WO2022040280A1

WO2022040280A1 - Microbials and antibiotics

Info

Publication number: WO2022040280A1
Application number: PCT/US2021/046452
Authority: WO
Inventors: Michael R. King; Sona SON; Kyle LEISTIKOW
Original assignee: Microbial Discovery Group LLC
Current assignee: Microbial Discovery Group LLC
Priority date: 2020-08-21
Filing date: 2021-08-18
Publication date: 2022-02-24
Anticipated expiration: 2023-02-21
Also published as: MX2023002033A; EP4199735A4; CA3192065A1; CN116801727B; CN116801727A; BR112023003047A2; EP4199735A1; US20230346854A1; US12514885B2

Abstract

The invention relates to direct-fed microbials for use in E. coli inhibition in animals in combination with enrofloxacin. More particularly, the invention relates to isolated Bacillus strains 9, 57, 71, and 126, and strains having all of the identifying characteristics of these strains, for a use comprising the above-mentioned use.

Description

MICROBIALS AND ANTIBIOTICS

CROSS-REFERENCE TO RELATED APPLICATIONS

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

HELD OF THE DISCLOSURE

The invention relates to direct-fed microbials for use in E. coli inhibition in animals in combination with the antibiotic enrofloxacin. More particularly, the invention relates to isolated Bacillus strains 9, 57, 71, and 126 and strains having all of the identifying characteristics of these strains, for a use comprising the above-mentioned use in combination with the antibiotic enrofloxacin.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to direct-fed microbial (DFM) compositions and methods for E. coli inhibition in an animal. An animal’s gastrointestinal tract is constantly challenged by large numbers of bacteria, viruses, fungi, and protozoa found in feed, bedding, and the environment. The gastrointestinal tract has a sophisticated system to counter these potential pathogens consisting of physical, chemical, and immunological lines of defense. Beneficial bacteria are an important part of this system because they provide animals with bacteria that assist in establishment (or reestablishment) of a normal bacterial profile, they strengthen the animal’s immune system, and they help to fight disease.

Antibiotic resistance is an increasingly important issue in human medicine, as well as in animal agriculture. The Food and Drug Administration reported that in 2016 the U.S. swine industry purchased about 6.9 million pounds of medically important antibiotics, which emphasizes the importance of preventing the development of antibiotic resistant organisms in the animal agricultural industry. Methods are being developed in an effort to combat antibiotic resistance, but little has been done to utilize synergistic direct-fed microbials (e.g., probiotics) as tools to combat antibiotic resistance.

Applicants have developed direct- fed microbials that result in E. coli inhibition in animals in combination with enrofloxacin. The direct-fed microbials and enrofloxacin and compositions comprising the direct-fed microbials and enrofloxacin described herein offer a commercial benefit by providing E. coli inhibition in animals, such as agricultural animals.

Methods are provided for inhibiting E. coli in animals. In various embodiments, the animal can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal. In the embodiment where the animal is a poultry species, the poultry species can be selected from the group consisting of a broiler, a chicken, a layer, a breeder, a turkey, a turkey poult, a gosling, a duckling, a guineakeet, a pullet, a hen, a rooster, a cockerel, and a capon. In the embodiment where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

In one embodiment, a method of feeding an animal is provided. The method comprises the steps of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B- 67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering to the animal an antibiotic, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal and wherein the antibiotic is enrofloxacin.

In another embodiment, a method of controlling a detrimental effect of E. coli is provided. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to the animal, and controlling the detrimental effect of E. coli in the animal wherein the antibiotic is enrofloxacin. In yet another embodiment, a method of feeding an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B- 67868), a strain having all of 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.

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 titled “DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS” and the EXAMPLES are applicable to any of the following embodiments of the invention described in the numbered clauses below.

1. A method of feeding an animal, the method comprising the steps of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering to the animal an antibiotic, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal and wherein the antibiotic is enrofloxacin.

