Bacillus velezensis for reducing c. difficile abundance in a gut

Abstract

The present invention relates to preventing Clostridioides difficile infections using bacterial strains such as Bacillus velezensis. Described is also the use of Bacillus velezensis for reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

Description

[0001] BACILLUS VELEZENSIS FOR REDUCING C. DIFFICILE ABUNDANCE IN A GUT

[0002] Reference to a deposit of biological material

[0003] This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference. For complete information see last paragraph of the description.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to preventing Clostridioides difficile infections using bacterial strains.

[0006] BACKGROUND OF THE INVENTION

[0007] Clostridioides difficile (earlier designated Clostridium difficile) is a pathogen that can cause symptoms ranging from diarrhea to life-threatening colon inflammation. The gut microbiome has the capability of providing colonization resistance against C. difficile. Currently, most infection treatment approaches involve the use of broad-spectrum antibiotics, which disrupt the gut microbiome, leading to the loss of colonization resistance, thereby increasing the risk of recurrence of C. difficile infection. C. difficile is a leading cause of nosocomial antibiotic-associated diarrhoea, and while the species has been found to reside asymptomatically in the gut of healthy individuals, asymptomatic colonization can be a risk factor for the development of C. difficile infection.

[0008] There is a need to develop a method to accelerate the restoration of the gut microbiome after disruption of the gut microbiome to a state that can provide a resistance against C. difficile colonization.

[0009] SUMMARY OF THE INVENTION

[0010] The invention provides the use of Bacillus velezensis for reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

[0011] The invention furthermore provides one or more uses selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained, b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted, c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject, d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject;, e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject, and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject. Further provided by the invention is a method of reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects, the method comprising providing a Bacillus velezensis strain to the subject. Also provided herein is a method of reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject, the method comprising providing a Bacillus velezensis strain to the subject after disruption of the gut microbiome.

[0012] BRIEF DESCRIPTION OF DRAWINGS

[0013] Fig. 1 shows human fecal samples incubated for 6 h with vehicle control, clindamycin, C. difficile and / or B. velezensis DSM 33864 (Example 1) where A) shows C. difficile levels (Iog10) as CFU / mL after 6 hours of incubation with vehicle control, clindamycin, C. difficile and / or B. velezensis DSM 33864 as indicated. B) shows Bacillus levels as CFU / mL from human fecal incubation for 6 h with vehicle control, clindamycin, C. difficile or B. velezensis DSM 33864 as indicated.

[0014] Fig. 2 shows a second set of human fecal samples incubated for 6 h with clindamycin with / without C. difficile and / or B. velezensis DSM 33864 (Example 1) where A) shows C. difficile levels (Iog10) as CFU / mL after 6 hours of incubation with clindamycin, C. difficile and / or B. velezensis DSM 33864 as indicated, B) shows C. difficile toxin A (TcdA) as ng / mL in the same samples, C) shows C. difficile toxin B (TcdB) as ng / mL in the same samples, and D) shows centered log-ratio (Clr) transformed commensal Clostridia abundance by 16S rRNA gene amplicon sequences aggregated at phylogenetic class level in human fecal incubations after 0.5 h and 6 h.

[0015] Fig. 3 shows Shannon diversity index in human fecal incubations with / without clindamycin, C. difficile or B. velezensis DSM 33864 where A) shows the results after 0.5 h and B) shows the results 6 h.

[0016] Fig. 4 shows results obtained from mice colonized by C. difficile 24h after their commensal gut microbiome was disrupted with clindamycin (Example 2) where A) shows C. difficile levels (log 10) as CFU / mL after 24 hours in mice receiving vehicle control, clindamycin, B. velezensis DSM 33864 and / or C. difficile as indicated. B) shows C. difficile toxin A (TcdA) levels in cecum from mice receiving vehicle control, clindamycin, B. velezensis DSM 33864 and / or C. difficile as indicated. C) shows C. difficile toxin B (TcdB) levels in cecum from mice receiving vehicle control, clindamycin, B. velezensis DSM 33864 and / or C. difficile as indicated. D) shows gut microbiome composition of mice receiving vehicle control, clindamycin, B. velezensis DSM 33864 and / or C. difficile at indicated time points during the study. E) shows abundance of commensal Clostridia class bacteria as normalized values in samples of mice receiving vehicle control, clindamycin, B. velezensis DSM 33864 and / or C. difficile. F) shows Shannon diversity index per group assessed by 16S rRNA gene amplicon sequencing of mouse fecal samples timepoints T1-T4.

[0017] Fig. 5 shows results on the microbiome in fecal samples of healthy, non-antibiotic-treated mice receiving vehicle control or B. velezensis DSM 33864 (Example 3) where A) shows gut microbiome composition over time in fecal samples of mice receiving vehicle control or B. vele- zensis DSM 33864, B) shows alpha diversity over time as Shannon diversity index in fecal samples of mice receiving vehicle control or B. velezensis DSM 33864, and C) shows Clr transformed abundance of commensal Clostridia class bacteria as normalized values in fecal samples of mice after 14 days of daily, oral administration of vehicle control or B. velezensis DSM 33864.

[0018] Fig. 6 shows results on the microbiome in fecal samples of antibiotic-treated mice receiving vehicle control or B. velezensis DSM 33864 (Example 4) where A) shows gut microbiome composition over time in fecal samples of antibiotic-depleted microbiome of mice receiving vehicle control or B. velezensis DSM 33864. B) shows alpha diversity over time as Shannon diversity index in fecal samples of antibiotic-depleted microbiome of mice receiving vehicle control or B. velezensis DSM 33864. C) shows Clr transformed abundance of commensal Clostridia class bacteria as normalized values in fecal samples of antibiotic-depleted microbiome of mice receiving vehicle control or B. velezensis DSM 33864.

[0019] DEFINITIONS

[0020] The term “abundance” of a microorganism such as Clostridioides difficile is herein used in interchangeably with “load” of a microorganism and describes the number of said microorganisms occupying an object, organ, or organism.

[0021] The term “allowing natural reconstitution” in connection with the microbiome or commensal gut bacteria is herein used interchangeably with the term “not interfering with the natural reconstitution”. The term is used for the situation where the microbiome / commensal gut bacteria of a subject has been disrupted e.g. by antibiotic treatment and the microbiome subsequently is reconstituting to its pre-disruption state, where a bacterial strain such as e.g. Bacillus velenzis does not prevent, delay or disrupt the natural reconstitution of the microbiome in the gut of a subject.

