Microorganisms and antibiotics

Abstract

The present invention relates to direct fed microbial for Escherichia coli inhibition in animals in combination with enrofloxacin. More particularly, the present invention relates to isolated Bacillus strains 9, 57, 71 and 126 and strains having all the identifying characteristics of these strains for use including the above mentioned uses.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 068,678, filed August 21, 2020, under 35 U.S.SC §119(e), the disclosure of which is expressly incorporated herein by reference. Technical Field

[0003] This invention relates to direct-feed microorganisms for the inhibition of Escherichia coli in animals in combination with the antibiotic enrofloxacin. More particularly, this invention relates to isolated Bacillus strains 9, 57, 71, and 126 for the purposes described above, including in combination with the antibiotic enrofloxacin, and strains having all the identifying characteristics of these strains.

[0004] Invention Background and Overview

[0005] This invention relates to direct feeding microbial (DFM) compositions and methods for inhibiting Escherichia coli in animals. The gastrointestinal tract of animals is constantly attacked by a large number of bacteria, viruses, fungi, and protozoa found in feed, bedding, and the environment. The gastrointestinal tract possesses a complex system against these potential pathogens, consisting of physical, chemical, and immune defenses. Beneficial bacteria are an important part of this system because they provide animals with bacteria that help establish (or rebuild) a normal bacterial profile, enhance the animal's immune system, and help fight disease.

[0006] Antibiotic resistance is an increasingly important problem in human medicine and animal husbandry. The U.S. Food and Drug Administration reported that in 2016, the U.S. swine industry purchased approximately 6.9 million pounds of medically important antibiotics, highlighting the importance of preventing the development of antibiotic-resistant organisms in the livestock industry. Methods are being developed to combat antibiotic resistance, but little is being done in utilizing synergistic direct feeding of microorganisms (such as probiotics) as a tool to combat antibiotic resistance.

[0007] The applicant has developed a direct-feeding microorganism that, in combination with enrofloxacin, results in inhibition of Escherichia coli in animals. The direct-feeding microorganism and enrofloxacin described herein, as well as compositions comprising the direct-feeding microorganism and enrofloxacin, provide commercial benefits by providing inhibition of Escherichia coli in animals, such as agricultural animals.

[0008] Methods for inhibiting *E. coli* in animals are provided. In various implementation schemes, the animals may be selected from poultry species, pig species, goat species, cattle species, sheep species, horse species, and companion animals. In implementation schemes where the animal is a poultry species, the poultry species may be selected from broiler chickens, chickens, laying hens, breeding chickens, turkeys, turkey chicks, goslings, ducklings, guinea fowl, young hens, hens, roosters, young roosters, and capons. In implementation schemes where the animal is a pig species, the pig species may be selected from growing-finishing pigs, nursery pigs, sows, and breeding pigs.

[0009] In one embodiment, a method of feeding an animal is provided. The method includes the steps of: administering to the animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains possessing all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains possessing all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains possessing all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), and strains possessing all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868). All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals, wherein the Bacillus strains and antibiotics cause Escherichia coli inhibition in animals, and wherein the antibiotic is enrofloxacin.

[0010] In another embodiment, a method for controlling the harmful effects of *Escherichia coli* is provided. The method includes the step of administering to an animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated *Bacillus* strains selected from: *Bacillus* strain 9 (NRRL No. B-67866), strains possessing all the identification characteristics of *Bacillus* strain 9 (NRRL No. B-67866), *Bacillus* strain 57 (NRRL No. B-67870), strains possessing all the identification characteristics of *Bacillus* strain 57 (NRRL No. B-67870), *Bacillus* strain 71 (NRRL No. B-67867), strains possessing all the identification characteristics of *Bacillus* strain 71 (NRRL No. B-67867), *Bacillus* strain 126 (NRRL No. B-67868), and strains possessing all the identification characteristics of *Bacillus* strain 126 (NRRL No. B-67868). All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals and control of the harmful effects of Escherichia coli in animals, wherein the antibiotic is enrofloxacin.

[0011] In yet another implementation, a method for feeding animals is provided. The method includes the following steps: administering to an animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof; and administering to the animal an antibiotic, wherein the antibiotic is enrofloxacin.

[0012] The following clauses and combinations thereof provide various additional illustrative aspects of the invention described herein. The various embodiments described in any other section of this patent application (including the section entitled "Detailed Description of Illustrative Embodiments" and the examples) are applicable to any of the following embodiments of the invention described in the following numbered clauses.

[0013] 1. A method of feeding animals, the method comprising the steps of: administering to the animals a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated strains of the genus Bacillus selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868 ... All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals, wherein the Bacillus strains and antibiotics cause Escherichia coli inhibition in animals, and wherein the antibiotic is enrofloxacin.

[0014] 2. The method of Clause 1, wherein the animals are selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals.

[0015] 3. The method of any one of clauses 1 to 2, wherein Escherichia coli inhibition prevents Escherichia coli disease in animals.

[0016] 4. The method of any one of clauses 1 to 2, wherein Escherichia coli inhibition reduces Escherichia coli disease in animals.

[0017] 5. The method of any one of clauses 1 to 4, wherein the animal is a pig species, and the pig species is selected from growing-finishing pigs, nursery pigs, sows and breeding pigs.

[0018] 6. The method of any one of clauses 1 to 5, wherein the Bacillus strain produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0019] 7. The method of any one of clauses 1 to 6, further comprising the step of administering to an animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, or a combination thereof.

[0020] 8. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0021] 9. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0022] 10. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0023] 11. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0024] 12. The method of any one of clauses 1 to 11, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / g of feed composition is applied to the feed composition.

[0025] 13. The method of any one of clauses 1 to 11, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g feed composition is applied to the feed composition.

