Therapeutic and preventive applications of microorganisms

Abstract

The present invention relies on the discovery that certain microorganisms in the intestines and feces of fecal microbiota transplant (FMT) donors can affect the results of FMT treatment. Therefore, new methods for identifying subjects as suitable donors to optimize FMT results, and pre-treating donors and / or recipients for optimized FMT results are provided. Also provided are kits and compositions for improving FMT results, including for specific purposes, such as for weight loss or cholesterol reduction.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 785,640, filed on December 27, 2018, the contents of which are hereby incorporated by reference in their entirety for all purposes. Background of the Invention

[0004] Fecal microbiota transplantation (FMT) is a very effective method for treating various conditions, including gastrointestinal disorders and Clostridium difficile infection (CDI), particularly in patients with recurrent CDI. FMT is also referred to as fecal transplantation, which relates to the process of transplanting fecal material containing microorganisms from healthy individuals into the gastrointestinal tract of recipients. The target of FMT is to repair the intestinal microbial flora destroyed by the disease such as CDI by various ways of infusing the feces of healthy individuals, such as by colonoscopy, enema, orogastric tube, or by introducing (or reintroducing) healthy bacterial groups by mouth in the form of capsules containing freeze-dried material obtained from healthy donors. Except CDI, FMT is increasingly used to treat other intestinal and extraintestinal diseases, including other gastrointestinal diseases, such as inflammatory bowel disease (IBD), antibiotic-resistant bacterial infections, diarrhea, constipation, irritable bowel syndrome, autism, depression, obesity, diabetes, alopecia, etc. In addition, FMT has been used to treat some neurological conditions, such as multiple sclerosis and Parkinson's disease. Recent studies have revealed that certain microorganisms, such as certain bacteria and fungi, present in the donor material used for FMT can significantly affect the efficacy of FMT and directly affect whether additional health benefits related to metabolic regulation may be conferred to the FMT recipient. Given the prevalence of various conditions and diseases treatable by FMT in the human population and their significant economic impact, there is an urgent need to develop new and improved methods to increase the therapeutic efficacy of FMT and optimize treatment outcomes, particularly on the basis of donor and / or recipient specificity. The present invention meets this need and other related needs. SUMMARY OF THE INVENTION

[0006] The present invention relates to new methods and compositions, which can be used to optimize fecal microbiota transplantation (FMT) treatment, in particular to maximize the health benefits conferred to FMT recipients. In particular, the inventors have found that when certain microorganisms (e.g., bacteria, fungi, viruses) are present in the transplant for FMT recipients and subsequently in the gastrointestinal (GI) tract of the recipient after receiving FMT treatment (particularly at elevated levels), significant health benefits can be achieved in FMT recipients, such as weight loss, higher insulin sensitivity, lower blood cholesterol, reduced presence of multidrug-resistant bacteria, reduced adverse conditions after hematopoietic stem cell transplantation such as acute graft-versus-host disease (aGvHD), and reduced GI tract conditions such as ulcerative colitis (UC) or inflammatory bowel disease (IBD) or Crohn's disease; and certain other microorganisms present in the FMT recipient GI tract (particularly at higher levels) are associated with a lack of or greatly reduced health benefits of this nature or with a substantially ineffective therapeutic effect. These discoveries allow the inventors to design methods and compositions that can improve the effectiveness of FMTs with respect to these health benefits. Thus, the present invention provides novel methods for identifying suitable FMT donors who provide fecal material for FMT after appropriate processing. The method comprises the step of determining the level of one or more bacteria, fungi or viruses shown in Tables 1a, 1b, 2, 3a, 3b, 4, 5, 6a, 6b, 7a, 7b, 8a, 8b, 10a, 10b, 11, 15 or 16 in a fecal sample obtained from a FMT donor candidate.

[0007] In the first aspect, the present invention provides a method for identifying a suitable FMT donor, comprising a step of determining the level of one or more bacteria shown in Tables 1a, 4, 6a, 10b, 11 or 15 in a stool sample obtained from a candidate. In some embodiments, the level of one or more bacteria is a percentage relative abundance. In some embodiments, when the level of one or more bacteria is found to be greater than 0.1%, the candidate is considered to be a suitable FMT donor. In some embodiments, the level of one or more bacteria shown in Tables 1b, 6b or 10a is not greater than 0.01%. In some embodiments, the method also includes the step of obtaining fecal material from a suitable candidate for FMT. In some embodiments, the method also includes the step of determining the total bacterial load in the stool sample. In some embodiments, when the level of one or more bacteria shown in 1b, 6b or 10a is greater than 0.01%, the candidate is identified as an inappropriate FMT donor.

[0008] In some embodiments, the level of one or more bacteria shown in Table 1a, 1b, 4, 6a, 6b, 10a, 10b, 11, 15 or 16 in a first stool sample obtained from a first candidate and a second stool sample obtained from a second candidate is determined. In some embodiments, the first candidate has a higher level of one or more bacteria shown in Table 1a, 4, 6a, 10b, 11 or 15 than the second candidate and is considered to be a more suitable FMT donor than the second candidate. In some embodiments, the first candidate has a lower level of one or more bacteria shown in Table 1b, 6b or 10a than the second candidate and is considered to be a more suitable FMT donor than the second candidate.

[0009] In one example, when a donor is screened for its potential as a suitable FMT donor, particularly for the purpose of helping the recipient lose weight, the presence of beneficial bacteria such as Bifidobacterium bifidum, Roseburia intestinalis, and Sutterella wadsworthensis in their stool samples should reach or exceed a certain relative amount threshold: for example, at least about 0.3% or at least 0.7% of Bifidobacterium bifidum, at least about 3.5% or at least about 6% of Roseburia intestinalis, or at least about 1% of Sutterella wadsworthensis. In contrast, the presence of harmful bacteria such as Blautia hydrogenotrophica or Peptostreptococcus is preferably as low as possible (e.g., less than about 0.001%) or even undetectable. On the other hand, if a candidate donor has insufficient amounts of beneficial bacteria in his or her stool sample (e.g., less than about 0.1% each of Bifidobacterium bifidum, Roseburia enterica, and Waldersartella), but too many harmful bacteria (e.g., at least about 0.01%, at least about 0.05%, or at least about 0.07% hydrogenotrophic Blautia or Peptostreptococcus), he or she should be excluded from use as a donor, particularly when weight loss is a goal during the proposed FMT process.

[0010] In a related aspect, a method for improving the effectiveness of FMT is provided, the method comprising introducing an effective amount of one or more bacteria shown in Table 1a, 4, 6a, 10b, 11 or 15 into a composition intended for transplantation before FMT. In some embodiments, after the introduction step, the level of each of the one or more bacteria shown in Table 1a, 4, 6a, 10b, 11 or 15 is greater than 0.1% of the total bacteria in the composition. In some embodiments, the method further comprises the step of using the composition to perform FMT. In some embodiments, the method further comprises the step of introducing an effective amount of an antibacterial agent that inhibits the growth of one or more bacteria shown in Table 1b, 6b or 10a in the composition. In some embodiments, after the introduction step, the level of each of the one or more bacteria shown in Table 1b, 6b, 10a is less than 0.01% of the total bacteria in the composition. In some embodiments, the method further comprises the step of using the composition to perform FMT.

[0011] In another related aspect, a method for improving the effectiveness of FMT is provided by administering an effective amount of an antibacterial agent that inhibits the growth of one or more bacteria shown in Table 1b, 6b or 10a to the FMT recipient before FMT. In some embodiments, the level of one or more bacteria shown in Table 1b, 6b or 10a in a stool sample from the FMT recipient is determined before administering the antibacterial agent. In some embodiments, the level of one or more bacteria shown in Table 1b, 6b or 10a in a stool sample from the FMT recipient is determined after administering the antibacterial agent. In some embodiments, the method further includes the step of administering an effective amount of an agent that can reduce the total bacterial load in a stool sample taken from the recipient before FMT.

[0012] In yet another related aspect, a kit is provided, comprising: (1) a first composition comprising donor feces; and (2) a second composition comprising: (i) an effective amount of one or more bacteria shown in Table 1a, 4, 6a, 10b, 11, 15 or 16; or (ii) an effective amount of an antibacterial agent that inhibits the growth of one or more bacteria shown in Table 1b, 6b or 10a. In some embodiments, the first composition comprises donor feces that has been dried, frozen and placed in a capsule for oral administration. In some embodiments, the kit also comprises an effective amount of an antibacterial agent that inhibits the growth of one or more bacteria shown in Table 1b, 6b or 10a in the second composition, or an effective amount of an antibacterial agent that reduces the total bacterial load in the third composition. In addition, the kit may include printed instructions to guide the user to properly use the kit.

[0013] In some embodiments, in the above methods, the level of one or more bacteria shown in Table 1a, 1b, 4, 6a, 6b, 10a, 10b, 11 or 15 is determined by quantitative polymerase chain reaction (PCR).

[0014] In a second aspect, the present invention provides a method for identifying a suitable FMT donor. The method includes determining the level of one or more fungi shown in Table 2, 3a, 5, 7a or 16 in a fecal sample obtained from a candidate. In some embodiments, the level of one or more fungi is a percentage relative abundance. In some embodiments, the level of one or more fungi shown in Table 2, 3a, 5, 7a or 16 is greater than 0.5%, and the candidate is identified as a suitable FMT donor. In some embodiments, the level of one or more fungi shown in Table 3b or 7b is not more than 0.05%. In some embodiments, the method also includes the step of obtaining fecal material from a suitable candidate for FMT. In some embodiments, the level of one or more fungi shown in Table 3b or 7b is greater than 0.05%, and the candidate is identified as an unsuitable FMT donor. In some embodiments, the method also includes the step of determining the total fungal load in the fecal sample.

[0015] In some embodiments, the level of one or more fungi shown in Table 2, 3a, 3b, 5, 7a, 7b, or 16 is determined in a first stool sample obtained from a first candidate and a second stool sample obtained from a second candidate. In some embodiments, the first candidate has a higher level of one or more fungi shown in Table 2, 3a, 5, 7a, or 16 than the second candidate and is considered to be a more suitable FMT donor than the second candidate. In some embodiments, the first candidate has a lower level of one or more fungi shown in Table 3b or 7b than the second candidate and is considered to be a more suitable FMT donor than the second candidate.

[0016] In a related aspect, a method for improving the effectiveness of FMT is provided, the method comprising introducing an effective amount of one or more fungi shown in Table 2, 3a, 5, 7a or 16 into a composition intended for transplantation before FMT. In some embodiments, after the introduction step, the level of one or more fungi shown in Table 2, 3a, 5, 7a or 16 is greater than 0.5% of the total fungi in the composition. In some embodiments, the method further comprises the step of using the composition to perform FMT. In some embodiments, the method further comprises the step of introducing an effective amount of an antifungal agent that inhibits the growth of one or more fungi shown in Table 3b or 7b in the composition. In some embodiments, after the introduction step, the level of one or more fungi shown in Table 3b or 7b is less than 0.05% of the total fungi in the composition. In some embodiments, the method further comprises the step of using the composition to perform FMT.

[0017] In another related aspect, a method for improving the effectiveness of FMT is provided, the method comprising administering to the FMT recipient an effective amount of an antifungal agent that inhibits the growth of one or more fungi shown in Table 3b or 7b before the FMT. In some embodiments, the level of one or more fungi shown in Table 3b or 7b in a stool sample from the FMT recipient is determined before the antifungal agent is administered. In some embodiments, the level of one or more fungi shown in Table 3b or 7b in a stool sample from the FMT recipient is determined after the antifungal agent is administered. In some embodiments, the method further comprises administering to the recipient an effective amount of an agent that reduces the total fungal load in a stool sample taken from the recipient before the FMT.

[0018] In another related aspect, a kit is provided, comprising: (1) a first composition comprising donor feces; and (2) a second composition comprising the following substances: (i) an effective amount of one or more fungi shown in Table 2, 3a, 5, 7a or 16; or (ii) an effective amount of an antifungal agent that inhibits the growth of one or more fungi shown in Table 3b or 7b. In some embodiments, the first composition comprises donor feces that has been dried, frozen and placed in a capsule for oral administration. In some embodiments, the kit also includes an effective amount of an antifungal agent that inhibits the growth of one or more fungi shown in Table 3b or 7b in the second composition, or an effective amount of an antifungal agent that reduces the total fungal load in the third composition. In addition, the kit may include printed instructions to guide the user to properly use the kit.

[0019] In some embodiments, in the above methods, the level of one or more fungi shown in Tables 2, 3a, 3b, 5, 7a, 7b, or 16 is determined by quantitative polymerase chain reaction (PCR).

[0020] In a third aspect, the invention provides a method for identifying a suitable FMT donor. The method includes determining the level of one or more viruses shown in Table 8b in a fecal sample obtained from a candidate. In some embodiments, the level of one or more viruses is a percentage relative abundance. In some embodiments, the level of one or more viruses shown in Table 8b is greater than 0.1%, and the candidate is identified as a suitable FMT donor. In some embodiments, the level of one or more viruses shown in Table 8a is not more than 0.1%. In some embodiments, the method also includes obtaining fecal material from the candidate for the step of FMT. In some embodiments, the method also includes obtaining fecal material from the candidate for the step of FMT. In some embodiments, the level of one or more viruses shown in Table 8a is greater than 0.1%, and the candidate is identified as an unsuitable FMT donor. In some embodiments, the method also includes determining the step of the total viral load in the fecal sample.

[0021] In some embodiments, the level of one or more viruses shown in Table 8a or 8b in the first stool sample obtained from the first candidate and the second stool sample obtained from the second candidate is determined. In some embodiments, the first candidate has one or more viruses shown in Table 8b at a higher level than the second candidate, and is considered to be a more suitable FMT donor than the second candidate. In some embodiments, the first candidate has one or more viruses shown in Table 8a at a lower level than the second candidate, and is considered to be a more suitable FMT donor than the second candidate.

[0022] In a related aspect, a method for improving FMT effectiveness is provided. The method includes the step of introducing one or more viruses shown in the table 8b of an effective amount into the composition intended for transplantation before FMT. In some embodiments, after the introduction step, the level of one or more viruses shown in Table 8b is greater than 0.1% of the total viruses in the composition. In some embodiments, the method also includes the step of using the composition to carry out FMT. In some embodiments, the method also includes the step of introducing an effective amount of an antifungal agent for the growth of one or more fungi shown in the inhibition table 8a in the composition. In some embodiments, after the introduction step, the level of one or more fungi shown in Table 8a is less than 0.1% of the total viruses in the composition. In some embodiments, the method also includes the step of using the composition to carry out FMT.

[0023] In another related aspect, a method for improving the effectiveness of FMT is provided, the method comprising administering to the FMT recipient an effective amount of an antiviral agent that inhibits the growth of one or more viruses shown in Table 8a before the FMT. In some embodiments, the level of one or more viruses shown in Table 8a in a stool sample from the FMT recipient is determined before the antiviral agent is administered. In some embodiments, the level of one or more viruses shown in Table 8a in a stool sample from the FMT recipient is determined after the antiviral agent is administered. In some embodiments, the method further comprises administering to the recipient an effective amount of an agent that can reduce the total viral load in a stool sample taken from the recipient before the FMT.

[0024] In yet another related aspect, a kit is provided, comprising: (1) a first composition comprising donor feces; and (2) a second composition comprising: (i) an effective amount of one or more viruses shown in Table 8b; or (ii) an effective amount of an antiviral agent that inhibits the growth of one or more viruses shown in Table 8a. In some embodiments, the first composition comprises donor feces that has been dried, frozen, and placed in a capsule for oral administration. In some embodiments, the kit also comprises an effective amount of an antiviral agent that inhibits the growth of one or more viruses shown in Table 8a in the second composition, or an effective amount of an antiviral agent that reduces the total viral load in the third composition. In addition, the kit may include printed instructions to guide the user in the proper use of the kit.

[0025] In some embodiments, in the above methods, the level of one or more viruses shown in Table 8a or 8b is determined by quantitative polymerase chain reaction (PCR).