[0022] The term “assisted recovery” in connection with the microbiome means recovery of the microbiome, i.e. bringing the microbiome back to normal after it has been disrupted, where the recovery is obtained using assistance such as from microbial replacement therapy e.g. fecal transplantation.

[0023] The term “Clostridioides difficile" is herein used interchangeably with “C. difficile" and describes a Gram-positive species of spore-forming bacteria which are anaerobic bacteria that are human pathogens. Clostridioides difficile was until 2016 designated “Clostridium difficile", a term that is still often used, and is also known as Peptoclostridium difficile, C. difficile, and C. diff.

[0024] The term “commensal bacteria” is herein used for bacteria that act on the immune system of a host to induce protective responses that prevent colonization and invasion by pathogens. The term “commensal Clostridia” is herein used for commensal bacteria that consist of grampositive, rod-shaped bacteria in the phylum Firmicutes and make up a substantial part of the total bacteria in the gut microbiome.

[0025] The term “ex vivo” is herein used for in vitro tests or experiments that the mimic the gut microbiome conditions in humans. An example of ex vivo test is provided in Example 1.

[0026] The term “gut” is herein used to describe the gastrointestinal (Gl) tract and is the tract or passageway of the digestive system of a human or animal such as a mammal. It contains all the major organs of the digestive system, including the esophagus, stomach, and intestines (small intestine (duodenum, jejunum, and ileum) and large intestine (colon, rectum, anal canal).

[0027] The term “microbiome” is herein used interchangeably with “microbiota” for all the bacteria, viruses, fungi, archaea, and eukaryotes that populate the animal or human body. An indication of the change in the composition of the microbiome of a human or an animal may e.g. be obtained by comparing results of microbiome compositional analysis I microbiome analysis from different timepoints as described in the examples herein.

[0028] The term “microbiome in the gut” is used interchangeably with “gut microbiome”, “microbiota in the gut” and “gut microbiota” to mean the bacteria, viruses, fungi, archaea, and eukaryotes that populate the gut of an animal or human body. An indication of the change in the composition of the microbiome in the gut of a human or an animal may e.g. be obtained by comparing results of gut microbiome compositional analysis I gut microbiome analysis from different timepoints as described in the examples herein.

[0029] The term “pathogen” is herein used for organisms such as bacteria, fungi, protozoa, worms, vira or prions that can cause infectious disease and are specially adapted and endowed with mechanisms for overcoming the normal body defences. Some pathogens are “opportunistic pathogens” which means they are potentially infectious agents that rarely cause disease in individuals with healthy immune systems but may cause infectious disease with immunocompromised and / or unhealthy individuals. “Pathogenic bacteria” are thus bacteria that are pathogens. In one aspect, pathogens or pathogenic bacteria are pathogenic to animals or humans. Examples of pathogenic bacteria include but are not limited to Clostridioides difficile, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia ga- rinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamyd- ophila psittaci, Clostridioides botulinum, Clostridioides difficile, Clostridioides perfringens, Clostridioides tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira nogu- chii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus sapro- phyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolit- ica, Yersinia pseudotuberculosis. In one aspect, the pathogenic bacteria, such as pathogenic Clostridioides difficile, are enterotoxigenic bacteria e.g. enterotoxigenic Clostridioides difficile.

[0030] The term “reconstitution” in connection with the microbiome or microbiota is herein used interchangeably with the term “restore” and is used for the situation where the microbiome / microbiota of a subject has been disrupted and the microbiome subsequently is re-establishing to its predisruption state.

[0031] The term “subject” is herein used to describe a human or animal such as e.g. a companion animal (e.g. dog, cat, rabbit), farmed animal (e.g. ruminant, poultry, swine) or rodent (e.g. mouse, rat).

[0032] The term “toxins” as herein used in connection with Clostridiodes difficile toxins means substances created by Clostridiodes difficile that are toxic, i.e., poisonous to humans and / or animals.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Herein described is the use of one or more Bacillus velezensis strains to reduce the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in a subject.

[0035] The inventors of the present invention have surprisingly found that B. velezensis is able to reduce the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects. It was thus found that B. velezensis does not colonize a healthy, complete microbiome but requires antibiotic microbiome disruption to open a niche environment and then outcompetes opportunistic bacteria like C. difficile. Also, the inventors have found that supplementation of B. velezensis to a subject can be used for reducing the abundance of C. difficile in the gut without interfering with the natural or assisted recovery of the microbiome following antibiotic treatment. In one aspect, assisted recovery of the microbiome is assisted using fecal microbiota transplant (FMT) therapy.

[0036] In one aspect, a Bacillus velezensis strain is used in or is for use in reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or the reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

[0037] In another or further aspect, a Bacillus velezensis strain is used in or is for one or more uses selected from a group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

[0038] The Clostridioides difficile strain may e.g. be a pathogenic Clostridioides difficile.

[0039] In one aspect, the Bacillus velezensis strain works simultaneously with the natural barrier to C. difficile colonization provided by the host microbiome.

[0040] In one aspect, the reduction of the abundance of Clostridioides difficile is reduction of the colony forming unit of Clostridioides difficile in the gut of the subject.

[0041] The Bacillus velezensis strain may be provided by any route to the subject, such as e.g. orally, sublingually, rectally, topically, parenterally, subcutaneously or intradermally. In one aspect, the Bacillus velezensis strain is orally provided to the subject.

[0042] The Bacillus velezensis strain may be provided to the subject in any composition. In one aspect, the Bacillus velezensis strain is provided to the subject in a composition. In a further aspect, the composition is selected from the group consisting of a supplement, a nutritional product, a drink, a dietary supplement, a meal replacement, a food product, a food additive, a supplement to a food product, and / or other edible product. In an alternative or yet further aspect, the composition comprises a combination with other strains, enzymes, and / or botanicals.

[0043] In one aspect, the abundance of commensal Clostridia is not changed relative to the abundance of commensal Clostridia of the subject before the Bacillus velezensis strain was provided to the subject.

[0044] In one aspect, the Bacillus velezensis strain outcompetes Clostridioides difficile in the gut that has a disrupted commensal gut bacteria.

[0045] In one aspect, the commensal gut bacteria control the colonization of the Bacillus velezensis strain in an undisrupted and / or healthy gut.