[0026] 14. The method of any one of clauses 1 to 11, wherein the Bacillus strain is at a concentration greater than about 7.0 × 10⁻⁶. 4 The dosage of CFU / g of feed composition is applied to the feed composition.

[0027] 15. The method of any one of clauses 1 to 14, further comprising the step of administering to an animal an enzyme selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0028] 16. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0029] 17. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0030] 18. The method of any one of clauses 1 to 17, wherein the feed composition is administered to the animal daily.

[0031] 19. The method of Clause 1, wherein the animal is selected from chickens, pigs, horses, ponies, cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0032] 20. A method for controlling the harmful effects of *Escherichia coli*, the method comprising the step of administering to an animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated *Bacillus* strains selected from: *Bacillus* strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of *Bacillus* strain 9 (NRRL No. B-67866), *Bacillus* strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of *Bacillus* strain 57 (NRRL No. B-67870), *Bacillus* strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of *Bacillus* strain 71 (NRRL No. B-67867), *Bacillus* strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of ... All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals and control of the harmful effects of Escherichia coli in animals, wherein the antibiotic is enrofloxacin.

[0033] 21. The method of Article 20, wherein the animals are selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals.

[0034] 22. The method of any one of clauses 20 to 21, wherein the harmful effect of controlling Escherichia coli is to inhibit Escherichia coli disease in animals.

[0035] 23. The method of any one of clauses 20 to 21, wherein the harmful effect of controlling Escherichia coli is to reduce Escherichia coli disease in animals.

[0036] 24. The method of any one of Articles 20 to 23, wherein the animal is a pig species and the pig species is selected from growing-finishing pigs, nursery pigs, sows and breeding pigs.

[0037] 25. The method of any one of clauses 20 to 24, wherein a strain of Bacillus produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0038] 26. The method of any one of clauses 20 to 25, further comprising the step of administering to an animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, or a combination thereof.

[0039] 27. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0040] 28. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0041] 29. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0042] 30. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0043] 31. The method of any one of clauses 20 to 30, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / g of feed composition is applied to the feed composition.

[0044] 32. The method of any one of clauses 20 to 30, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g of feed composition is applied to the feed composition.

[0045] 33. The method of any one of clauses 20 to 30, wherein the Bacillus strain is at a concentration greater than about 7.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is applied to the feed composition.

[0046] 34. The method of any one of clauses 20 to 33, further comprising the step of administering to an animal an enzyme selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0047] 35. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is applied during lactation.

[0048] 36. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0049] 37. The method of any one of clauses 20 to 36, wherein the feed composition is administered to the animal daily.

[0050] 38. The method of Article 20, wherein the animal is selected from chickens, pigs, horses, ponies, cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0051] 39. A method of feeding animals, the method comprising the steps of: administering to the animals a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated strains of the genus Bacillus selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof; and administering to the animals an antibiotic, wherein the antibiotic is enrofloxacin.

[0052] 40. The method of Article 39, wherein the animals are selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals.

[0053] 41. The method of any one of Articles 39 to 40, wherein the animal is a pig species and the pig species is selected from growing-finishing pigs, nursery pigs, sows and breeding pigs.

[0054] 42. The method of any one of clauses 39 to 41, wherein a strain of Bacillus produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0055] 43. The method of any one of clauses 39 to 42, further comprising the step of administering to an animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, or a combination thereof.

[0056] 44. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0057] 45. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0058] 46. ​​The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0059] 47. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0060] 48. The method of any one of clauses 39 to 47, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / g of feed composition is applied to the feed composition.

[0061] 49. The method of any one of clauses 39 to 47, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g of feed composition is applied to the feed composition.

[0062] 50. The method of any one of clauses 39 to 47, wherein the Bacillus strain is at a concentration greater than about 7.0 × 10⁻⁶. 4 The dosage of CFU / g of feed composition is applied to the feed composition.

[0063] 51. The method of any one of clauses 39 to 50, further comprising the step of administering to an animal an enzyme selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0064] 52. The method of any one of clauses 39 to 51, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0065] 53. The method of any one of clauses 39 to 51, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0066] 54. The method of any one of clauses 39 to 53, wherein the feed composition is administered to the animal daily.

[0067] 55. The method of Article 39, wherein the animal is selected from chickens, pigs, horses, ponies, cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0068] 56. The method of any one of clauses 1 to 55, wherein the Bacillus strain has the following functions: maintaining the microbial balance in the animal gut, improving animal performance or health, maintaining the gut health of the animal, reducing harmful pathogens in the animal gut, reducing odor, reducing harmful pathogens in the animal urine or feces, and maintaining the growth of beneficial bacteria in the animal gut. Brief description of the attached diagram

[0070] Figure 1A-B shows the percentage inhibition values ​​for 3 out of 15 E. coli strains, with improved inhibition observed when Bacillus supernatant was combined with enrofloxacin. IGS.

[0071] Figure 2 This is a photograph showing the RAPD PCR patterns of Bacillus strain 9 (NRRL No. B-67866) and Bacillus strain 57 (NRRL No. B-67870).

[0072] Figure 3 This is a photograph showing the RAPD PCR patterns of Bacillus strain 71 (NRRL No. B-67867) and Bacillus strain 126 (NRRL No. B-67868).

[0073] Detailed Explanatory Implementation Plan

[0074] Methods for inhibiting *E. coli* in animals are provided. In various implementation schemes, the animals may be selected from poultry species, pig species, goat species, cattle species, sheep species, horse species, and companion animals. In implementation schemes where the animal is a poultry species, the poultry species may be selected from broiler chickens, chickens, laying hens, breeding chickens, turkeys, turkey chicks, goslings, ducklings, guinea fowl, young hens, hens, roosters, young roosters, and capons. In implementation schemes where the animal is a pig species, the pig species may be selected from growing-finishing pigs, nursery pigs, sows, and breeding pigs.

