Method of culturing segmented filamentous bacteria in vitro

1. An in vitro method of culturing a segmented filamentous bacterium (SFB) strain, comprising co-culturing said SFB strain with a eukaryotic host cell, wherein the culture is performed at an O 2 level of less than 5% in a rich tissue culture liquid medium comprising bacterial medium components and iron, and wherein the co-culture is performed until the SFB strain releases intracellular offspring or spores.

2. The method according to claim 1 , wherein the SFB strain is a wild-type strain or a genetically modified strain.

3. The method according to claim 2 , wherein the genetically modified SFB strain expresses an antigen of a diarrheal pathogen selected from the group consisting of Shigella , enterotoxic Escherichia coli , and attaching and effacing lesion (A/E)-inducing enteropathogenic Escherichia coli.

4. The method according to claim 3 , wherein the antigen is Shigella IpaB protein or IpaD protein.

5. The method according to claim 3 , wherein the antigen is an enterotoxic Escherichia coli antigen selected from colonization factor antigen I, adhesin CfaE, a heat-labile toxin, and nontoxic B subunit of heat-labile toxin.

6. The method according to claim 3 , wherein the antigen is an attaching and effacing lesion (A/E)-inducing enteropathogenic Escherichia coli intimin, or a fragment encoding amino adds 363 to 808 of the 94 kDa beta-intimin antigen.

7. The method according to claim 2 , wherein the SFB strain is isolated from a mammal.

8. The method according to claim 7 , wherein the SFB strain is isolated from a mammal selected from the group consisting of a human and a mouse.

9. An in vitro method of culturing a SFB strain, comprising the steps of:

a) growing eukaryotic host cells on a solid culture medium;

b) transferring the eukaryotic host cells grown in step a) into a eukaryotic host cell-SFB liquid culture medium to provide a cell culture;

c) challenging the cell culture of step b) with a SFB strain; and

d) co-culturing the eukaryotic host cells and the SFB strain in the liquid culture medium at an O 2 level of less than 5%, wherein the co-culture is performed until the SFB strain releases intracellular offspring or spores.

10. The method according to claim 9 , wherein the eukaryotic host cell is an epithelial cell or a cancer cell.

11. The method according to claim 9 , wherein the eukaryotic host cells are gastrointestinal tract cells or carcinoma cells.

12. The method according to claim 9 , wherein the eukaryotic host cells are selected from the group consisting of human Caco-2 cell line cells, human TC7 cell line cells, human HeLa cell line cells, mouse mICcl2 cell line cells, and mouse CMT93 cell line cells.

13. The method according to claim 9 , wherein in step a) the eukaryotic host cells are grown at an O 2 level of from 0% to 5%.

14. The method according to claim 9 , wherein in step a) the eukaryotic host cells are grown at an O 2 level of from 0.5 to 3%.

15. The method according to claim 9 , wherein in step a) the eukaryotic host cells are grown at an O 2 level of from 1 to 2.5%.

16. The method according to claim 9 , wherein in step b) the eukaryotic host cell-SFB liquid culture medium is a rich tissue culture medium comprising bacterial medium components and iron.

17. The method according to claim 16 , wherein the liquid culture medium comprises brain-heart infusion and a yeast/peptone/casein amino-acid mixture.

18. The method according to claim 16 , wherein the liquid culture medium is supplemented with from 1% to 5% decomplemented fetal calf serum.

19. The method according to claim 16 , wherein the liquid culture medium is supplemented with from 1% to 3% decomplemented fetal calf serum.

20. The method according to claim 16 , wherein the liquid culture medium is supplemented with 2% decomplemented fetal calf serum.

21. The method according to claim 16 , wherein the liquid culture medium further comprises sugars, retinoic acid and/or nucleotides.

22. The method according to claim 16 , wherein the iron is in the form of Fe 2+ /Fe 3+ , 3×Fe 2+ , 3×Fe 2+ or hemin.

23. The method according to claim 16 , wherein the iron concentration is from 0.015 to 0.05 mM.

24. The method according to claim 16 , wherein the iron concentration is from 0.02 to 0.04 mM.

25. The method according to claim 9 , wherein the liquid culture medium comprises DMEM/F12 advanced medium, supplemented with fetal calf serum, non-essential amino acids, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, brain-heart infusion, a yeast/peptone/casein amino-acid mixture and iron.

26. The method according to claim 9 , wherein in step c) the SFB strain is in the form of a filament or an intracellular offspring.

27. The method according to claim 9 , wherein in step c), the SFB strain is directly contacted with the eukaryotic host cells.

28. The method according to claim 27 , wherein the co-culture of the eukaryotic host cells and the SFB strain is performed at an O 2 level of from 0.5 to 3%.

29. The method according to claim 9 , wherein the co-culture of the eukaryotic host cells and the SFB strain is performed at an O 2 level of from 0% to 5%.

30. The method according to claim 9 , wherein the co-culture of the eukaryotic host cell and the SFB is performed at an O 2 level of from 1 to 2.5%.

31. The method according to claim 9 , wherein iron is at a concentration of from 0.015 to 0.05 mM.

32. The method according to claim 9 , further comprising recovering the cultured SFB strain, wherein the cultured SFB strain is recovered in a form of at least one of a filament, an intracellular offspring and a spore.

33. The method according to claim 9 , wherein the SFB strain is a wild-type strain or a genetically modified strain.

34. The method of claim 9 , wherein the eukaryotic host cells grown in step a) are grown until a cell confluence of at least 20% is obtained.

35. The method of claim 9 , wherein the eukaryotic host cells grown in step a) are grown until a cell monolayer is obtained.

36. The method of claim 9 , wherein the eukaryotic host cells grown in step a) are plated on a plate.

37. The method of claim 9 , wherein the eukaryotic host cells grown in step a) are plated on a tissue culture well or transwell, prior to step b).

38. The method of claim 9 , wherein the seeding density of the eukaryotic host cells in a) is between 1×10 4 and 6×10 4 cells per cm 2 .

39. The method according to claim 9 , wherein the SFB strain is added in step c) when the culture density of the eukaryotic host cells is from 0.5+10 5 to 3×10 5 cells per cm 2 .

40. The method according to claim 9 , wherein the ratio in step c) between the eukaryotic host cells and the SFB strain is from 0.1 to 100.

41. The method according to claim 40 , wherein the ratio between the eukaryotic host cells and the SFB strain is from 0.3 to 60.

42. The method according to claim 9 , wherein the co-culture is performed for a duration selected from the group consisting of 1, 2, 3, 4, 5 and 6 days.

43. The method according to claim 9 , wherein d) comprises applying conditions to induce spore formation, selected from adding an oxidative stress and short-term culture in the presence of an antibiotic.

44. A food product comprising the recovered SFB strain made by the method of claim 32 .

45. The food product according to claim 44 , wherein the SFB strain is alive.