[0046] In one aspect, B. velezensis is used for subjects experiencing or at high risk of recurrent C. difficile infections. A test of whether the Bacillus velezensis strain reduces the colonization of Clostridioides difficile may be done in vitro, ex vivo or in vivo. In one aspect, the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vitro. In one aspect, the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested ex vivo. In one aspect, the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vivo.

[0047] In one aspect, the disruption of the commensal gut bacteria is caused by one or more antibiotics and / or fecal microbiota transplants. In one aspect, the one or more antibiotics is a semi-synthetic lincosamide antibiotic. In one aspect, the semi-synthetic lincosamide antibiotic is clindamycin.

[0048] In one aspect wherein the Bacillus velezensis strain reduces the abundance of Clostridioides difficile or Clostridioides difficile toxins in the gut that has a disrupted commensal gut bacteria, the reconstitution of the commensal gut bacteria is not affected following the disruption.

[0049] In one aspect, the Bacillus velezensis strain allows the natural reconstitution of the commensal gut bacteria as determined by testing the reconstitution in mice.

[0050] Further described herein is a method of reducing the abundance of Clostridioides difficile or Clostridioides difficile toxins in the gut of a subject, the method comprising providing Bacillus velezensis strain to the subject after disruption of the commensal gut bacteria.

[0051] In one aspect of the method, the reduction of the abundance of Clostridioides difficile is reduction of the colony forming unit of Clostridioides difficile in the gut of the subject.

[0052] The Bacillus velezensis strain of the method may be provided to the subject in any composition. In one aspect, the Bacillus velezensis strain of the method is provided to the subject in a composition. In a further aspect, the composition is selected from a group consisting of a supplement, a nutritional product, a drink, a dietary supplement, a meal replacement, a food product, a food additive, a supplement to a food product, and / or an edible product. In an alternative or yet further aspect, the composition comprises a combination with other strains, enzymes, and / or botanicals.

[0053] In one aspect of the method, the abundance of commensal Clostridia is not changed relative to the abundance of commensal Clostridia of the subject before the Bacillus velezensis strain was provided to the subject.

[0054] In one aspect of the method, the Bacillus velezensis strain outcompetes Clostridioides difficile in the gut that has a disrupted commensal gut bacteria.

[0055] In one aspect of the method, the commensal gut bacteria control the colonization of the Bacillus velezensis strain in an undisrupted and / or healthy gut.

[0056] In one aspect of the method, B. velezensis is used for subjects experiencing or at high risk of recurrent C. difficile infections.

[0057] A test of whether the Bacillus velezensis strain used in the method reduces the colonization of Clostridioides difficile may be done in vitro, ex vivo or in vivo. In one aspect of the method, the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in-vitro. In one aspect of the method, the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested ex vivo. In one aspect of the method, the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vivo.

[0058] In one aspect of the method, the disruption of the commensal gut bacteria is caused by one or more antibiotics and / or fecal microbiota transplant.

[0059] In one aspect of the method, the one or more antibiotics is a semi-synthetic lincosamide antibiotic. In one aspect of the method, the semi-synthetic lincosamide antibiotic is clindamycin.

[0060] In one aspect of the method, wherein the Bacillus velezensis strain reduces the abundance of Clostridioides difficile or Clostridioides difficile toxins in the gut that has a disrupted commensal gut bacteria, the reconstitution of the commensal gut bacteria is not affected following the disruption.

[0061] In one aspect of the method, the Bacillus velezensis strain allows the natural reconstitution of the commensal gut bacteria as determined by testing the reconstitution in mice.

[0062] PREFERRED EMBODIMENTS

[0063] 1. Use of a Bacillus velezensis strain for reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

[0064] 2. The use according to embodiment 1 , which is in the form of one or more uses selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

[0065] 3. Use of a Bacillus velezensis strain, for one or more uses selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

[0066] 4. The use according to embodiment 1 or 2, wherein Clostridioides difficile is pathogenic Clostridioides difficile.

[0067] 5. The use according to any one of the preceding embodiments, wherein the reduction of the abundance of Clostridioides difficile is reduction of the colony forming unit of Clostridioides difficile in the gut of the subject.

[0068] 6. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain is provided orally to the subject.

[0069] 7. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain is provided to the subject in a composition.

[0070] 8. The use according to embodiment 7, wherein the composition is selected from the group consisting of a supplement, a nutritional product, a drink, a dietary supplement, a meal replacement, a food product, a food additive, a supplement to a food product, and / or other edible products.

[0071] 9. The use according to embodiment 7 or 8, wherein the composition comprises a combination with other strains, enzymes, and / or botanicals.

[0072] 10. The use according to any one of the preceding embodiments, wherein the abundance of commensal Clostridia is not changed relative to the abundance of commensal Clostridia of the subject before the Bacillus velezensis strain was provided to the subject.

[0073] 11. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain outcompetes Clostridioides difficile in the gut that has a disrupted microbiome.

[0074] 12. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vitro.

[0075] 13. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested ex vivo. 14. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vivo.

[0076] 15. The use according to any one of the preceding embodiments, wherein the disruption of the gut microbiome is caused by one or more antibiotics and / or fecal microbiota transplants.

[0077] 16. The use according to embodiment 15, wherein the one or more antibiotics is a semisynthetic lincosamide antibiotic.

[0078] 17. The use according to embodiment 16, wherein the semi-synthetic lincosamide antibiotic is clindamycin.

[0079] 18. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain reduces the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject that has a disrupted gut microbiome without affecting the reconstitution of the gut microbiome following the disruption.

[0080] 19. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain allows the natural reconstitution of the commensal gut bacteria when tested in mice.

[0081] 20. The use according to any one of the preceding embodiments, wherein the Bacillus velezensis strain is Bacillus velezensis strain deposited as DSM 33864.

[0082] 21. A method of reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects, the method comprising providing a Bacillus velezensis strain to the subject.

[0083] 22. A method of reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject, the method comprising providing a Bacillus velezensis strain to the subject after disruption of the gut microbiome.

[0084] 23. The method according to embodiment 21 or 22, wherein Clostridioides difficile is pathogenic Clostridioides difficile.

[0085] 24. The method according to any one of embodiments 21 to 23, wherein the reduction of the abundance of Clostridioides difficile is reduction of the colony forming unit of Clostridioides difficile in the gut of the subject.

[0086] 25. The method according to any one of embodiments 21 to 24, wherein the Bacillus velezensis strain is provided orally to the subject.

[0087] 26. The method according to any one of embodiments 21 to 25, wherein the Bacillus velezensis strain is provided to the subject in a composition.