[0075] In one embodiment, a method of feeding an animal is provided. The method includes the steps of: administering to the animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains possessing all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains possessing all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains possessing all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), and strains possessing all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868). All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals, wherein the Bacillus strains and antibiotics cause Escherichia coli inhibition in animals, and wherein the antibiotic is enrofloxacin.

[0076] In another embodiment, a method for controlling the harmful effects of *Escherichia coli* is provided. The method includes the step of administering to an animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated *Bacillus* strains selected from: *Bacillus* strain 9 (NRRL No. B-67866), strains possessing all the identification characteristics of *Bacillus* strain 9 (NRRL No. B-67866), *Bacillus* strain 57 (NRRL No. B-67870), strains possessing all the identification characteristics of *Bacillus* strain 57 (NRRL No. B-67870), *Bacillus* strain 71 (NRRL No. B-67867), strains possessing all the identification characteristics of *Bacillus* strain 71 (NRRL No. B-67867), *Bacillus* strain 126 (NRRL No. B-67868), and strains possessing all the identification characteristics of *Bacillus* strain 126 (NRRL No. B-67868). All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals, wherein the antibiotic is enrofloxacin, and the harmful effects of Escherichia coli in animals.

[0077] In yet another embodiment, a method of feeding an animal is provided. The method includes the step of administering to the animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to the animal, wherein the antibiotic is enrofloxacin.

[0078] The following clauses and combinations thereof provide various additional illustrative aspects of the invention described herein. The various embodiments described in this section, entitled “Detailed Description of Illustrative Embodiments,” apply to any of the following embodiments of the invention described in the following numbered clauses.

[0079] 1. A method of feeding animals, the method comprising the steps of: administering to the animals a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated strains of the genus Bacillus selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868 ... All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals, wherein the Bacillus strains and antibiotics cause Escherichia coli inhibition in animals, and wherein the antibiotic is enrofloxacin.

[0080] 2. The method of Clause 1, wherein the animals are selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals.

[0081] 3. The method of any one of clauses 1 to 2, wherein Escherichia coli inhibition prevents Escherichia coli disease in animals.

[0082] 4. The method of any one of clauses 1 to 2, wherein Escherichia coli inhibition reduces Escherichia coli disease in animals.

[0083] 5. The method of any one of clauses 1 to 4, wherein the animal is a pig species, and the pig species is selected from growing-finishing pigs, nursery pigs, sows and breeding pigs.

[0084] 6. The method of any one of clauses 1 to 5, wherein the Bacillus strain produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0085] 7. The method of any one of clauses 1 to 6, further comprising the step of administering to an animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, or a combination thereof.

[0086] 8. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0087] 9. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0088] 10. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0089] 11. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0090] 12. The method of any one of clauses 1 to 11, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / g of feed composition is applied to the feed composition.

[0091] 13. The method of any one of clauses 1 to 11, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g of feed composition is applied to the feed composition.

[0092] 14. The method of any one of clauses 1 to 11, wherein the Bacillus strain is at a concentration greater than about 7.0 × 10⁻⁶. 4 The dosage of CFU / g of feed composition is applied to the feed composition.

[0093] 15. The method of any one of clauses 1 to 14, further comprising the step of administering to an animal an enzyme selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0094] 16. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0095] 17. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0096] 18. The method of any one of clauses 1 to 17, wherein the feed composition is administered to the animal daily.

[0097] 19. The method of Clause 1, wherein the animal is selected from chickens, pigs, horses, ponies, cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0098] 20. A method for controlling the harmful effects of *Escherichia coli*, the method comprising the step of administering to an animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated *Bacillus* strains selected from: *Bacillus* strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of *Bacillus* strain 9 (NRRL No. B-67866), *Bacillus* strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of *Bacillus* strain 57 (NRRL No. B-67870), *Bacillus* strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of *Bacillus* strain 71 (NRRL No. B-67867), *Bacillus* strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of ... All identifying characteristics of strains and combinations thereof (No. B-67868), and administration of antibiotics to animals and control of the harmful effects of Escherichia coli in animals, wherein the antibiotic is enrofloxacin.

[0099] 21. The method of Article 20, wherein the animals are selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals.

[0100] 22. The method of any one of clauses 20 to 21, wherein the harmful effect of controlling Escherichia coli is to inhibit Escherichia coli disease in animals.

[0101] 23. The method of any one of clauses 20 to 21, wherein the harmful effect of controlling Escherichia coli is to reduce Escherichia coli disease in animals.

[0102] 24. The method of any one of Articles 20 to 23, wherein the animal is a pig species and the pig species is selected from growing-finishing pigs, nursery pigs, sows and breeding pigs.

[0103] 25. The method of any one of clauses 20 to 24, wherein a strain of Bacillus produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0104] 26. The method of any one of clauses 20 to 25, further comprising the step of administering to an animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, or a combination thereof.

[0105] 27. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0106] 28. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0107] 29. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0108] 30. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0109] 31. The method of any one of clauses 20 to 30, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / g of feed composition is applied to the feed composition.

[0110] 32. The method of any one of clauses 20 to 30, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g of feed composition is applied to the feed composition.

[0111] 33. The method of any one of clauses 20 to 30, wherein the Bacillus strain is at a concentration greater than about 7.0 × 10⁻⁶. 4 The dosage of CFU / g of feed composition is applied to the feed composition.

[0112] 34. The method of any one of clauses 20 to 33, further comprising the step of administering to an animal an enzyme selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0113] 35. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is applied during lactation.

[0114] 36. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0115] 37. The method of any one of clauses 20 to 36, wherein the feed composition is administered to the animal daily.