2. The method of clause 1 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

3. The method of any one of clauses 1 to 2 wherein the E. coli inhibition prevents E. coli disease in the animal.

4. The method of any one of clauses 1 to 2 wherein the E. coli inhibition reduces E. coli disease in the animal. 5. The method of any one of clauses 1 to 4 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

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

7. The method of any one of clauses 1 to 6 further comprising the step of administering to the animal another different bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

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

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

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

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

12. The method of any one of clauses 1 to 11 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 5.0 x 10¹² CFU/gram of the feed composition.

13. The method of any one of clauses 1 to 11 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition.

14. The method of any one of clauses 1 to 11 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10⁴ CFU/gram of the feed composition.

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

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.

17. The method of any one of clauses 1 to 15 wherein the animal is a sow and the Bacillus strain is administered during gestation. 18. The method of any one of clauses 1 to 17 wherein the feed composition is administered daily to the animal.

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

20. A method of controlling a detrimental effect of E. coli, the method comprising the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B- 67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to the animal, and controlling the detrimental effect of E. coli in the animal wherein the antibiotic is enrofloxacin.

21. The method of clause 20 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

22. The method of any one of clauses 20 to 21 wherein controlling the detrimental effect of the E. coli is inhibiting E. coli disease in the animal.

23. The method of any one of clauses 20 to 21 wherein controlling the detrimental effect of the E. coli is reducing E. coli disease in the animal.

24. The method of any one of clauses 20 to 23 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

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

26. The method of any one of clauses 20 to 25 further comprising the step of administering to the animal another different bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

27. The method of any one of clauses 20 to 26 wherein the strain administered is Bacillus strain 9 (NRRL No. B-67866). 28. The method of any one of clauses 20 to 26 wherein the strain administered is Bacillus strain 57 (NRRL No. B-67870).

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

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

31. The method of any one of clauses 20 to 30 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 5.0 x 10¹² CFU/gram of the feed composition.

32. The method of any one of clauses 20 to 30 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition.

33. The method of any one of clauses 20 to 30 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10⁴ CFU/gram of the feed composition.

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

35. The method of any one of clauses 20 to 34 wherein the animal is a sow and the Bacillus strain is administered during lactation.

36. The method of any one of clauses 20 to 34 wherein the animal is a sow and the Bacillus strain is administered during gestation.

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

38. The method of clause 20 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

39. A method of feeding an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of 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.

40. The method of clause 39 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

41. The method of any one of clauses 39 to 40 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

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

43. The method of any one of clauses 39 to 42 further comprising the step of administering to the animal another different bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

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

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

46. The method of any one of clauses 39 to 43 wherein the strain administered is Bacillus strain 71 (NRRL No. B-67867).

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

48. The method of any one of clauses 39 to 47 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 5.0 x 10¹² CFU/gram of the feed composition.

49. The method of any one of clauses 39 to 47 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition.

50. The method of any one of clauses 39 to 47 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10⁴ CFU/gram of the feed composition.

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

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.

53. The method of any one of clauses 39 to 51 wherein the animal is a sow and the Bacillus strain is administered during gestation.

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

55. The method of clause 39 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

56. The method of any one of clauses 1 to 55 wherein the Bacillus strain has an effect selected from the group consisting of maintaining microbial balance in the gut of the animal, improving animal performance or health, maintaining gut health in the animal, reducing detrimental pathogens in the gut of the animal, odor reduction, reducing detrimental pathogens in the urine or feces of the animal, and preserving the growth of beneficial bacteria in the gut of the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-B show percent inhibition values from 3 of 15 E. coli showing an improved inhibition response when Bacillus supernatant and Enrofloxacin are combined.IGS.

Fig. 2 is a photograph displaying RAPD PCR profiles for Bacillus strain 9 (NRRL No. B-67866) and Bacillus strain 57 (NRRL No. B-67870).