[0088] 27. The method according to embodiment 26, wherein the composition is selected from a group consisting of a supplement, a nutritional product, a drink, a dietary supplement, a meal replacement, a food product, a food additive, a supplement to a food product, and / or other edible products. 28. The method according to any one of embodiments 21 to 27, wherein the composition comprises a combination with other strains, enzymes, and / or botanicals.

[0089] 29. The method according to any one of embodiments 21 to 28, wherein the abundance of commensal Clostridia is not changed relative to the abundance of commensal Clostridia of the subject before the Bacillus velezensis strain was provided to the subject.

[0090] 30. The method according to any one of embodiments 21 to 29, wherein the Bacillus velezensis strain outcompetes Clostridioides difficile in the gut that has a disrupted gut microbiome.

[0091] 31. The method according to any one of embodiments 21 to 30, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vitro.

[0092] 32. The method according to any one of embodiments 21 to 31 , wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested ex vivo.

[0093] 33. The method according to any one of embodiments 21 to 32, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vivo.

[0094] 34. The method according to any one of embodiments 21 to 33, wherein the disruption of the gut microbiome is caused by one or more antibiotics and / or fecal microbiota transplant.

[0095] 35. The method according to any one of embodiments 21 to 34, wherein the one or more antibiotics is a semi-synthetic lincosamide antibiotic.

[0096] 36. The method according to any one of embodiments 21 to 35, wherein the semi-synthetic lincosamide antibiotic is clindamycin.

[0097] 37. The method according to any one of embodiments 21 to 36, wherein the Bacillus velezensis strain reduces the abundance of Clostridioides difficile or Clostridioides difficile toxins in the gut that has a disrupted gut microbiome without affecting the reconstitution of the commensal gut bacteria following the disruption.

[0098] 38. The method according to any one of embodiments 21 to 37, wherein the Bacillus velezensis strain allows the natural reconstitution after disruption of the gut microbiome when tested in mice.

[0099] 39. The method according to any one of embodiments 21 to 38, wherein the Bacillus velezensis strain is Bacillus velezensis strain deposited as DSM 33864.

[0100] 40. A Bacillus velezensis strain for use in reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

[0101] 41. The Bacillus velezensis strain according to embodiment 40, which is in the form of one or more uses selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and g. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

[0102] 42. A Bacillus velezensis strain for use in one or more applications selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

[0103] 43. The use according to embodiment 40 or 41 , wherein the Clostridioides difficile is pathogenic Clostridioides difficile.

[0104] 44. The Bacillus velezensis strain according to any one of embodiments 40 to 43, wherein the reduction of the abundance of Clostridioides difficile is reduction of the colony forming unit of Clostridioides difficile in the gut of the subject.

[0105] 45. The Bacillus velezensis strain according to any one of embodiments 40 to 44, wherein the Bacillus velezensis strain is provided orally to the subject.

[0106] 46. The Bacillus velezensis strain according to any one of embodiments 40 to 45, wherein the Bacillus velezensis strain is provided to the subject in a composition.

[0107] 47. The Bacillus velezensis strain according to embodiment 46, wherein the composition is selected from the group consisting of a supplement, a nutritional product, a drink, a dietary supplement, a meal replacement, a food product, a food additive, a supplement to a food product, and / or other edible products.

[0108] 48. The Bacillus velezensis strain according to embodiment 46 or 47, wherein the composition comprises a combination with other strains, enzymes, and / or botanicals.

[0109] 49. The Bacillus velezensis strain according to any one of embodiments 40 to 48, wherein the abundance of commensal Clostridia is not changed relative to the abundance of commensal Clostridia of the subject before the Bacillus velezensis strain was provided to the subject.

[0110] 50. The Bacillus velezensis strain according to any one of embodiments 40 to 49, wherein the Bacillus velezensis strain outcompetes Clostridioides difficile in the gut that has a disrupted microbiome.

[0111] 51. The Bacillus velezensis strain according to any one of embodiments 40 to 50, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vitro.

[0112] 52. The Bacillus velezensis strain according to any one of embodiments 40 to 51 , wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested ex vivo.

[0113] 53. The Bacillus velezensis strain according to any one of embodiments 40 to 52, wherein the Bacillus velezensis strain reduces the colonization of Clostridioides difficile when tested in vivo.

[0114] 54. The Bacillus velezensis strain according to any one of embodiments 40 to 53, wherein the disruption of the gut microbiome is caused by one or more antibiotics and / or fecal microbiota transplants.

[0115] 55. The Bacillus velezensis strain according to embodiment 54, wherein the one or more antibiotics is a semi-synthetic lincosamide antibiotic.

[0116] 56. The Bacillus velezensis strain according to embodiment 55, wherein the semi-synthetic lincosamide antibiotic is clindamycin.

[0117] 57. The Bacillus velezensis strain according to any one of embodiments 40 to 56, wherein the Bacillus velezensis strain reduces the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject that has a disrupted gut microbiome without affecting the reconstitution of the gut microbiome following the disruption.

[0118] 58. The Bacillus velezensis strain according to any one of embodiments 40 to 57, wherein the Bacillus velezensis strain allows the natural reconstitution of the commensal gut bacteria when tested in mice.

[0119] 59. The Bacillus velezensis strain according to any one of embodiments 40 to 58, wherein the Bacillus velezensis strain is Bacillus velezensis strain deposited as DSM 33864. EXAMPLES

[0120] Deposit of Biological Material

[0121] The following biological material has been deposited under the terms of the Budapest Treaty with the Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, and given the following accession number:

[0122] Deposit Accession Number Date of Deposit Country of origin

[0123] Bacillus velezensis DSM 33864 19-05-2021 Vietnam

[0124] The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0125] B. velezensis DSM 33864 spore preparation

[0126] B. velezensis DSM 33864 was previously classified as B. amyloliquefaciens and is also known as SG277. Sporulation of B. velezensis DSM 33864 was induced using Difco Sporulation broth

[0127] (DSM). In brief, a single bacterial colony was transferred from Difco agar to 25 mL of Difco Sporulation broth and incubated 8 h at 37°C while shaking at 150 rpm. After 8 h, 10 mL of culture was poured into tray and incubated 72 h at 37°C. Bacterial lawn was harvested and resuspended in

[0128] 10 mL water. After washing and centrifugation (4.500 g, 10 min) spores were diluted to a concentration of 1x1010spores / mL in ddH2O and stored at 4°C until use.

