is well known and understood by those of skill in the art. As used herein, the term spore refers to a microorganism in its dormant, protected state. Stable: The term "stable" is a term that is known in the art, and in a preferred aspect, stable is intended to mean the ability of the microorganism to remain in a spore form until it is administered to an animal to improve the health of the animal.

Swine: The term "swine" or "pigs" means domesticated pigs kept by humans for food, such as their meat. Swine includes members of the genus Sus, such as Sus scrofa domesticus or Sus domesticus and include piglets, growing pigs, and sows.

Vegetable protein: The term "vegetable protein" refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.

Detailed Description of the Invention

It has been surprisingly found that the addition of direct fed microbes (DFM) from Bacillus species to animal feed can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in mono-gastric animal such as pigs and/or poultry and at the same time improve the body weight gain and/or feed conversion rate (in challenged and unchallenged chickens).

Bacillus strains of the invention

The invention relates to the following aspect with respect to Bacillus subtilis subspecies and Bacillus strain(s):

Aspect 1 : A Bacillus subtilis subspecies or one or more Bacillus strain(s) comprising one or more of the features selected from the group consisting of

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2;

ii) a rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4;

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8; and

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 such as at least 94.8%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% and such as at least 99% sequence identity. In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 such as at least 99.5%, such as at least 99.6%, such as at least 99.7%, such as at least 99.8% and such as at least 99.9% sequence identity.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more

Bacillus strain(s) comprises an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 such as at least 96.8%, such as at least 97%, such as at least 98% and such as at least 99% sequence identity.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 such as at least 99.75%, such as at least 99.8% and such as 99.9% sequence identity.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 such as at least 97.6%, such as at least 97.8%, such as at least 98%, such as at least 99% and such as at least 99.5% sequence identity.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% and such as at least 99% sequence identity.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8. In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more

Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more

Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 1 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments is non-hemolytic (e.g., determined as described in Example 8).

In a preferred embodiment the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments has a high compatibility with monensin such as being compatible with at least 2.3 μg/ml monensin as determined in Example 12. It is even more preferred that the Bacillus subtilis subspecies is compatible with at least 2.4 μςΛηΙ monensin as determined in Example 12 (such as at least 2.5 g/ml monensin as determined in Example 12, such as at least 2.6 μςΛηΙ monensin as determined in Example 12 or such as at least 2.7 μςΛηΙ monensin as determined in Example 12).

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments has antimicrobial activity against Clostridium perfringens. The effect against Clostridium perfringens can be determined as described in Example 6.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments has antimicrobial activity against E. coli. The effect against E. coli can be determined as described in Example 7.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments has antimicrobial activity against Clostridium perfringens and E. coli.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments is sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline (e.g., as determined as described in Example 9).

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments has a 16S rDNA with more than 98% (such as more than 98.5%, such as more than 99%, such as more than 99.5%) sequence identity to SEQ ID NO: 9.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments is a Bacillus subtilis strain.

The Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to

Aspect 1 or any of the above embodiments is in one embodiment the Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In a further embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 1 or any of the above embodiments is the Bacillus strain having deposit accession number DSM 29870.

The invention relates in one embodiment to a Bacillus having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. In an embodiment, the invention relates to a Bacillus having deposit accession number DSM 29870.

The invention also relates to a biologically pure culture of the Bacillus strain according to Aspect 1. In a further embodiment the invention relates to a biologically pure culture of the Bacillus strain having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. In a further embodiment, the invention relates to a biologically pure culture of the Bacillus strain having deposit accession number DSM 29870.

The invention also relates to an isolated Bacillus strain according to Aspect 1. In a further embodiment the invention also relates to an isolated Bacillus strain having deposit accession number DSM 29870 or an isolated strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

The invention also relates to a spore of Bacillus strain according to Aspect 1. In a further embodiment the invention also relates to a spore of Bacillus strain having deposit accession number DSM 29870 or a spore having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. The spore is preferably a stable spore.

Compositions of the invention

The invention relates to a composition comprising spores (such as stable spores) of the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to the invention.

More specifically the invention relates to the following aspects with respect to compositions comprising the Bacillus subtilis subspecies or the Bacillus strain(s):

Aspect 2: A composition comprising a Bacillus subtilis subspecies or one or more Bacillus strain(s) comprising one or more of the features selected from the group consisting of:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2;

ii) a rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4;

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8; and

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 such as at least 94.8%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% and such as at least 99% sequence identity. In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 such as at least 99.5%, such as at least 99.6%, such as at least 99.7%, such as at least 99.8% and such as at least 99.9% sequence identity.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more

Bacillus strain(s) comprises an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 such as at least 96.8%, such as at least 97%, such as at least 98% and such as at least 99% sequence identity.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 such as at least 99.75%, such as at least 99.8% and such as 99.9% sequence identity.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 such as at least 97.6%, such as at least 97.8%, such as at least 98%, such as at least 99% and such as at least 99.5% sequence identity.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% and such as at least 99% sequence identity.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ I D NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8. In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 2 the composition comprising a Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more

Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8.

In one embodiment of Aspect 2 the composition comprising a Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more

Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 or

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 or

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 or

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) are non-hemolytic (e.g., determined as described in Example 8).

In a preferred embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) has a high compatibility with monensin such as being compatible with at least 2.3 μg/ml monensin as determined in Example 12. It is even more preferred that the Bacillus subtilis subspecies is compatible with at least 2.4 μg/ml monensin as determined in Example 12 (such as at least 2.5 μg/ml monensin as determined in Example 12, such as at least 2.6 μς/ηιΙ monensin as determined in Example 12 or such as at least 2.7 μςΛηΙ monensin as determined in Example 12).

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) have antimicrobial activity, e.g., against Clostridium perfringens. The effect against Clostridium perfringens can be determined as described in Example 6.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) have antimicrobial activity against E. coli. The effect against E. coli can be determined as described in Example 7.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) have antimicrobial activity against Clostridium perfringens and E. coli.

In one embodiment of Aspect 2 the Bacillus subtilis subspecies or the one or more Bacillus strain(s) is sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline (e.g., as determined as described in Example 9).

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 2 or any of the above embodiments has a 16S rDNA with more than 98% (such as more than 98.5%, such as more than 99%, such as more than 99.5%) sequence identity to SEQ ID NO: 9.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 2 or any of the above embodiments is a Bacillus subtilis strain.

In one embodiment, the Bacillus subtilis subspecies or the one or more Bacillus strain(s) according to Aspect 2 or any of the above embodiments can in one embodiment be the Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.

The invention relates in one embodiment to a composition comprising a Bacillus having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

The invention also relates to a composition comprising a biologically pure culture of the Bacillus strain according to Aspect 2. In a further embodiment the invention relates to a composition comprising a biologically pure culture of the Bacillus strain having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

The invention also relates to a composition comprising an isolated Bacillus strain according to Aspect 2. In a further embodiment the invention also relates to a composition comprising an isolated Bacillus strain having deposit accession number DSM 29870 or an isolated strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

In one embodiment of Aspect 2 the Bacillus spores of the composition are present as dried spores (such as, e.g., spray-dried spores). In one embodiment of Aspect 2 the Bacillus spores of the composition are present as stable spores. The composition according to Aspect 2 can also be a liquid composition and/or comprise culture supernatant comprising the Bacillus strain(s) of the invention.

In one embodiment of Aspect 2 the composition further comprises a carrier. The carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, cellulose farigel, cassava cores, sodium aluminium silicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000 and carbopol.

In a preferred embodiment of Aspect 2 the composition further comprises calcium carbonate and sodium aluminium silicate.

In a preferred embodiment of Aspect 2 the composition further comprises Calcium carbonate, sodium aluminium silicate and sucrose.

In another preferred embodiment of Aspect 2 the composition further comprises one or more carriers such as one or more carriers selected from the group consisting of Calcium carbonate, sodium sulphate, starch, farigel and cassava cores.

In another preferred embodiment of Aspect 2 the composition further comprises one or more flowability agents such as sodium aluminium silicate and/or colloidal amorphous silica (e.g., Sipernat 50S).

In another preferred embodiment of Aspect 2 the composition further comprises one or more binder such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillus DSM 29870, calcium carbonate and sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacillus DSM 29870, Calcium carbonate, sodium aluminium silicate and sucrose. In a preferred embodiment the composition comprises Bacillus DSM 29870 and one or more carriers such as one or more carriers selected from the group consisting of Calcium carbonate, sodium sulphate, starch, farigel and cassava cores.