[0026] Related compositions with improved efficacy that can be used in FMT may include: (1) donor fecal material containing live fecal microorganisms, and (2) an antibacterial or antifungal or antiviral agent that specifically inhibits the growth or proliferation of one or more bacteria / fungi / viruses shown in Tables 1b, 3b, 6b, 7b, 8a or 10a, but does not show such suppressive or inhibitory effects on other bacteria, fungi or viruses. Rather than being a broad-spectrum antibacterial, antifungal or antiviral agent, such a specific antibacterial, antifungal, antiviral agent may be a short polynucleotide property (e.g., a small inhibitory RNA, microRNA, small RNA, lncRNA or antisense oligonucleotide) that is capable of disrupting the expression of key genes in the life cycle of one or more bacteria, fungi or viruses shown in Tables 1b, 3b, 6b, 7b, 8a or 10a, which can specifically target only these species without targeting other closely related fungi.

[0027] In addition, various methods for therapeutic applications are provided: For example, a method for weight loss in a subject is provided, comprising the step of introducing into the gastrointestinal tract of the subject an effective amount of: (1) one or more bacteria shown in Table 1a, or (2) one or more fungi shown in Table 2 or 3a. On the other hand, a method for weight loss in a subject is provided, comprising the step of introducing into the gastrointestinal tract of the subject an effective amount of an inhibitor that inhibits: (1) one or more bacteria shown in Table 1b, or (2) one or more fungi shown in Table 3b. In some cases, the introducing step is performed by means of FMT. Optionally, the step of administering to the individual an effective amount of a broad-spectrum antibacterial agent or an antifungal agent, respectively, is performed before the step of introducing beneficial bacteria or fungi, such as by FMT.

[0028] Likewise, methods of inhibiting multidrug-resistant bacteria in a subject are provided, comprising the step of introducing into the gastrointestinal tract of the subject an effective amount of: (1) one or more bacteria shown in Table 4 or 15, or (2) one or more fungi shown in Table 5 or 16. In some embodiments, the bacteria are carbapenem-resistant Enterobacteriaceae (CRE). In some embodiments, the bacteria are vancomycin-resistant Enterococci (VRE). In some cases, the introducing step is performed by means of FMT. Optionally, the step of administering an effective amount of a broad-spectrum antibacterial agent or an antifungal agent to the subject is performed before the step of introducing beneficial bacteria or fungi, such as by FMT.

[0029] In addition, methods of treating acute graft-versus-host disease (aGvHD) in a subject are provided, comprising the step of introducing into the gastrointestinal tract of the subject an effective amount of: (1) one or more bacteria shown in Table 6a, or (2) one or more fungi shown in Table 7a. In another aspect, methods of treating acute graft-versus-host disease (aGvHD) in a subject are provided, comprising the step of introducing into the gastrointestinal tract of the subject an effective amount of an inhibitor of: (1) one or more bacteria shown in Table 6b, or (2) one or more fungi shown in Table 7b. In some cases, the introducing step is performed by means of FMT. Optionally, the step of administering to the individual an effective amount of a broad-spectrum antibacterial agent or an antifungal agent, respectively, is performed prior to the step of introducing the beneficial bacteria or fungi, such as by FMT.

[0030] In addition, methods for treating ulcerative colitis are provided, comprising the step of introducing into the gastrointestinal tract of a subject an effective amount of: (1) one or more bacteria shown in Table 10b, or (2) one or more viruses shown in Table 8b. In another aspect, methods for treating ulcerative colitis are provided, comprising the step of introducing into the gastrointestinal tract of a subject an effective amount of an inhibitor that inhibits: (1) one or more bacteria shown in Table 10a, or (2) one or more viruses shown in Table 8a. In some cases, the introducing step is performed by means of FMT. Optionally, the step of administering to the subject an effective amount of a broad-spectrum antibacterial agent or an antiviral agent, respectively, is performed prior to the step of introducing the beneficial bacteria or viruses, such as by FMT.

[0031] In addition, a method of treating Crohn's disease in a subject is provided, comprising the step of introducing into the gastrointestinal tract of the subject an effective amount of one or more fungi shown in Table 11, for example, by means of FMT. Optionally, the step of administering to the subject an effective amount of a broad-spectrum antifungal agent is performed prior to the step of introducing, such as by FMT.

[0032] Finally, a method for weight loss in a subject is provided, comprising introducing an effective amount of any one, two or three of Wardsartella, intestinal Rosebairnella or Mitsuokella multacida into the gastrointestinal tract of the subject, for example, the subject can receive all three, i.e., an effective amount of each of Wardsartella, intestinal Rosebairnella and Mitsuokella multacida. A method for reducing total cholesterol and low-density lipoprotein cholesterol in a subject is also provided, comprising introducing an effective amount of any one, two or three of Wardsartella, intestinal Rosebairnella or Mitsuokella multacida into the gastrointestinal tract of the subject, for example, the subject can receive all three, i.e., an effective amount of each of Wardsartella, intestinal Rosebairnella and Mitsuokella multacida. The cholesterol-lowering method can be independent of weight loss in the subject, or implemented simultaneously with weight loss. These methods can be performed by means of FMT. Optionally, a step of administering an effective amount of a broad-spectrum antibacterial agent such as an antibiotic to the subject is performed before the introduction step such as FMT is performed. For use in the method, a composition is provided comprising an effective amount of any one, two or three of Waldersartella, Roseburia intestinalis or Pseudomonas aeruginosa, for example, such a composition can be derived from donor fecal material and has been dried, frozen and placed in a capsule for oral administration.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1a "Beneficial" bacteria present in FMT donors for inducing weight loss identified by correlation (linear regression). Five species were identified by linear regression to be positively correlated with weight loss. These species are provided in Table 1a. Figure 1b"Unfavorable" bacteria present in FMT donors for inducing weight loss identified by correlation (linear regression). 17 species were identified from linear regression as negatively correlated with weight loss. These species are provided in Table 1b. Figures 1c and 1d "Favourable" and "unfavourable" bacteria present in FMT donors used to induce weight loss identified by LEfSe analysis. Figure 1c uses the median as a cut-off point and the LefSe plot shows bacteria with significant differences in abundance between "favourable" and "less favourable" donors. Figure 1d uses the mean as a cut-off point and the LefSe plot shows bacteria with significant differences in abundance between "favourable" and "less favourable" donors. These species are provided in Tables 1a and 1b.

[0035] Figure 2 "Favourable" fungi in FMT donors for inducing weight loss. The LEfSe plots show that the relative abundance of Cercospora beticola and Kazachstania naganishii in controls is significantly higher than in obese subjects. These species are provided in Table 2.

[0036] Figure 3 Fungi in receptors associated with successful and unsuccessful weight loss. The LEfSe plots show the relative abundance of fungi that are significantly higher in the “weight loss” or “non-weight loss” groups. These species are provided in Tables 3a and 3b.

[0037] Figure 4a "Favourable" bacteria in FMT donors for the treatment of CRE identified in a cross-sectional study. Classification cladogram from LEfSe analysis of metagenomic sequencing comparing bacterial characteristics in patients with CRE and healthy controls. Comparison of bacterial characteristics with the linear discriminant analysis effect size (LEfSe) model. Only classifications that met a linear discriminant analysis (LDA) threshold of >2 were considered significant. The relative abundance of 33 species (black) in healthy controls was significantly higher. These species are provided in Table 4. Figure 4b "Favourable" bacteria in FMT donors for the treatment of CRE identified in a prospective pilot study. Comparison of taxonomic properties of bacteria in FMT donors and recipients before and after FMT based on metagenomic sequencing. Relative abundance of bacterial taxa present in fecal samples of donors and recipients before and after FMT. R1 and R2 refer to fecal samples collected from FMT recipients 1 and 2, respectively. Columns are marked by the letter D followed by the sample collection day, with the first FMT performed on day 0. These species are provided in Table 4.

[0038] Figure 5"Favourable" fungi in donors of FMT for the treatment of CRE identified in a prospective pilot trial. Comparison of taxonomic characteristics of fungi in FMT donors and recipients with CRE before and after FMT based on metagenomic sequencing. Relative abundance of fungal taxa present in fecal samples of donors and recipients before and after FMT. R1 and R2 refer to fecal samples collected from FMT recipients 1 and 2, respectively. Columns are marked by the letter D and the subsequent sample collection day, with the first FMT performed on day 0. Detailed relative abundance of selected species is shown in Table 5.

[0039] Figure 6a "Favourable" and "unfavourable" bacteria in FMT donors for the treatment of acute graft-versus-host disease. Comparison of the taxonomic characteristics of bacteria in FMT donors and recipients suffering from aGvHD before and after FMT based on metagenomic sequencing. Bar graphs represent the relative abundance of bacterial classification in stool samples from donors and recipients before and after FMT. The stool samples collected from recipients are marked by the sample collection day, where 0 represents the date of the first FMT. The stool samples from donors for FMT are marked as D4 and D8. Donor D8 collected 5 samples on different dates. Before FMT, Corynebacterium jeikeium (71.2%) dominated the intestinal bacterial community of recipients, and it dropped significantly to <0.1% after the first FMT. After receiving repeated FMT, the transplantation of 7 bacteria was observed in patients, and these species accounted for a large proportion of the patient's bacterial community. These species are Alistipes onderdonkii, Alistipes putredinis, Clostridium bolteae, Clostridium nexile, Clostridium symbiosum, Eggerthella unclassified, Ruminococcus gnavu. These species are listed in Table 6a and Table 6b. Figure 6b"Favourable" and "unfavourable" bacteria in FMT donors for the treatment of acute graft-versus-host disease identified by LEfSe analysis.LEfSe graph shows the relative abundance of 4 bacteria with significant differences when patients have diarrhea or no diarrhea.Before and after FMT, a total of 22 stool samples were collected from patients with aGvHD.Samples were classified according to whether the patient had or did not have diarrhea on the stool collection day.Diarrhea was defined as bowel opening with 2 or more bowels per day, while non-diarrhea was defined as bowel opening with 0 or 1 bowel movement per day.LEfSe analysis was used to identify bacteria with significant differences between stool samples from "diarrhea" or "non-diarrhea" groups.The classification that only meets the LDA threshold of>2 is considered significant.Eubacterium rectale in the diarrhea group is significantly higher.Alistipes putredinis, Alistipes onderdonkii and Clostridium hathewayi in the non-diarrhea group are significantly higher. These species are listed in Tables 6a and 6b.

[0040] Figure 7 "Favorable" and "unfavorable" fungi in FMT donors for the treatment of aGvHD identified from heat maps. Comparison of the taxonomic characteristics of fungi in FMT donors and recipients suffering from aGvHD before and after FMT based on metagenomic sequencing. Relative abundance of fungal classifications present in fecal samples of recipients suffering from aGVHD before and after FMT of donors and FMT. Fecal samples collected from recipients are marked by sample collection day, where 0 represents the date of the first FMT. Fecal samples from donors for FMT are marked as D4 and D8. Donor D8 collected 5 samples on different dates. Transplantation of fungi from donors after FMT in patients with aGvHD was assessed by heat maps. 44 fungi were transplanted after FMT treatment of patients. Blue to red tones represent the relative abundance of the increase as shown in the legend. The classification mark on the right side of the figure represents the mark of the indicated species. Representative species are listed in Tables 7a and 7b.

[0041] Figure 8Differential viral classifications at the family, genus, and species level between the mucosa of a healthy population and patients with UC. Differentially enriched viral families (a), genera (b), and species (c) between healthy and UC mucosa were determined by DESeq analysis with FDR correction (only those differential classifications with adjusted p-values ​​< 0.05 and |Log2(fold change between groups)|> 2 are shown). For the viral classification names, black classifications are colored to represent prokaryotic viruses, while those in gray represent eukaryotic viruses. For the viral abundance box plots, the boxes extend from the 1st quartile to the 3rd quartile (25th to 75th percentiles), with the median shown by a vertical line. These classifications are listed in Tables 8a and 8b.

[0042] Fig. 9 Mucosal virosome enterotypes in healthy subjects and patients with UC. Fig. 9 a Virome enterotype clustering of viral community structures from mucosa of healthy cohort and patients with UC based on partition around medoids (PAM) algorithm and principal coordinate analysis (PCoA). The inset shows the ratio of healthy individuals and UC subjects in each enterotype group. Fig. 9 b Heat map of the presence of differentially expressed viruses contributing to the clustering of enterotypes of the two mucosal virus groups. Differential species were identified by concordant DEseq and random forest analysis. Virus abundance was calculated based on Log 10 RPKM values ​​are colored darker. Only those species with effect sizes > 2 and FDR-adjusted P values ​​< 0.05 determined by DEseq and random forest algorithms are shown. These species are listed in Tables 9a and 9b.

[0043] Fig.10 Altered bacterial microbiota in UC mucosa. Fig.10 a Comparison of bacterial alpha diversity based on Simpson diversity and Chao1 richness in the mucosa of controls and UC subjects. Statistical significance was determined by t-test, *P<0.05, **P<0.01. For box plots, the boxes extend from the 1st quartile to the 3rd quartile (25th to 75th percentiles), with the median shown by the vertical line. The bacterial composition in healthy and UC mucosa is plotted as relative abundance at the phylum (b), family (c), and genus (d) levels. e, Differentially enriched bacterial taxa between the mucosa of healthy individuals and UC subjects were determined by Lefse analysis with FDR correction. Only those taxa with adjusted P values ​​<0.05 and LDA effect sizes >2 are shown. These taxa are listed in Tables 10a and 10b.

[0044] Fig.11Study timeline of mouse model. Mice were fed a high-fat diet for 1 month, followed by a normal diet for 1 month and antibiotic treatment for 7 days. The mice were then divided into 3 groups and orally gavaged with 100 mg / kg of waldersartella, 100 mg / kg of roseborgia, 100 mg / kg of chlamydomonas, and 100 mg / kg of chlamydomonas. 9 cfu / ml) or culture medium.

[0045] Fig.12 Administration of S. waldensis significantly reduced body weight gain, whereas administration of R. intestinalis showed a trend toward reduced body weight gain. The effect was particularly evident after day 15 (after 3 oral gavages). Abbreviations: SW - S. waldensis; RI - R. intestinalis

[0046] Fig.13 Both S. waldensis and R. enterica reduced body weight gain at day 20 and day 45. The reduced body weight gain persisted for 20 days after cessation of treatment (at day 45). Body weights at day 20 and 45 were compared to day 0 and presented as a percentage of body weight gain. Abbreviations: SW - S. waldensis; RI - R. enterica

[0047] Fig.14 Mean food intake showed a decreasing trend after administration of combination, MM, RI and SW, in contrast to administration of medium alone, which showed an increasing trend. The effect of food intake reduction was most significant in RI and SW. Mean food intake was calculated as grams of food intake / mouse day.

[0048] Fig.15 After 15 days, the average food efficiency was reduced in mice given combination, MM, RI and SW compared to those given medium. The average food efficiency was calculated as the body weight gain (g) in 5 days / total food intake (g) in these 5 days for each mouse.

[0049] Fig.16 Administration of S. waldensis alone reduced LDL-C, TC, and TG. Administration of R. enterica alone reduced LDL-C and TC. Administration of a combination of S. waldensis, R. enterica, and P. polyacidum reduced LDL-C and TC. All results at day 45 were compared to a control administered with medium. Abbreviations: SW - S. waldensis; RI - R. enterica; MM: P. polyacidum; LDL-C: low-density lipoprotein cholesterol; TC: total cholesterol; TG: total triglycerides.

[0050] Fig.17 Sample collection timeline for donors and recipients. Relative sample collection times and results for CRE for each recipient are shown.

[0051] Fig.18The relative abundance of the bacteria Ruminococcus sp_5_1_39BFAA, Collinsella tanakaei and Eubacterium sicaeum in three FMT recipients and their corresponding donors before and after FMT was changed. The relative abundance of Ruminococcus sp_5_1_39BFAA, Collinsella tanakaei and Eubacterium sicaeum was undetectable before FMT and increased to a level similar to that of the donors or even higher after FMT.

[0052] Fig.19 LEfSe analysis comparing bacterial composition before (black) and after (grey) FMT. All taxa that were significantly different with LDA scores > 2.0 are shown.