[0129] Example 1 - Bacillus velezensis DSM 33864 reduced C. difficile levels

[0130] To mimic a more complex environment in vitro, the ability of B. velezensis DSM 33864 to reduce

[0131] C. difficile levels was tested through an ex vivo human fecal fermentation. Here, pooled human fecal samples from healthy donors were treated with or without clindamycin, C. difficile and / or B. velezensis DSM 33864 and samples collected after 0.5 and 6 hours of incubation.

[0132] Human fecal fermentation assay and fluorescence in situ hybridization assay

[0133] Fecal fermentations were conducted in 4 replicates. All fecal donors were healthy females between ages 18-65 years. Samples were tested negative for the following pathogens: Adenovirus, Astrovirus, Norovirus Genotyp 1 , Norovirus Genotype 2, Rotavirus, Sapovirus, Campylobacter spp., C. difficile toxins A / B, Escherichia coli 0157, Salmonella spp., Shigella spp., Enteroinvasive E. Coli (EIEC), Shiga Toxin-producing E. coli (STEC), Yersinia enterocolitica and Hepatitis A. Fecal matter from each donor was first normalized by mixing 10 g fecal material, 16 mL of 50% glycerol (v / v) and 14 mL water (cell grade) to a total of 40 mL fecal slurry. For the fermentations, a pool of fecal matter from four donors was used. Each fecal fermentation had a final volume of 10 mL, composed of 1 mL fecal slurry spiked with the components specified in Table 1. In addition to the fecal slurry components were added in the following final concentrations: 0,45 mg / mL clindamycin (Sigma-Aldrich, Germany, CAS-N° 24729-96-2), 105CFUs / mL C. difficile, 108CFUs / mL of B. velezensis DSM 33864 spores. All fecal matter preparations and fermentations were conducted under strictly anaerobic conditions. Fecal fermentations were incubated for 6 h at 37°C and samples for C. difficile and Bacillus CFU quantification, microbiome profiling and Fluorescence In Situ Hybridization (FISH) assays were collected at Oh and 6h of incubation. Material for microbiome compositional analysis was sampled from the fermentation tubes and centrifuged 5 min at 11 ,000 x g. Supernatant thereof was aspirated and DNA was extracted from the remaining pellet following the procedure described below on 16S rRNA gene amplicon sequencing. Supernatant was furthermore used to quantify C. difficile toxins A and B (TcdA and TcdB) by ELISA (tgcBIOMICS) following the manufacturer’s instructions.

[0134] C. difficile and Bacillus CFUs per mL were quantified in the fecal fermentations at the indicated time points by a serial dilution of the samples then streaked in selective media either for C. difficile (Brazier’s Clostridium difficile Selective Medium, OXOID GmbH, Germany) or for Bacillus genus (Bacillus ChromoSelect Agar NutriSelect® Plus, Merck KGaA, Germany). The selective plates for C. difficile were incubated at 37°C under strictly anaerobic conditions for 24 to 48h. Selective plate media for the Bacillus genus were incubated at room temperature under aerobic conditions for 24h to 48h. Identification of all colonies was confirmed by MALDI-TOF mass spectrometry and CFU counts were adjusted to colonies identified only as either C. difficile or Bacillus genera. Calculation of CFUs / mL for each replica and condition was performed considering the dilution factors.

[0135] Table 1 : Tested conditions for human fecal fermentation assay

[0136] 16S rRNA gene amplicon sequencing Microbial DNA was extracted using NucleoSpin® 96 Soil (Macherey-Nagel) by adding lysis buffer directly to mouse fecal pellets in sterile tubes and this mix was transferred to beads tube provided in the extraction kit. Amplification of the 16s RNA gene by PCR from human fecal media and mouse fecal pellets from the microbiome safety study was done with the universal bacterial 16S rDNA primers, which target the V3-V4 region (341.2FDI and 805.2RDI). The following PCR program was used: 98 °C for 2 min, 9x (98° C for 30 s, 52 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min followed by a hold at 4°C followed by a hold at 4°C. Indices were added in a subsequent PCR with the following PCR program: 98 °C for 2 min, 9x (98°C for 30 s, 55 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min and a hold at hold at 4°C.

[0137] The PCR product was purified using AM Pure beads (Beckman Coulter) and samples were pooled in equimolar amounts based on DNA concentrations determined fluorometrically Sequencing was done on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2x 300 bp paired-end sequencing. Sequence data was processed using dada2. Primer sequences were first removed from raw reads (dada2::p-trim-left-f / r, 19 bases from the forward read and 20 from the reverse read). Reads were then trimmed at the 3’ ends based on quality scores, but keeping a minimum read lengths of 279 and 209 bp for forward and reverse reads, respectively (dada2::filterAndTrim). All trimmed reads with more than two expected errors were discarded. The remaining reads were dereplicated into unique sequences and then denoised separately for forward and reverse reads. In the denoising step, a less abundant sequence can be assigned to a closely related more abundant sequence based on comparison to a data- adjusted error matrix. In that case, the low abundance sequence is considered a sequencing error of the higher abundant sequence. Denoised forward and reverse reads were then merged, thereby discarding read pairs without sufficient overlap (12 bp) or with any mismatch in the overlap. Finally, suspected chimeras were removed from the generated abundance table by internal abundance and sequence comparisons. The default taxonomic assignment of the detected amplicon sequence variants (ASVs) was done using a custom naive Bayesian classifier algorithm comparing the ASV sequences to the SILVA reference database (v138). Singleton ASVs were removed from downstream analyses.