In a preferred embodiment the composition comprises Bacillus DSM 29870 and one or more flowability agents such as sodium aluminium silicate and/or colloidal amorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacillus DSM 29870 and one or more binder such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

In one embodiment the composition comprises one or more coccidiostats wherein the composition, e.g., is a premix.

In a preferred embodiment the composition according to Aspect 2 the composition comprises from 10 ⁵ to 10 ¹² CFU/g of isolated Bacillus spores such as from 10 ⁵ to 10 ⁷ CFU/g of isolated Bacillus spores, such as 10 ⁷ to 10 ⁹ CFU/g of isolated Bacillus spores, such as from 10 ⁹ to 10 ¹¹ CFU/g of isolated Bacillus spores, such as 10 ¹¹ to 10 ¹² CFU/g of isolated Bacillus spores or any combination of these intevals.

In yet another preferred embodiment the composition according to Aspect 2 is an animal feed such as an animal feed additive. In one embodiment the animal feed is characterized in that at least 70% (such as at least 80% or at least 90%) of the Bacillus spores survive gastric stability in a mono-gastric animal such as chickens.

The composition according to Aspect 2 can be an animal feed which further comprises one or more components selected from the list consisting of: one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

The composition according to Aspect 2 can be an animal feed or an animal feed additive wherein the bacterial count of each Bacillus spore is 1x10 ³ and 1x10 ¹³ CFU/animal/day, preferably between 1x10 ⁵ and 1x10 ¹¹ CFU/animal/day, more preferably between 1x10 ⁶ and 1x10 ¹⁰ CFU/animal/day and most preferably between 1x10 ⁷ and 1x10 ⁹ CFU/animal/day.

The composition according to Aspect 2 can be a mono-gastric animal feed. The mono-gastric animal can be selected from the group consisting of pigs, swine, piglets, sows, poultry, turkeys, ducks, chicken, broilers, layers, chicks, fish and crustaceans. In one embodiment the animal is not a human being. Mono-gastric animals include in one embodiment, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broilers, chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.

In a preferred embodiment, the composition according to Aspect 2 is an animal feed or animal feed additive wherein the Bacillus strain improves gut health of chickens with infection of Clostridium perfringens by having antimicrobial activity against Clostridium perfringens.

In a preferred embodiment, the composition according to Aspect 2 is an animal feed or animal feed additive for treatment of necrotic enteritis or treatment of a Clostridium perfringens infection (e.g., for treatment of mono-gastric animals including swine and poultry such as chickens).

In an embodiment to any of the aforementioned embodiments, the Bacillus spore kills/inhibits at least 40% (such as at least 45%, at least 50%, at least 60%, at least 70% or at least 80%) of Clostridium perfringens after 24 hours, e.g., determined as described in Example 6.

In another embodiment of the invention, the composition such as the animal feed further comprises concentrate. In another embodiment of the invention the composition such as the animal feed further comprises forage. In another embodiment of the invention the composition such as the animal feed further comprises one or more additional microbes. In another embodiment of the invention the composition such as the animal feed further comprises one or more enzymes. In another embodiment of the invention the composition such as the animal feed further comprises one or more vitamins. In another embodiment of the invention the composition such as the animal feed further comprises one or more minerals. In another embodiment of the invention the composition such as the animal feed further comprises one or more amino acids. In another embodiment of the invention the composition such as the animal feed further comprises one or more other feed ingredients.

In an embodiment to any of the aforementioned embodiments, the composition also improves the health of the mono-gastric animal when fed to said animal. In another embodiment to any of the aforementioned embodiments, the composition also increases the egg yield of poultry when fed to said poultry. In an embodiment to any of the aforementioned embodiments, the composition increases the meat yield of the mono-gastric animal when fed to said animal.

In a preferred embodiment, the composition such as the animal feed comprises one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1x10 ⁴ and 1x10 ¹⁸ CFU/kg of composition, preferably between 1x10 ⁷ and 1x10 ¹⁶ CFU/kg of composition, more preferably between 1x10 ¹⁰ and 1x10 ¹⁵ CFU/kg of composition and most preferably between 1x10 ¹¹ and 1x10 ¹⁴ CFU/kg of composition.

In a preferred embodiment, the bacterial count of each of the bacterial strains in the animal feed additive is between 1x10 ⁴ and 1x10 ¹⁸ CFU/kg of composition, preferably between 1x10 ⁷ and 1x10 ¹⁶ CFU/kg of composition, more preferably between 1x10 ¹⁰ and 1x10 ¹⁵ CFU/kg of composition and most preferably between 1x10 ¹¹ and 1x10 ¹⁴ CFU/kg of dry matter.

In a preferred embodiment, the bacterial count of each of the bacterial strains in the animal feed is between 1x10 ⁴ and 1x10 ¹⁴ CFU/kg of dry matter, preferably between 1x10 ⁶ and 1x10 ¹² CFU/kg of dry matter, and more preferably between 1x10 ⁷ and 1x10 ¹¹ CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10 ⁸ and 1x10 ¹⁰ CFU/kg of dry matter.

In a preferred embodiment, the composition such as the animal feed has a bacterial count of each Bacillus spore between 1x10 ³ and 1x10 ¹³ CFU/animal/day, preferably between 1x10 ⁵ and 1x10 ¹¹ CFU/animal/day, more preferably between 1x10 ⁶ and 1x10 ¹⁰ CFU/animal/day and most preferably between 1x10 ⁷ and 1x10 ⁹ CFU/animal/day.

In still yet another embodiment of the invention, the one or more bacterial strains are present in the composition in form of a spore such as a stable spore. In still a further embodiment of the invention, the stable spore will germinate in the intestine and/or stomach of the mono-gastric animal.

In one embodiment, the one or more bacterial strains are stable when subjected to pressures applied/achieved during an extrusion process for pelleting. In a particular embodiment, the one or more bacterial strains are stable at pressures ranging from 1 bar to 40 bar, particularly 10 bar to 40 bar, more particularly 15 bar to 40 bar, even more particularly 20 bar to 40 bar, still even more particularly 35 bar to 37 bar, even still more particularly 36 bar.

In a particular embodiment, the one or more bacterial strains are stable at high temperatures. In particular, the bacterial strains are stable when they are subjected to temperatures achieved during an extrusion process for pelleting. In an even more particular embodiment, the one or more bacterial strains are stable at temperatures ranging from 80°C to 120°C, particularly temperatures ranging from, 90°C to 120°C, even more particularly temperatures ranging from 95°C to 120°C.

In another aspect, the invention relates to a composition such as an animal feed composition comprising a carrier, such as forage and one or more of the bacteria cultures having characteristics substantially identical to that of the strain having the deposit accession number DSM 29870. In an embodiment, the animal feed composition comprises a carrier and the strain having the deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

In another embodiment, the animal feed composition is for feeding to a mono-gastric animal. Mono-gastric animals include, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.

In an embodiment, the animal feed composition further comprises one or more additional microbes. In a particular embodiment, the animal feed composition further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

In a particular embodiment, animal feed composition further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, or any combination thereof.

In a particular embodiment, the animal feed composition further comprises one or more types of yeast. The one or more types of yeast can be selected from the group consisting of Saccharomycetaceae, Saccharomyces (such as S. cerevisiae and/or S. boulardii), Kluyveromyces (such as K. marxianus and K. lactis), Candida (such as C. utilis, also called Torula yeast), Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii), Phaffia yeasts and Basidiomycota.

In an embodiment to any of the aforementioned embodiments the composition further comprises a carrier. The carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium aluminium silicate, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, farigel, cassava cores, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol. and cellulose. Animal Feed

In one aspect, the animal feed comprising the Bacillus subtilis subspecies or the Bacillus strain(s) according to Aspect 1 or the Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant of DSM 29870 and further comprises one or more of concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and/or other feed ingredient(s) (such as in a premix). In a specific embodiment the animal feed further comprises forage.

Forage as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Crops suitable for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.

Roughage is generally dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).

Examples of concentrates are feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, saff lower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

In one embodiment, the forage and one or more microbes are mixed with a concentrate. In another embodiment, the forage and one or more microbes are mixed with a premix. In a further embodiment, the forage and one or more microbes are mixed with vitamins and/or minerals. In a further embodiment, the forage and one or more microbes are mixed with one or more enzymes. In a further embodiment, the forage and one or more microbes are mixed with other feed ingredients, such as colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides, anti-fungal polypeptides and amino acids.