[0053] Fig. 20 Analysis of fungal composition of donors and recipients. (a) Alpha diversity (Shannon diversity) of donors, recipients and recipients after FMT at different times. (b) Principal coordinate analysis (PCoA) based on Bray-Curtis distance of recipients. The figure shows the distance between donors and CRE recipients over time. (c) LEfSe analysis comparing the mycobiome composition before (grey) and after (black) FMT. (d) Heatmap showing the significantly dominant species present in fecal samples before and after FMT.

[0054] definition

[0055] The term "fecal microbiota transplant (FMT)" or "fecal transplant" refers to a medical procedure in which fecal material containing live fecal microorganisms (bacteria, fungi, viruses, etc.) obtained from a healthy individual is transferred into the gastrointestinal tract of a recipient to restore a healthy intestinal microbiota that has been disrupted or destroyed by any of a variety of medical conditions. Typically, fecal material from a healthy donor is first processed into a form suitable for transplantation, which can be performed by direct deposition in the lower gastrointestinal tract, such as by colonoscopy or by nasal cannula or by oral administration of a packaged material containing processed (e.g., dried and frozen) fecal material. FMT is used to treat a variety of medical conditions, including obesity, metabolic syndrome, gastrointestinal diseases such as inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), antibiotic-resistant bacterial infections such as Clostridium difficile infection (CDI) or conditions caused by multidrug-resistant organisms including carbapenem-resistant Enterobacteriaceae (CRE) or vancomycin-resistant Enterococci (VRE), as well as autism, depression, obesity, diabetes, alopecia, acute graft-versus-host disease (aGvHD), and also certain neurological conditions such as multiple sclerosis and Parkinson's disease.

[0056] As used herein, the term "inhibiting" or "inhibition" refers to any detectable negative effect on a target biological process, such as RNA / protein expression of a target gene, biological activity of a target protein, cell signal transduction, cell proliferation, etc. In general, inhibition is reflected as a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of a target process (e.g., growth or proliferation of certain types of microorganisms, e.g., one or more of the bacteria shown in Tables 1b, 6b, or 10a; or fungi shown in Tables 3b or 7b; or viruses shown in Table 8a), or any of the downstream parameters described above, when compared to a control. "Inhibition" also includes a 100% reduction, i.e., complete elimination or abrogation of a target biological process or signal. Other related terms such as "suppressing," "suppression," "reducing," and "reduction" are used in a similar manner in the present disclosure to refer to varying levels of reduction (e.g., a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level) up to complete elimination of a target biological process or signal. On the other hand, terms such as "activate," "activating," "activation," "increase," "increasing," "promote," "promoting," "enhance," "enhancing," or "enhancement" are used in the present disclosure to include positive changes in various levels of a target process or signal (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or greater, such as a 3, 5, 8, 10, 20-fold increase, compared to a control level of, for example, one or more of the bacteria shown in Tables 1a, 4, 6a, 10b, 11 or 15, or the fungi shown in Tables 2, 3a, 5, 7a or 16, or the viruses shown in Table 8b).

[0057] The term "antibacterial / antifungal / antiviral agent" refers to any substance capable of inhibiting, suppressing or preventing the growth or proliferation of bacteria, fungi or viruses, particularly those shown in Tables 1b, 3b, 6b, 7b, 8a and 10a, respectively. Known agents with antibacterial activity include various antibiotics that broadly inhibit the proliferation of a broad spectrum of bacteria, as well as agents such as reactive oligonucleotides, small inhibitory RNAs that can inhibit the proliferation of specific bacteria. The term "antibacterial / antifungal / antiviral agent" is similarly defined to include agents having a broad spectrum of activity that kills almost all bacteria, fungi or viruses, respectively, and agents that specifically inhibit the proliferation of target bacteria, fungi or viruses, respectively.

[0058] When used in the context of describing a particular bacterium or fungus or virus (e.g., any of those shown in any of Tables 1-11) relative to all bacteria or fungi or viruses, respectively, when present in the same environment, "percent relative abundance" refers to the relative amount of the bacterium or fungus or virus expressed as a percentage of the amount of all bacteria or fungi or viruses. For example, the percent relative abundance of a particular fungus can be determined by comparing the amount of DNA specific to that species in a given sample (e.g., as determined by quantitative polymerase chain reaction) with the amount of all fungal DNA in the same sample (e.g., as determined by quantitative polymerase chain reaction (PCR) and sequencing based on internal transcribed spacer 2 or ITS2 sequences).

[0059] When used in the context of describing the presence of a specific bacterium or fungus or virus (e.g., any of those shown in Tables 1-11, 15, and 16) in feces, "absolute abundance" refers to the amount of DNA derived from bacteria or fungi or viruses, respectively, out of the amount of all DNA in a fecal sample. For example, the absolute abundance of a fungus can be determined by comparing the amount of DNA specific to that fungus in a given sample (e.g., determined by quantitative PCR) with the amount of all fecal DNA in the same sample.

[0060] As used herein, the "total bacterial / fungal / viral load" of a stool sample refers to the amount of all bacterial / fungal / viral DNA in the amount of all DNA in the stool sample, respectively. For example, the absolute abundance of fungi can be determined by comparing the amount of fungal-specific DNA in a given sample (e.g., 18S rDNA determined by quantitative PCR) with the amount of all fecal DNA in the same sample.

[0061] As used herein, the term "effective amount" refers to an amount of a substance that produces a desired effect (e.g., an inhibitory or suppressive effect on the growth or proliferation of one or more harmful bacteria or fungi or viruses (e.g., bacteria shown in Tables 1b, 6b or 10a) to which the substance (e.g., antibacterial agent) is used or administered. The effect includes preventing, inhibiting or delaying any relevant biological process in the process of bacterial / fungal / viral proliferation to any detectable extent. The exact amount will depend on the nature of the substance (active agent), the mode of use / administration, and the purpose of the administration, and will be determined by those skilled in the art using known techniques and the present invention. In another case, when an "effective amount" of one or more beneficial bacteria or fungi or viruses (e.g., those listed in Tables 1a, 2, 3a, 4, 5, 6a, 7a, 8b, 10b, 11, 15 or 16) is artificially introduced into a composition intended for FMT, it means that the amount of the relevant bacteria / fungi / virus introduced is sufficient to confer health benefits to the FMT recipient, such as weight loss, improved sensitivity to insulin, reduced blood cholesterol levels, inhibition of drug-resistant bacterial infections, and / or reduction of acute graft-versus-host disease (aGvHD) and GI tract diseases such as IBD.

[0062] As used herein, the term "about" refers to a range of values ​​that is + / -10% of a specified value. For example, "about 10" refers to a range of values ​​of 9-11 (10+ / -1). DETAILED DESCRIPTION OF THE INVENTION

[0064] I. Introduction

[0065] The present invention provides a new method for evaluating the possibility of effective FMT before the scheme is carried out and improving the effectiveness of the FMT program in terms of giving certain health benefits to the recipient. In its research, the inventor found that the presence and relative abundance of certain bacteria, fungi and viruses in the feces of the recipient's gastrointestinal tract and the donor are directly related to the results of FMT. For example, it is found that the presence of the bacteria shown in Table 1a, 4, 6a, 10b, 11 or 15 (especially at elevated levels) gives health benefits to the FMT recipient, while the presence of the bacteria shown in Table 1b, 6b or 10a (especially at elevated levels) tends to adversely affect the FMT results. Similarly, the presence and relative abundance of certain fungi and viruses have been identified to be relevant to the desired FMT results. Thus, detection of one or more "beneficial" bacteria / fungi / viruses of those shown in Tables 1a, 2, 3a, 4, 5, 6a, 7a, 8b, 10b, 11, 15 or 16 in the feces of potential donors (particularly at elevated levels), and on the other hand, detection of one or more "harmful" bacteria / fungi / viruses of those shown in Tables 1b, 3b, 6b, 7b, 8a, 10a or 16 (particularly at elevated levels), can be used to guide donor selection, while analysis of these relevant bacteria / fungi / virus levels in FMT recipients can determine whether the subject is ready for FMT directly or should be treated with antibacterial / fungal / viral agents that inhibit such undesirable microorganisms prior to FMT to optimize treatment outcomes. II. FMT Donor / Recipient Selection and Preparation

[0066] Patients with CDI, especially recurrent CDI, are generally considered recipients for FMT treatment. In addition to CDI, other diseases and conditions, including those of the digestive or nervous systems, such as colitis, irritable bowel syndrome (IBS), Crohn's disease, acute graft-versus-host disease (aGvHD), infections caused by multidrug-resistant bacteria such as CRE or VRE, multiple sclerosis, Parkinson's disease, diabetes, and obesity, are also suitable for FMT treatment.

[0067] Fecal material used in FMT is obtained from healthy donors and then processed into an appropriate form for the intended mode of delivery in the upcoming FMT procedure. Until recently, the general criteria for FMT donors have been simple, namely that the donor is a healthy individual without any known disease or disorder, particularly in the digestive tract, although preference is often given to members of the same family as the recipient.

[0068] The inventors have found in their studies that an elevated presence of one or more "beneficial" bacteria, those shown in Table 1a, in the GI tract of a recipient or in the feces of a donor (which is processed for transplantation) can confer significant health benefits after FMT treatment in patients, such as weight loss, improved insulin sensitivity, and reduced blood / serum / plasma cholesterol levels in the recipient. In contrast, when an elevated presence of other "harmful" bacteria, those shown in Table 1b, is found in the GI tract of a recipient or in the feces of a donor for FMT, such health benefits are not conferred after FMT treatment. Similarly, an elevated presence of one or more "beneficial" fungi, those shown in Table 2 or 3a, in the GI tract of a recipient or in the feces of a donor for FMT can confer the same or similar health benefits in a patient after FMT treatment, whereas no such health benefits are obtained when an elevated presence of "harmful" fungi, those shown in Table 3b, is found in the GI tract of a recipient or in the feces of a donor for FMT after FMT.

[0069] This revelation enables the initial screening of individuals who are suitable FMT donors, as well as the initial screening of patients who are likely candidates for successful and beneficial FMT treatment, particularly in the context of treating obesity or metabolic syndrome, including insulin insensitivity and / or type II diabetes: if the stool of the candidate donor contains minimal or elevated levels of any one or more of the bacteria shown in Table 1a (e.g., each being greater than 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8% or 2% of the total bacteria), then The candidate is considered suitable as an FMT donor, and its feces can be directly retrieved for processing and subsequent use in FMT; on the other hand, if the candidate's fecal sample shows no or only low levels of these beneficial bacteria (e.g., each is no more than 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8% or 2% of the total bacteria), then the candidate is considered not to be a directly suitable FMT donor, and its fecal material should not be collected for FMT without necessary adjustments before possible use. One possible way to improve the donor's fecal material before processing for FMT is to artificially introduce one or more beneficial bacteria (e.g., those in Table 1a) so as to increase the presence of such bacteria in the fecal material for FMT (e.g., each species is greater than 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8% or 2% of the total bacteria).

[0070] Conversely, the inventors have also revealed that the absence or reduced presence of certain unwanted or harmful bacteria in fecal material for FMT, including one or more of those shown in Table 1b, tends to produce significant health benefits in patients after FMT treatment, such as weight loss in recipients, improved insulin sensitivity, and reduced blood / serum / plasma cholesterol levels. Therefore, if a fecal sample of a candidate donor has been tested and shown to contain reduced or undetectable levels of one or more of the bacteria shown in Table 1b (e.g., each being no greater than 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.4%, or 0.5% of total bacteria), the individual is considered a suitable donor and his or her fecal sample can be directly collected and processed for FMT. On the other hand, if the stool of an individual who is a potential donor has been tested and found to contain a large presence (particularly at elevated levels) of one or more bacteria shown in Table 1b (e.g., each being greater than 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.4% or 0.5% of total bacteria), then the individual is considered unsuitable as a donor of fecal material for FMT and therefore a stool sample thereof should not be collected for FMT.

[0071] Ideally, the desired FMT composition prepared from donor fecal material and intended for use in FMT for the treatment of obesity or metabolic syndrome or type II diabetes has high levels of one or more beneficial bacteria (e.g., those shown in Table 1a) and low levels of one or more harmful bacteria (e.g., those shown in Table 1b). Therefore, although the composition intended for FMT can be improved to address the problem of insufficient levels of beneficial bacteria and excessive presence of harmful bacteria, respectively, a possible improvement is to increase the level of one or more beneficial bacteria (e.g., those shown in Table 1a), for example, by supplementing the transfer material intended for FMT or by directly introducing sufficient amounts of such beneficial bacteria in the GI tract of the recipient, optionally while suppressing the level of one or more harmful bacteria (e.g., those shown in Table 1b), to maximize the potential health benefits that the recipient may obtain from the FMT procedure.

[0072] In the context of FMT recipients, if a patient is proposed to receive FMT treatment for obesity or metabolic syndrome or type II diabetes, and their stool sample shows elevated levels of one or more harmful bacteria shown in Table 1b (e.g., each greater than 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.4% or 0.5% total bacteria), the patient may be considered unsuitable for immediate FMT because treatment may have a reduced chance of producing health benefits, such as reduced weight, increased insulin sensitivity, and reduced blood cholesterol. The patient may first receive an antibacterial treatment to reduce the levels of harmful bacteria in their gastrointestinal tract, particularly those shown in Table 1b, before starting FMT treatment, during which the patient will receive donor fecal material that is enriched with one or more beneficial bacteria (those shown in Table 1a) and / or has suppressed levels of one or more harmful bacteria (e.g., those shown in Table 1b). On the other hand, if the patient proposed for FMT treatment does not have a significant presence, particularly an elevated presence (e.g., each being no greater than 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.4% or 0.5% of total bacteria) of any one or more of the bacteria shown in Table 1b, the patient can immediately begin FMT treatment without undergoing additional preparation or pretreatment steps.

[0073] In addition, the inventors have also identified "beneficial" and "harmful" fungi for FMT treatment of obesity or metabolic syndrome or type II diabetes, which can guide the selection of suitable FMT donors and recipients in the same manner as described above: For example, a desired FMT composition prepared from donor fecal material and intended for FMT for the treatment of obesity or metabolic syndrome or type II diabetes has an elevated level of one or more beneficial fungi (e.g., those shown in Tables 2 or 3a, each of which is not less than 0.5%, 1%, 1.5%, 2%, 2.5%, 3% of the total fungi). , 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14% or 15%) and / or lower levels of one or more harmful bacteria (e.g., those shown in Table 3b, each of which is no greater than 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3% or 3.5% of the total fungi). Additionally, preparation of the FMT donor / donor fecal material or recipient can be performed to manipulate the levels of beneficial fungi (causing an increase) and / or the levels of harmful fungi (causing a decrease) in order to optimize the health benefits from the FMT procedure, similar to the manipulation of beneficial and / or harmful bacteria prior to FMT described above.

[0074] In the case of FMT treatment for the inhibition or eradication of multidrug-resistant bacteria such as carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE), the inventors have identified "beneficial" bacteria and fungi that, when present at elevated levels in FMT donor feces, allow the procedure to achieve improved therapeutic efficacy. These bacteria and fungi are shown in Tables 4 and 5, respectively. The desired FMT composition prepared from donor fecal material and intended for use in FMT for the treatment of multidrug-resistant bacteria has elevated levels of one or more beneficial bacteria or fungi (e.g., those shown in Tables 4, 5, 15 or 16, each being not less than 0.02%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5% or 3% of total bacteria or total fungi). In addition, preparation of the FMT donor / donor fecal material or recipient can be performed to manipulate the levels of (cause an increase in) beneficial bacteria or fungi in order to maximize the efficacy and health benefits from the FMT procedure, similar to the manipulation of beneficial bacteria prior to FMT described above, for example, by artificially introducing added and effective amounts of one or more "beneficial" bacteria and / or fungi of Tables 4, 5, 15 or 16 in the transfer material to be used for FMT or directly into the GI tract of the recipient.