[0138] Amplification of the 16s RNA gene by PCR from the mouse colonization study was carried out using primers of the V3-V4 region (S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21). Indices were added in a subsequent PCR using the Nextera Index Kit V2 (Illumina) and PCR products were pooled based on concentration and resulting library cleaned with AM Pure beads (Beckman Coulter). Sequencing was done on an Illumina MiSeq desktop sequencer using the MiSeq Reagents Kit V3 (Illumina) for 2x 300 bp paired-end sequencing. An adjusted dada2 pipeline was used for bioinformatics processing of the sequence data into the ASV (amplicon sequence variant) abundance table. In the first step, primer sequences were removed from raw reads using cutadapt. Reads without perfect primer match or with ambiguous bases (e.g., Ns) as well as reads shorter than 275 bp were filtered out. In an additional filtering and trimming step (dada2::filterAndTrim command), reads were first trimmed at the 3 prime ends based on quality scores, but keeping a minimum read length of 250 and 220 bp for forward and reverse reads, respectively. All trimmed reads with more than 1 expected error were discarded. The remaining reads were dereplicated into unique sequences and then denoised separately for forward and reverse reads. In the denoising step a less abundant sequence can be assigned to a closely related more abundant sequence based on comparison to a data-adjusted error matrix. In that case, the low abundance sequence is considered a sequencing error of the higher abundant sequence. Denoised forward and reverse reads were then merged thereby discarding read pairs without sufficient overlap (20 bp) or with any mismatch in the overlap. Finally, suspected chimeras were removed from the generated abundance table by internal abundance and sequence comparisons. The default taxonomic assignment of the detected ASVs was done using a naive Bayesian classifier algorithm comparing the ASV sequences to the SILVA reference database (v138). For improving the taxonomic assignment of the ASVs, amplicons corresponding to the used primers in the study were in-silico extracted from current versions of three reference databases (SILVA, RDP and UHGG). For each ASV, we then used reference amplicons matching perfectly to the ASV or the amplicons with the highest sequence identity to the ASV to check / improve the default assignment of the ASV.

[0139] All ASV count tables generated from human fecal media and both mouse studies were analyzed using R. Zero imputation was performed using zCompositions, and counts were subsequently center-log transformed using compositions. Ordinations were performed using Principal Component Analysis (PCA) on the subsequent pseudo-counts. Alpha diversity was calculated according to the Shannon Diversity Index. All tests for differences between groups were performed using permutational multiple analysis of variance (perMANOVA) with Euclidean distance matrices. Results

[0140] In fecal samples incubated with clindamycin and C. difficile for 6 h, the C. difficile load was reduced by the addition of B. velezensis DSM 33864 (Fig 1A and 2A). Bacillus counts increased only where B. velezensis DSM 33864 was added to clindamycin-treated fecal samples (Fig 1 B). B. velezensis DSM 33864 furthermore reduced C. difficile toxins A (TcdA) and C. difficile toxins B (TcdB) in the fecal samples containing clindamycin and C. difficile (Fig 2B and 2C), 16S rRNA gene amplicon sequencing showed no measurable change in relative abundance of commensal Clostridia, i.e. Clostridia not mapping to the added C. difficile, by B. velezensis DSM 33864 (Fig 2D). The fecal microbiome composition was similar in all conditions shortly after the addition of clindamycin, C. difficile and / or B. velezensis DSM 33864 although addition of B. velezensis DSM 33864 increased the Shannon diversity at 0.5 h (Fig 3A), which was explained by an increase in Bacillus abundance (data not shown). As expected, clindamycin treatment altered the fecal microbiome composition after 6 h of incubation as did addition of C. difficile, which was not modified further by adding B. velezensis DSM 33864 (Fig 3B). Example 2 - B. velezensis DSM 33864 reduced C. difficile colonization in mice

[0141] After demonstrating that B. velezensis DSM 33864 reduced C. difficile load in vitro, we investigated the effects in vivo. Here, we used a model where mice were colonized by C. difficile 24h after their commensal gut microbiome was disrupted with clindamycin. One group of mice additionally received B. velezensis DSM 33864 by oral gavage before and after C. difficile administration.

[0142] Experiment

[0143] To evaluate the efficacy of B. velezensis DSM 33864 on reduction of C. difficile colonization, a study of non-fatal and asymptomatic murine colonization model using 38 female C57BL / 6 mice (Charles River) aged 7-8 weeks was carried out. During the acclimatization and study, the mice were fed non-restricted irradiated diet, provided autoclaved tap water, and housed in cages containing wood fiber bedding, cardboard tunnels, Bed-r’Nests (Datesand) and chew blocks. After 31 days of acclimation mice were single caged (using HEPA-filtered independently ventilated cages) to avoid reinfection by coprophagia and were randomly allocated in 4 groups.

[0144] Mice were dosed by intragastric gavage (0.2 mL) with 30 mg / kg body weight of clindamycin (clindamycin hydrochloride C5269, Sigma) in 0.2 mL deionized distilled water (ddH2O) to deplete the commensal microbiome, alternatively with 0.2 mL water as control. After 5 h the mice were orally gavaged with 1x109spores of B. velezensis DSM 33824 in 0.2 mL PBS or an equal volume of PBS as vehicle control. The gavage with B. velezensis DSM 33824 or vehicle was repeated 17 h after clindamycin gavage. C. difficile 360 spores (TcdA+TcdB+; Histodenz purified, prevalidated by CFU viability counting and for infectivity within mice) were diluted to a concentration of 1x103spores / mL in ddH2O using a screw cap glass serological test tube. The spore dose concentration was validated prior to challenge using viable counts on Brain Heart Infusion (BHISS) (37 g / L, 5 g yeast extract / L) agar containing 0.1 % sodium taurocholate. C. difficile (1x102spores in 0.1 mL) or an equal volume water was dosed to mice by oral gavage 24 hours after the clindamycin dose. 1 h following C. difficile administration the mice were gavaged with B. velezensis DSM 33824 or vehicle a third time, which was repeated 6 hours following C. difficile administration. The study was terminated 24 h after C. difficile administration, equivalent to 48 h after the single dose of clindamycin.

[0145] During the study body weight, feed and water intake was monitored. Feces pellets were collected at several time points (T1 (-26 h), T2 (-21 h), T3 (-1 h) and T4 (22 h)) for microbiome analysis. The mice were terminated by cervical dislocation followed by necropsy. Ceca were removed by dissection, stored on ice during collection, and then frozen at -20 °C until subsequent analysis of C. difficile and toxin quantification. Murine cecum samples were centrifuged and pellets were suspended in 5 volumes of 80% (v / v) ethanol, vortexed vigorously for 5 m, and incubated for 1 hour at room temperature before serial dilution and plating on selective media (CH ROM ID® C. difficile, bioMerieux, France). Plates were incubated for 24 h at 37°C under anaerobic conditions. Colonies were counted and C. difficile CFU per g of sample calculated.

[0146] TcdA and TcdB were quantified from cecum content from the murine C. difficile colonization study. The wet weight of material was determined, and samples were suspended in 5 volumes of toxin extraction buffer (50 ml of buffer contains 1 ml Fetal Bovine Serum, 2 Pierce Protease Inhibitor tablets, 100 pL PMSF, 50 pL Benzamide, 500 pL 100x Penicillin-Streptomycin, 500 pL EDTA and 47.85 mL PBS). Samples were then macerated, and toxins extracted for 2h at 4°C with gentle agitation. Tubes were centrifuged (microfuge, 4°C, 15 min at 10,000 rpm) and the supernatant filtered through a 0.2 pm Corning syringe filter. Filtered supernatant was stored at 4°C prior to use. Samples were analyzed for the presence of TcdA and TcdB by ELISA (tcgBIOMICS) and the amount of toxins per g of sample determined by following the manufacturer’s instructions.