[0116] 38. The method of Article 20, wherein the animal is selected from chickens, pigs, horses, ponies, cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0117] 39. A method of feeding an animal, the method comprising the steps of: administering to the animal a feed composition or drinking water containing an effective amount of an additive, said additive comprising isolated strains of the genus Bacillus selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof; and administering to the animal an antibiotic, wherein the antibiotic is enrofloxacin.

[0118] 40. The method of Article 39, wherein the animals are selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals.

[0119] 41. The method of any one of Articles 39 to 40, wherein the animal is a pig species and the pig species is selected from growing-finishing pigs, nursery pigs, sows and breeding pigs.

[0120] 42. The method of any one of clauses 39 to 41, wherein a strain of Bacillus produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0121] 43. The method of any one of clauses 39 to 42, further comprising the step of administering to an animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, or a combination thereof.

[0122] 44. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0123] 45. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0124] 46. ​​The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0125] 47. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0126] 48. The method of any one of clauses 39 to 47, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / g of feed composition is applied to the feed composition.

[0127] 49. The method of any one of clauses 39 to 47, wherein the Bacillus strain is at approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g of feed composition is applied to the feed composition.

[0128] 50. The method of any one of clauses 39 to 47, wherein the Bacillus strain is at a concentration greater than about 7.0 × 10⁻⁶. 4 The dosage of CFU / g of feed composition is applied to the feed composition.

[0129] 51. The method of any one of clauses 39 to 50, further comprising the step of administering to an animal an enzyme selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0130] 52. The method of any one of clauses 39 to 51, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0131] 53. The method of any one of clauses 39 to 51, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0132] 54. The method of any one of clauses 39 to 53, wherein the feed composition is administered to the animal daily.

[0133] 55. The method of Article 39, wherein the animal is selected from chickens, pigs, horses, ponies, cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0134] 56. The method of any one of clauses 1 to 55, wherein the Bacillus strain has the following functions: maintaining the microbial balance in the animal gut, improving animal performance or health, maintaining the gut health of the animal, reducing harmful pathogens in the animal gut, reducing odor, reducing harmful pathogens in the animal urine or feces, and maintaining the growth of beneficial bacteria in the animal gut.

[0135] In various embodiments, the animals to which the feed additives, feed compositions, or drinking water described herein, and antibiotics (wherein the antibiotic is enrofloxacin) are administered may be poultry species, pig species, goat species, cattle species, sheep species, horse species, and companion animals. In embodiments where the animal is a companion animal, the companion animal may be, for example, a dog species or a cat species. In embodiments where the animal is a pig species, the pig species may be selected from growing-finishing pigs, nursery pigs, sows, and breeding pigs. In various exemplary embodiments, the animals may be selected from chickens (e.g., broiler chickens or laying hens), pigs, horses, ponies, dairy cows, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish (e.g., tilapia, catfish, halibut, or salmon), crustaceans (e.g., shrimp or crab), and combinations thereof. In another embodiment, the feed additives, feed compositions, or drinking water described herein, and antibiotics (wherein the antibiotic is enrofloxacin) may be administered to the animal.

[0136] In various embodiments of the methods or compositions described herein, the Bacillus strain and the antibiotic (wherein the antibiotic is enrofloxacin) may have an effect selected from: maintaining the microbial balance in the animal's gut, preventing or reducing Escherichia coli disease in animals, improving animal performance or health, maintaining the animal's gut health, reducing harmful pathogens in the animal's gut, reducing odor, reducing harmful pathogens in animal urine or feces, and maintaining the growth of beneficial bacteria in the animal's gut. In any of the embodiments described herein, the Bacillus strain may be a Bacillus subtilis strain or a Bacillus licheniformis strain.

[0137] In any of the embodiments described herein, Bacillus strains may be administered alone or in any combination with enrofloxacin, or may be in the form of any of the compositions described herein. In one embodiment, one or more strains are administered with enrofloxacin in the same composition or separate compositions. The Bacillus strains described herein with enrofloxacin may also be used in combination with other different microbial strains, including other Bacillus strains or Lactobacillus strains.

[0138] In one embodiment of the invention, an effective amount of a Bacillus strain can be applied to inhibit Escherichia coli in animals. As used herein, "inhibit Escherichia coli" can mean reducing E. coli disease, preventing E. coli disease, maintaining normal microbial balance in animals, reducing the number of harmful E. coli organisms in animals, reducing the activity of E. coli in animals, or reducing the symptoms of E. coli disease in animals, or a combination thereof. "Effective amount" refers to the amount of a Bacillus strain (e.g., strains 9, 57, 71, and 126) that can inhibit E. coli or control the harmful effects of E. coli through any mechanism as described below.

[0139] In the embodiments described herein in which the composition of the present invention comprising Bacillus strains 9, 57, 71 and / or 126 or strains having their identifying characteristics, and enrofloxacin is administered to animals, any other effective method of administration known to those skilled in the art may be employed, such as in pastes, gels, liquid drench, top dresses, powders, liquids, pellets or capsules.

[0140] The feed composition may contain Bacillus strains 9, 57, 71, and / or 126, or strains having their identifying characteristics, mixed with an animal feed blend (including any animal feed blend known in the art or any animal feed blend described herein). As used herein, "feed composition" or "animal feed composition" refers to a feed composition containing Bacillus strains 9, 57, 71, and / or 126, or strains having their identifying characteristics, mixed with an animal feed blend and optionally any other components that can be used in the feed composition (including other bacterial strains, such as other different Bacillus or Lactobacillus strains). In one embodiment, the feed composition may be in the form of ground meal.