[0075] In the case of FMT treatment for conditions related to cell or tissue transplantation such as acute graft-versus-host disease (aGvHD), the inventors have identified certain "beneficial" bacteria and fungi that are associated with improved therapeutic efficacy when present at elevated levels in the feces of the recipient after FMT treatment. These bacteria and fungi are shown in Tables 6a and 7a, respectively. On the other hand, certain "harmful" bacteria and fungi have also been identified, see Tables 6b and 7b. They are present at elevated levels in the feces of the recipient after FMT treatment, which is associated with reduced or insufficient therapeutic results. Therefore, when selecting potential FMT donors, donors whose stool samples contain elevated levels of any one or more "beneficial" bacteria and / or fungi (shown in Table 6a or 7a) compared to the average level (e.g., greater than 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14% or 15% of total bacteria or total fungi) or the corresponding levels found in the stool sample of another potential donor will be considered more suitable donors. In contrast, suitable donors should also have lower levels of one or more or all of the "harmful" bacteria and / or fungi shown in Table 6b or 7b in their stool samples. In addition, preparation of the FMT donor / donor fecal material or recipient may be performed to manipulate the levels of beneficial bacteria or fungi (causing an increase) and / or the levels of harmful bacteria or fungi (causing a decrease) in order to maximize the efficacy and health benefits from the FMT procedure, similar to the manipulation of beneficial bacteria prior to FMT described above, for example, by artificially introducing an added and effective amount of one or more of the "beneficial" bacteria and / or fungi of Table 6a or 7a in the transfer material to be used for FMT or directly into the GI tract of the recipient. The manner of inhibiting, particularly specifically inhibiting, one or more or all of the harmful bacteria and / or fungi shown in Table 6b or 7b in the GI tract of the recipient after FMT may be applied to increase the efficacy or benefit of the treatment.

[0076] In the case of GI tract diseases or conditions such as inflammatory bowel disease (IBD), particularly ulcerative colitis (UC), the inventor has identified certain bacteria and viruses, whose presence, particularly with elevated levels, is associated with the presence of disease. These bacteria and viruses are shown in Tables 8a and 10a, respectively. On the other hand, some other bacteria and viruses have also been identified, referring to Tables 8b and 10b, respectively, compared to healthy controls, observed to be absent or present at reduced levels in individuals with disease. Therefore, these specific bacteria and viruses can be used to guide the selection of FMT donors for the purpose of providing materials for the treatment of GI diseases such as IBD (which include UC and CD): one or more bacteria and / or viruses shown in Tables 8a or 10a of lower levels tend to indicate that a candidate is suitable as an FMT donor. On the contrary, one or more bacteria and / or viruses shown in Tables 8b or 10b of higher levels tend to indicate that a candidate is suitable as an FMT donor. In addition, for a potential donor whose level of one or more bacteria and / or viruses shown in Table 8a or 10a in its fecal sample is lower than the corresponding level found in the fecal sample of another candidate, it will be considered as a more suitable donor than the second candidate. On the other hand, when a potential donor has a level of one or more bacteria and / or viruses shown in Table 8b or 10b in its fecal sample that is higher than the corresponding level found in the fecal sample of another candidate, it will be considered as a more suitable donor than the second candidate. For example, to introduce additional and effective amounts of one or more bacteria and viruses shown in Table 8b and 10b, or to reduce the amount of bacteria and viruses shown in Table 8a and 10a in the GI tract of the FMT material and / or the recipient, further manipulation of these related bacteria and / or viruses in the GI tract of the FMT material and / or the recipient can be used to improve the therapeutic effect of the FMT treatment strategy for treating IBD, particularly UC.

[0077] In the case of FMT treatment of GI tract disorders or conditions such as Crohn's disease (CD), which is another type of inflammatory bowel disease (IBD), the inventors have identified certain "beneficial" bacteria that, when present at elevated levels in FMT donor feces, allow the procedure to achieve improved therapeutic efficacy. These bacteria are shown in Table 11. The desired FMT composition prepared from donor fecal material and intended for FMT for the treatment of CD has elevated levels of one or more beneficial bacteria (e.g., those shown in Table 11, each reaching no less than 0.1%, 0.2%, or 0.5% of total bacteria). In addition, preparations for FMT donors / donor fecal material or recipients can be performed to manipulate the levels of one or more beneficial bacteria (causing an increase) so as to maximize the efficacy and health benefits from the FMT procedure, which is similar to the manipulation of beneficial bacteria before FMT described above, for example, by artificially introducing an added and effective amount of one or more "beneficial" bacteria of Table 11 in the transfer material to be used for FMT or directly to the GI tract of the recipient.

[0078] Various methods for determining the levels of all bacteria or fungi or viruses in a sample have been reported in the literature, for example, bacterial polynucleotide sequence amplification (e.g., by PCR) and sequencing using sequence similarity in the commonly shared 16S rDNA bacterial sequence. On the other hand, the level of any given bacterium can be determined by amplification and sequencing of its unique genomic sequence. Percent abundance is often used as a parameter indicating the relative level of bacteria in a given environment.

[0079] III. Therapeutic approaches using beneficial microorganisms

[0080] The inventors' discoveries reveal a direct correlation between (1) certain "beneficial" bacteria (e.g., those shown in Tables 1a, 4, 6a, 10, 11, or 15), "beneficial" fungi (e.g., those shown in Tables 2, 3a, 5, 7a, or 16), "beneficial" viruses (e.g., those shown in Table 8b), or certain "harmful" bacteria (e.g., those shown in Tables 1b, 6b, or 10a), "harmful" fungi (e.g., those shown in Tables 3b or 7b), "harmful" viruses (e.g., those shown in Table 8a) in an individual's feces or GI tract or transferred material derived from donor feces for FMT, and (2) the presence / absence of disease or significant health benefits conferred to the FMT recipient by way of FMT treatment, such as weight loss, improved insulin sensitivity, reduced blood cholesterol levels, suppression of multidrug-resistant bacteria such as CRE and VRE, and alleviation of certain conditions such as aGvHD, UC, or CD. This discovery not only allows the design of a preliminary screening process to identify suitable donors and recipients to obtain therapeutic efficacy and / or health benefits from the FMT procedure, it also enables different approaches to enhance or optimize the potential health benefits conferred by the FMT procedure by modulating (increasing or decreasing) the levels of one or more beneficial or harmful bacteria, fungi or viruses as shown in the table herein in the donor fecal material and recipient prior to FMT.

[0081] As discussed in the above sections, when the stool of a candidate donor is tested and found to contain elevated levels of one or more harmful bacteria or fungi or viruses of those shown in Tables 1b, 3b, 6b, 7b, 8a or 10a (e.g., greater than 0.01%, 0.02%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 3.5% of the total bacteria, total fungi or total viruses in the stool sample, respectively), the candidate is considered unsuitable as an FMT donor and his stool should not be collected for FMT without pretreatment or improvement because, without improvement, such fecal material is unlikely to produce health benefits to the recipient of FMT treatment, such as weight loss, increased insulin sensitivity or lowered blood cholesterol, clearance of multidrug-resistant bacteria, reduction of aGvHD, UC or CD. Similarly, when the stool of a proposed FMT donor is tested and found to contain insufficient levels of one or more of the beneficial bacteria or fungi or viruses shown in Tables 1a, 2, 3a, 4, 5, 6a, 7a, 8b, 10b, 11, 15 or 16 (e.g., less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.6%, 7.7%, 7.8%, 7.9%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 10. 5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, or 15%), the proposed donor is considered an unsuitable FMT donor intended to confer health benefits such as weight loss, sensitivity to insulin response, reduced blood cholesterol levels, elimination of multidrug-resistant bacteria, and alleviation of aGvHD, UC, or CD, and their fecal material should not be used directly for FMT due to the lack of prospect of conferring such beneficial health effects unless the fecal material is adequately improved. In these situations where a lack of health benefit from FMT treatment is expected, improvements can be readily made based on the inventors' discoveries, for example, fecal material from a donor can be modified to increase the level of one or more beneficial bacteria (e.g., by increasing the level of one or more bacteria shown in Tables 1a, 4, 6a, 10b, 11 or 15 to greater than 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.5%, 3%, 3.5% or 4% of the total bacteria by introducing additional amounts of such bacteria), and / or reduce the level of one or more harmful bacteria (e.g., by reducing the level of one or more bacteria shown in Tables 1b, 6b or 10a to less than 0.01%, 0.02%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the total bacteria by using specific inhibitors of such bacteria).Patients who will soon undergo FMT treatment may be prepared using conditioning regimens with similar intended goals to maximize their likelihood of obtaining health benefits such as those described above and herein.

[0082] First, for patients who have been considered for FMT but have also been deemed unsuitable FMT recipients due to elevated levels of one or more of the harmful bacteria or fungi or viruses shown in Tables 1b, 3b, 6b, 7b, 8a or 10a found in their stool samples (which indicates a reduced chance of a beneficial FMT outcome), steps can be taken prior to commencing FMT to reduce the levels of such bacteria or fungi or viruses so that a much greater chance of achieving a beneficial outcome of the FMT procedure can be obtained. For example, generally or specifically, prior to commencing an FMT procedure intended for the treatment of obesity / patient weight manipulation or for the treatment of metabolic syndrome or for the treatment of type II diabetes, an effective amount of an antibacterial agent capable of inhibiting the growth or proliferation of the bacteria shown in Table 1b can be administered to the patient so that the levels of such harmful bacteria in the patient's digestive tract and feces are significantly reduced (e.g., each being no greater than 0.01%, 0.02%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the total bacteria). Similarly, generally or specifically, prior to commencing an FMT program intended for the treatment of obesity / patient weight manipulation or for the treatment of metabolic syndrome or for the treatment of type II diabetes, an effective amount of an antifungal agent capable of inhibiting the growth or proliferation of the fungi shown in Table 3b may be administered to the patient such that the level of such harmful fungi in the patient's digestive tract and feces is significantly reduced (e.g., each to no greater than 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or 1.5% of the total fungi).

[0083] For the treatment of aGvHD, generally or specifically, prior to initiating an FMT procedure intended to treat aGvHD, an effective amount of an antibacterial agent capable of inhibiting the growth or proliferation of the bacteria shown in Table 6b can be administered to the patient so that the levels of such harmful bacteria in the patient's digestive tract and feces are significantly reduced (e.g., each is no greater than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% of the total bacteria). Similarly, generally or specifically, prior to initiating an FMT procedure intended to treat aGvHD, an effective amount of an antifungal agent capable of inhibiting the growth or proliferation of the fungi shown in Table 7b may be administered to a patient such that the levels of such harmful fungi in the patient's digestive tract and feces are significantly reduced (e.g., each to no greater than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1% of total fungi).

[0084] For the treatment of ulcerative colitis (UC), generally or specifically, before starting a FMT program intended to treat UC, an effective amount of an antiviral agent capable of inhibiting the growth or proliferation of the viruses shown in Table 8a can be administered to the patient, so that the level of such harmful bacteria in the patient's digestive tract and feces is significantly reduced (e.g., each is no more than 0.01%, 0.02%, 0.05% or 0.1% of the total viruses). Similarly, generally or specifically, before starting a FMT program intended to treat UC, an effective amount of an antibacterial agent capable of inhibiting the growth or proliferation of the bacteria shown in Table 10a can be administered to the patient, so that the level of such harmful bacteria in the patient's digestive tract and feces is significantly reduced (e.g., each is no more than 0.01%, 0.02%, 0.05% or 0.1% of the total bacteria).

[0085] To this end, the patient's level of harmful bacteria or fungi or viruses will be determined at least twice before their FMT procedure: once during the initial screening phase, and a second time after the initial level is deemed too high for a beneficial FMT outcome and after the patient has been given an antibacterial agent. Once the level of harmful bacteria or fungi or viruses is confirmed to be reduced to a level or percentage that will allow for a satisfactory FMT outcome, the patient is considered ready for FMT treatment.

[0086] Secondly, for a candidate who has been deemed unsuitable for use as an FMT donor due to low levels of one or more of the beneficial bacteria or fungi or viruses shown in Tables 1a, 2, 3a, 4, 5, 6a, 7a, 8b, 10b, 11, 15 or 16 in their feces, they may be disqualified as a donor in favor of another individual whose fecal sample shows a more favorable bacterial, fungal or viral profile. In this alternative, their fecal material may still be used, and the expected unsatisfactory FMT results can be remedied by supplementing the donor fecal material with an effective amount of beneficial bacteria or fungi or viruses. For example, one or more of the bacteria shown in Table 1a can be introduced into donor fecal material from an external source prior to it being processed for FMT to treat obesity or metabolic syndrome or type II diabetes so as to increase the level of bacteria in the fecal material (e.g., to at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8% or 2% of the total bacteria in the fecal material). Similarly, one or more fungi shown in Tables 2 or 3a can be introduced into donor fecal material from an external source prior to it being processed for FMT to treat obesity or metabolic syndrome or type II diabetes so as to increase the fungal levels in the fecal material (e.g., to at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the total fungi in the fecal material).

[0087] In the case of eradication of multidrug-resistant bacteria such as CRE or VRE, one or more bacteria shown in Table 4 or 15 can be introduced from an external source into the donor fecal material before it is processed for FMT so as to increase the bacterial level in the fecal material (e.g., to at least 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, or 3%). Similarly, one or more fungi shown in Table 5 or 16 can be introduced from an external source into the donor fecal material before it is processed for FMT so as to increase the fungal level in the fecal material (e.g., to at least 0.01%, 0.02%, 0.05%, or 0.1%) of the total fungi in the fecal material.

[0088] In the case of treating aGvHD, one or more bacteria shown in Table 6a can be introduced into the donor fecal material from an external source prior to it being processed for FMT so as to increase the level of bacteria in the fecal material (e.g., to at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% of the total bacteria in the fecal material). Similarly, one or more of the fungi shown in Table 7a can be introduced into the donor fecal material from an external source prior to it being processed for FMT so as to increase the level of fungi in the fecal material (e.g., to at least 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, or 15% of the total fungi in the fecal material).

[0089] In the case of treating ulcerative colitis (UC), one or more bacteria shown in Table 10b can be introduced into the donor fecal material from an external source before it is processed for FMT to increase the bacterial level in the fecal material (e.g., to at least 0.1%, 0.2%, or 0.5% of the total bacteria in the fecal material). Similarly, one or more viruses shown in Table 8b can be introduced into the donor fecal material from an external source before it is processed for FMT to increase the viral level in the fecal material (e.g., to at least 0.1%, 0.2%, or 0.5% of the total viruses in the fecal material).

[0090] In the case of treating Crohn's disease (CD), one or more bacteria shown in Table 11 can be introduced into the donor fecal material from an external source prior to it being processed for FMT in order to increase the bacterial level in the fecal material (e.g., to at least 0.1%, 0.2%, or 0.5% of the total bacteria in the fecal material).

[0091] Conversely, for a candidate who has been deemed unsuitable for use as an FMT donor due to elevated levels of one or more of the harmful bacteria or fungi or viruses shown in Tables 1b, 3b, 6b, 7b, 8a or 10a in their feces, they may be disqualified as a donor in favor of another individual whose fecal sample exhibits more favorable bacterial characteristics. In this alternative, their fecal material may still be used, and the expected unsatisfactory FMT results may be remedied by treating the candidate donor or their fecal material with an effective amount of an antibacterial or antifungal or antiviral agent capable of inhibiting the growth or proliferation of such harmful bacteria or fungi or viruses, respectively, which may be administered in substantially the same manner as described above and herein to inhibit or clear the recipient's GI tract of unwanted bacteria, fungi or viruses. Optionally, the stool sample collected from the donor after treatment can be further improved by artificially adding one or more beneficial bacteria / fungi / viruses of those shown in Tables 1a, 2, 3a, 4, 5, 6a, 7a, 8b, 10b, 11, 15 or 16 to a large amount (e.g., each greater than 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14% or 15% of the total bacteria, fungi or viruses, respectively). Because the donor's body, especially the gastrointestinal tract, contains a large collection of microorganisms, many of which are important for the health of the intestinal microbiota and the success of FMT, useful antibacterial / antifungal / antiviral agents for this purpose are preferably not broad-spectrum agents that kill all bacteria, fungi or viruses, respectively. Instead, they can be agents that narrowly and precisely target unwanted bacteria, fungi or viruses without significantly affecting other bacteria, fungi or viruses, including those closely related. Although the agent can have any compound properties, small polynucleotides (e.g., siRNA, miRNA, miniRNA, lncRNA or antisense DNA / RNA) may be most effective in achieving the specific task of disrupting the expression of one or more key genes in the life cycle of the targeted bacteria, fungi or viruses so as to specifically inhibit the proliferation of only the target species without significantly affecting other closely related species.