[0147] Results

[0148] C. difficile was measurable in 80% of the ceca from mice which were pre-treated with clindamycin but did not receive B. velezensis DSM 33864, which was associated with increased toxin levels (Fig 4A-C). Both C. difficile and associated toxin levels in cecum were reduced by oral gavage of B. velezensis DSM 33864 in clindamycin-treated mice (Fig 4A-C).

[0149] The overall gut microbiome composition was similar between the groups at the start of the study, after which clindamycin drastically shifted the microbiome composition from T3 (-1 h) (Fig 4D) and reduced Shannon diversity index (Fig 4F). Neither C. difficile nor B. velezensis DSM 33864 impacted the clindamycin-disrupted fecal microbiome composition in the mice (Fig 4E). To investigate the effects of introducing B. velezensis DSM 33864 on the commensal gut microbiome composition, we assessed amplicon sequence variants (ASVs) annotated into the Clostridia class. Of the 190 Clostridia strains in the data set, only C. difficile was significant between the groups. Commensal Clostridia were affected by clindamycin, but not by C. difficile or B. velezensis DSM 33864 in clindamycin-administered mice (Fig 4E).

[0150] Example 3 - Commensal gut microbes were not affected by B. velezensis DSM 33864 in healthy mice

[0151] To further investigate potential effects of B. velezensis DSM 33864 on the commensal gut microbiome, we conducted a study with healthy, non-antibiotic-treated mice.

[0152] Mouse study of gut microbiome composition impact, experiment

[0153] Potential microbiome compositional changes inferred by B. velezensis DSM 33864 were monitored at pre-defined time points during the 14 days daily oral gavage period followed by 14 days of wash-out period. A mouse study to evaluate potential influence of B. velezensis DSM 33864 on the commensal gut microbiome composition was conducted using 30 female C57BI / 6Ncrl mice (Charles River). The mice were co-housed with three mice in each cage containing bedding (Tapvei), nesting material (Tapvei), wood brick (Tapvei), mouse igloo (Bio-Serv), Diamond Twist (Envigo), and a handling tube (Datesand). The mice were fed a soy, wheat, corn fixed formula free of alfalfa and fish or animal meal, and deficient in nitrosamines (1314 Fortified diet, Altromin) ad libitum and had nonrestricted access to drinking water during the acclimatization period and the study.

[0154] Following 18 days of acclimatization the mice were allocated randomly to 2 groups of which one group received a daily dose of either B. velezensis DSM 33824 (1 x106spores / dose) and one group received PBS vehicle by oral gavage in a fixed volume of 0.2 mL. The mice received daily oral administration of the B. velezensis DSM 33824 or vehicle for 14 days, followed by a 14-day wash out period. Mice were weighed upon arrival and monitored weekly during the four weeks of the study. From four hours prior to the first administration of B. velezensis DSM 33864 or vehicle, food consumption was recorded once weekly for each cage.

[0155] Fecal pellets were collected from all mice in the mornings prior to any oral gavage a total of 6 timepoints, snap-frozen on dry ice and stored at -80 °C. The first fecal sample was collected three days prior the start of the study and prior to any oral gavage with clindamycin or B. velezensis DSM 33824. The second fecal sample was collected 24 hours after the dose of clindamycin or vehicle and prior to the second B. velezensis DSM 33864 dose. From there a fecal sample was collected weekly. Mice were euthanized by placement in a chamber with atmospheric air upon which a mixture of 70% CO2 and 30% O2 was applied with a steadily increasing concentration of CO2.

[0156] Results

[0157] All mice in the study had a similar baseline microbiome composition (Day -3), which remained stable during the study in both the vehicle-control group as well as the group given B. velezensis DSM 33864 (Day 1-28, fig 5A). Shannon diversity of the microbiome composition did not change over time regardless of whether the microbiome was sampled during the 14 days of daily vehicle or B. velezensis DSM 33864 gavage (Day 1-14) or during the following 14-days of wash-out (Day 21-28, fig 5B). In mice with a healthy microbiome, the B. velezensis DSM 33864 did not increase the relative Bacillus abundance. Furthermore, B. velezensis DSM 33864 did not impact commensal Clostridia abundance (fig 5C).

[0158] Mouse study of B. velezensis DSM 33864 safety

[0159] To evaluate the safety of repeated dosing of B. velezensis DSM 33864 a study in both female and male mice with a 28-days repeat dose was conducted. A total of 54 C57BI / 6Ncrl mice (Charles River) (27 Male and 27 females) were used in the study. The study was performed in accordance with principles and considerations described in the OECD Guideline No. 407: Repeated Dose 28-Day Oral Toxicity Study in Rodents and in compliance with Principles of Good Laboratory Practice (GLP).

[0160] The mice were co-housed in cages with enrichment, feed and drinking water as described for the mouse study of gut microbiome composition. The mice were randomly allocated to 3 groups with 9 mice per sex in each. Following 19 days of acclimatization the mice received B. velezensis DSM 33864 in a dosage of 1x106or 1x108spores / day in 0.2 mL PBS by oral gavage or equal volume PBS for 28-days. During the 28-day treatment period clinical signs were monitored daily, body weight and food consumption recorded weekly. Mice were euthanized by placement in a chamber with atmospheric air upon which a mixture of 70% CO2 and 30% O2 was applied with a steadily increasing concentration of CO2. Death was confirmed and the animals were bled before proceeding with necropsy. At termination blood samples were collected by retro-orbital bleeding. Samples for hematology and coagulation parameters were collected from 5 males and 4 females from each group. For hematology, at least 150 pL K3EDTA stabilized blood was taken and samples were diluted 1 :1 with ABX Diluent before analysis and the results were corrected where appropriate. For coagulation tests, 250 pL citrate stabilized blood was taken. At least 500 pL blood was taken for clinical chemistry in tubes with clotting activator for serum. The samples collected from the remaining 4 males and 5 females in each group. A macroscopic examination was performed after opening the cranial, thoracic, and abdominal cavities by observing the appearance of the organs and tissues in situ. Any macroscopic change was recorded with details of the location, color, shape and / or size in Provantis version 9.3.0.0 (Instem). All tissues were fixed in phosphate buffered neutral 4 % formaldehyde except for the eyes and testes (Modified Davidson’s fixative). The lungs were infused with fixative at necropsy.