In particular embodiments, the animal feed may comprise Bacillus stain DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant of DSM 29870 and 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0- 20% whey.

The animal feed may comprise Bacillus stain DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant of DSM 29870 and vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

In a particular embodiment the animal feed consists of or comprises milk (e.g., from sow), e.g., for feeding of piglets. In another particular embodiment the animal feed consists of or comprises milk replacement, e.g., for feeding of piglets.

Premix

In an embodiment, the animal feed may include a premix, comprising, e.g., vitamins, minerals, enzymes, preservatives, antibiotics, other feed ingredients or any combination thereof which are mixed into the animal feed.

Vitamins and Minerals

In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals. Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so- called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D- panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc.

Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.

Enzymes

In another embodiment, the animal feed compositions or animal feed additive described herein optionally include one or more enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: expasy.ch/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch, 2000, The ENZYME database, Nucleic Acids Res. 28: 304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, xylanase, galactanase, mannanase, dextranase and alpha-galactosidase, in families based on amino acid sequence similarities has been proposed a few years ago. They currently fall into 90 different families: See the CAZy(ModO) internet site (Coutinho and Henrissat, 1999, Carbohydrate-Active Enzymes server at URL: afmb.cnrs- mrs.fr/~cazy/CAZY/index.html (corresponding papers: Coutinho, P.M. & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrated database approach. In "Recent Advances in Carbohydrate Bioengineering", H.J. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., The Royal Society of Chemistry, Cambridge, pp. 3-12; Coutinho, P.M. & Henrissat, B. (1999) The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In "Genetics, Biochemistry and Ecology of Cellulose Degradation"., K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo, pp. 15-23).

Thus the composition of the invention may also comprise at least one other enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof.

In a particular embodiment, the composition of the invention comprises a phytase (EC

3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P and HiPhos™ (DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue (AB Enzymes), the Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in, e.g., WO 98/28408, WO 00/43503, and WO 03/066847.

In a particular embodiment, the composition of the invention comprises a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium) and Axtra® XB (Xylanase/beta-glucanase, DuPont)

In a particular embodiment, the composition of the invention comprises a protease

(EC 3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).

Amino Acids

The composition of the invention may further comprise one or more amino acids.

Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

Other feed ingredients

The composition of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides and anti-fungal polypeptides.

Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein. Examples of aroma compounds/flavorings are creosol, anethol, deca-, undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1 , Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.

Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

The composition of the invention may further comprise at least one amino acid. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

Manufacturing

Animal diets can, e.g., be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. The bacteria cultures and optionally enzymes can be added as solid or liquid formulations. For example, for mash feed a solid or liquid culture formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) culture preparation may also be added before or during the feed ingredient step. Typically a liquid culture preparation comprises the culture of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The enzyme may also be incorporated in a feed additive or premix.

The enzyme may be added to the feed mix as a granule, which is optionally pelleted or extruded. The granule typically comprises a core particle and one or more coatings, which typically are salt and/or wax coatings. The core particle can either be a homogeneous blend of an active compound optionally together with salts (e.g., organic or inorganic zinc or calcium salt) or an inert particle with an active compound applied onto it. The active compound is the culture of the invention optionally combined with one or more enzymes. The inert particle may be water soluble or water insoluble, e.g. , starch, a sugar (such as sucrose or lactose), or a salt (such as NaCI, Na ₂ S0 ₄ ). The salt coating is typically at least 1 μηι thick and can either be one particular salt or a mixture of salts, such as Na ₂ S0 ₄ , K ₂ S0 ₄ , MgS0 ₄ and/or sodium citrate. Other examples are those described in, e.g., WO 2008/017659, WO 2006/034710, WO 97/05245, WO 98/54980, WO 98/55599, WO 00/70034 or polymer coating such as described in WO 01/00042.

Alternatively, the protease can be prepared by freezing a mixture of liquid culture solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture.

Methods of treatment of C. perfringens infections and/or necrotic enteritis

In one embodiment, the invention relates to a method for treatment of C. perfringens infections and/or necrotic enteritis in an animal such as a mono-gastric animal including poultry using the composition according to Aspect 2. In one embodiment the animal is not a human being. In a further embodiment, animal feed comprising the Bacillus strains according to Aspect 1 is fed to a mono-gastric animal in an effective amount. Mono-gastric animals include, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.

The animal feed can further comprise one or more components selected from the list consisting of concentrate; forage; one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

In one embodiment, the method comprises administrering to the animal feed the Bacillus strains having the deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof in an effective amount.

In another embodiment of the method, the animal feed further comprises concentrate. In another embodiment of the method, the animal feed further comprises forage. In another embodiment of the method, the animal feed further comprises one or more additional microbes. In another embodiment of the method, the animal feed further comprises one or more enzymes. In another embodiment of the method, the animal feed further comprises one or more vitamins. In another embodiment of the method, the animal feed further comprises one or more minerals. In another embodiment of the method, the animal feed further comprises one or more amino acids. In another embodiment of the method, the animal feed further comprises one or more other feed ingredients.

In an embodiment to any of the aforementioned embodiments, the method also improves the health of the mono-gastric animal feed. In another embodiment to any of the aforementioned embodiments, the method also increases the egg yield of poultry. In an embodiment to any of the aforementioned embodiments, the method also increases the meat yield of the mono-gastric animal.

In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1x10 ⁴ and 1x10 ¹⁴ CFU/kg of forage, preferably between 1x10 ⁶ and 1x10 ¹² CFU/kg of forage, and more preferably between 1x10 ⁷ and 1x10 ¹¹ CFU/kg of animal feed. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1x10 ⁸ and 1x10 ¹⁰ CFU/kg of animal feed.

In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1x10 ³ and 1x10 ¹³ CFU/animal/day, preferably between 1x10 ⁵ and 1x10 ¹¹ CFU/animal/day, more preferably between 1x10 ⁶ and 1x10 ¹⁰ CFU/animal/day and most preferably between 1x10 ⁷ and 1x10 ⁹ CFU/animal/day.

In another aspect, the invention covers the method for treatment of C. perfringens infections comprising:

(a) feeding a mono-gastric animal a feed; and

(b) administering to an animal the strain having the deposit accession number DSM 29870; or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof;

wherein the Bacillus strain antimicrobial activity against Clostridium perfringens, and wherein step (a) occurs before, after, or simultaneously with step (b).

In a preferred embodiment of the method, the animal feed is fed to a mono-gastric animal. In an embodiment of the method, the mono-gastric animal is, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), or fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.

In another embodiment of the method, the animal feed further comprises one or more components selected from the list consisting of concentrate; forage; one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

In another embodiment of the method, the animal feed further comprises concentrate. In another embodiment of the method, the animal feed further comprises forage. In another embodiment of the method, the animal feed further comprises one or more additional microbes. In another embodiment of the method, the animal feed further comprises one or more enzymes. In another embodiment of the method, the animal feed further comprises one or more vitamins. In another embodiment of the method, the animal feed further comprises one or more minerals. In another embodiment of the method, the animal feed further comprises one or more amino acids. In another embodiment of the method, the animal feed further comprises one or more other feed ingredients.

In still yet another embodiment of the method, the one or more bacterial strains are present in the form of a stable spore. In still a further embodiment of the method, the stable spore will germinate in the rumen of the ruminant.

In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1x10 ⁴ and 1x10 ¹⁴ CFU/kg of forage, preferably between 1x10 ⁶ and 1x10 ¹² CFU/kg of forage, and more preferably between 1x10 ⁷ and 1x10 ¹¹ CFU/kg of forage. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1x10 ⁸ and 1x10 ¹⁰ CFU/kg of forage.

In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1x10 ⁵ and 1x10 ¹⁵ CFU/animal/day, preferably between 1x10 ⁷ and 1x10 ¹³ CFU/animal/day, and more preferably between 1x10 ⁸ and 1x10 ¹² CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1x10 ⁹ and 1x10 ¹¹ CFU/animal/day.

In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each Bacillus spore is between 1x10 ⁵ and 1x10 ¹⁵ CFU/animal/day, preferably between 1x10 ⁷ and 1x10 ¹³ CFU/animal/day, and more preferably between 1x10 ⁸ and 1x10 ¹² CFU/animal/day.