[0141] Any animal feed blend, including those known in the art and described herein, can be used according to the methods and compositions described in this patent application, such as rapeseed meal, cottonseed meal, soybean meal, corn flour, barley, wheat, silage, and semi-dry silage. In various embodiments, the animal feed blend may be supplemented with Bacillus strains 9, 57, 71, and / or 126, or strains possessing their identifying characteristics, but other components may optionally be added to the animal feed blend, including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0142] In various illustrative embodiments, optional components of the animal feed blend include sugars and complex carbohydrates, such as water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides. Other optional components include dried distillers grains solubles, fats (e.g., crude fat), phosphorus, sodium bicarbonate, limestone, salts, phytates, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, oil derived from fishmeal, raw seeds (e.g., flaxseed), antioxidants, and starch. In another embodiment, minerals may be added in the form of a mineral premix.

[0143] Optional amino acid components that can be added to animal feed blends include arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl ester hydrochloride, alanine, aspartic acid, monosodium glutamate, glycine, proline, serine, cysteine ​​ethyl ester hydrochloride, and their analogues and salts. Optional vitamins include thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, niacin, nicotinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, and E. In another embodiment, vitamins can be added in the form of a vitamin premix. In yet another embodiment, protein components can be added to the animal feed blends and include proteins derived from meat meal, bone meal, or fish meal, liquid or powdered eggs, fish solubles, crude protein, etc.

[0144] In the method implementations described herein, the antibiotic used is enrofloxacin. In various implementations, the antibiotic dosage can be approximately 0.1 mg / lb feed to approximately 1000 mg / lb feed, approximately 0.1 mg / lb feed to approximately 900 mg / lb feed, approximately 0.1 mg / lb feed to approximately 800 mg / lb feed, approximately 0.1 mg / lb feed to approximately 700 mg / lb feed, approximately 0.1 mg / lb feed to approximately 600 mg / lb feed, approximately 0.1 mg / lb feed to approximately 500 mg / lb feed, approximately 0.1 mg / lb feed to approximately 400 mg / lb feed. 0 mg / lb feed, approximately 0.1 mg / lb feed to approximately 300 mg / lb feed, approximately 0.1 mg / lb feed to approximately 200 mg / lb feed, approximately 0.1 mg / lb feed to approximately 100 mg / lb feed, approximately 0.1 mg / lb feed to approximately 90 mg / lb feed, approximately 0.1 mg / lb feed to approximately 80 mg / lb feed, approximately 0.1 mg / lb feed to approximately 70 mg / lb feed, approximately 0.1 mg / lb feed to approximately 60 mg / lb feed, approximately 0.1 mg / lb feed / pound of feed to approximately 50 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 40 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 30 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 20 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 10 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 9 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 8 mg / pound of feed, approximately 0.1 mg / pound of feed to approximately 7 mg / pound of feed, approximately 0.1 mg / lb feed to approximately 6 mg / lb feed, approximately 0.1 mg / lb feed to approximately 5 mg / lb feed, approximately 0.1 mg / lb feed to approximately 4 mg / lb feed, approximately 0.1 mg / lb feed to approximately 3 mg / lb feed, approximately 0.1 mg / lb feed to approximately 2 mg / lb feed, approximately 0.1 mg / lb feed to approximately 1 mg / lb feed, approximately 0.1 mg / lb feed to approximately 0.5 mg / lb feed, approximately 0.1 mg / lb feed to approximately 0.3 mg / lb feed, approximately 0.5 mg / lb feed to approximately 1 mg / lb feed, approximately 1 mg / mL to approximately 1000 mg / mL drinking water, approximately 1 mg / mL to approximately 900 mg / mL drinking water, approximately 1 mg / mL to approximately 800 mg / mL drinking water, approximately 1 mg / mL to approximately 700 mg / mL drinking water, approximately 1 mg / mL to approximately 600 mg / mL drinking water, approximately 1 mg / mL to approximately 500 mg / mL drinking water, approximately 1 mg / mL to approximately 400 mg / mL drinking water, approximately 1 mg / mL to approximately 300 mg / mL drinking water, approximately 1 mg / mL to approximately 200 mg / mL drinking water, approximately 1 mg / mL to approximately 100 mg / mL drinking water, approximately 50 mg / mL to approximately 100 mg / mL drinking water. mg / mL drinking water, approximately 50 mg / mL to approximately 90 mg / mL drinking water, approximately 50 mg / mL to approximately 80 mg / mL drinking water, approximately 50 mg / mL to approximately 70 mg / mL drinking water, approximately 50 mg / mL to approximately 60 mg / mL drinking water, approximately 1000 ppm, approximately 900 ppm, approximately 800 ppm, approximately 700 ppm, approximately 600 ppm, approximately 500 ppm, approximately 400 ppm, approximately 300 ppm, approximately 200 ppm, approximately 100 ppm, approximately 90 ppm, approximately 80 ppm, approximately 70 ppm, approximately 60 ppm, approximately 50 ppm, approximately 40 ppm, approximately 30 ppm, approximately 20 ppm, or approximately 10 ppm.

[0145] In another illustrative embodiment, one or more enzymes may be added to animal feed blends, feed compositions, feed additives, or bacterial strains. In various embodiments, the enzymes that may be added include galactosidase, phytase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzymes, combinations thereof, and any other enzymes that improve the effectiveness of the feed composition or feed additive in inhibiting or controlling the harmful effects of *E. coli*. In yet another embodiment, yeast, fungi (e.g., *Aspergillus* or *Trichoderma*), or micronutrients may be added to animal feed, feed compositions, feed additives, or bacterial strains. Any of the above-mentioned components suitable for addition to additives for animal drinking water may be added as a component of the additives for animal drinking water described herein.