[0092] Soon after the completion of the FMT procedure, the recipient can be further monitored based on daily continuous testing of fecal samples for levels of beneficial and harmful bacteria, fungi or viruses (until 5 days after the FMT), while also monitoring clinical symptoms of the condition being treated and the expected health benefits to assess the FMT results and the corresponding microbial levels in the recipient: in the case of treatment of obesity / patient weight manipulation, the levels of bacteria shown in Tables 1a and 1b and the levels of fungi shown in Tables 2, 3a and 3b can be monitored in conjunction with the observation of health benefits achieved such as weight loss, improved insulin sensitivity and lowered blood cholesterol; in the case of treatment of conditions caused by multidrug-resistant bacteria such as CRE or VRE and eradication of such harmful bacteria, the levels of bacteria shown in Tables 2, 3a and 3b can be monitored in conjunction with the observation of health benefits achieved such as reduction in related symptoms and reduction in CRE or VRE. In the case of treating aGvHD, the levels of the bacteria shown in Tables 6a and 6b and the levels of the fungi shown in Tables 7a and 7b may be monitored in conjunction with the observation of a health benefit achieved, such as a reduction in aGvHD-related symptoms; in the case of treating ulcerative colitis (UC), the levels of the bacteria shown in Tables 10a and 10b and the levels of the viruses shown in Tables 8a and 8b may be monitored in conjunction with the observation of a health benefit achieved, such as a reduction in UC-related symptoms; and in the case of treating Crohn's disease (CD), the levels of the bacteria shown in Table 11 may be monitored in conjunction with the observation of a health benefit achieved, such as a reduction in CD-related symptoms.

[0093] IV. Kits and Compositions for Improved FMT

[0094] The present invention also provides novel kits and compositions that can be used to improve the therapeutic efficacy and health benefits delivered by various therapeutic and / or preventive treatment regimens involving FMT. For example, in a kit for treating a patient in need of FMT (e.g., for obesity / weight control, inhibition or eradication of multidrug-resistant bacteria such as CRE or VRE, alleviation of acute graft-versus-host disease, and alleviation of IBD such as UC or CD), a first composition is intended for transplantation into a patient or FMT recipient, and a second composition is used to: (1) increase the level of one or more beneficial bacteria (those shown in Tables 1a, 4, 6a, 10b, 11 or 15), fungi (those shown in Tables 2, 3a, 5, 7a or 16), or viruses shown in Table 8b; or (2) reduce the level of one or more harmful bacteria (those shown in Tables 1b, 6b or 10a), fungi (those shown in Tables 3b or 7b), or viruses shown in Table 8a - the composition may be intended to be added to the first composition, or it may be intended to be administered to a recipient, e.g., placed directly in the GI tract. The first composition comprises fecal material from a donor that has been processed, formulated and packaged into a suitable form according to the mode of delivery in the FMT procedure, which may be by direct placement in the lower gastrointestinal tract of the recipient (e.g., in a wet or semi-moist form) or by oral administration (e.g., freeze-dried packaged). In some cases, the second composition may comprise a sufficient or effective amount of one or more beneficial bacteria (those shown in Tables 1a, 4, 6a, 10b, 11 or 15), fungi (those shown in Tables 2, 3a, 5, 7a or 16), or viruses shown in Table 8b so that they can be added to the first composition prior to FMT in order to optimize the prospects of achieving therapeutic efficacy and / or conferring a health benefit to the recipient. In other cases, the second composition comprises an antibacterial or antifungal or antiviral agent capable of inhibiting the growth / proliferation of one or more harmful bacteria (those shown in Table 1b, 6b or 10a), fungi (those shown in Table 3b or 7b), or viruses shown in Table 8a, which may be a broad-spectrum agent that kills bacteria, fungi or viruses, or a specific inhibitor of harmful bacteria or fungi or viruses, and one or more pharmaceutically acceptable excipients, so that the FMT recipient composition can be administered shortly before the procedure, simultaneously during the procedure, or immediately after the procedure. The composition is formulated for the intended method of delivering the antibacterial, antifungal or antiviral agent, for example, by injection (intravenous, intraperitoneal, intramuscular or subcutaneous injection) or by oral administration or by local placement (e.g., suppository). The first and second compositions are typically stored separately in two different containers of the kit. In some cases, there are compositions for increasing beneficial bacteria / fungi / viruses and for inhibiting harmful bacteria / fungi / viruses, and they are provided in separate containers as the second and third components of the kit.Typically, the kit will also include printed material that provides detailed instructions to the user of the kit, such as information providing schedules and dosage arrangements for administering the first and second (and optionally third) compositions to a recipient.

[0095] In another aspect of the invention, an alternative composition for FMT with increased efficacy can be designed that contains at least these two components: (1) donor fecal material containing live fecal microorganisms, and (2) an antibacterial agent that specifically inhibits the growth or proliferation of one or more harmful bacteria (e.g., those shown in Table 1b, 6b or 10a), but does not exhibit such a suppressive or inhibitory effect on other bacteria, particularly those shown in Table 1a, 4, 6a, 10b, 11 or 15; or an antifungal agent that specifically inhibits the growth or proliferation of one or more harmful fungi (e.g., those shown in Table 3b or 7b), but does not exhibit such a suppressive or inhibitory effect on other fungi, particularly those shown in Table 2, 3a, 5, 7a or 16; or an antiviral agent that specifically inhibits the growth or proliferation of one or more harmful viruses (e.g., those shown in Table 8a), but does not exhibit such a suppressive or inhibitory effect on other bacteria, particularly those shown in Table 8b.

[0096] Component (2) is preferably not a broad-spectrum bactericidal / fungal / viral agent; instead, it should be an antibacterial / fungal / viral agent that specifically targets harmful bacteria / fungi / viruses (e.g., those shown in Tables 1b, 3b, 6b, 7b, 8a or 10a). For example, it can have a short polynucleotide nature, such as a small inhibitory RNA, microRNA, small RNA, lncRNA or antisense oligonucleotide, which can destroy the expression of at least one key gene in the life cycle of the targeted harmful bacteria / fungi / virus, so that the agent can specifically target only the bacteria / fungi / virus without significantly affecting other closely related bacteria / fungi / viruses. Component (2) is particularly useful in situations where the feces of the donor contains too high a level of one or more harmful bacteria / fungi / viruses to allow a satisfactory FMT result, because it can locally and specifically inhibit the proliferation of such unwanted bacteria / fungi / viruses to ensure the success of the FMT, even if the initial quality of the donor fecal material is not satisfactory. Example

[0097] The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that a variety of noncritical parameters can be changed or modified to produce essentially the same or similar results.

[0098] introduction

[0099] The purpose of the present invention is to determine whether and how donor and recipient fecal bacteria, virus groups and fungi groups affect the effectiveness of FMT in various human conditions. These conditions include, but are not limited to: obesity or malnutrition, metabolic diseases, type 2 diabetes, inflammatory bowel disease, irritable bowel syndrome, antibiotic-resistant infection and graft-versus-host disease. The practical use of the present invention includes optimized donor selection, FMT implementation before and during FMT implementation of donor / patient fecal bacteria, virus and fungi characteristic analysis in recipient and donor, implementation before FMT according to specific bacteria, virus group and fungi level / abundance (including and reaching species level) to patient classification, and subsequently before FMT treatment, these recipients are subjected to continuous antifungal or antibacterial treatment. Determined "favorable" bacteria, virus group and fungi standard set have been provided for establishing donor feces, donor feces bank and derived products thereof, thereby optimizing diagnosis and treatment.

[0100] Example 1 Obesity and Weight Manipulation

[0101] A. Experimental Models of Obesity and Weight Manipulation

[0102] background

[0103] Worldwide, the prevalence of obesity is increasing at an alarming rate. In China, it is estimated that more than 100 million people suffer from diabetes. Similarly, the prevalence of overweight and obese individuals in China has increased fourfold from 3.7% to 19.0% over the past two decades. Currently, non-drug interventions based on diet and physical exercise have limited long-term success in producing sustained weight loss. Alternative approaches such as weight loss medications or bariatric surgery are limited by the incomplete resolution of the disease, side effects, high costs, and morbidity associated with surgery. Existing therapies are insufficient to reduce the morbidity and mortality of obesity.

[0104] Recently, increasing evidence has shown that obesity is associated with changes in the composition of the gut microbiota and that the obese microbiome is more efficient in obtaining energy from the diet. In addition, there are differences in the bacterial composition between lean and obese animals. Therefore, changing the composition of the gut microbiota has become a new therapeutic approach to reduce weight and improve insulin sensitivity. In murine and human studies, Bacteroidetes and Firmicutes taxa dominated the microbiota (92.6%), but obese individuals had a lower proportion of Bacteroidetes and higher levels of Firmicutes compared to lean controls. Colonization of germ-free mice with "obese microbiota" resulted in a significantly greater increase in total body fat than colonization with "lean microbiota", suggesting that the gut microbiota may serve as another contributor to the pathophysiology of obesity. Obesity and lean phenotypes can also be induced in germ-free mice by transferring fecal microbiota from human donors. Finally, animal studies suggest a causal relationship, as adverse phenotypes can be transferred via fecal transplants. These data have led to the use of microbiota therapy as a potential treatment for metabolic syndrome and obesity.

[0105] Fecal microbiota transplantation (FMT) can restore intestinal microbial ecology and has been shown to be a breakthrough in the treatment of recurrent Clostridium difficile infection. In addition, clinical trials are being conducted to evaluate its use in other conditions, including inflammatory bowel disease, irritable bowel syndrome, diabetes, non-alcoholic fatty liver disease and hepatic encephalopathy. Early results in humans show that when FMT from thin donors is transplanted into subjects with metabolic syndrome, a significant increase in insulin sensitivity and an increase in intestinal microbial diversity, including a significant increase in butyrate-producing bacterial strains, is caused. The therapy is generally well tolerated and appears to be safe. Whether FMT can effectively treat obesity remains to be determined. In addition, FMT from donors to malnourished or underweight individuals can manipulate microbiota, leading to prevention or reversal of weight loss. The efficacy of FMT in obesity and / or malnutrition varies according to the selection of optimal / favorable donors based on their microbiota characteristics (including but not limited to bacteria, virome or fungi). Compatibility between donor and recipient microbial strains may also be the reason for successful transplant results.

[0106] method

[0107] Human subjects

[0108] Fecal samples were obtained from 13 healthy donors (3 females, 7 males and 3 participants without reported gender, aged 21-67 years, body mass index BMI 18.3-23.0). The 13 healthy donors included healthy Chinese individuals. These donors were D4, D8, D9, which were FMT donors for human clinical trials; and donors 1-9 and donor 19, which were randomly selected.

[0109] Animal Models and Human-to-Mouse FMT

[0110] Age and sex-matched C57 mice were fed a high-fat diet for 4 weeks to establish an obesity model. If their body weight exceeds 20% of the control, it is considered that the obesity model is successfully established. Then twice a week, the obese mice were gavaged with feces or PBS (control) from 13 healthy human donors. Its diet was changed from a high-fat diet to a normal diet. Body weight was measured twice a week. After the 2nd FMT, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were carried out. Plasma glucose levels and lipid profiles were measured at the end of the study, including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C).

[0111] Fecal DNA isolation and metagenomic sequencing

[0112] Using DNeasy blood and tissue kit (QIAGEN), extract the fecal sample DNA collected from 13 donors according to the manufacturer's scheme, and further purify.Metagenomic sequencing was performed by first constructing a paired end library with an insert size of 350bp according to the manufacturer's instructions (illumina), and sequencing on the NovaSeq Illumina sequencer.Using MetaPhlan2, the community composition was calculated using default settings.In R package vegan (R package vegan), bacterial taxonomy summary, microbial diversity rarefaction analysis, composition differences (difference values ​​represented by unweighted UniFrac distance) were calculated.

[0113] It was found that when all donors were analyzed together, the percentage of body weight loss in the FMT group was not significantly higher than that in the PBS control group. Those

[0114] The body weight of mice 3 days after the second FMT / PBS gavage was compared with the body weight of mice before the first FMT / PBS gavage. Mice were divided into FMT group (n=39) and PBS control group (n=6). Compared with baseline, the median percentage weight loss in the FMT group was 13.17% (IQR: 6.08%-18.89%), while in the PBS control group it was 11.36% (IQR: 8.43%-15.61%). Observe the trend of higher percentage weight loss by FMT compared to control, but not significant.

[0115] Weight loss effects through FMT vary depending on the donor

[0116] In order to analyze the influence of donors, mice were grouped according to the donors. 13 groups of mice received FMT from different donors (each group included 3 mice), and 1 control group received PBS (6 mice). 11 groups achieved a higher percentage of weight loss compared to the control. The mice receiving feces from donor M9 achieved the highest amount of weight loss [average percentage weight loss 19%; standard deviation (SD) 4.9%]. Although FMT induced weight loss overall, the effect was different according to the donor. Therefore, it is necessary to select the best donor.

[0117] Human-to-mouse FMT is associated with improved insulin sensitivity in obese mice

[0118] A total of 60 mice were included in the insulin tolerance test (ITT), 54 received FMT, and 6 received PBS. Compared with the PBS control group, the mice in the FMT group showed a significantly reduced ITT curve area (AUC) (P < 0.05), which indicates an increased ability to remove glucose from the bloodstream. These data show that FMT improves insulin sensitivity in obese mice.

[0119] Blood serum total cholesterol in obese mice receiving FMT from human feces compared to those receiving PBS The levels of TC and LDL were significantly reduced.

[0120] Blood serum TC and LDL levels were measured in a total of 33 mice (10 FMT groups of 3 mice each, each receiving FMT from 10 different donors, and 1 control group of 3 mice, receiving PBS) after the second FMT / PBS gavage. All FMT groups showed significantly reduced TC and LDL levels compared to the PBS control group, regardless of the donor (p<0.001).

[0121] Bacterial profiling of human donor feces shows great variation in bacterial composition and abundance

[0122] Comparison among the 13 donors revealed large differences in bacterial alpha diversity (Shannon diversity index and Simpson diversity index) as well as the relative abundance of bacteria at the phylum level.

[0123] "Good" and "bad" bacteria for weight loss

[0124] Weight loss in obese mice shows significant correlation with 22 bacterial species

[0125] The relative abundance of specific bacteria was calculated from the metagenomic sequencing results. In order to identify specific bacteria associated with the donor effect of FMT on weight loss, the relative abundance of specific bacteria observed in each group of mice (n=3) receiving the second FMT from 13 different donors was associated with the average percentage weight loss. 5 bacteria were found to have a significant positive correlation with the average percentage weight loss, and 17 bacteria were found to have a significant negative correlation with the average percentage weight loss.

[0126] Bacterial criteria for selecting "favorable" FMT donors for inducing weight loss

[0127] In an alternative analysis, 13 donors were classified as "favorable" donors and "unfavorable" donors. "Favourable donors" were defined as donors that could induce more weight loss than the mean (13.17%) or median (13.67%) percentage weight loss between the 13 mouse groups, and the remaining were defined as "unfavorable donors". Linear discriminant analysis (LDA) effect size (LEfSe) analysis was used to analyze the relative abundance of bacteria between the two groups (linear discriminant analysis effect size>2, adjusted p value<0.05 was considered significant).

[0128] The inventors identified 13 bacterial taxa that were over-represented in "favorable" donors (favorable bacteria for weight loss) and 7 bacterial taxa that were over-represented in "unfavorable" donors (unfavorable bacteria for weight loss).

[0129] A summary of the above identified "favorable" and "unfavorable" bacteria for weight loss and their relative abundance is shown in Tables 1a and 1b.