The invention relates in a further embodiment to use of the animal feed composition to improve one or more performance parameters in an animal, wherein the performance parameters are selected from the list consisting of improving the feed conversion ratio, improving the body weight gain, improving the feed efficiency, improving the European Production Efficacy Factor and improving the health. Preferred embodiments

Preferred embodiments of the invention are described in the set of items herein below (ITEM SET I and ITEM SET II). ITEM SET I:

1. A Bacillus strain comprising one or more of the features selected from the group consisting of:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and/or

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and/or

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and/or

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

2. A Bacillus strain comprising:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4.

3. A Bacillus strain comprising:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8. 4. A Bacillus strain comprising:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%. 5. A Bacillus strain comprising:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8.

6. A Bacillus strain comprising:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

7. A Bacillus strain comprising:

i) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

ii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

8. A Bacillus strain comprising:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a gyrA gene with at least 89.5% sequence identity to SEQ I D NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8.

9. A Bacillus strain comprising:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%. 10. A Bacillus strain comprising:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

11. A Bacillus strain comprising:

i) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

ii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

12. The Bacillus strain according to item 1 , wherein the Bacillus strain comprises:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2 and

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4 and

iii) a gyrA gene with at least 89.5% sequence identity to SEQ ID NO: 7 or a gyrA gene product with at least 97.4% sequence identity to SEQ ID NO: 8 and

iv) a genome with an Average Nucleotide Identity to the genome sequence of DSM 29870 of at least 95%.

13. The Bacillus strain according to any of the previous items, wherein the Bacillus strain is non-hemolytic, e.g., as determined in Example 8.

14. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has antimicrobial activity. 15. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens, e.g., as determined in Example 6.

16. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has antimicrobial activity against E. coli, e.g., as determined in Example 7. 17. The Bacillus strain according to any of the previous items, wherein the Bacillus strain is sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracyclin, e.g., as determined in Example 9. 18. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has a high compatibility with monensin such as being compatible with at least 2.3 μg/ml monensin as determined in Example 12 (such as at least 2.4 μg/ml monensin, such as at least 2.5 μg/ml monensin, such as at least 2.6 μg/ml monensin or such as at least 2.7 μg/ml monensin as determined in Example 12).

19. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has a 16S rDNA with more than 98% sequence identity to SEQ ID NO: 9.

20. The Bacillus strain according to any of the previous items, wherein the Bacillus strain is a Bacillus subtilis strai n .

21. The Bacillus strain according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.

22. A Bacillus having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

23. A composition comprising spores of a Bacillus strain according to any of items 1 to 22.

24. The composition according to item 23, wherein the Bacillus spores of the composition are present as dried spores.

25. The composition according to any of items 23 to 24 which further comprises a carrier.

26. The composition according to item 25, wherein the carrier comprises one or more of the following compounds: water, glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium aluminium silicate, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, farigel, cassava cores, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol and cellulose.

27. The composition according to any of items 23 to 26, wherein the composition comprises from 10 ⁵ to 10 ¹² CFU/g of isolated Bacillus spores.

28. An animal feed or an animal feed additive comprising the composition of any of items 23 to 27. 29. The animal feed or animal feed additive of item 28, wherein at least 70% (such as at least 80% or at least 90%) of the Bacillus spores survive gastric stability in a mono-gastric animal such as chickens.

30. The animal feed or animal feed additive of any of items 28 to 29 which further comprises one or more components selected from the list consisting of:

one or more enzymes;

one or more additional microbes;

one or more vitamins;

one or more minerals;

one or more amino acids; and

one or more other feed ingredients.

31. The animal feed or animal feed additive of any of items 28 to 30, wherein the bacterial count of each Bacillus spore is 1x10 ⁴ and 1x10 ¹⁸ CFU/kg of composition, preferably between 1x10 ⁶ and 1x10 ¹⁶ CFU/kg of composition, and more preferably between 1x10 ⁸ and 1x10 ¹⁴ CFU/kg of composition.

32. The animal feed or animal feed additive of any of items 28 to 31 , wherein the animal feed composition is a mono-gastric animal feed.

33. The animal feed or animal feed additive of any of items 28 to 32, wherein the animal feed or animal feed additive improves gut health of chickens with infection of Clostridium perfringens by having antimicrobial activity against Clostridium perfringens. 34. The mono-gastric animal feed according to item 32, wherein the mono-gastric animal is selected from the group consisting of pigs, swine, piglets, sows, poultry, turkeys, ducks, chicken, broilers, layers, chicks, fish and crustaceans. 35. The composition according to any of items 23 to 34 for treatment of necrotic enteritis or treatment of a Clostridium perfringens infection. 36. The composition according to item 35 for treatment of mono-gastric animals.

37. A method of treating a Clostridium perfringens infection or for treating necrotic enteritis in one or more animals comprising administrating to the one or more animals the composition of any of items 23 to 36 to the animal.

38. Use of the composition according to any of items 23 to 27 or the animal feed or animal feed additive of any of claims 28 to 33 to improve one or more performance parameters in an animal, wherein the performance parameters are selected from the list consisting of improving the feed conversion ratio, improving the body weight gain, improving the feed efficiency, improving the European Production Efficacy Factor and improving the health.

39. A biologically pure culture of the Bacillus strain according to any of items 1 to 22.

40. An isolated Bacillus strain according to any of items 1 to 22.

41. An isolated Bacillus strain selected from the group consisting of Bacillus strain DSM 29870, a strain having all of the identifying characteristics of Bacillus DSM 29870 and a mutant thereof. 42. The isolated Bacillus strain according to item 41 , wherein the identifying characteristics can one or more (such as all) of the characteristics selected from the group consisting of i) non-hemolytic, e.g., as determined in Example 8,

ii) antimicrobial activity against Clostridium perfringens, e.g., as determined in Example 6, iii) antimicrobial activity against E. coli, e.g., as determined in Example 7,

iv) sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracyclin, e.g., as determined in Example 9, and v) high compatibility with monensin such as being compatible with at least 2.3 μg/ml monensin as determined in Example 12 (such as at least 2.4 μg/ml monensin, such as at least 2.5 μg/ml monensin, such as at least 2.6 μg/ml monensin or such as at least 2.7 μg/ml monensin as determined in Example 12). 43. The Bacillus strain according to any of items 1 to 22, the composition according to any of items 23 to 27 or the animal feed or animal feed additive of any of claims 28 to 33 for treatment of a Clostridium perfringens infection and/or for treatment of necrotic enteritis in one or more animals.

ITEM SET II:

1. A Bacillus strain comprising one or more of the features selected from the group consisting of:

i) a gyrB gene with at least 94.7% sequence identity to SEQ ID NO: 1 or a gyrB gene product with at least 99.4% sequence identity to SEQ ID NO: 2;

ii) an rpoB gene with at least 96.6% sequence identity to SEQ ID NO: 3 or an rpoB gene product with at least 99.7% sequence identity to SEQ ID NO: 4;

and

iii) a genome with an Average Nucleotide Identity to the genome sequence of DSM

29870 of at least 95%.

2. The Bacillus strain according to item 1 , wherein the Bacillus strain is non-hemolytic and/or is compatible with monensin (such as with at least 2.4 μg/ml monensin as determined in Example 12).

3. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens or E. coli. 4. The Bacillus strain according to any of the previous items, wherein the Bacillus strain is sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracyclin.

5. The Bacillus strain according to any of the previous items, wherein the Bacillus strain has a 16S rDNA with more than 98% sequence identity to SEQ ID NO: 9.

6. The Bacillus strain according to any of the previous items, wherein the Bacillus strain is a Bacillus subtilis strain. 7. A Bacillus having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. 8. A composition comprising spores of a Bacillus strain according to any of items 1 to 7.

9. The composition according to item 8 which further comprises a carrier. 10. The composition according to item 9, wherein the carrier comprises one or more of the following compounds: glycerol, ethylene glycol, 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose.

11. The composition according to any of items 8 to 10, wherein the composition comprises from 10 ⁵ to 10 ¹² CFU/g of isolated Bacillus spores.

12. The composition according to any of items 8 to 11 , wherein the composition is an animal feed or animal feed additive.

13. The composition according to any of items 8 to 11 for treatment of necrotic enteritis or treatment of a Clostridium perfringens infection. 14. A method of treating a Clostridium perfringens infection or for treating necrotic enteritis in one or more animals comprising administrating the composition according to any of items 8 to 13 to the animals.