Fig. 3 is a photograph displaying RAPD PCR profiles for Bacillus strain 71 (NRRL No. B-67867) and Bacillus strain 126 (NRRL No. B-67868).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Methods are provided for inhibiting E. coli in animals. In various embodiments, the animal can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal. In the embodiment where the animal is a poultry species, the poultry species can be selected from the group consisting of a broiler, a chicken, a layer, a breeder, a turkey, a turkey poult, a gosling, a duckling, a guineakeet, a pullet, a hen, a rooster, a cockerel, and a capon. In the embodiment where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig. In one embodiment, a method of feeding an animal is provided. The method comprises the steps of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B- 67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering to the animal an antibiotic, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal and wherein the antibiotic is enrofloxacin.

In another embodiment, a method of controlling a detrimental effect of E. coli is provided. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of 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, and controlling the detrimental effect of E. coli in the animal.

In yet another embodiment, a method of feeding an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B- 67868), a strain having all of 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. The following clauses, and combinations thereof, provide various additional illustrative aspects of the invention described herein. The various embodiments described in this section titled “DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS” are applicable to any of the following embodiments of the invention described in the numbered clauses below.

4. The method of any one of clauses 1 to 2 wherein the E. coli inhibition reduces E. coli disease in the animal.

5. The method of any one of clauses 1 to 4 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

8. The method of any one of clauses 1 to 7 wherein the strain administered is Bacillus strain 9 (NRRL No. B-67866). 9. The method of any one of clauses 1 to 7 wherein the strain administered is Bacillus strain 57 (NRRL No. B-67870).

17. The method of any one of clauses 1 to 15 wherein the animal is a sow and the Bacillus strain is administered during gestation.

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

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

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

32. The method of any one of clauses 20 to 30 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition. 33. The method of any one of clauses 20 to 30 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10⁴ CFU/gram of the feed composition.

42. The method of any one of clauses 39 to 41 wherein the Bacillus strain produces an enzyme selected from the group consisting of an a-galactosidase, a protease, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof. 43. The method of any one of clauses 39 to 42 further comprising the step of administering to the animal another different bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

In various embodiments, the animal to which a feed additive, a feed composition, or drinking water, and an antibiotic, wherein the antibiotic is enrofloxacin, as described herein is administered can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal. In the embodiment where the animal is a companion animal, the companion animal can be, for example, a canine species or a feline species. In the embodiment where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig. In various exemplary embodiments, the animal can be selected from the group consisting of a chicken (e.g., a broiler or a layer), a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish (e.g., a tilapia, a catfish, a flounder, or a salmon), a crustacean (e.g., a shrimp or a crab), and combinations thereof. In another embodiment, the feed additive, feed composition, or drinking water, and the antibiotic wherein the antibiotic is enrofloxacin, as described herein, can be administered to an animal and the animal is a human.

In various embodiments of the methods or compositions described herein, the Bacillus strain and the antibiotic, wherein the antibiotic is enrofloxacin, can have an effect selected from the group consisting of maintaining microbial balance in the gut of the animal, preventing or reducing E. coll disease in the animal, improving animal performance or health, maintaining gut health in the animal, reducing detrimental pathogens in the gut of the animal, odor reduction, reducing detrimental pathogens in the urine or feces of the animal, and preserving the growth of beneficial bacteria in the gut of the animal. In any of the embodiments described herein, the Bacillus strain can be a Bacillus subtilis strain or a Bacillus licheniformis strain.

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

In one embodiment of the invention, an effective amount of the Bacillus strain can be administered to inhibit E. coli in the animal. As used herein, “inhibit E. coli” can mean reducing E. coli disease, preventing E. coli disease, maintaining the normal microbial balance in the animal, reducing the number of detrimental E. coli organisms in the animal, reducing the activity of E. coli in the animal, or reducing the symptoms of E. coli disease in the animal, or combinations thereof. By “effective amount” is meant an amount of the Bacillus strain (e.g., strain 9, 57, 71, and 126) capable of E. coli inhibition or capable of controlling a detrimental effect of E. coli, as described below, by any mechanism.