[0130] B. Human Studies of FMT for Obesity and Weight Manipulation

[0131] method

[0132] Cross-sectional studies in humans

[0133] Fecal samples from 23 obese subjects and 15 controls were collected to compare their microbial profiles. Obese subjects were defined as having a BMI ≥ 28 kg / m 2 , while controls were defined as those with a BMI ≥ 18.5 kg / m 2 and <23kg / m 2 .

[0134] Randomized controlled trials of fecal microbiota transplantation (FMT)

[0135] Among the obese subjects recruited in the cross-sectional study, 12 subjects who also had type 2 diabetes were also recruited. The clinical trial title was "A randomized placebo-controlled study of fecal microbiota transplant (FMT) to impact body weight and glycemic control in obese subjects with type 2 diabetes mellitus" (reference number on ClinicalTrials.gov: NCT03127696). In this study, subjects were randomly divided into 3 arms: 1) FMT together with a lifestyle modification program (LMP), 2) FMT alone, and 3) a sham trial together with LMP. The primary outcome was defined as a 5% weight loss compared to baseline, which was the weight at randomization before receiving any intervention. Stool samples were collected at baseline. Body weight was measured at weeks 0, 4, 8, 12, 16, 20, 24, and 52.

[0136] FMT Procedure

[0137] Volunteers from the general population, spouses or partners, first-degree relatives, other relatives, friends, and other people known or unknown to the subject were recruited as donors to provide stool for FMT. To ensure that the donors were healthy and suitable for stool donation, the donors were first screened using a questionnaire, followed by stool and blood tests to rule out any infection. In addition, only those with a BMI < 23 kg / m 2 of lean donors. Shortly after the donors donated stool at the Prince of Wales Hospital, the stool was diluted with sterile saline (0.9%). The solution was mixed and filtered through a filter. The resulting supernatant was then stored as a frozen FMT solution for future FMTs. For subjects randomized to receive an FMT, FMT solution from a single donor or mixed stool from multiple donors was used. For subjects randomized to receive a sham test, normal saline was infused. During the procedure, 100-200 ml of FMT solution or sterile saline was injected into the distal duodenum or jejunum over 2-3 minutes via OGD.

[0138] Lifestyle Modification Program (LMP)

[0139] The subjects randomized to LMP were seen by a dietitian at weeks 0, 1, 2, 4, 6, 8, and 12. The dietitian provided guidance and correction to the subjects in their eating habits, physical activity patterns, and other lifestyle habits. The subjects also recorded their dietary history for 7 days before each consultation with the dietitian.

[0140] Fecal DNA extraction and metagenomic sequencing

[0141] use Fecal DNA was isolated using the RSC PureFood GMO and Authentication kit according to the protocol. DNA libraries were constructed through a process of end pairing, purification, and PCR amplification. After DNA library construction, the DNA library was sequenced by Novogene in Beijing, China using an Illumina Novaseq 6000 using a paired-end 150 bp sequencing strategy.

[0142] Read quality control and preprocessing

[0143] Raw sequence reads were quality screened by Fastp and adapters were removed. Human host contamination reads were removed by Kneaddata using default parameters (reference database: GRCh38 p12). For metagenomic data tables, clean reads were processed in Kraken2 by inclusion in the NCBI fungal Refseq database (downloaded on September 20, 2018). Bracken was used to estimate species relative abundance tables for downstream analysis.

[0144] Statistical analysis

[0145] The linear discriminant analysis effect size (LEfSe) model was used to identify species with different relative abundances between groups. Only classifications that met an LDA threshold of >2 were considered significant. For species identified by the LEfSe model, alternative analyses were performed to verify differences in relative abundance between groups using the Wilcoxon rank sum test. P values ​​< 0.05 were considered statistically significant.

[0146] Discover

[0147] The relative abundance of Cercospora betae and Saccharomyces najakazakhstan in controls was significantly higher than that in obese subjects. Those ones

[0148] In a cross-sectional study, fecal fungal communities were compared between 23 obese subjects and 15 controls [median age of obese subjects was 52 years (IQR: 47–60 years) and controls was 57 years (IQR: 54–60 years); 60% of obese subjects were male and 69.6% of controls were female]. By LEfSe analysis, the relative abundance of two fungi, Cercospora betae and Saccharomyces naja, was found to be significantly higher in controls than those in obese subjects. The median abundance was 0.045 (IQR: 0.038-0.050) in obese subjects and 0.056 (IQR: 0.045-0.063) in controls (p=0.016). The median abundance in obese subjects was 0.004 (IQR: 0.003-0.006) and in controls was 0.006 (IQR: 0.004-0.007) (p=0.044). These two fungi are beneficial for weight loss (Table 2). Subjects who achieved weight loss after FMT or LMP and those who did not The relative abundance of four fungi was significantly different between subjects who lost weight

[0149] Twelve subjects from the randomized controlled trial who reached 16 weeks of follow-up were included in the analysis. To identify fungi that could predict weight loss, subjects were divided into "weight loss" and "non-weight loss" groups, and their baseline stool fungal profiles were compared. Subjects were assigned to the "weight loss" group if their weight loss at 16 weeks was greater than 5% from baseline, and to the "non-weight loss" group if their weight loss at 16 weeks was less than 5% from baseline. Three subjects were assigned to the "weight loss" group (median percentage weight loss of 5.3%), and nine subjects were assigned to the "non-weight loss" group (median percentage weight loss of 0.2%).

[0150] By LEfSe analysis, it was found that the "weight loss" group had a significantly higher level of 1 fungus, Komagataella phaffii, than the "non-weight loss" group. The higher relative abundance of this species in baseline fecal samples was able to predict successful subsequent weight loss by FMT or LMP intervention. Supplementation of this "favorable species" can promote weight loss (Table 3a).

[0151] On the other hand, LEFSe analysis also showed that the relative abundance of three fungi in the "non-weight loss" group was significantly higher than that in the "weight loss" group. These fungi were Torulaspora delbrueckii, Encephalitozoon romaleae, and Kluyveromyces lactis. The higher relative abundance of these "unfavorable" species in baseline fecal samples was able to predict resistance to weight loss by FMT or LMP (Table 3b).

[0152] Example 2 Multidrug-resistant organisms (Carbapenem-resistant Enterobacteriaceae (CRE) and Vancomycin-resistant Enterococci (VRE))

[0153] FMT in CRE: bacterial and fungal determinants of disease and FMT outcomes

[0154] background

[0155] Multidrug-resistant organisms (MDROs) pose an increasing public health threat to the global population. The incidence of various MDROs, including carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE), has been increasing worldwide and has become epidemic in some countries or regions. For example, in countries or regions adjacent to Hong Kong, China, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae are prevalent and widespread in China, while IMP-producing Enterobacteriaceae are widespread in Taiwan Province of China and Japan. Data from the Hospital Authority showed that the number of carbapenemase-producing Enterobacteriaceae (CPE) cases increased from 36 in 2012 to 134 in 2015. Recently, a large outbreak of VRE involving >200 patients was reported in a tertiary hospital in Hong Kong, China. In some countries or regions, the spread of MDROs is not limited to the hospital setting but is also prevalent in the population.

[0156] The primary site for most MDRO colonization and persistence is in the gastrointestinal tract. Carriage can persist for months, with up to 40% of individuals still colonized one year after discharge. MDRO outbreaks have been reported in hospitals and long-term care facilities. Approximately 10% of patients colonized with MDROs will develop a clinical infection caused by the same organism. These MDRO-caused infections are associated with significant morbidity and mortality rates of up to 50%, in part due to limited options for safe and effective antimicrobial therapy. Currently, control of MDRO transmission is primarily through infection control measures, and there is no proven therapy to eradicate MDRO intestinal colonization.

[0157] There is increasing evidence that the gut microbiota plays an important role in controlling enteric pathogen colonization and infection. The composition and diversity of the gut microbiota in MDRO carriers differ from those in non-carriers. Intestinal commensal bacteria help regulate innate immunity of the mucosa, for example by inducing the production of antimicrobial proteins by epithelial cells. Flagellin has been shown to induce the production of RegIIIγ, an antimicrobial protein, and reduce VRE colonization in mice. Administration of obligate anaerobic commensal bacteria to mice has also been shown to significantly reduce VRE colonization. Preliminary evidence, mainly from anecdotal reports, suggests that fecal microbiota transplantation (FMT) in MDRO human carriers is safe and potentially effective in clearing intestinal colonization of various MDROs, including CRE and VRE, even in immunocompromised patients.

[0158] Using metagenomic sequencing, the microbial profiles of healthy controls and FMT recipients were evaluated to identify microorganisms associated with CRE colonization. The microbial profiles of FMT donors and those of recipients before and after FMT were also compared to identify species associated with CRE clearance. These species play an important role in donor or microbial product selection to improve FMT efficacy and as predictive markers for successful CRE clearance after FMT.

[0159] method

[0160] Human clinical trials of FMT for eradication of CRE and VRE

[0161] Patients aged ≥18 years, with two or more stool or rectal swabs positive for CRE or VRE one week apart, and who had not received antimicrobial therapy for at least 48 hours before FMT infusion were enrolled in the clinical trial. Patients with active CRE or VRE infection requiring antimicrobial therapy, pregnancy, active gastrointestinal infection or inflammatory conditions, recent intra-abdominal surgery, short bowel syndrome, or medications that alter gastrointestinal motility were excluded. CRE was defined as the presence of any Enterobacteriaceae resistant to any carbapenem. VRE was defined as the presence of enterococci resistant to vancomycin. In this study, patients received 2 FMTs using frozen donor stool samples. Within 2-3 minutes, 100-200 ml of FMT solution or sterile saline was injected into the distal duodenum or jejunum via OGD. Stool samples were collected from patients in advance before and after FMT.

[0162] Human subjects without CRE or VRE

[0163] Fecal samples from 2 subjects without CRE or VRE were included as controls.

[0164] Fecal DNA extraction and metagenomic sequencing

[0165] DNA libraries were constructed by a process of end pairing, purification, and PCR amplification. Sequencing libraries were prepared using the NexteraXT DNA library preparation kit (Illumina, California, USA) according to the manufacturer's instructions. Sequencing was performed on a NovoSeq PE150 (Novogene, Tianjin, China; standard 2×150 bp run), generating 12G of raw data per sample.

[0166] Read quality control and preprocessing

[0167] Raw sequence reads were quality screened by Fastp and adapters were removed. Human host contamination reads were removed by Kneaddata using default parameters (reference database: GRCh38 p12). For metagenomic data tables, clean reads were processed in Kraken2 by subclassification into the NCBI fungal Refseq database (downloaded on September 20, 2018). Species relative abundance tables were estimated using Bracken for downstream analysis.

[0168] Statistical analysis

[0169] A linear discriminant analysis effect size (LEfSe) model was used to identify species with different relative abundances between groups. Only classifications that met a linear discriminant analysis (LDA) threshold of >2 were considered significant.

[0170] Discover

[0171] Fecal microbiota transplantation for eradication of intestinal colonization with carbapenem-resistant Enterobacteriaceae

[0172] Two patients recruited into the clinical trial successfully cleared CRE after FMT. Recipient 1 (female, age 90 years) underwent 2 FMTs 5 days apart, on Day 0 and Day 5. CRE tested negative on Day 11 and remained negative until Week 5 after the first FMT. She then developed a foot ulcer infection and received antibiotic treatment from Week 6 to Week 19 after the first FMT. Recipient 1 tested positive for CRE at Weeks 14 and 19. At Week 22, it tested negative again after completing 4 courses of Augmentin. Recipient 2 (male, age 70 years) received two FMTs on consecutive days, and all results of CRE follow-up tests at Weeks 1, 3, and 6 were negative.

[0173] Fecal samples were collected before and after FMT from recipient 1 and recipient 2. Both recipients received FMT from the same donor.

[0174] "Favorable" and "unfavorable" bacteria in selecting FMT donors for the treatment of CRE

[0175] Bacterial profiles of patients with CRE differ significantly from those of healthy controls

[0176] In a cross-sectional study, fecal bacterial profiles were compared between the CRE group, which consisted of fecal samples from recipient 1 (R1) and recipient 2 (R2) before FMT, and the healthy control group, which consisted of fecal samples from FMT donors and 2 other healthy controls recruited to the Hong Kong Health Gut Microbiota Survey, China.

[0177] The relative abundance of 40 species identified by metagenomic sequencing was significantly different (LDA > 2) between the CRE and control groups using LEfSe analysis, representing bacteria that are “favorable” and “unfavorable” for the elimination of CRE.

[0178] Bacterial characteristics in donors and CRE recipients before and after FMT

[0179] In a prospective pilot trial, the bacterial α diversity in the two recipients before and after FMT was consistently lower in the subjects compared to the donors. However, the bacterial α diversity of R1 showed an increase on the 18th day after FMT, and the results in R2 showed that the α diversity levels on the 3rd and 8th days were greater than on the 14th day. The relative abundance of single bacteria before and after FMT was compared (in receptor 1, on the 7th day vs the 18th day; in receptor 2, on the 3rd day vs the 18th day). For receptor 2, there were 2 stool sample collection time points before FMT. For receptor 2, the 3rd day was selected because it was a time point closer to FMT. The 18th day was selected as the time point after FMT for the two recipients because the bacterial characteristics began to stabilize at that time. The inventors identified bacteria with an increased multiple change of>1.0 in receptor 1 or receptor 2. These species appear at very low levels or are not even detected in stool samples before FMT. Transplanting these species from donors may be related to the removal of CRE. Lachnospiraceae_bacterium_5_1_63FAA showed the largest increase after FMT in R1 and the second largest increase in R2, with fold changes of 1659.5 and 16.9, respectively.

[0180] A summary of the above identified “favorable” bacteria for CRE clearance and their relative abundance is shown in Table 4.

[0181] Fecal fungal characteristics in donors and CRE recipients before and after FMT

[0182] In the prospective pilot trial, changes in fungal alpha diversity after FMT in R1 and R2 showed opposite trends, indicating that the effect of FMT on fungal diversity varied among patients. The relative abundance of individual fungal species before and after FMT was analyzed.

[0183] Although short-term donor engraftment with fungal FMT was not as pronounced as with bacteria, some fungi showed clear changes in relative abundance. The fungal communities of recipients after FMT were more structurally similar to the donor characteristics than before FMT. Fungi with a fold change increase >1.0 in either Recipient 1 or Recipient 2 are listed below. In particular, Encephalitozoon hellem showed the highest fold change in both recipients, increasing 9.31-fold in R1 and 81-fold in R2, respectively. Engraftment and increase of these fungi after FMT may be determinants of successful CRE decolonization (Table 5).

[0184] Example 3 Acute Graft Versus Host Disease (AGVHD)

[0185] FMT in aGvHD: bacterial and fungal determinants of disease and FMT outcomes

[0186] background

[0187] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most promising tumor immunotherapies for hematological disorders. Acute graft-versus-host disease (aGvHD) is a serious complication of allo-HSCT, which occurs in 35%-45% of recipients and is associated with a mortality rate of 15%-25%. In particular, involvement of the lower gastrointestinal (GI) tract is associated with a high mortality rate. Glucocorticoids are used as first-line therapy for aGVHD, but only about half of the patients respond, and second-line treatment has not yet been established. The one-year survival rate of aGvHD is less than 30%. So far, there has been no satisfactory improvement in patient survival for refractory GvHD.

[0188] There is increasing evidence that changes in the composition of intestinal microbiota are closely related to the occurrence of GvHD. Multiple factors contribute to the reduction of microbiota diversity in patients with allo-HSCT, including repeated use of antibiotics, chemotherapy and / or radiotherapy, and changes in nutritional patterns. Allogeneic bone marrow transplantation (BMT) leads to the loss of overall diversity and the amplification of lactobacilli and the loss of clostridium, which increases the risk of subsequent GVHD. In addition, after allo-HSCT, enterococci were observed in recipients and were associated with the severity of GI-aGVHD. It has also been reported that the altered viruses are associated with intestinal GVHD. For example, the unexpected association of picobirnavirus with early post-transplant GVHD5 has been observed.