15. Use of the composition according to any of items 8 to 13 to improve one or more performance parameters in an animal, wherein the performance parameters are selected from the list consisting of improving the feed conversion ratio, improving the body weight gain, improving the feed efficiency, improving the European Production Efficacy Factor and improving the health. Examples

Example 1 : Identification, characterization and deposit of the biological material

The following biological material was deposited under the terms of the Budapest Treaty at Leibniz-lnstitut DSMZ-Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH, InhoffenstraBe 7 B, 38124 Braunschweig Germany, and given the following accession number: Table 1.1 : Deposit of Biological Material

Bacillus subtilis DSM 29870 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected at Jamaica in 1990. The strain was identified as Bacillus subtilis based on 16S rDNA sequencing.

The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposits represent a substantially pure culture of the deposited strain. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Sequencing of 16S rDNA gene

DNA was extracted from a culture of DSM 29870 using QiaAmp DNA Blood Mini Kit

(Qiagen art 51 106). The kit was used as recommended for extraction of DNA from gram positive bacteria.

16S rDNA was amplified in a total volume of 50 μΙ by mixing: 10 pmol of each of Primer 16S-F and 16S-R (Table 1.2), 0.2 mM of each nucleotide, 2.5 units Ampli taq, 1 x Ampli taq buffer and 5 μΙ DNA template.

The following PCR program: 94°C min 2 min (94°C 30 s, 52°C 30 s, 72°C 1 min)x35, 72°C 10 min was applied on a Perkin Elmer PCR machine. PCR product was sequenced using primer 794-R, 357-F, 1390-R and 1000-F (Table 1.2) on an ABI Prism sequencer. Table 1.2: Primers:

Degeneration of primer: R is A or G. The 16 S rDNA sequence is shown in SEQ ID NO: 9, the sequence was analysed by BLAST against EMBL database and showed identity to 16 S rDNA sequences of Bacillus subtilis. Example 2: Whole genome sequencing and Taxonomic placement of Bacillus strain DSM 29870

Summary:

Average nuclear identity (AN I) and phylogenetics, reveal that the Bacillus strain DSM 29870 is a novel subspecies of Bacillus subtilis.

Materials and Methods:

Genome sequencing of DSM 29870

DNA was extracted from a culture of DSM 29870 using QiaAmp DNA Blood Mini Kit (Qiagen art 51 106). The kit was used as recommended for extraction of DNA from gram positive bacteria. Genomic DNA was fragmented using a Covaris M220™ ultrasonicator (Covaris, Inc., Woburn, MA) and was used to generate TruSeq™ library ((lllumina, Inc., San Diego, CA) using the Apollo 324™ library prep system (Wafergen Biosystems, Inc., Fremont, CA). The prepared libraries were sequenced using a MiSeq desktop sequencer (lllumina, Inc., San Diego, CA) with MiSeq Reagent Kit v3 (600 cycles) to generate ~ 360 bp forward and 240 bp reverse paired end reads. Sequences were trimmed and denovo assembled in CLC Genomics Workbench 7.0.4 (CLC Bio, Aarhus, Denmark) using the respective modules.

The following main trimming parameters were used: Ambiguous trim = Yes, Ambiguous limit = 2, Quality trim = Yes, Quality Limit = 0.01 , Minimum number of nucleotides = 50, Save broken pairs = Yes. The following main denovo assembly parameters were used: Mapping mode = Map reads back to contigs, Update contigs = Yes, Automatic bibble size = Yes, Minimum contig length = 200, Perform scaffolding = yes, Mismatch cost = 2, Insertion cost = 3, Deletion cost = 3, Length fraction = 0.8, Similarity graction = 0.95.

Benchmark Genome sequences:

Complete and draft genomes of the public Bacillus strains were downloaded from

NCBI genomes on December 29, 2014.

Core genome phylogenetics:

Prodigal version 2.6 [Hyatt et al., 2010, Prodigal: prokaryotic gene recognition and translation initiation site identification; BMC Bioinformatics 1 1 : 119] was used to determine protein sequences. PhyloPhlan [Segata et al., 2013, PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes, Nat. Commun. 4: 2304] was used to generate a core genome phylogenetic tree based on phylogenetic signals from 400 most conserved protein sequences.

Dendroscope 3 [Huson et al. , 2-12. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks, Syst. Biol. 61 : 1061-1067] was used to visualize the tree as a rectangular phylogram with midpoint rooting along with bootstrap support (Figure 3).

ANI estimations:

An open source Ruby script (ani.rb) for ANI estimation was obtained from the Kostas lab (enveomics.blogspot.com/2013/10/anirb.html) and used for pair-wise ANI estimations.

Detailed methods:

All the scripts and programs executed in command line were either run on Ubuntu version 10.04 or version 12.04 (Canonical Ltd.).

Core genome phylogenetics:

All the fasta files of the genomes were moved to the same folder and then prodigal version 2.6 was used to determine protein sequences as given in the following example:

Example of prodigal script execution

prodigal -i RO_NN-1_CP002906.fasta -a ./Protein_files/RO_NN-1_CP002906.faa -c - m

All the protein sequences were then moved to a folder in the input folder of the Phylophlan working directory (For example under Protein_files) and then phylophlan was executed as in the following example:

Example of PhyloPhlan script execution

./phylophlan. py -nproc 14 -u Protein_files

The newick file generated in output folder was then used as an input for Dendroscope 3 for midpoint rooting and the image was exported in the desired format after the font sizes were adjusted and the desired tree rendering method was selected.

ANI estimations:

Fasta files of the genomes of each strain were compared pairwise using ani.rb.

Example of ani script execution:

ruby ./ani.rb -1 O52BCU_EFGB10.fasta -2 RO_NN-1_CP002906.fasta

The results were output on screen and copied into word document and then summarized as a table. Results and discussion:

ANI estimations:

Average nuclear identity (ANI) is a distance based approach to delineate species based on pair-wise comparisons of genome sequences [Goris et al., 2007, "DNA-DNA hybridization values and their relationship to whole-genome sequence similarities", Int. J. Syst. Evol. Microbiol. 57: 81-91]. See the definition of ANI for further details.

Table 2.1 shows pair-wise ANI estimations of Strain DSM 29870 compared to other Bacillus species. Data shows that Strain DSM 29870 is most identical (>93%) to Bacillus subtilis spizezenii TU-B-10 and Bacillus vallismortis. The identity is lower in all other comparisons with species within the Bacillus genus. The ANI value is less than 95% in all comparisons and therefore it is difficult to make a definitive statement of taxonomic placement using ANI alone. ANI values less than 95% but higher than 93% are also observed between Bacillus amyloliquefaciens plantarum and amyloliquefaciens subspecies. Therefore it is quite possible that Strain DSM 29870 is a subspecies of Bacillus subtilis.

Table 2.1 : Two-way pairwise ANI of Bacillus strain 052BCU compared to other bacterial

Phylogenetics:

Phylogenetic analysis placed Strain DSM 29870 close to Bacillus subtilis subspecies spizezenii and Bacillus vallismortis (Figure 3). Figure 3 is zoomed in to show Strain DSM 29870 relationship to its nearest neighbors. See Figure 2 for the complete phylogram. Conclusion:

The Average Nuclear Identity (ANI) value is less than 95% in all comparisons and therefore it is difficult to make a definitive statement of taxonomic placement using ANI alone. However, in combination with phylogenetics, we conclude that the bacterial strain DSM 29870 is distinct to known hitherto known Bacillus strains to a level that it at least represents a novel subspecies of Bacillus subtilis. Example 3: GyrB analysis

Background

Comparative analysis of the gyrB gene encoding the subunit B of the DNA gyrase protein was previously shown to be an efficient tool for taxonomic characterization of members of the Bacillus subtilis group. [International Journal of Systematic and Evolutionary Microbiology (2007), 57: 1846-1850].

The gyrB gene sequence of Bacillus subtilis strain 168 deposited as CP010052_13 in the EMBL database was used to find the gyrB gene in the genome sequence of DSM 29870.

With a Pearl Script the EMBL:CP010052_13 was used for a BlastN analysis that gave the location - contig number, position and orientation - of the gyrB gene in the genome sequence of DSM 29870. The gyrB gene sequence of DSM 29870 was subsequently manually extracted from the genome sequence.

For the comparative analysis we selected to use only a partial gyrB gene. The partial sequence of the gyrB gene of Bacillus subtilis DSM 29870 is shown in SEQ ID NO: 1. The sequence was translated into amino acid sequence (SEQ ID NO: 2). The amino acid sequence of the partial gyrB gene product cover amino acids 49-614 of the gyrB gene product in Bacillus subtilis type strain UNIPROT:P05652 [Moriya et al., 1985, "Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region." Nucleic Acids Res. 13: 2251-2265].