In embodiments described herein wherein the compositions of the present invention comprising Bacillus strains 9, 57, 71, and/or 126, or strains having their identifying characteristics, with enrofloxacin are administered to an animal, the compositions are preferably administered to animals orally in a feed composition or in drinking water, but any other effective method of administration known to those skilled in the art may be utilized such as in a paste, a gel, a liquid drench, a top dress, a powder, a liquid, a pellet, or a capsule. In one illustrative embodiment, the Bacillus strains 9, 57, 71, and/or 126, or strains having their identifying characteristics, and enrofloxacin, are provided in the form of an additive for addition to the drinking water of an animal, or enrofloxacin is added to the drinking water separately.

In another illustrative embodiment, the Bacillus strains 9, 57, 71, and/or 126, or strains having their identifying characteristics, and enrofloxacin, are provided in the form of a feed additive for addition to a feed composition, or the enrofloxacin is administered separately. The feed composition may contain Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics, in a mixture with an animal feed blend, including any art-recognized animal feed blend or any animal feed blend described herein. As used herein, “feed composition” or “animal feed composition” means a feed composition comprising Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics, in a mixture with an animal feed blend, and, optionally any other components that could be used in a feed composition, including other bacterial strains, such as other different Bacillus strains or Lactobacillus strains. In one embodiment, the feed composition may be in the form of a ground meal.

Any animal feed blend, including those known in the art and those described herein, may be used in accordance with the methods and compositions described in this patent application, such as rapeseed meal, cottonseed meal, soybean meal, cornmeal, barley, wheat, silage, and haylage. In various embodiments, the animal feed blend can be supplemented with Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics, but other ingredients may optionally be added to the animal feed blend, including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains. In various illustrative embodiments, optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides. Other optional ingredients include dried distillers grain solubles, fat (e.g., crude fat), phosphorous, sodium bicarbonate, limestone, salt, phytate, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, an oil derived from fish meal, raw seed (e.g., flaxseed), an antioxidant, and starch. In another embodiment, minerals may be added in the form of a mineral premix.

Optional amino acid ingredients that may be added to the animal feed blend are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HC1, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HC1, and analogs, and salts thereof. Vitamins that may be optionally added are thiamine HC1, riboflavin, pyridoxine HC1, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. In another embodiment, vitamins may be added in the form of a vitamin premix. In yet another embodiment, protein ingredients may be added to the animal feed blend and include protein obtained from meat meal, bone meal, or fish meal, liquid or powdered egg, fish solubles, crude protein, and the like.

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

In another illustrative embodiment, one or more enzymes may be added to the animal feed blend or to the feed composition or feed additive or the bacterial strain. In various embodiments, the enzymes that may be added include a galactosidase, a phytase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, combinations thereof, and any other enzyme that improves the effectiveness of the feed composition or feed additive for E. coli inhibition or controlling a detrimental effect of E. coli. In yet another embodiment, yeast, fungi (e.g., Aspergillus or Trichoderma), or micronutrients may be added to the animal feed, feed composition, or feed additive, or the bacterial strain. Any of the ingredients described above that are suitable for addition to an additive for the drinking water of the animal may be added as a component of the additive for the drinking water of the animal as described herein.