[0146] In various illustrative embodiments, Bacillus strains (e.g., Bacillus strains 9, 57, 71, and / or 126, or strains possessing their identifying characteristics), or any other distinct bacterial strain added besides Bacillus strains 9, 57, 71, and / or 126 or strains possessing their identifying characteristics, can be in quantities of approximately 1.0 × 10⁻⁶. 3 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / gram of feed composition, or approximately 1.0 × 10⁻⁶ CFU / gram, is... 3 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / g of feed composition is administered in the feed composition. In other embodiments, Bacillus strains (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126, or strains having their identifying characteristics) are administered at a dose greater than about 1.0 × 10⁻⁶. 3 The dosage of CFU / g feed composition is greater than approximately 1.1 × 10⁻⁶. 3 The dosage of CFU / g feed composition is greater than approximately 1.25 × 10⁻⁶. 3 The dosage of CFU / g feed composition is greater than approximately 1.5 × 10⁻⁶. 3 The dosage of CFU / g feed composition is greater than approximately 1.75 × 10⁻⁶. 3 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 2.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 3.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 4.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 5.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 6.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 7.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 8.0 × 10⁻⁶. 4 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 5 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 6 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 7 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 8 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 9The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 10 The dosage of CFU / g feed composition is greater than approximately 1.0 × 10⁻⁶. 11 The dosage of CFU / gram feed composition, or at a concentration greater than approximately 1.0 × 10⁻⁶ CFU / gram, is... 12 The dosage of CFU / g of feed composition is administered in the feed composition. In another embodiment, Bacillus strains (e.g., Bacillus strains 9, 57, 71 and / or 126, or strains having their identifying characteristics), or any other different bacterial strains added besides Bacillus strains 9, 57, 71 and / or 126 or strains having their identifying characteristics, may be administered at approximately 1.0 × 10⁻⁶. 2 CFU / g feed composition up to approximately 5.0 × 10⁻⁶ 12 The dosage of CFU / gram feed composition, or approximately 1.0 × 10⁻⁶ CFU / gram, is... 2 CFU / g feed composition up to approximately 1.0 × 10⁻⁶ 7 The dosage of CFU / gram feed composition, or at a concentration greater than approximately 1.0 × 10⁻⁶ CFU / gram, is... 2 The dosage of CFU / gram of feed composition is administered in the feed composition. In another embodiment, in which the strain is administered in animal drinking water, any of the dosages described herein may be expressed in CFU / mL of drinking water.

[0147] In various embodiments, the Bacillus strains used according to the methods or compositions described herein (e.g., Bacillus strains 9, 57, 71 and / or 126, or strains having their identifying characteristics) may be selected from Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof. Bacillus strains 9, 57, 71, and 126 were deposited on October 10, 2019, at the Agricultural Research Service Culture Collection (NRRL), USDA, 1815 North University Street, Peoria, Illinois 61604-3999, and assigned accession numbers B-67866, B-67870, B-67867, and B-67868, respectively. The deposits were made in accordance with the Budapest Treaty on the Preservation of Microorganisms, which is internationally recognized for use in patent proceedings. NRRL No. B-67866, NRRL No. B-67870, NRRL No. B-67867, and NRRL No. B-67868 are equivalent to Bacillus strains 9, 57, 71, and 126 mentioned in this application, respectively. The preservation certificates identify the strains as MDG9, MDG57, MDG71, and MDG126, respectively. These four strains are Bacillus subtilis strains.

[0148] In one aspect, any of these strains may be administered alone or in combination as a feed composition (e.g., a complete feed containing an animal feed blend) or as animal drinking water. In one embodiment, multiple strains are administered in combination in a single composition. In another embodiment, multiple strains are administered in combination in separate compositions. In yet another embodiment, any of these strains or combinations thereof are administered in combination with enrofloxacin described herein in the same or separate compositions.

[0149] In another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strains 9, 57, 71, and / or 126, or strains having their identifying characteristics) may be administered to animals together with another different bacterial strain selected from another Bacillus strain, lactic acid bacteria strains, and combinations thereof. In yet another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strains 9, 57, 71, and / or 126, or strains having their identifying characteristics) may be administered to animals together with any other bacterial strain that effectively inhibits Escherichia coli or controls its harmful effects in animals.

[0150] The term "strain possessing all the identifying characteristics" of Bacillus strains 9, 57, 71, or 126, as used herein, can refer to a mutant strain possessing all the identifying characteristics of Bacillus strains 9, 57, 71, or 126 (e.g., a DNA fingerprint based on DNA analysis corresponding to the DNA fingerprint of Bacillus strains 9, 57, 71, or 126; enzyme activity corresponding to Bacillus strains 9, 57, 71, or 126; antimicrobial activity corresponding to Bacillus strains 9, 57, 71, or 126; antibiotic sensitivity and tolerance profile corresponding to Bacillus strains 9, 57, 71, or 126; or a combination of these identifying characteristics). In an alternative embodiment, the mutation can be a naturally occurring mutation or a genetically engineered mutation. In another embodiment, a strain “having” all the identifying characteristics of Bacillus strain 9, 57, 71, or 126 can be, for example, a strain produced by isolating one or more plasmids from Bacillus strain 9, 57, 71, or 126 and introducing the one or more plasmids into another bacterium (such as another Bacillus strain), provided that the one or more plasmids contain DNA that provides the identifying characteristics of Bacillus strain 9, 57, 71, or 126 (e.g., a DNA fingerprint based on DNA analysis corresponding to the DNA fingerprint of Bacillus strain 9, 57, 71, or 126).

[0151] In one alternative embodiment, the feed composition or drinking water may be administered to the animal during lactation and / or pregnancy. The time periods for administering the feed composition or drinking water described above are non-limiting examples, and it should be understood that any time period or administration regimen determined to be effective in inhibiting or controlling harmful effects on Escherichia coli, or a combination thereof, may be used.

[0152] In implementation schemes relating to “controlling the harmful effects of Escherichia coli”, controlling harmful effects can refer to reducing E. coli disease, preventing E. coli disease, maintaining normal microbial balance in animals, reducing the number of harmful E. coli in animals, reducing the activity of E. coli in animals, or reducing the symptoms of E. coli disease in animals, or a combination thereof. “Effective amount” refers to the amount of Bacillus strains (e.g., Bacillus strains 9, 57, 71, and / or 126) that are capable of controlling the harmful effects of E. coli through any mechanism as described herein.