The Amino acid and DNA sequences were analyzed by BLAST [Altschul et al., 1990, "Basic local alignment search tool", J. Mol. Biol. 215: 403-410]. The amino acid sequence showed 99.3% identity to the closest related strain of Bacillus subtilis, the DNA sequence showed 94.6 % identity to the closest relative, Bacillus subtilis subsp. Spizizenii. This indicates that Bacillus subtilis DSM 29870 is a novel subspecies of Bacillus subtilis.

Example 4: rpoB analysis

Background

The rpoB gene encoding the RNA polymers beta subunit was previously used as a phylogenetic marker. The extensive use of rpoB was reviewed [Adekambi et al., 2009, "The rpoB gene as a tool for clinical microbiologists", Trends in Microbiology 17(1): 37-45]. It was used to discriminate sub-groups/closely related Bacillus species [Qi et al., 2001 , "Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis", Appl. Environ. Microbiol. 67(8): 3720-3727].

The rpoB gene sequence of Bacillus subtilis wild type Marburg strain deposited in the

EMBL database under L24376 [Boor et al., 1995, "Genetic and transcriptional organization of the region encoding the beta subunit of Bacillus subtilis RNA polymerase", J. Biol. Chem. 270(35): 20329-20336] was used to find the rpoB gene in the genome sequence of DSM 29870.

With a Pearl Script the EMBL:L24376 was used for a BlastN analysis that gave the location - contig number, position and orientation - of the rpoB gene of DSM 29870. The rpoB gene sequence of DSM 29870 was manually extracted from the genome sequence. For the comparative analysis we selected to use only a partial rpoB gene. The partial sequence of the rpoB gene of Bacillus subtilis DSM 29870 is shown in SEQ ID NO: 3.

The sequence was translated into the amino acid sequence. The amino acid sequence of the partial rpoB gene product is shown in SEQ ID NO: 4. It covers the amino acids equivalent to 1 to 1 193 of the rpoB gene product in Bacillus subtilis of EMBL: L24376 [Boor et al., 1995, "Genetic and transcriptional organization of the region encoding the beta subunit of Bacillus subtilis RNA polymerase", J. Biol. Chem. 270(35): 20329-20336].

The amino acid and DNA sequences were analyzed by BLAST [Altschul et al., 1990, "Basic local alignment search tool." J. Mol. Biol. 215: 403-410]. The amino acid sequence showed 99.4% identity to the closest related strain of Bacillus subtilis, the DNA sequence showed 96.5% identity to the closest relative, Bacillus subtilis subsp. Spizizenii. This indicates that Bacillus subtilis DSM 29870 is a novel subspecies of Bacillus subtilis.

Example 5: Comparative analysis of GyrA extracted from the genome sequence

Background

Partial sequence of the gyrA gene encoding the alpha subunit of the DNA gyrase protein has previously been used as a phylogenetic marker to discriminate members of the Bacillus subtilis group. Twelve strains of Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. spizizenii and Bacillus vallismortis were sequenced, and it was shown that Bacillus subtilis subsp. subtilis could be discriminated from Bacillus subtilis subsp. spizizenii based on this gene sequence [Chun & Bae, 2000, "Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences", Antonie van Leeuwenhoek 78: 123- 127,]. The partial gyrA gene sequence of Bacillus amyloliquefaciens KCTC 1660 ^T deposited in EMBL as AF272015 was used to identify and extract the gyrA gene from the genome of DSM 29870.

Data analysis

With a Pearl Script the EMBLAF272015 was used for a BlastN analysis that gave the location - contig number, position and orientation - of the partial gyrA gene of DSM 29870. The partial gyrA gene sequence of DSM 29870 was manually extracted from the genome sequence. The partial sequence of the gyrA gene of Bacillus subtilis DSM 29870 is shown in SEQ ID NO: 7. The sequence was translated into amino acid sequence. The amino acid sequence of the partial gyrA gene product covers the amino-acids shown in SEQ ID NO: 8. The Amino acid and DNA sequences were analyzed by BLAST [Altschul et al., 1990, "Basic local alignment search tool", J. Mol. Biol. 215: 403-410]. The amino acid sequence show 97.3% identity to the closest related strain of Bacillus subtilis subsp spizizenii TU-B10, the DNA sequence showed 89.4% identity to the closest relative, Bacillus subtilis subsp. spizizenii ATCC6633, this indicates that Bacillus subtilis DSM 29870 is a novel subspecies of Bacillus subtilis.

Example 6: Determination of inhibition of growth of Clostridium perfringens

Clostridium perfringens strains, 23 and 48 (both are netB positive) [Gholamiandekhordi et al., 2006, "Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different diseasestatus", Vet. Microbiol. 1 13: 143- 152] were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35°C under static anaerobic conditions. 250 μΙ_ of the overnight culture of Clostridium perfringens was added to 250 mL of tryptic soy agar supplemented with 0.6% yeast extract at 40°C and poured into rectangular petri plates (Nunc part 267060). The inoculated agar was then allowed to cool at room temperature after which an 8mm diameter well was made in the agar. Plates were stored in absence of oxygen until use.

Bacillus DSM 29870 was grown overnight in tryptic soy broth at 35°C under aerobic conditions. 1000 μΙ_ of the Bacillus culture was collected and fractionated into cell-free supernatant and cells by centrifugation. 20 μΙ_ of cell-free supernatant or 100x diluted cells in phosphate buffered saline were added directly to the wells in the Clostridium perfringens inoculated agar plates. A control well contained 20 μΙ_ of phosphate buffer saline. The plates were incubated for 18 hours at 35°C under anaerobic conditions.

Inhibition of the Clostridium perfringens strain was noted by a circular clearing zone around the well of interest. The phosphate buffer saline well was considered a negative control based on lack of clearing zone around the well.

Cell-free supernatant and 100x diluted cells of Bacillus strain DSM 29870 was able to consistently inhibit growth of C. perfringens strains 23 and 48 in vitro. Inhibition was also seen by competitor strain "CloSTAT", for both supernatant and cells, but was not seen with competitor strain GalliproTect. "CloSTAT" is a strain of Bacillus amyloliquefaciens that was isolated from the commercial DFM product CloSTAT, Kemin. "GalliproTect" is a strain of Bacillus licheniformis that was isolated from the commercial product Gallipro Tect, Chr. Hansen. Example 7: Determination of inhibition of growth of Escherichia coli

Escherichia coli strains, ATCC10536 or ATCC25922, were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35°C under static anaerobic conditions. 100 μΙ_ of the overnight culture of Escherichia coli was added to 250 ml_ of tryptic soy agar supplemented with 0.6% yeast extract at 40°C and poured into rectangular petri plates (Nunc part 267060). The inoculated agar was then allowed to cool at room temperature after which an 8 mm diameter well was made in the agar.

Bacillus DSM 29870 was grown overnight in tryptic soy broth at 35°C under aerobic conditions. 1000 μΙ_ of the Bacillus culture was collected and fractionated into cell-free supernatant and cells by centrifugation. 20 μΙ_ of cell-free supernatant or 100x diluted cells in phosphate buffer saline were added directly to the wells in the Escherichia coli inoculated agar plates. A control well contained 20 μΙ_ of phosphate buffer saline. The plates were incubated for 18 hours at 30°C under aerobic conditions.

Inhibition of the Escherichia coli strain was noted by a circular clearing zone around the well of interest. The phosphate buffer saline well was considered a negative control based on lack of clearing zone around the well.

Cell-free supernatant and 100x diluted cells of Bacillus strains DSM 29870 was able to consistently inhibit growth of E. coli strains ATCC10535 and ATCC25922 in vitro. Inhibition was also seen by competitor strain CloSTAT, for both supernatant and cells.

Example 8: Non-hemolytic

Hemolysis was tested according to the Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition, EFSA Journal 201 1 ; 9(11):2445.

Sheep blood agar plates were purchased as ready to use (Becton Dickenson art 254053 or 254087). Alternatively, the agar plates can be prepared by adding 5% defibrinated sheep blood (obtained from Statens Serum Institute, Denmark) to TS-agar (Oxoid CM 131). Agar should be autoclaved at 121 °C for 20 minutes and cooled down to about 40°C before adding the blood immediately before pouring the plates.