[0189] There is increasing evidence that an altered intestinal microbiota is associated with acute graft-versus-host disease (aGvHD) in stem cell transplantation (SCT). Fecal microbiota transplantation (FMT) represents a promising treatment for aGVHD. Recently, several small case series have pointed to the potential of FMT as a treatment for refractory aGVHD. In three consecutive patients with refractory GI-aGvHD, FMT improved gastrointestinal symptoms, reduced diarrhea, and reconstituted the intestinal microbiota. In patients after allo-HSCT, administration of FMT capsules reduced the occurrence of aGvHD and improved survival.

[0190] To date, there is limited experience with the use of FMT in HSCT recipients. Although more carefully designed clinical studies are necessary to confirm the safety and efficacy of FMT for aGvHD, this life-threatening disease requires immediate attention in some patients. Most studies have focused on the restoration of bacterial diversity, while the role of bacteriophages and fungi in FMT efficacy is less well known. In this study, we provide the first case of successful treatment of aGvHD (grade IV) in an adolescent using FMT, and use metagenomics to characterize the serial fecal bacterial, virome, and fungal profiles in recipients after FMT to investigate potential associations between altered microbiome dynamics and aGvHD treatment.

[0191] In a young boy with severe, life-threatening grade 4 intestinal aGVHD refractory to corticosteroids and biologic therapy, repeated FMTs (4 times total) restored the diversity of the microbiome and caused disease improvement. This is the first case of FMT for pediatric GI-GVHD, which highlights the restoration of bacterial, viral, and fungal diversity and their stable engraftment. Potential donor and recipient bacterial and fungal characteristics that influence favorable FMT outcomes were identified.

[0192] Case presentation

[0193] A 14-year-old male with myelodysplastic syndrome with haplotype 7 received an HLA sibling allogeneic hematopoietic stem cell transplant (allo-HSCT) on December 21, 2017. He developed stage II skin GvHD (rash 25%-50%), intestinal aGvHD (stage IV, with significant functional impairment), and overall grade IV life-threatening GvHD shortly after allo-HSCT. He had diarrhea, vomiting, and abdominal pain. He received his first FMT 77 days after allo-HSCT, followed by three more FMTs on days 5, 13, and 25 after the first FMT. The first three FMTs were from a single donor D8, and the fourth FMT was from a different single donor D4. During the course of the four FMTs, the patient also received methylprednisolone, cyclosporine A, infliximab, ruxolitinib, budesonide (Entocort), octreotide, antibiotics, antivirals, and antifungals.

[0194] The day before receiving the first FMT, the patient had a stool volume of 465 ml and 5 bowel movements. On the second day of the first FMT, the stool volume decreased to 0 and the bowel movement decreased to 1. On the 5th day after the first FMT, the stool volume increased to 293 ml, with blood and watery consistency, and the bowel movement increased to two. The patient received a second FMT from the same donor on the 7th day. On the 11th day, the stool volume decreased to 100 ml and the bowel movement decreased to 1. The stool was well-formed and the patient had only mild abdominal cramps. Thereafter until the 35th day, the stool volume was maintained at about 100 ml per day, and bowel movements were one or zero per day. Immunosuppressants were slowly stopped. Until the last follow-up on the 117th day after the first FMT, the patient remained stable.

[0195] FMT Procedure

[0196] Donor feces were obtained from the feces bank of the Center for Gut Microbiota Research at the Chinese University of Hong Kong.

[0197] method

[0198] Stool from donors and recipients

[0199] Patients were given FMTs on days 0, 5, 13, and 25. Clinical data in recipients were collected daily from day 0 to day 25 (when patients underwent 4 FMTs), and then monthly until day 117. For this analysis, 1 pre-FMT stool sample and 3, 4, 6, and 8 stool samples after the 1st, 2nd, 3rd, and 4th FMTs, respectively, were analyzed for bacterial, viral, and fungal characteristics by metagenomic sequencing.

[0200] Fecal samples from 2 FMT donors D4 and D8 for FMT were collected for metagenomic sequencing. Fecal samples from donor D8 were collected from 5 different time points within 2 weeks. These samples were labeled D8-26, D8-27, D8-28, and D8-29. Feces from donor D4 were collected from a single time point and labeled D4. All of these samples were analyzed for bacterial, viral, and fungal characteristics separately. In the 1st and 2nd FMT, feces from donor D8 collected on different days were used. The distribution of fecal samples for the 4 FMTs is listed below:

[0201] 1st FMT: D8-26 and D8-27

[0202] 2nd FMT: D8-28 and D8-29

[0203] 3rd FMT: D8-34

[0204] 4th FMT: D4

[0205] Fecal DNA extraction and metagenomic sequencing

[0206] use Fecal DNA was isolated using the RSC PureFood GMO and Authentication kit according to the protocol. DNA libraries were constructed through a process of end pairing, purification, and PCR amplification. After DNA library construction, the libraries were sequenced by Novogene in Beijing, China using an Illumina Novaseq 6000 using a paired-end 150bp sequencing strategy. Bacterial and fungal metagenomic sequencing generated 10-12G of raw data.

[0207] Read quality control and preprocessing

[0208] The original sequence was read by Fastp for quality screening and adapter removal. By Kneaddata, the human host contaminant was removed using default parameters (reference database: GRCh38 p12). For the metagenome data table, clean reads were processed in Humann2 for bacterial analysis and in Kraken2 for fungal and viral analysis. The bacterial taxonomy summary, the sparseness analysis of microbial diversity, and the composition differences (the difference values ​​represented by the unweighted UniFrac distance) were calculated in the R package vegan.

[0209] Quantitative PCR for detection of total fungal load in total human fecal DNA

[0210] The total fungal load in human feces was quantified by TaqMan qPCR analysis (Premix Ex TaqTM, TaKaRa) of human fecal DNA extracted using primer 36: fungal-quantification-F 5′-GGRAAACTCACCAGGTCCAG-3′; fungal-quantification-R 5′-GSWCTATCCCCAKCACGA-3′, and probe: 5′-TGGTGCATGGCCGTT-3′.

[0211] Statistical analysis

[0212] The linear discriminant analysis effect size (LEfSe) model was used to identify species with different relative abundances between groups. Only classifications that met an LDA threshold of >2 were considered significant. For species identified by the LEfSe model, alternative analyses were performed to verify the differences in relative abundance between groups by the Wilcoxon rank sum test. P values ​​< 0.05 were considered statistically significant.

[0213] Discover

[0214] Bacterial changes after FMT

[0215] Increased alpha diversity of fecal bacterial communities in patients with aGvHD after FMT

[0216] The Shannon and Simpson indices were used to estimate the alpha diversity of bacterial communities in recipients with aGvHD and FMT donors (D4 and D8) after FMT (after the 1st, 2nd, 3rd, and 4th FMT). After the first FMT, the alpha diversity in recipient feces increased significantly compared with that before FMT, and after the 2nd, 3rd, and 4th FMT, the alpha diversity reached a level similar to that of donors D4 and D8.

[0217] Restoration of bacterial community structure in aGvHD patients after FMT

[0218] Principal component analysis (PCA) was used to investigate the differences in community structure between fecal samples of recipients and donors before and after FMT. After repeated FMT, the fecal bacterial communities gradually approached those of the donors, indicating that the bacterial community structure of the patients was restored and became similar to those of the donors.

[0219] Seven bacterial species transferred from donors became dominant in FMT recipients

[0220] The bacterial transplantation from donors in the patients suffering from aGvHD after FMT was evaluated by bar graph.Corynebacterium jessenii (71.2%) is dominant in the intestinal bacterial community of patients, and drops to less than 0.1% after the first FMT.7 kinds of bacterial transplantations were observed in patients after receiving repeated FMT, and these species occupied a larger proportion of the bacterial community of patients.These species are: Alistipes onderdonkii, Alistipes putredinis, Clostridium baumannii, Clostridium truncatum, Clostridium symbioticum, Eggerthella unclassified, Ruminococcus gnavu.

[0221] The relative abundance of the four bacteria was significantly different when patients had diarrhea or did not have diarrhea

[0222] A total of 22 stool samples were collected from patients with aGvHD before and after FMT. In order to identify stool bacteria with different relative abundances when patients suffer from diarrhea or no diarrhea, these 22 stool samples were classified according to whether the patient suffered from or did not suffer from diarrhea on the day of stool collection. Diarrhea is defined as 2 or more bowel movements per day, and non-diarrhea is defined as 0 or 1 bowel movement per day. LEfSe analysis is used to identify bacteria with significant differences in relative abundance between stool samples from "diarrhea" or "non-diarrhea" groups. The relative abundance of Eubacterium rectum is significantly higher in the diarrhea group, while the relative abundance of Alistipes putredinis, Alistipes onderdonkii and Clostridium hasawei in the non-diarrhea group is significantly higher. "Favourable" donors should contain Alistipes putredinis, Alistipes onderdonkii and Clostridium hasawei of higher relative abundance, and Eubacterium rectum of lower relative abundance.

[0223] aGvHD symptoms significantly associated with five bacterial species

[0224] To identify specific bacteria associated with treatment outcomes, the relative abundance of specific bacteria was correlated with GvHD symptoms including bowel movement frequency, stool volume, and vomiting volume by linear regression. One bacterium was found to be significantly positively correlated with bowel movement frequency and stool volume. "Favorable" donors should have a lower relative abundance of Corynebacterium jessenii or no Corynebacterium jessenii. On the other hand, four bacteria showed a significant negative correlation with vomiting symptoms. "Favorable" donors should have a higher relative abundance of these bacteria (listed below): Alistipes putredinis; Clostridium baumannii; Clostridium hassawayii; Clostridium truncatum.

[0225] A summary of the above identified "favorable" and "unfavorable" bacteria for treating aGvHD and their relative abundance is shown in Tables 6a and 6b.

[0226] Fungal changes after FMT

[0227] Fecal fungal diversity increases in aGvHD after FMT

[0228] The Shannon and Simpson indices were used to estimate the alpha diversity of fungal communities in recipients with aGvHD and FMT donors (donors 4 and 8) after FMT. After receiving the first FMT, the alpha diversity increased significantly compared to those before FMT. After the subsequent 3 FMTs, the alpha diversity of fungi continued to increase steadily and reached a level similar to that of donors D4 and D8.

[0229] Total fungal load increases in aGvHD patients after FMT

[0230] Total fungal load is measured by qPCR.Total fungal load in patient increases to the level similar to donor after accepting FMT from donor D8, then declines again after a few days and keeps low level after accepting FMT for the second time.Level in patient increases again after accepting FMT for the third time.Total fungal load slightly reduces after the fourth FMT utilizing feces from donor D4 (it has slightly lower total fungal load compared to donor D8).

[0231] The relative abundance of 2 fungi decreased significantly after FMT, while 12 fungi transferred from donors became abundant in FMT recipients. The dominant fungus

[0232] The fungal transplantation from donors in patients with GvHD after FMT was evaluated by bar graph. Fusarium oxysporum (61.44%) and Botrytis cinereal (29.13%) dominated the intestinal fungal community of patients and decreased significantly after the first FMT. These are considered "unfavorable" fungi. After receiving repeated FMT, 12 species were transplanted into patients and occupied a large proportion of the patient's fungal community. These species are: Schizosaccharomyces pombe, Eremothecium sinecaudum, Cercospora exigua, Fusarium venenatum, Thermothelomyces thermophile, Colletotrichum higginsianu, Fusarium verticillioides, Thielavia terrestris, Candida dubliniensis, Eremothecium gossypii, Fusarium pseudograminearum and Neurospora crassa. These are considered "favorable" fungi.

[0233] aGvHD symptoms were significantly associated with 21 fungal species

[0234] To identify specific fungi associated with treatment outcomes, the relative abundance of specific fungi was correlated with GvHD symptoms including bowel movement frequency, stool volume, and vomiting volume by linear regression. Two fungi were found to show significant positive correlations with bowel movement frequency and stool volume. Based on these findings, "favorable" donors should contain lower relative abundances of these two fungi, Fusarium oxysporum and Botrytis cinerea. On the other hand, 19 fungi showed significant negative correlations with at least one symptom. "Favorable" donors should contain higher relative abundances of these fungi.

[0235] A summary of the above identified "favorable" and "unfavorable" fungi for the treatment of aGvHD and their relative abundance is shown in Table 7a and Table 7b.

[0236] Example 4 Inflammatory Bowel Disease Virus Group

[0237] Mucosal virome in healthy subjects and ulcerative colitis

[0238] background

[0239] Ulcerative colitis (UC), a subtype of inflammatory bowel disease (IBD), is a relapsing, relapsing inflammatory disease that affects the entire large intestine, typically beginning in the rectum and spreading upward. The incidence of UC continues to increase, especially in developing and newly industrialized countries. Although the etiology of IBD is unclear, IBD is thought to result from an exacerbated immune response to the intestinal microbiota in genetically susceptible individuals. Fecal virome alterations in IBD have been studied in humans and mice and are characterized by the proliferation of phages from the order Caudoviridae. However, the composition and function of the mucosal virome in health or IBD are not clear, and there is limited knowledge about mucosal bacterial alterations in UC. To begin to address these challenges, the inventors enriched virus-like particles (VLPs) from the rectum of healthy individuals and patients with UC, performed ultra-deep virome metagenomic sequencing and 16S rDNA sequencing to determine the mucosal virome, bacteriome, and virus-bacteria interactions in subjects with UC compared to healthy controls. This study is the first and largest study to characterize the mucosal virome in health and UC to date.

[0240] method

[0241] Study subjects

[0242] This study recruited 48 healthy subjects from Hong Kong, China, for 63 patients with UC, 20 healthy subjects from Beijing, China, for 20 patients with UC, and 8 healthy subjects from Xiangshan County, Zhejiang Province, China, for 8 patients with UC (all Chinese) with informed consent. Patient inclusion criteria included subjects aged ≥18 years with UC confirmed by endoscopy, radiology, and histology. Controls consisted of individuals undergoing colonoscopy for polyps or colorectal cancer screening or gastrointestinal symptom investigation, as well as friends and spouses or partners of patients in local hospitals, or any individuals interested in participating in this study. Rectal biopsy samples from study subjects were collected endoscopically and then stored at -80°C for downstream mucosal virome and bacterial analysis.

[0243] Virus-like particle (VLP) enrichment and sequencing

[0244] Virus-like particles (VLPs) were enriched from rectal biopsies of patients with UC and healthy subjects using a protocol modified from previously described methods. Qualified libraries were amplified on cBot to generate clusters on flow cells (TruSeq PE Cluster Kit V3–cBot–HS, Illumina). Paired-end sequencing of amplified flow cells was performed on the HiSeq Xten System (TruSeq SBS KIT-HS V3, Illumina) (BGI, Shenzhen, China; standard 2X 150bp runs), generating 20–60 million raw sequences per sample (5–8G raw data).

[0245] Sequence processing and quality control

[0246] Raw reads were screened by SOAPnuke (v 1.5.3) developed by BGI (website: soap.genomics.org.cn / ) as follows: (i) remove adapters, (ii) remove reads with N bases exceeding 3% of the reads, (iii) remove reads with bases with a quality lower than 20 exceeding 40% of the reads, and (iv) remove all dimers. Human sequences were removed from the quality-trimmed data set by DeconSeq (v0.4.3) using default parameters and the human reference sequence GRCh38.

[0247] De novo contig assembly and taxonomic annotation

[0248] Contigs were assembled using IDBA (v 1.1.1) with a maximum kmer length of 120, with a maximum contig length of 1,000 bp. Assembled contigs were clustered at the 95% identity level using CD-HIT

[23] to generate unique contig associations.

[0249] Virome abundance data analysis

[0250] In order to estimate the abundance of overlapping groups and calculate sequence diversity, all reads were compared with the selected overlapping groups obtained using Bowtie2 (v 2.2.9). Mapped sequence counts, overlapping group lengths and total sequence counts were used to standardize sequence counts and represent the RPKM (reads per million per kilobase) for overlapping groups for each sample. These values ​​were used to generate viral abundance tables at different classification levels.

