Stool collection method and sample preparation method for transplanting fecal microbiota

1. A method for the preparation of a homogenous mixture of fecal microbiota originating from at least two preselected donors comprising the following steps:

a) taking at least one sample of fecal microbiota from said at least two preselected donors,

b) placing the sample obtained in a) in an oxygen-impermeable collection device within a time period of less than 5 minutes after taking the sample,

c) performing quality control of the samples taken based on quality criteria, and excluding samples that do not meet the quality criteria,

d) adding an aqueous saline solution comprising at least one cryoprotectant and/or a bulking agent to each of the samples retained after the quality control step c),

e) filtering the samples obtained at the end of step d) to form a series of inocula,

f) grouping said inocula to form a mixture of inocula, and

g) homogenization of said mixture obtained in step f) to obtain a homogenous mixture,

wherein steps b) and d) to g) are carried out under anaerobic conditions.

2. The method according to claim 1 , wherein the homogenous mixture of fecal microbiota originates from at least four donors.

3. The method according to claim 1 , wherein the donors are preselected according to the following preselection criteria:

i) age ranging from 18 to 60 years,

ii) body mass index (BMI) ranging from 18 to 30,

iii) absence of personal medical history of infectious diseases, metabolic and neurological disorders, or depression,

iv) absence of taking medicaments capable of degrading the composition of the intestinal microbiota,

v) absence of occurrence of symptoms associated with a gastrointestinal disease,

vi) absence of travel in tropical countries,

vii) absence of at-risk sexual behavior,

viii) absence of wound, piercing and/or tattoo,

ix) absence of chronic fatigue,

x) absence of allergic reaction, and

xi) optionally, having a varied diet.

4. The method according to claim 1 , wherein the quality criteria of the samples of step c) comprise:

consistency of the sample ranging from 1 to 6 on the Bristol scale, and

absence of blood or urine in the sample.

5. The method according to claim 4 , wherein the quality criteria of the samples of step c) further comprise:

absence of the following bacteria: Campylobacter, Clostridium difficile, Salmonella, Yersinia enterocolitica, Vibrio sp., Shiga toxin-producing E. coli (STEC) stx1/stx2, multi-resistant bacteria, bacteria producing extended-spectrum beta-lactamases (ESBL) glycopeptide/vancomycin resistant Enterococci (GRE/VRE) and Listeria monocytogenes,

absence of the following parasites: Cryptosporidium parvum, Cyclospora sp., Entamoeba histolytica, Giardia lamblia, Blastocystis hominis , Helminths, Strongyloides stercoralis, Isospora sp., Microsporidia and Dientamoeba fragilis,

absence of the following viruses: adenovirus F40/41, astrovirus, norovirus, rotavirus A, sapovirus and picornavirus, and

absence of the following bacteria: enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) It/st, Shigella /enteroinvasive E. coli (EIEC) and Plesiomonas shigelloides.

6. The method according to claim 5 , wherein picornavirus is aichi virus and/or enterovirus.

7. The method according to claim 4 , wherein the quality criteria of the samples of step c) further comprise:

absence of the following bacteria: Campylobacter, Clostridium difficile, Salmonella, Yersinia enterocolitica, Vibrio sp., Shiga toxin-producing E. coli (STEC) stx1/stx2, multi-resistant bacteria, bacteria producing extended-spectrum beta-lactamases (ESBL) glycopeptide/vancomycin resistant Enterococci (GRE/VRE), Listeria monocytogenes , and carbapenem-resistant bacteria,

absence of the following parasites: Cryptosporidium parvum, Cyclospora sp., Entamoeba histolytica, Giardia lamblia, Blastocystis hominis , Helminths, Strongyloides stercoralis, Isospora sp., Microsporidia and Dientamoeba fragilis,

absence of the following viruses: adenovirus F40/41, astrovirus, norovirus, rotavirus A, sapovirus and picornavirus, and

absence of the following bacteria: enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) It/st, Shigella /enteroinvasive E. coli (EIEC) and Plesiomonas shigelloides.

8. The method according to claim 7 , wherein picornavirus is aichi virus and/or enterovirus.

9. The method according to claim 1 , wherein the at least one cryoprotectant and/or bulking agent is a polyol, a di-, tri- or polysaccharide or mixture thereof and a bulking agent.

10. The method according to claim 1 , wherein the aqueous saline solution comprises maltodextrin and trehalose.

11. The method according to claim 1 , wherein the filtration in step e) is carried out with a filter comprising pores of diameter less than or equal to 0.5 mm.

12. The method according to claim 1 , wherein the time between the beginning of step a) and the end of step g) is less than 76 hours.

13. The method according to claim 1 , wherein step g) of homogenization is carried out at a temperature ranging from 2° C. to 25° C.

14. The method according to claim 1 , further comprising

h) transferring the homogenized mixture obtained in step g):

i) to inoculum pouches for storage at a temperature of approximately −50° C. to −80° C., or for storage at a temperature ranging approximately from 2 to 6° C. for use of the mixture within approximately 16 hours, or for storage at a temperature ranging from 10 to 25° C. for use of the mixture within approximately 4 hours, or

ii) to a lyophilization device for lyophilization.

15. The method according to claim 1 , wherein the homogenous mixture of fecal microbiota comprises the following bacterial genera Blautia, Faecalibacterium, Alistipes, Eubacterium, Bifidobacterium, Ruminococcus, Clostridium, Coprococcus, Odoribacter, Roseburia , Holdemanella, Anaerostipes, Oscillibacter, Subdoligranulum and Butyrivibrio.