The Bacillus strains were taken from the preservation at -80°C and streaked on TSA agar plates, which were incubated at 30°C overnight or until growth appeared. From a single colony as little as possible of the material was used to streak a line on ¼ of an agar plate. The plate was incubated at 30°C for 72 hours. Hemolysis/clearing zones of the Bacillus strains to be tested were compared with the positive and negative control. As positive control Bacillus subtilis ATCC 21332 was used. As negative control Bacillus subtilis 168 was used. In a screening of 599 independent isolates of Bacillus 223 strains (37%) were hemolysis negative. In another screening 21 of 65 independent isolates of Bacillus (32%) were hemolysis negative. Both screenings exclusively comprised strains that based on identification by 16S rDNA sequencing belong to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus and Bacillus amyloliquefaciens (or Bacillus species that are difficult to discriminate from these species based on 16 S rDNA sequencing).

The non-Hemolytic Bacillus strains therefore seem to be common and fairly abundant in nature, but only comprising a minority of the natural strains. The non-hemolytic strains seem to be more abundant in Bacillus licheniformis, while most Bacillus amyloliquefaciens strains appear hemolytic. The following strain from this screening was hemolysis negative and selected for further studies: Bacillus DSM 29870.

Example 9: Sensitivity to antibiotics

The minimal inhibitory concentrations (MIC) of eight antibiotics against Bacillus strains DSM 29870 were determined using broth micro dilution essentially as described in the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). Only modification was that volumes were changed; 90 μΙ MHB with bacteria was added to 10 μΙ antibiotics dilutions, cfu's of bacteria and concentration of antibiotics were changed so final concentrations and final cfu's matched the guideline. The plates were incubated for 20-24 h instead of 16-20 h. The control strain recommended in the CLSI standard Staphylococcus aureus ATCC 29213 was used as control strain.

Materials

Strains:

Bacillus subtilis DSM 29870

S. aureus ATCC 29213

Antibiotics:

Chloramphenicol (Sigma C1919, 10 mg/ml, solubilized in 96% ethanol)

Clindamycin (Sigma PHR1159, 10 mg/ml, solubilized in water)

Erythromycin (ABBOTICIN from Amdipharm 40501 , 50 mg/ml, solubilized in 96% ethanol) Gentamycin (Biomedicals inc., 190057, 10 mg/ml, solubilized in water)

Kanamycin (Sigma, CAS no. 25389-94-0, 50 mg/ml, solubilized in water)

Streptomycin (Sigma, S1277, 10 mg/ml, solubilized in water)

Tetracycline (Sigma, T3383, 10 mg/ml, solubilized in water)

Vancomycin (Sigma, V-8138, 10 mg/ml, solubilized in water) Mueller Hinton Broth 2 (Sigma/Fluka 90922)

0.9% NaCI (Sigma/RdH 31434/Merck 106404)

Tryptone soya agar plates (Oxoid CM 131)

Microtiter plates: Costar plate, polypropylene, round bottom, Corning 3879

Method

Preparation of bacteria:

A few colonies of Bacillus spp. (<1 day old) were inoculated into Mueller Hinton Broth 2 (MHB) and incubated for around 4 hours at 37°C. OD ₆₀₀ (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.25 (equivalent to McFarland 0.5) in MHB. For the control strain direct colony suspension was used. A few colonies of S. aureus ATCC 29213 (<1 day old) were suspended in MHB and OD ₆₀₀ (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.10-0.12 (equivalent to McFarland 0.5) in MHB. The bacterial suspensions were diluted 200x in MHB.

Preparation of assay plates:

Antibiotics were diluted to the concentration of 640 μg/ml in MHB. A two fold dilution series was prepared in MHB down to the concentration 0.625 μg/ml. 10 μΙ of each dilution and of each antibiotic was pipetted into a microtiter plates. Later, when the antibiotics were mixed with the suspension of bacteria, the samples were diluted 10x (10 μΙ_ sample in a total volume of 100 μΙ). This resulted in the final test range of 0.06-64 μg/ml.

If the plates were not used right away the plates are stored in the freezer at -20°C until usage.

90 μΙ of the bacterial suspensions were added to the assay plates. The assay plates were incubated in a plastic bag with at wet cloth at 37°C for 20-24 h. The MIC was determined as the lowest concentration of antibiotic that completely inhibited growth of bacteria as detected by the unaided eye. CFU estimation:

A 10-fold dilution series in 0.9% NaCI was made to the 10 ^"3 of the cultures inoculated into the microtiter plates. 2x 100 ul from the 10 ^"3 dilution were plated onto two TSA plates. The plates were incubated overnight at 37°C. Number of CFU/ml was counted.

Three biological replicates of the assay were performed for Bacillus DSM 29870. Results:

The MIC values obtained for B. subtilis DSM 29870 showed that the breakpoint values were equal to or below the breakpoint values given in the EFSA guideline (EFSA Journal 2012; 10(6):2740).

As a control S. aureus ATCC 29213 was tested in parallel and had MIC values within the ranges given by the CLSI standard (M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014).

The amount of bacteria inoculated into the assay plates was measured (CFU/ml). In general the CFU/ml was very close to the target value of 1.5*10 ⁵ CFU/ml. However, the CFU/ml for the Bacillus strains was most possibly higher than the actual value, since the bacteria tend to aggregate and one aggregated will only result in one colony forming unit (Tables 9.1 and 9.2).

Table 9.1 : MIC results for B. subtilis DSM 29870

ourna ; :

Table 9.2: MIC results for S. aureus ATCC 29213

M100-S24 Performance Standards Antimicrobial Susceptibility Testing; informational Supplement, 2014 Example 10 gyrB analysis

PCR amplification of gyrB

For the PCR amplification 1 μΙ_ of genomic DNA (see Example 2) was mixed with 12.5 μΙ_ Reddymix Extensor PCR solution (Thermo Fisher Scientific, Surrey, UK), 1 μΙ_ of each of 10 μΜ solutions of primers 3AF and 4R2 (Table 10.1) and 9.5 μΙ distilled water. For negative control PCR reactions 1 μΙ_ MQ water was added instead of DNA.

Table 10.1 : Primers:

Degeneration of primers: M is A or C, S is G or C, Y is C or T and R is A or G. The PCR thermal cycler (DNA Engine DYAD BIORAD) was programmed to run; 92°C for 2 min, 40*[94°C for 30 s, 52°C for 30 s, 72°C for 60 s], 72°C for 10 min. The PCR product was evaluated by agarose gel electrophoresis on FlashGel cassette (Lonza, Rockland, ME USA), using FlashGel DNA marker 100 bp-4000 bp, to estimate size of the amplicon. The PCR amplification of gyrB gene was successful when a band of about 1700 nt was seen on the gel. Purification of PCR products

The PCR purification was done with Multiscreen PCR 96 well filterplate (Millipore, Ireland); The entire volume of the PCR reaction was taken to one well in a 96 well plate, MilliQ water up to 100 μΙ_ was added. The plate was subjected to vacuum until it was dry (20- 25 s) 100 μΙ_ milli water was added, the vacuum step was repeated. The PCR product was eluted by addition of 50 μΙ_ elution buffer (elution buffer was water) and pipetting the PCR product away from the top of the filter. The purified PCR product was stored at -22°C.

Sequencing of the genes

The DNA concentration of the purified PCR product was quantified on NanoDrop

1000 Spectrophotometer (Thermo Fisher, Waltham, MA, USA). Four sequence reactions were set up each using 10 ng PCR product, 1 uL primer of 10 mM of one of the primers C-F, D-R, BS-F or BS-R (Table 10.2) and distilled water to 12 μΙ_. The solutions were mixed and used for Sanger sequencing on an ABI Prism sequencer [Sanger, »DNA sequencing with chain-terminating inhibitors, « Proceedings of the National Academy of Sciences of the United States of America, p. 5463-5467, December 1977].

Table 10.2: Primers:

Degenerations of primers: Y is C or T, I is inosine, S is G or C and M is A or C, N is A, C, G or T.