[0153] In one embodiment, the feed additive for addition to the animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, using an automated micronutrient delivery system, or by manual weighing and addition, to achieve any dosage of Bacillus strains 9, 57, 71, and / or 126, or strains having their identifying characteristics, as described herein for administration to animals in the form of a complete feed composition. Mixing can also be carried out by any other suitable method known in the art for combining directly fed microorganisms with the animal feed blend to obtain a homogeneous mixture. In various embodiments, mixing can be carried out within any suitable time period (e.g., from about 1 minute to about 4 minutes). In embodiments where Bacillus strains 9, 57, 71, and / or 126, or strains possessing their identifying characteristics, are in the form of an additive for animal drinking water, Bacillus strains 9, 57, 71, and / or 126, or strains possessing their identifying characteristics, may be in, for example, powder, liquid, or pellet form, and may be mixed with drinking water using any suitable method known in the art to achieve any dosage of Bacillus strains 9, 57, 71, and / or 126, or strains possessing their identifying characteristics, as described herein for administration to animals in animal drinking water. Bacillus strains 9, 57, 71, and / or 126, or strains possessing their identifying characteristics, may also be fed directly to animals orally (i.e., via oral placement) in the form of powder, freeze-dried composition, gel, top-dressing, liquid, capsule, paste, oral solution, or pellets. Any of these embodiments may also be used for mixing and administering enrofloxacin as described herein.

[0154] In another embodiment of the invention, a composition is provided comprising Bacillus strain 9 (NRRL No. B-67866), a strain having all the identification characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), a strain having all the identification characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identification characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identification characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0155] In one embodiment, a commercial package is provided comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0156] In another embodiment, a feed additive for animal feed is provided, comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identification characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identification characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identification characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identification characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0157] In yet another embodiment, an additive for animal drinking water is provided, comprising isolated Bacillus strain 9 (NRRL No. B-67866), strains having all the identification characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identification characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identification characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identification characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0158] In another illustrative aspect of the invention, an animal feed composition is provided comprising isolated Bacillus strains selected from: Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0159] In one illustrative aspect, the strains used to be added to commercial packaging, feed additives, additives for animal drinking water, or feed compositions may be concentrates (e.g., approximately 1 × 10⁻⁶). 8 Up to approximately 5×10 9 CFU / g or superconcentrate (e.g., approximately 1×10⁻⁶ CFU / g) or ultraconcentrate (e.g., approximately 1×10⁻⁶ CFU / g) 10 Up to approximately 5×10 12 In the form of CFU / g. In another embodiment, the strains used to be added to commercial packaging, feed additives, feed compositions, or additives for animal drinking water can be in dry form (e.g., powder), granular form, liquid form, oral solution, powder, freeze-dried composition, topical application, paste, capsule form, or gel form, or any other suitable form.

[0160] In another illustrative embodiment, one or more strains in the commercial packaging, feed additive, additive for animal drinking water, or feed composition may further comprise a carrier for one or more Bacillus strains. The carrier may be selected from wheat bran, rice husk, salt, mineral oil, dextrin (e.g., maltodextrin), whey, sugar, sucrose, limestone, yeast culture, dry starch, sodium aluminosilicate, silica, polypropylene glycol, polysorbate 80, vegetable oil, and combinations thereof. The carrier is exogenously added to the bacterial strain (i.e., it is not naturally occurring or not present in nature with the bacterial strain). In another embodiment, the feed additive, additive for animal drinking water, or feed composition may further comprise a binder, such as clay, yeast cell wall components, aluminum silicate, dextran, or other known binders. In another embodiment, the commercial packaging, feed additive, additive for animal drinking water, or feed composition may further comprise inorganic / organic binders, essential oils, and / or organic acids. The binder is exogenously added to the bacterial strain (i.e., it is not naturally occurring or not present in nature with the bacterial strain).

[0161] In some other embodiments, commercial packaging, feed additives, additives for animal drinking water, or feed compositions comprising Bacillus strain 9 (NRRL No. B-67866), strains having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), strains having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), strains having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), and strains having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, in containers for commercial use.

[0162] In various implementations, the container may be, for example, a bag (e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a 1-pound bag, or a 1-kilogram bag), a pouch, a barrel, a bottle, or a box. In illustrative terms, containers used for commercial packaging, feed additives, additives for animal drinking water, or feed compositions containing strains of Bacillus spp. 9 (NRRL No. B-67866), strains having all the identifying characteristics of strains of Bacillus spp. 9 (NRRL No. B-67866), strains of Bacillus spp. 57 (NRRL No. B-67870), strains having all the identifying characteristics of strains of Bacillus spp. 57 (NRRL No. B-67870), strains of Bacillus spp. 71 (NRRL No. B-67867), strains having all the identifying characteristics of strains of Bacillus spp. 71 (NRRL No. B-67867), strains of Bacillus spp. 126 (NRRL No. B-67868), strains having all the identifying characteristics of strains of Bacillus spp. 126 (NRRL No. B-67868), and combinations thereof may contain plastic, metal, foil, paper, fiber, or cardboard (e.g., plastic drums, paper bags, foil bags, fiberboard drums, etc.). Commercial packaging, feed additives, additives for animal drinking water, or feed compositions may further include instructions for the use of one or more strains of Bacillus.

[0163] In one respect, the commercial packaging, feed additives, additives for animal drinking water, or feed compositions described herein may further contain exogenously added nutrients (i.e., nutrients not present in nature with bacterial strains), said nutrients being selected from vitamins, enzymes, water-soluble or water-insoluble monosaccharides, disaccharides or polysaccharides, fats, phosphorus, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants, and starch.