[0161] Results

[0162] In the 28-day repeat dose safety study in mice with daily oral administration of B. velezensis DSM 33864, mice receiving either 1x106or 1x108spores / animal / day of B. velezensis DSM 33864 showed no treatment-related clinical signs or differences in feed intake or body weight compared to the control group. Further assessment of selected hematological, clinical chemistry, bone marrow smears, macroscopic and microscopic examinations of selected organs and tissue did not show any adverse effects compared to the concurrent control group. The 28-day oral gavage with B. velezensis DSM 33864 established the no-observed-adverse-effect level (NOAEL) in the study to be 1x108CFU / animal / day.

[0163] Collectively, these studies suggest that daily oral B. velezensis DSM 33864 administration did not impact the gut microbiome or caused any adverse effects in healthy, non-antibiotic-treated mice without C. d / / c / 7e-challenge. Example 4 - Post-antibiotic reconstitution of the commensal microbiome was not affected by B. velezensis DSM 33864

[0164] We investigated whether B. velezensis DSM 33864 impacted the reconstitution of the commensal gut microbiome composition in a model where mice received a single-dose of clindamycin followed daily oral gavage of B. velezensis DSM 33864 for 14 days, followed by a 14 days washout period.

[0165] Experiment

[0166] A mouse study to evaluate potential influence of B. velezensis DSM 33864 on the antibiotic-depleted gut microbiome composition was conducted using 30 female C57BI / 6Ncrl mice (Charles River) that were housed and fed as described in Example 3.

[0167] Following 18 days of acclimatization the mice received 30 mg / kg body weight of clindamycin and were then allocated to 2 groups of 15 mice of which one group received a daily dose of either B. velezensis DSM 33824 (1 x106spores / dose) and one group received PBS vehicle by oral gavage in a fixed volume of 0.2 mL. The mice received daily oral administration of the B. velezensis DSM 33824 or vehicle for 14 days, followed by a 14-day wash out period. At the start of the study, four hours prior to the first administration of B. velezensis DSM 33864 or vehicle, animals received either a single dose of clindamycin (30 mg / kg), or vehicle by oral gavage. Mice were weighed upon arrival and monitored weekly during the four weeks of the study. From four hours prior to the first administration of B. velezensis DSM 33864 or vehicle, food consumption was recorded once weekly for each cage.

[0168] Fecal pellets were collected, and mice were euthanized as described in Example 3.

[0169] Results

[0170] The baseline fecal microbiome composition was similar between the groups prior to treatment (Day -3, Fig. 6Aa). Clindamycin induced a drastic shift in the overall fecal microbiome composition and impacted the Shannon diversity index in samples obtained the day after clindamycin-treat- ment (Day 1 , Fig. 6A-B).

[0171] Daily oral gavage with B. velezensis DSM 33864 (at 1x106spores / animal / day) did not impact the effect of clindamycin on the fecal microbiome composition. This was observed by a similar response to clindamycin in the groups additionally receiving daily B. velezensis DSM 33864 following the single-clindamycin dose (Day 1-28, Fig. 6A-B). Over time, the gut microbiome composition reconstituted at the same rate as the vehicle-treated group, thus showing no delay in the natural return of the microbiome composition or Shannon diversity (1-28 d, Fig. 6A-B). The relative abundance of Bacillus or commensal Clostridia were not affected in this study after daily oral B. velezensis DSM 33864 administration for 14 days following clindamycin-disruption of the microbiome composition (14 d, Fig. 6C). This indicated that, in addition to B. velezensis DSM 33864 not affecting already present commensal Clostridia, the strain did not hinder the return and growth of the commensal strains of this bacterial genus.

Claims

CLAIMS1 . A Bacillus velezensis strain for use in one or more applications selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

2. A Bacillus velezensis strain for use in reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

3. Use of a Bacillus velezensis strain, for one or more uses selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and f. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

4. Use of a Bacillus velezensis strain for reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects.

5. The use or Bacillus velezensis strain according to anyone of the preceding claims, which is in the form for one or more uses selected from the group consisting of: a. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are maintained; b. reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject while the commensal gut bacteria of the subject are reconstituted; c. reducing colonization of Clostridioides difficile in the gut of a subject after disruption of the gut microbiome of the subject; d. allowing the natural reconstitution of the microbiome in the gut of a subject after disruption of the gut microbiome of the subject; e. preventing Clostridioides difficile infection of a subject during or after disruption of the gut microbiome of the subject; and h. restoring the abundance of commensal gut bacteria in the gut of a subject after disruption of the gut microbiome of the subject.

6. The use or Bacillus velezensis strain according to any one of the preceding claims, wherein the Clostridioides difficile is pathogenic Clostridioides difficile.

7. The use or Bacillus velezensis strain according to any one of the preceding claims, wherein the reduction of the abundance of Clostridioides difficile is reduction of the colony forming unit of Clostridioides difficile in the gut of the subject.

8. The use or Bacillus velezensis strain according to any one of the preceding claims, wherein the Bacillus velezensis strain is provided to the subject in a composition.

9. The use or Bacillus velezensis strain according to any one of the preceding claims, wherein the Bacillus velezensis strain outcompetes Clostridioides difficile in the gut that has a disrupted microbiome.

10. The use or Bacillus velezensis strain according to any one of the preceding claims, wherein the disruption of the gut microbiome is caused by one or more antibiotics and / or fecal microbiota transplants.

11. The use or Bacillus velezensis strain according to any one of the preceding claims, wherein the Bacillus velezensis strain is Bacillus velezensis strain deposited as DSM 33864.

12. A method of reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject without impacting commensal gut bacteria or reconstitution of the gut microbiome in healthy or antibiotic-treated subjects, the method comprising providing a Bacillus velezensis strain to the subject.

13. A method of reducing the abundance of Clostridioides difficile and / or Clostridioides difficile toxins in the gut of a subject, the method comprising providing a Bacillus velezensis strain to the subject after disruption of the gut microbiome.

14. The method according to any one of claims 10 to 12, wherein the Bacillus velezensis strain is provided to the subject in a composition.

15. The method according to any one of claims 10 to 14, wherein the Bacillus velezensis strain is Bacillus velezensis strain deposited as DSM 33864.

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