The sequence traces were assembled to a partial gyrB gene using the SeqMan program of the Lasergene (DNA STAR 7, Lasergene). The sequence of the entire PCR product minus the primer region of Bacillus subtilis DSM 29870 is shown in SEQ ID NO 32. The sequence was translated into amino acid sequence (SEQ ID NO: 33). The amino acid sequence of the partial gyrB gene product cover amino acids 49-614 of the gyrB gene product in Bacillus subtilis type strain UNIPROT:P05652 [Moriya et al., 1985, "Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region", Nucleic Acids Res. 13: 2251-2265]. The Amino acid and DNA sequences were analyzed by BLAST [Altschul et al., 1990, "Basic local alignment search tool", J. Mol. Biol. 215: 403-410]. The amino acid sequence showed 99.2% identity to the two closest related sequences which were from Bacillus subtilis subsp. spizizenii strains ATCC663 and W23, respectively. The DNA sequence showed 94.7 % identity to the closest related sequence which was from Bacillus subtilis subsp. spizizenii TU-B-10, this indicates that Bacillus subtilis DSM 29870 is a novel subspecies of Bacillus subtilis. Example 11 rpoB analysis

Background

The rpoB gene encoding the RNA polymers beta subunit was previously used as a phylogenetic marker to discriminate sub-groups/closely related species in Bacillus [Qi et al., 2001 , "Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis", Appl. Environ. Microbiol. 67(8): 3720-3727]. By comparison of rpoB genes from Bacillus amyloliquefaciens and Bacillus subtilis primers were designed and a similar sequenced based evaluation was used to show that DSM 29870 is a novel subspecies of Bacillus subtilis.

PCR amplification of rpoB

For the PCR amplification 1 μΙ_ of genomic DNA (see Example 2) was mixed with 12.5 μΙ Reddymix Extensor PCR solution (Thermo Fisher Scientific, Surrey, UK), 1 μΙ_ of each of 10 μΜ solutions of primers rpoB-PCR-F and rpoB-PCR-R (Table 11.1) and 9.5 μΙ distilled water. For negative control PCR reactions 1 μΙ_ MQ water was added instead of DNA.

Table 1 1.1 : Primers:

The PCR thermal cycler (DNA Engine DYAD BIORAD) was programmed to run; 92°C for 2 min, 40*[94°C for 30 s, 52°C for 30 s, 72°C for 60 s], 72°C for 10 min. The PCR product was evaluated by agarose gel electrophoresis on FlashGel cassette (Lonza, Rockland, ME USA), using FlashGel DNA marker 100-4000 bp, to estimate size of the amplicon. The PCR amplification of rpoB gene was successful when a band of about 3600 nt was seen on the gel (theoretical size is 3639 nt based on embl:L24376). Purification of PCR products

The PCR purification was done with Multiscreen PCR 96 well filterplate (Millipore, Ireland); The entire volume of the PCR reaction was taken to one well in a 96 well plate, MilliQ water up to 100 μΙΙ_ was added. The plate was subjected to vacuum until it was dry (20-25 s) 100 μΙ_ milli water was added, the vacuum step was repeated. The PCR product was eluted by addition of 50 μΙ_ elution buffer (elution buffer was water) and pipetting the PCR product away from the top of the filter. The purified PCR product was stored at -22°C.

Sequencing of the genes

The DNA concentration of the purified PCR product was quantified on NanoDrop

1000 Spectrophotometer (Thermo Fisher, Waltham, MA, USA). Eight sequence reactions were set up each using 10 ng PCR product, 1 uL primer of 10 mM of one of the primers rpoB-seq-F1 , rpoB-seq-F2, rpoB-seq-F3, rpoB-seq-F4, rpoB-seq-R1 , rpoB-seq-R2, rpoB- seq-R3, and rpoB-seq-R4 and distilled water to 12 μΙ_. The solutions were mixed and used for Sanger sequencing on an ABI Prism sequencer.

Table 1 1.2: Primers:

The sequence traces were assembled to a partial rpoB gene using the SeqMan program of the Laser Gene (DNA STAR 7, lasergene) the sequence of the entire PCR product minus the primer region of Bacillus subtilis DSM 29870 is shown in SEQ ID NO: 34. The sequence was translated into amino acid sequence (SEQ ID NO:35). The amino acid sequence of the partial gyrB gene product cover amino acids 49-614 of the rpoB gene product in Bacillus subtilis type strain UNIPROT:P05652 [Moriya et al., 1985, "Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region", Nucleic Acids Res. 13: 2251-2265]. The amino acid and DNA sequences were analyzed by BLAST [Altschul et al., 1990, "Basic local alignment search tool", J. Mol. Biol. 215: 403-410. The amino acid sequence showed 99.3% identity to the 3 closest related sequences of Bacillus subtilis subsp. spizizenii (strain ATCC 23059, NRRL B-14472, W23) and ATCC6633 and a third that was poorly annotated in UniProt database. The DNA sequence showed 96.5 identity to the closest related sequence which was from Bacillus subtilis subsp. spizizenii TU-B-10. This indicates that Bacillus subtilis DSM29870 is a novel subspecies of Bacillus subtilis. Example 12: Determination of Monensin Compatibility

Monensin compatibility of DSM 29870 was determined using a modified broth micro dilution similar to the method described in the Example 9. Briefly, a single colony of Bacillus spp. (from overnight tryptic soy agar plates) was inoculated into Mueller Hinton Broth (MHB) and cultured overnight. Sterile media was then inoculated with the overnight culture and allowed to grow for 4 hours to test bacteria in log growth phase. Cultures were then diluted once more 1 :200 into fresh MHB and 90 μΙ_ of this inoculated broth was added to the diluted monensin at the indicated concentrations. Prior art strains were also tested for comparison: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA- 6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439.

Strains:

Bacillus subtilis DSM 29870.

Bacillus licheniformis N N019785.

Bacillus amyloliquefaciens NN062266 (NRRL B-50013).

Bacillus subtilis NN062267 (NRRL B-50104).

Bacillus subtilis NN062278 (PTA-6507).

Bacillus amyloliquefaciens NN062319 (FERM BP-1096).

Bacillus subtilis NN062440.

Bacillus licheniformis NN062441 (DSM 17236).

Bacillus amyloliquefaciens NN062439.

Materials:

Monensin sodium salt (Sigma, CAS no. 22373-78-0, solubilized in 96% ethanol).

Mueller Hinton Broth (Becton, Dickinson and Company, 275730).

Tryptic soy agar (Becton, Dickinson and Company, 236920).

Micro titer plates: Costar plate, polypropylene, flat bottom, Corning, 3628.

Borosilicate glass tubes: Kimbale, 16 x 125 mm, 73500-16125.

Adhesive gas permeable seals: Thermo Scientific, AB-0718.

Preparation of bacteria:

Bacillus spp. were grown overnight on tryptic soy agar plates (40 g/L) at 37°C. Mueller Hinton broth (21 g/L) was dissolved in water and autoclaved in glass tubes containing 5 mL of broth each. A single colony of Bacillus spp. (from overnight plates) was inoculated into Mueller Hinton Broth (MHB) and incubated overnight at 37°C shaking at 200 rpm. A 5 mL glass tube of fresh, sterile media was then inoculated with 25 mL of overnight culture and allowed to grow for 4 hours at 37°C. Cultures were then diluted once more 1 :200 into fresh MHB. 90 μΙ_ of this inoculated broth was then added to the diluted antibiotic at the indicated concentrations. Preparation of assay plates:

Monensin was diluted into 96% ethanol to a concentration of 800 μg/mL. This solution was then diluted 10-fold into sterile phosphate buffer to a concentration of 80 μg/mL. A two fold dilution series was prepared in MHB down to the concentration 2.5 μg/mL. 10 μΙ of each dilution and of each antibiotic was pipetted into a microtiter plate. Later, when the antibiotics were mixed with the suspension of bacteria, the samples were diluted 10x (10 μΙ_ sample in a total volume of 100 μΙ). This resulted in the final test range of 0.25-8 μg/ml.

90 μΙ of the bacterial suspensions were added to the assay plates. The assay plates were then covered with an adhesive glass permeable seal and incubated overnight at 37°C shaking at 200 rpm. The maximum compatible concentration was determined similar to a MIC analysis as the concentration above that which inhibited 80% of bacteria as detected by the unaided eye.

Results:

A potential challenge of delivering Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore it is necessary to determine the compatibility of strains with commonly-used feed antibiotics in order to identify any potential conflicts with use as a direct fed microbial. Therefore, the monensin compatibility DSM 29870 was determined along with prior art strains. DSM 29870 has a higher level of compatibility with monensin than the prior art strains included herein: NN019785, NN062266 (NRRL B- 50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439.

Table 12.1 : Monensin compatibility results

NN062278 PTA-6507 Bacillus subtilis Eviva Pro (Dupont) 1.4

FERM BP- Bacillus

NN062319 Calsporin (Calpis) 2.2

1096 amyloliquefaciens

NN062440 Bacillus subtilis GalliPro Max (Chr. Hansen) 0.4

NN062441 DSM 17236 Bacillus licheniformis GalliPro Tect (Chr. Hansen) 0.9

Bacillus

NN062439 Clostat (Kemin) 2.1 amyloliquefaciens