In various illustrative embodiments, the Bacillus strain (e.g., Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics), or any other different bacterial strains added in addition to Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics, can be administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 5.0 x 10¹² CFU/gram of the feed composition or at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition. In other embodiments, the Bacillus strain (e.g., Bacillus strain 2, 3, 4, 5, 9, 57, 71, and/or 126, or strains having their identifying characteristics) is administered in the feed composition at a dose greater than about 1.0 x 10³ CFU/gram of the feed composition, at a dose greater than about 1.1 x 10³ CFU/gram of the feed composition, at a dose greater than about 1.25 x 10³ CFU/gram of the feed composition, at a dose greater than about 1.5 x 10³ CFU/gram of the feed composition, at a dose greater than about 1.75 x 10³ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 2.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 3.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 4.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 5.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 6.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 7.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 8.0 x 10⁴ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10⁵ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10⁶ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10⁷ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10⁸ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10⁹ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10¹⁰ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10¹¹ CFU/gram of the feed composition, or at a dose greater than about 1.0 x 10¹² CFU/gram of the feed composition. In another embodiment, the Bacillus strain (e.g., Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics), or any other different bacterial strains added in addition to Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics, can be administered in the feed composition at a dose of about 1.0 x 10² CFU/gram of the feed composition to about 5.0 x 10¹² CFU/gram of the feed composition or at a dose of about 1.0 x 10² CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition, or at a dose greater than about 1.0 x 10² CFU/gram of the feed composition. In another embodiment, any of the dosages described herein can be in CFU/ml of drinking water in embodiments where the strains are administered in the drinking water of the animal.

In various embodiments, the Bacillus strain (e.g., Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics) for use in accordance with the methods or compositions described herein can be selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of 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), National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, 1815 North University Street, Peoria, Illinois 61604- 3999, and were given accession numbers B-67866, B-67870, B-67867, and B-67868, respectively. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. 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, respectively, as referred to in the application. The deposit certificates refer to the strains as MDG9, MDG57, MDG71, and MDG126, respectively. These four strains are Bacillus subtilis strains.

In one aspect, any of these strains can be administered alone or in combination in the form of a feed composition (e.g., a complete feed comprising an animal feed blend) or drinking water for an animal. 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, is administered in combination with enrofloxacin, as described herein in the same or separate compositions.

In another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics) can be administered to the animal along with another different bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof. In yet another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain 9, 57, 71, and/or 126, or strains having their identifying characteristics) can be administered to the animal along with any other bacterial strain effective to inhibit or control detrimental effects of E. coli in the animal.

As used herein “a strain having all of the identifying characteristics of’ Bacillus strain 9, 57, 71, or 126, can be a mutant strain having all of the identifying characteristics of Bacillus strain 9, 57, 71, or 126 (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain 9, 57, 71, or 126, enzyme activities that correspond to Bacillus strain 9, 57, 71, or 126, antimicrobial activity that corresponds to Bacillus strain 9, 57, 71, or 126, antibiotic sensitivity and tolerance profiles that correspond to Bacillus strain 9, 57, 71, or 126, or combinations of these identifying characteristics). In alternate embodiments, the mutation can be a natural mutation, or a genetically engineered mutation. In another embodiment, “a strain having all of the identifying characteristics of’ Bacillus strain 9, 57, 71, or 126, can be a strain, for example, 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, as long as 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 that corresponds to the DNA fingerprint of Bacillus strain 9, 57, 71, or 126).

The feed composition or drinking water comprising Bacillus strain 9, 57, 71, or 126, or strains with their identifying characteristics, and enrofloxacin, may be administered to the animal for any time period that is effective to inhibit E. coli or control a detrimental effect of E. coli, or combinations thereof. For example, in one embodiment the feed composition or drinking water may be provided to the animal daily. In an alternate embodiment, the feed composition or drinking water may be administered to the animal during lactation and/or during gestation. The time periods for administration of the feed composition or drinking water described above are non-limiting examples and it should be appreciated that any time period or administration schedule determined to be effective to inhibit E. coli or control a detrimental effect of E. coli, or combinations thereof, may be used.

In embodiments involving “controlling a detrimental effect of E. coli" controlling a detrimental effect can mean reducing E. coli disease, preventing E. coli disease, maintaining the normal microbial balance in the animal, reducing the number of detrimental E. coli in the animal, reducing the activity of E. coli in the animal, or reducing the symptoms of E. coli disease in the animal, or combinations thereof. By “effective amount” is meant an amount of the Bacillus strain (e.g., Bacillus strain 9, 57, 71, and/or 126) capable of controlling a detrimental effect of E. coli as described herein, by any mechanism.

