METHODS OF REDUCING OR PREVENTING CLOSTRIDIOIDES DIFFICILE COLONIZATION

20230200397

17/996394

April 16, 2021

Embodiments of the present disclosure relate to a method of preventing and/or reducing C. difficile colonization of a surface. In certain embodiments, the aptamer can be used to kill and/or deactivate a C. difficile spore. In certain embodiments, the aptamer can be used to increase the sporicidal activity of a sporicidal agent. Embodiments of the present disclosure relate to the use of aptamers which specifically bind to C. difficile surface proteins, for example proteins located on the surface of a C. difficile spore.

LIV PROCESS, INC. (Ardmore, PA, US)

1. A method for killing or inactivating Clostridioides difficile (C. difficile) spores, comprising contacting the spores with an agent selected from the group consisting of a sporicidal agent and a sporostatic agent; and an aptamer capable of specifically binding to C. difficile.

2. A method for enhancing a sporicidal effect against C. difficile of a sporicidal agent, the method comprising contacting C. difficile spores with an aptamer capable of specifically binding to C. difficile spores and an agent selected from the group consisting of a sporicidal agent and a sporostatic agent.

3. The method according to claim 1 or claim 2, wherein the agent is a sporicidal agent.

4. The method according to claim 1 or claim 2, comprising locating the aptamer and the sporicidal agent at a location suspected of comprising C. difficile spores.

5. The method according to claim 4, wherein the location comprises a surface and the method comprises contacting the surface with the sporicidal agent prior to, at essentially the same time, or subsequent to contacting the surface with the aptamer.

6. The method according to claim 5, which comprises: contacting the surface with the aptamers for a predetermined period of time configured to enable the aptamer to bind to the C. difficile spore to form aptamer-spore complex, and contacting the surface comprising the aptamer-spore complex with the sporicidal agent.

7. The method according to claim 5 or claim 6, further comprising contacting the surface with a GH neutraliser.

8. The method according to claim 5 or claim 6, comprising submerging the C. difficile spores with a composition comprising the aptamer and/or submerging the spores in the agent.

9. The method according to claim 5 or claim 6, wherein the method comprises spraying the C. difficile spores with a composition comprising the aptamer and/or spraying the C. difficile spores with the sporicidal agent.

10. The method according to claim 5 or claim 6 wherein the method comprises applying a composition comprising the aptamer by means of a cloth and/or applying the sporicidal agent by means of a cloth and/or a wipe.

11. The method according to claim 1 or claim 2, wherein the method is performed at a temperature of about 5° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C. or 90° C.

12. The method according to any one of the preceding claims, wherein a C. difficile spore coat surface protein or an exosporium layer protein.

13. The method according to any one of the preceding claims, wherein the C. difficile protein comprises CdeC, CdeM, CotA, CotE or CotE Chitinase.

14. The method according to claim 13, wherein the C. difficile protein is a CdeC protein having an amino acid sequence as set forth in SEQ. ID. NO. 18.

15. The method according to claim 13, wherein the C. difficile protein is a CdeM protein having an amino acid sequence as set forth in SEQ. ID. NO. 19.

16. The method according to claim 13, wherein the C. difficile protein is a CotA protein having an amino acid sequence as set forth in SEQ. ID. NO. 15.

17. The method according to claim 13, wherein the C. difficile protein is a CotE protein having an amino acid sequence as set forth in SEQ. ID. NO. 16.

18. The method according to claim 13, wherein the C. difficile protein is a CotE Chitinase protein having an amino acid sequence as set forth in SEQ. ID. NO. 17.

19. The method according to claim 12, wherein the aptamer comprises an aptamer which comprises or consists of: (a) a nucleic acid sequence selected from any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (b) a nucleic acid sequence having at least 85% identity for example 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; or (d) a nucleic acid sequence having at least about 20 consecutive nucleotides of a sequence having at least 85% identity with any one of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55.

20. The method according to claim 12, wherein the aptamer is a single stranded DNA aptamer.

21. The method according to claim 12, wherein the aptamer comprises an aptamer selected from: a) an aptamer which specifically binds to a CdeC; b) an aptamer which specifically binds to CdeM, c) an aptamer which specifically binds to CotA, d) an aptamer which specifically binds to CotE; e) an aptamer which specifically binds to CotE Chitinase; and f) a combination of any of (a) to (e).

22. The method according to claim 4, comprising contacting the location with a plurality of aptamers, wherein each aptamer is capable of specifically binding to a C. difficile spore, wherein the plurality of aptamers comprises at least two aptamers capable of specifically binding to the same epitope of a C. difficile spore and/or at least two aptamers each aptamer capable of specifically binding to a different epitope of a C. difficile spore.

23. The method according to claim 22, wherein the plurality of aptamers comprises at least two aptamers selected from the group consisting of an aptamer comprising, consisting essentially of, or consisting of: (a) a nucleic acid sequence selected from any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (b) a nucleic acid sequence having at least 85% identity for example 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (d) a nucleic acid sequence having at least about 20 consecutive nucleotides of a sequence having at least 85% identity with any one of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55, or (e) a combination of any of (a) to (d).

24. The method according to claim 22 or claim 23, wherein the plurality of aptamers comprises at least two aptamers selected from the group consisting of: a) an aptamer which specifically binds to a CdeC; b) an aptamer which specifically binds to CdeM, c) an aptamer which specifically binds to CotA, d) an aptamer which specifically binds to CotE; e) an aptamer which specifically binds to CotE Chitinase; and f) a combination of any of (a) to (e).