[0164] In one embodiment, the exogenously added nutrient is an enzyme, and the enzyme is selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, phytase, and combinations thereof.

[0165] In one embodiment, any of the compositions described herein can be a dietary nutrient composition (e.g., a probiotic composition).

[0166] In the embodiments of the methods and compositions described herein, a synergistic effect occurs between Bacillus strains and enrofloxacin.

[0167] The following examples are for illustrative purposes only. These examples are not limiting and are not intended to limit the invention in any way.

[0168] Example 1

[0169] Bacillus test for Baytril (15) resistant Escherichia coli

[0170] Four Bacillus strains were tested using the broth-based AST method against 15 drug-resistant Escherichia coli species. All four Bacillus strains were observed to be more effective than Mecadox in inhibiting the growth of these drug-resistant E. coli. TM It is more effective when done alone.

[0171] When combined with the same concentration of antibiotics and administered via supernatant, Bacillus strains 9 (NRRL No. B-67866), 57 (NRRL No. B-67870), 71 (NRRL No. B-67867), and 126 (NRRL No. B-67868) consistently improved and effectively inhibited (>50% inhibition) antibiotic-resistant pathogens. Among 15 enrofloxacin-resistant Escherichia coli isolates: when combined with antibiotics, Bacillus strains 9 and 71 effectively inhibited 14 of the 15 isolates. When combined with antibiotics, Bacillus strains 57 and 126 effectively inhibited 15 of the 15 isolates.

[0172] The goal of this project was to identify Bacillus strains that exhibited strong inhibitory potential against target pathogens when combined with antibiotics. This synergistic effect was defined as the percentage of inhibition against antibiotic-resistant pathogens achieved when the Bacillus supernatant was combined with an antibiotic, compared to tests on the supernatant and antibiotics separately against the same isolates. Four Bacillus strains demonstrated consistently strong inhibitory activity against Baytril-resistant Escherichia coli and were tested. These four Bacillus strains (BS 9, 57, 71, and 126) were identified as strains (NRRL No. B-67866), (NRRL No. B-67870), (NRRL No. B-67867), and (NRRL No. B-67868), respectively.

[0173] Example 2

[0174] RAPD-PCR DNA Map

[0175] Random amplified polymorphic DNA PCR (RAPD-PCR) is used to identify the genetic variability of each strain. By using... Tissue and Blood Single Column Kit The preparation of DNA for RAPD-PCR reaction was carried out in Venlo, The Netherlands. Figure 2 and 3 The RAPD-PCR results for strains 9, 57, 71, and 126 are shown, where the unlabeled lanes are molecular weight ladder markers. The results show that all strains are unique to each other.

Claims

1. Use of an additive in the manufacture of a feed composition or drinking water for use in a method of feeding an animal the additive to enhance the efficacy of antibiotic treatment and inhibit the growth of antibiotic resistant E. coli, the method comprising the steps of: administering to the animal a feed composition or drinking water comprising an effective amount of the additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 having Accession No. NRRL No. B-67866, Bacillus strain 57 having Accession No. NRRL No. B-67870, Bacillus strain 71 having Accession No. NRRL No. B-67867, Bacillus strain 126 having Accession No. NRRL No. B-67868, and combinations thereof, and administering to the animal an antibiotic, wherein the antibiotic is enrofloxacin.

2. The use of claim 1, wherein the animal is selected from the group consisting of a swine species, a goat species, a bovine species, a sheep species, a horse species.

3. The use of claim 1, wherein the animal is a poultry species.

4. The use of claim 1, wherein the animal is a companion animal.

5. The use of claim 1, wherein the animal is a swine species, and the swine species is selected from the group consisting of a growing-finishing pig, a nursery pig.

6. The use of claim 1, wherein the animal is a sow.

7. The use of claim 1, wherein the animal is a breeding pig.

8. The use of claim 1, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

9. The use of claim 1, wherein the strain is Bacillus strain 9 having Accession No. NRRL No. B-67866.

10. The use of claim 1, wherein the strain is Bacillus strain 57 having Accession No. NRRL No. B-67870.

11. The use of claim 1, wherein the strain is Bacillus strain 71 having Accession No. NRRL No. B-67867.

12. The use of claim 1, wherein the strain is Bacillus strain 126 having Accession No. NRRL No. B-67868.

13. The use of claim 1, wherein the Bacillus strain is included in the feed composition at a dosage of 1.0 x 10 3 CFU / gram of the feed composition to 5.0 x 10 12 CFU / gram of the feed composition.

14. The use of claim 1, wherein the Bacillus strain is included in the feed composition at a dosage of 1.0 x 10 3 CFU / gram of the feed composition to 1.0 x 10 7 CFU / gram of the feed composition.

15. The use of claim 1, wherein the Bacillus strain is included in the feed composition at a dosage of greater than 7.0 x 10 4 CFU / gram of the feed composition.

16. The use of claim 1, wherein the feed composition or drinking water further comprises an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, xylanase, cellulase, phytase, and combinations thereof.

17. The use of claim 1, wherein the feed composition or drinking water further comprises an NSP enzyme.

18. The use of claim 1, wherein the feed composition or drinking water further comprises an arabinoxylanase.

19. The use of claim 1, wherein the animal is a sow, and the Bacillus strain is administered during lactation.

20. The use of claim 1, wherein the animal is a sow, and the Bacillus strain is administered during gestation.

21. The use of claim 1, wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

22. The use of claim 1, wherein the Bacillus strain has an effect selected from the group consisting of maintaining a microbial balance in the gut of the animal, improving animal performance or health, maintaining the gut health of the animal, reducing harmful pathogens in the gut of the animal, reducing odor, reducing harmful pathogens in the urine or feces of the animal, and preserving the growth of beneficial bacteria in the gut of the animal.

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