In one embodiment, the feed additive for addition to an animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, with an automated micro-nutrient delivery system, or, for example, by hand- weighing and addition to achieve any of the doses of Bacillus strain 9, 57, 71, and/or 126, or strains with their identifying characteristics, described herein, for administration to the animal in the form of a complete feed composition. The mixing can also be done by any other suitable method known in the art for combining direct-fed microbials with an animal feed blend to obtain a uniform mixture. In various embodiments, the mixing can be done for any suitable time period (e.g., about 1 to about 4 minutes). In the embodiment where Bacillus strain 9, 57, 71, and/or 126, or strains with their identifying characteristics, are in the form of an additive for the drinking water of the animal, the Bacillus strain 9, 57, 71, and/or 126, or strains with their identifying characteristics, can be in the form of, for example, a powder, a liquid, or pellets, and can be mixed with the drinking water using any suitable method known in the art to achieve any of the doses of Bacillus strain 9, 57, 71, and/or 126, or strains with their identifying characteristics, described herein, for administration to the animal in the drinking water of the animal. Bacillus strain 9, 57, 71, and/or 126, or strains with their identifying characteristics, can also be fed directly to the animal orally (i.e., by oral insertion) in the form of a powder, a freeze-dried composition, a gel, a top-dressing, a liquid, a capsule, a paste, a liquid drench, or a pellet. Any of these embodiments can also be applied to mixing and administration of enrofloxacin as described herein.

In additional embodiments of the invention, compositions are provided comprising Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B- 67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B- 67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B- 67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

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

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

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

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

In one illustrative aspect, the strains for addition to the commercial package, feed additive, additive for the drinking water of the animal, or the feed composition can be in the form of a concentrate (e.g., about 1 x 10⁸ to about 5 x 10⁹ CFU/g) or a superconcentrate (e.g., about 1 x 10¹⁰ to about 5 x 10¹² CFU/g). In another embodiment, the strains for addition to the commercial package, feed additive, feed composition, or additive for the drinking water of the animal can be in a dry form (e.g., a powder), a pelleted form, a liquid form, a liquid drench, a powder, a freeze-dried composition, in the form of a top-dressing, a paste, a capsule, or in the form of a gel, or any other suitable form.

In another illustrative embodiment, the strain or strains in the commercial package, the feed additive, additive for the drinking water of the animal, or feed composition can further comprise a carrier for the Bacillus strain or strains. The carrier can be selected from the group consisting of a bran, rice hulls, a salt, mineral oil, a dextrin (e.g., maltodextrin), whey, sugar, sucrose, limestone, yeast culture, dried starch, sodium silico aluminate, silicon dioxide, polypropylene glycol, polysorbate 80, vegetable oil, and combinations thereof. The carrier is exogenously added to the bacterial strain (i.e., not naturally present or not present in nature with the bacterial strain). In another embodiment, the feed additive, additive for the drinking water of the animal, or feed composition can further comprise a binder such as clay, yeast cell wall components, aluminum silicate, glucan, or other known binders. In another embodiment, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise inorganic/organic binders, essential oils, and/or organic acids. The binder is exogenously added to the bacterial strain (i.e., not naturally present or not present in nature with the bacterial strain).

In yet other embodiments, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B- 67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B- 67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, is in a container for commercial use.

In various embodiments the container can 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 drum, a bottle, or a box. In illustrative aspects, the container for the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, can comprise plastic, metal, foil, paper, fiber, or cardboard (e.g., a plastic pail, a paper bag, a foil bag, a fiber drum, etc.). The commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise instructions for use of one or more of the Bacillus strains.

In one aspect, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition described herein can further comprise an exogenously added nutrient component (i.e., a nutrient component not present with the bacterial strain in nature) selected from the group consisting of a vitamin, an antibiotic, an enzyme, a water- soluble or water-insoluble monosaccharide, disaccharide, or polysaccharide, a fat, phosphorous, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seed, an antioxidant, and a starch.