25. The method according to claim 22, wherein the plurality of aptamers comprises: an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 1; an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 5; and an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 6.

26. The method according to claim 1 or claim 2, wherein the method comprises determining the presence, absence and/or concentration of C. difficile at a location prior to contacting the location with the sporicidal agent and/or sporistatic agent, wherein the step of determining comprises contacting the location with an aptamer and a detection molecule for a time sufficient for an aptamer-spore complex to form.

27. The method according to claim 26, wherein the aptamer comprises a detectable label.

28. The method according to claim 27, wherein the detectable label is selected from a fluorophore, a nanoparticle, a quantum dot, an enzyme, a radioactive isotope, a pre-defined sequence portion, a biotin, a desthiobiotin, a thiol group, an amine group, an azide, an aminoallyl group, a digoxigenin, an antibody, a catalyst, a colloidal metallic particle, a colloidal non-metallic particle, an organic polymer, a latex particle, a nanofiber, a nanotube, a dendrimer, a protein, and a liposome.

29. The method according to claim 1 or claim 2, wherein the agent is a sporicidal solution comprising chlorhexidine gluconate and optionally isopropyl alcohol.

30. The method according to claim 1 or claim 2, wherein the agent is a sporicidal agent which comprises peracetic acid.

31. The method according to claim 30, wherein the agent is a peracetic acid generating agent, and wherein the method further comprises wetting a wipe comprising sodium percarbonate with an aqueous solution prior to contacting the location.

32. The method according to claim 31, wherein the sporicidal solution comprises hydrogen peroxide and optionally comprises a silver stabilised hydrogen peroxide.

33. The method according to claim 31, wherein sporicidal solution comprises chlorhexidine gluconate and 70% isopropyl alcohol complex.

34. The method according to claim 1 or claim 2, comprising locating a combination of sporicidal and/or sporostatic agents.

35. The method according to claim 34, wherein the combination comprises peracetic acid and hydrogen peroxide.

36. A composition comprising an aptamer capable of specifically binding to C. difficile and a sporicidal agent and/or a sporistatic agent.

37. The composition according to claim 36, wherein the composition is for use in killing and/or inactivation of C. difficile.

38. The composition according to claim 36 or claim 37, wherein the aptamer is selected from the group consisting of an aptamer comprising, consisting essentially of, or consisting of: (a) a nucleic acid sequence selected from any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (b) a nucleic acid sequence having at least 85% identity for example 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; or (d) a nucleic acid sequence having at least about 20 consecutive nucleotides of a sequence having at least 85% identity with any one of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55.

39. The composition according to claim 36 or claim 37, wherein the aptamer comprises at least one of: a) an aptamer which specifically binds to a CdeC; b) an aptamer which specifically binds to CdeM, c) an aptamer which specifically binds to CotA, d) an aptamer which specifically binds to CotE; e) an aptamer which specifically binds to CotE Chitinase; or f) a combination of any of (a) to (e).

40. The composition according to claim 39, wherein the combination comprises a plurality of aptamers, and the plurality of aptamers comprising aptamers comprising, consisting essentially of, or consisting of: (a) a nucleic acid sequence selected from any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (b) a nucleic acid sequence having at least 85% identity for example 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (d) a nucleic acid sequence having at least about 20 consecutive nucleotides of a sequence having at least 85% identity with any one of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55, or (e) a combination of any of (a) to (d).

41. The composition according to claim 40, wherein the plurality of aptamers comprises an aptamer comprising, consisting essentially of, or consisting of: a nucleic acid sequence as set forth in SEQ. ID. NO. 1; an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 5; and an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 6.

42. The composition according to claim 36 or claim 37, wherein the sporicidal agent comprises peracetic acid and/or an agent capable of generating peracetic acid.

43. The composition comprising: (1) one or more aptamers comprising, consisting essentially of, or consisting of: (a) a nucleic acid sequence selected from any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (b) a nucleic acid sequence having at least 85% identity for example 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one the nucleic acid sequences as set forth in any of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (d) a nucleic acid sequence having at least about 20 consecutive nucleotides of a sequence having at least 85% identity with any one of SEQ ID NOs: 1 to 14, 23 to 39, or 43 to 55; (e) a combination of any of (a) to (d); and (2) a sporicidal agent and/or a sporostatic agent.

44. The composition according to claim 43, which comprises a plurality of aptamers, wherein optionally the plurality of aptamers comprises an aptamer comprising, consisting essentially of, or consisting of: a nucleic acid sequence as set forth in SEQ. ID. NO. 1; an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 5; and an aptamer comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ. ID. NO. 6.

45. The composition according to claim 43 or claim 44, wherein the sporicidal agent comprises peracetic acid and/or agent capable of generating peracetic acid.

46. A kit for killing or inactivating C. difficile spores comprising: an aptamer of any one of claims 36 to 45; and a sporicidal agent and/or sporistatic agent of any one of claims 36 to 45.

47. A kit for enhancing a sporicidal effect against C. difficile spores comprising: an aptamer of any one of claims 36 to 45; and a sporicidal agent and/or sporistatic agent of any one of claims 36 to 45.

48. The kit according to claim 46 or claim 47 further comprising: a light source and viewing goggles for determining the presence, absence and/or concentration of C. difficile at the location prior to contacting the location with the sporicidal agent and/or sporistatic agent.

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