In one embodiment, the exogenously added nutrient component is an enzyme and the enzyme is selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

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

In embodiments of the methods and compositions described herein, synergism occurs between the Bacillus strains and enrofloxacin.

The following examples are for illustrative purposes only. The examples are non-limiting, and are not intended to limit the invention in any way.

EXAMPLE 1

Bacillus Testing on Baytril (15) resistant E. coli

Utilizing a broth AST method, four Bacillus strains were tested against 15 Baytril-resistant E. coli. All four Bacillus strains were observed to be more effective at inhibiting the growth of these resistant E. coli than Mecadox™ was capable of alone.

Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), and Bacillus strain 126 (NRRL No. B-67868), consistently improved and effectively inhibited (>50% inhibition) antibiotic resistant pathogens when the same concentration of antibiotic was administered in conjunction with Bacillus supernatant. Of the 15 Enrofloxacin resistant E. coli: when combined with antibiotics Bacillus strains 9 and 71 effectively inhibited 14/15 isolates. When combined with antibiotics Bacillus strains 57 and 126 effectively inhibited 15/15 isolates.

The goal of this project was to identify Bacillus strains that exhibit strong inhibition potential of target pathogens in combination with antibiotics. This synergy can be defined as a resulting higher percent inhibition of antibiotic-resistant pathogens when Bacillus supernatants are combined with antibiotics than when the supernatant and antibiotic are tested separately against the same isolates. Four Bacillus strains yielded consistent strong inhibition activity against Baytril-resistant E. coli and were tested. These four Bacillus strains are (BS 9, 57, 71, and 126), identified as strains (NRRL No. B-67866), (NRRL No. B-67870), (NRRL No. B-67867), (NRRL No. B-67868), respectively.

EXAMPLE 2

RAPD-PCR DNA Profiles The Randomly Amplified Polymorphic DNA PCR method (hereafter referred to as RAPD-PCR) was used to identify genetic variability of each strain. Preparation of the DNA to be used in the RAPD-PCR reaction was done by using the QIAGEN® Tissue and Blood single column kit (QIAGEN®, Venlo, The Netherlands). Figures 2 and 3 illustrate RAPD-PCR results for strains 9, 57, 71, and 126, with the unlabeled lanes being a molecular weight ladder.

The results show that all strains are unique from each other.

Claims

WHAT IS CLAIMED IS:

1. A method of feeding an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 (NRRL No. B-67866), a strain having all of the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No.B-67870), a strain having all of the identifying characteristics of Bacillus strain 57 (NRRL No.B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all of the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all of 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.

2. The method of claim 1 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

3. The method of claim 1 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

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

5. The method of claim 1 further comprising the step of administering to the animal another different bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

6. The method of claim 1 wherein the strain administered is Bacillus strain 9 (NRRL No. B-67866).

7. The method of claim 1 wherein the strain administered is Bacillus strain 57 (NRRL No. B-67870).

8. The method of claim 1 wherein the strain administered is Bacillus strain 71 (NRRL No. B-67867).

9. The method of claim 1 wherein the strain administered is Bacillus strain 126 (NRRL No. B-67868).

10. The method of claim 1 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 5.0 x 10¹² CFU/gram of the feed composition.

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11. The method of claim 1 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10³ CFU/gram of the feed composition to about 1.0 x 10⁷ CFU/gram of the feed composition.

12. The method of claim 1 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10⁴ CFU/gram of the feed composition.

13. The method of claim 1 further comprising the step of administering to the animal an enzyme selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

14. The method of claim 1 wherein the animal is a sow and the Bacillus strain is administered during lactation.

15. The method of claim 1 wherein the animal is a sow and the Bacillus strain is administered during gestation.

16. The method of claim 1 wherein the feed composition is administered daily to the animal.

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

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