[0251] Virome abundance data were imported into R 3.2.3. Diversity, evenness, and richness calculations were performed using phyloseq in R. Spearman correlations and their significance were calculated using the cor and cor.test functions in R, respectively. For virus-bacteria correlations in terms of alpha diversity and taxonomic abundance, Spearman correlations were calculated. Correlation plots were generated using the corrplot R package. Heatmaps were generated using the pheatmap R package.

[0252] Non-metric multidimensional scaling

[0253] Differences in mucosal viral community structure between controls and UC were performed via NMDS (non-metric multidimensional scaling) mapping based on Bray-Curtis dissimilarity between all subjects.

[0254] DESeq, random forest and LEfSe linear discriminant analysis

[0255] To compare the differences in the structure of the mucosal virome and bacterial microbiome between mucosal virome enterotypes and in virome function between UC patients and healthy family controls, differential analysis was performed. DEseq and random forests were performed in R by the DEseq and randomForest packages, respectively. Lefse analysis was performed on the Huttenhower lab Galaxy server (website: huttenhower.sph.harvard.edu / galaxy / ). DB-RDA analysis was also performed in R to describe the effects of medication and health on mucosal virome structure.

[0256] Virome functional analysis

[0257] Virome functions were annotated by HUMANN2 v0.9.4. Predicted functions were contracted by gene ontology terms and Pfam protein family identities, with abundance values ​​expressed as RPK (reads per kilobase). To establish the presence or absence of function in a sample, a strict RPK threshold of >10 was used to define presence.

[0258] Mucosal bacterial DNA extraction

[0259] Bacterial DNA was extracted from rectal biopsy samples using the Maxwell RSC Tissue DNA Kit (Promega, Madison, Wisconsin) according to the protocol.

[0260] 16S rRNA sequencing and quality control

[0261] Final fecal DNA samples were sequenced on an Illumina Hiseq 2500 platform (V4 region, 2×250 bp). Quality control and data analysis were performed in mothur (v 1.38.0) as previously described. Any sequence with unclear bases and any sequence longer than 275 bp were removed and compared against the non-redundant Greengenes database (v13.8) using the NAST algorithm.

[0262] 16S rRNA sequencing data analysis

[0263] The resulting sequences were classified against the Greengenes database and annotated at the deepest level of classification, as indicated by a pseudo-bootstrap confidence score of at least 80% averaged over 1,000 iterations of the naive Bayes classifier.

[0264] Discover

[0265] Alterations in the diversity of the mucosal virome in UC

[0266] In Hong Kong, China, the rectal viromes of 63 patients with UC were compared with those of 48 healthy subjects. An average of 56,632,558 ± 14,330,713 clean paired-end reads were obtained from enriched rectal VLP preparations. The mucosal virome composition in health and UC was investigated at the order, genus, and species levels.

[0267] Compared with healthy individuals, UC subjects had significantly higher viral abundance in the mucosa (Mann-Whitney test, p = 0.017) and reduced viral diversity, evenness, and richness (t-test, p = 0.013, 0.020, and 0.029, respectively). We next investigated changes in Caudovirales bacteriophages, the dominant mucosal viruses, and found that Caudovirales abundance was also amplified in UC (Mann-Whitney test, p = 0.003), while species diversity, evenness, and richness in Caudovirales were all decreased (t-test, p = 0.009, 0.017, and 0.018, respectively). Taken together, these findings suggest a dysregulated mucosal virome in patients with UC.

[0268] In UC patients, total viruses and Caudovirales phages were more enriched in the inflamed mucosa than in the non-inflamed mucosa (Mann-Whitney test, p=0.015 and 0.017). Comparing the differences in alpha diversity of the mucosal virome of non-inflamed mucosa of UC patients relative to those of healthy controls, it was found that the decrease in diversity, evenness, and abundance of total viruses and Caudovirales phages in the inflamed mucosa was more statistically significant relative to those of healthy controls. Therefore, it suggests that intestinal inflammation is an important contributing factor to the dysregulation of the mucosal virome in UC.

[0269] Different mucosal virome structures between healthy subjects and patients with UC

[0270] At the family level, Microviridae (single-stranded DNA phages), Mycoviridae, Podoviridae (double-stranded DNA phages from the order Caudovirales), and Penumoviridae (eukaryotic viruses) were more abundant in UC than in controls, while Anelloviridae (eukaryotic viruses) were higher in controls than in UC. At the genus level, Phix174microvirus, P1virus, Lambdavirus, T4virus, P22virus (all phages from the order Caudovirales), and Orthopneuovirus were enriched in UC, while giant viruses Coccolithovirus, Minivirus, and vertebrate-infecting viruses Orthopoxvirus (all eukaryotic viruses) were enriched in controls. Consistent with these observations, the abundance of more Caudovirales phages, including Escherichia phage and Enterobacteria phage, was expanded in UC mucosa relative to mucosa of healthy subjects. Taken together, these findings suggest that dysregulation of prokaryotic viruses, especially Caudovirales phages, is predominant in UC mucosa, whereas some eukaryotic viruses are more abundant in healthy controls, suggesting an association between bacterial dysregulation and phage expansion in UC, and an association between eukaryotic viral infection in healthy individuals and maturation of host immunity, which can protect the host from autoimmune diseases.

[0271] Using random forests, the inventors classified the mucosal virome in the Beijing and Xiangshan populations. All objects were classified as enterotype 1. Differential analysis of all enterotype 1 objects between healthy controls and UC in the Hong Kong population identified a group of different viruses. However, only two species were repeatable in the validation population, namely: Feldmannia species virus, which infects algae, and its presence in UC enterotype 1 objects was significantly higher than that in the control enterotype 1 objects in the Xiangshan population; and Pseudomonas virus, which was significantly higher in the control enterotype 1 objects than that in the UC enterotype 1 objects in the Beijing population. In summary, it shows that there is a significant geographical influence on the structure of the mucosal virome, which results in a large variation in the composition of the mucosal virome between different populations.

[0272] Functional alterations of the mucosal virome in UC

[0273] We then compared mucosal virome functions between healthy and UC. Although the abundance of the most abundant functions did not differ between healthy and UC, healthy individuals showed more abundant virome functions (according to GO and Pfam protein functions), whereas UC subjects showed a large number of functional abrogations. Despite this, several molecular functions were identified as being more abundant in UC than in the healthy mucosal virome, including: DNA template negative regulation of transcription, β-lactamase, glutamine amidotransferase, glycohydrolase, type II / IV secretion system, and multi-copper oxidase, all of which are related to phage lysis of the bacterial host and bacterial function. This result suggests that the enriched mucosal virome functions in UC are related to bacterial fitness, pathogenicity, and antibiotic resistance, and that there may be intensive genetic exchange between bacteria and phages in the UC mucosa.

[0274] Alterations in the mucosal bacteriome and cross-kingdom interactions between the viroome and the bacteriome at the mucosa

[0275] The bacterial microbiome changes in UC mucosa were further evaluated compared to healthy control mucosa. Bacterial diversity and richness increased significantly (t-test, p = 0.024 and 0.007, respectively), indicating that there was an expansion of different bacteria in the mucosa of UC patients. The UC mucosal bacterial group showed a different structure at the phylum, family, and genus levels compared to the control. Quantitative differential analysis identified many bacterial classifications that were different between healthy and UC mucosa. Among them, compared with the control, Firmicutes (phylum), Pseudomonadaceae, Ruminococcaceae, Thermus, unclassified Clostridiales, Vellonellaceae (family) and Coprococcus (genus) were greatly enriched in UC.

[0276] To characterize the association between the bacteriome and the virome in the mucosa, the correlation between the alpha diversity (diversity, evenness, and richness) of the bacteriome and the virome was assessed. In controls, there was a significant within-kingdom alpha diversity correlation. However, the significance of the within-kingdom alpha diversity correlation observed in controls was lost in UC, indicating that the mucosal bacterial microbiota is more imbalanced than the mucosal virome in UC.

[0277] Example 5 Crohn's disease and adherent invasive Escherichia coli (AIEC)

[0278] FMT in Crohn's disease: presence of AIEC and FMT outcomes

[0279] background

[0280] Crohn's disease (CD), one of the two major inflammatory bowel diseases (IBD), is a lifelong, disabling intestinal disease. In Hong Kong, China, the incidence of CD has increased sevenfold over the past decade. The cause of CD is unknown. Although genetic factors may play a role, strong evidence suggests that intestinal bacteria may underlie the development of the disease. FMT is currently being explored as a potential therapy for IBD. However, the mechanisms and factors that influence treatment outcomes are unclear. Adherent-invasive Escherichia coli (AIEC) can invade the intestinal epithelium and colonize the mucosa of IBD patients. They can also survive within macrophages without inducing cell death, allowing them to remain latent in the mucosa and cause damage over the long term. Because FMT directly alters the contents of the intestinal lumen, we hypothesized that FMT may fail to eradicate AIEC bacteria present within epithelial cells and that macrophages may continue to cause inflammation. Therefore, the aim of our study was to determine whether the presence of AIEC strains would render FMT ineffective and worsen disease outcomes.

[0281] method

[0282] Study subjects

[0283] Patients with ileal Crohn's disease (disease location was L1-ileum or L3-ileocolon according to the Montreal classification) were recruited in Hong Kong, China. Patients were aged ≥18 years and had a confirmed diagnosis of ileal Crohn's disease by endoscopy, radiology, and histology (ileum or ileocolon). Subjects without IBD were recruited as controls. These consisted of individuals ≥18 years old (who would undergo colonoscopy for polyps or colorectal cancer screening, or investigation of gastrointestinal symptoms), as well as friends and spouses or partners of patients at local hospitals, or any individual interested in participating in this study. Clinical data were collected using standardized data collection forms, and ileal biopsy samples were collected during ileocolonoscopy. The biopsy samples were then stored at -80°C for downstream analysis. Subjects were excluded if they took antibiotics, probiotics, or prebiotics within 3 months of biopsy sample collection. All subjects agreed to participate.

[0284] Quantification of mucosal E. coli levels and isolation of E. coli strains colonizing the ileal mucosa

[0285] The biopsy samples were thawed and weighed to calculate the weight of the isolated bacteria relative to the tissue. The bacteria associated with the intestinal mucosal adhesion biota on the mucosal surface were separated by gently shaking in PBS for 5 minutes. The internal bacteria were released by treating the biopsy samples with Triton 1X and Ultra-Turrax. The isolated bacteria were cultured overnight at 37°C. The colony forming units (CFU) were counted the next day to quantify the Escherichia coli associated with the mucosa. The mucosal Escherichia coli level was calculated by dividing the number of colony forming units by the weight of the mucosal biopsy sample.

[0286] For each patient, 48 bacterial colonies from mucosa-associated E. coli isolated on Drigalski medium and positive for lactose metabolism (E. coli differential signature) were cultured in 96-well microplates in Luria-Bertani medium supplemented with 15% glycerol and stored at -80°C.

[0287] Confirmation of AIEC positivity

[0288] By first separating the E. coli strains in the mucosal biopsy sample (as described above), then screening the main features of the AIEC strains in the isolated strains, and classifying based on these features of AIEC, determine that the AIEC positive representing the presence of AIEC. The main features of AIEC include adhesion, invasion and survival in macrophages to intestinal epithelial cells. E. coli strains with an invasion index greater than or equal to 0.1 relative to the initial inoculum and the ability to survive and reproduce in macrophages 24 hours after infection (% survival in macrophages greater than 100) are considered as AIEC.

[0289] Bacterial administration and fecal microbiota transplantation in a mouse model of DSS colitis

[0290] An acute DSS colitis mouse model was constructed using 8-9 week old male C57B / L6 wild-type mice (n=50) to study the effects of FMT on AIEC-infected mice. One group of mice was given only clean drinking water as a control (n=6). 44 mice were given drinking water supplemented with 2% DSS.

[0291] Among the 21 AIEC strains isolated from CD patients, the strain AIEC62d with the strongest invasive ability was selected for animal experiments. Starting from the first day of DSS treatment, mice were gavaged with 10 9 CFU dose of the AIEC strain (n=22), or the non-pathogenic E. coli strain K12 (n=22).

[0292] Feces were collected from healthy mice and diluted with sterile PBS at 1:10. After sedimentation, 200 μl of supernatant was administered to each mouse for 3 consecutive days as FMT treatment. Mice from AIEC group and K12 group (n=6, each group) were killed on the 7th day as the "before FMT" group. Then, fecal material from healthy mice or sterile PBS (n=8, each group) was administered to the remaining 32 mice for 3 consecutive days starting from the 7th day.

[0293] Quantification of fecal AIEC in mice

[0294] Counting was performed by inoculating PBS-resuspended feces on LB agar plates containing ampicillin (50 μg / mL) and erythromycin (25 μg / mL), because the AIEC strains tolerated these antibiotics, and such tolerant bacteria were not found in uninfected mice. On the second day, colony-forming units (CFU) were counted, and the CFU number per mg of feces was assessed.

[0295] Assessment of colitis severity in mice

[0296] The body weight of mice was measured daily. Fecal samples were collected on two days. Colon length and histological scores were measured at sacrifice to assess the severity of colitis. The colon length was measured from the cecum to the rectum. Blinded histological scoring of H&E-stained colon tissue was performed as follows. Four scores were assigned to each section based on the extent of epithelial damage and inflammation infiltrating into the mucosa, submucosa, and muscularis / serosa, resulting in a total score range of 0–12 for each mouse. The mean scores for the control and DSS-treated groups were then tabulated.

[0297] DNA extraction and 16S from mouse samples rRNA sequencing

[0298] Proximal colon tissue DNA was extracted to assess the mucosa-associated microbiota. Feces and tissues from mice sacrificed on day 7 were characterized as “before FMT,” while feces and tissues harvested on day 14 were characterized as “after FMT.” FMT was performed using the FMT kit according to the manufacturer’s instructions. DNA was extracted using a 16S tissue DNA purification kit and sent to Novogene (HK) Ltd. for library construction and sequencing of the V3-V4 region of 16S rRNA on an Illumina PE250 platform.

[0299] Bioinformatics analysis

[0300] The Mothur pipeline was used to convert the raw data into OUT tables. Sequence classification was performed at 97% similarity.

[0301] Discover

[0302] The levels of mucosal E. coli present in AIEC-positive CD patients were higher than those in AIEC-negative CD patients and healthy controls. higher

[0303] It has been reported that E. coli is over-represented in the mucosa of CD patients when compared to healthy controls. To evaluate whether E. coli is further over-represented in the mucosa of CD patients with AIEC, the total E. coli load was measured in the ileal mucosa taken from 56 CD subjects with ileal involvement and 24 healthy controls.

[0304] Among all subjects we examined, 37.5% (n=21) of CD patients and 8.3% (n=2) of healthy controls were AIEC positive. The total E. coli load in AIEC positive patients was significantly higher than that in AIEC negative patients and healthy controls (p=0.0323 and p<0.001 after logarithmic transformation, Turkey's multiple comparison test).

[0305] Fecal AIEC levels in mice only transiently decreased after FMT

[0306] The effect of FMT on AIEC levels in DSS colitis mice infected with AIEC was studied. The changes in AIEC load in mice before and after FMT were assessed in fecal samples. In AIEC mice treated with FMT, fecal AIEC load decreased immediately after mice were treated with FMT for 3 consecutive days, but then gradually increased. Comparison of AIEC mice with and without FMT found that the fecal AIEC load in the FMT group on the 9th and 12th days after AIEC infection was significantly lower than those in the non-FMT group. However, on the 14th day after AIEC infection, the AIEC load difference between the FMT group and the non-FMT group became significant. These data show that FMT alone may not be enough to eradicate AIEC in mice. The treatment of specific targeting AIEC should be combined to eradicate persistent AIEC infection.

[0307] The efficacy of FMT for colitis is compromised by AIEC infection

[0308] The effect of AIEC on the efficacy of FMT for the treatment of colitis was then investigated. The effects of FMT were compared between mice infected with AIEC and mice infected with K12, a non-pathogenic E. coli strain. The severity of colitis was assessed by body weight, colon length, and histological scores.

[0309] For body weight, the percentage of body weight recovery after FMT compared to baseline (day 0) was assessed. In K12-infected mice, the percentage of body weight recovery of mice treated with FMT on the 12th day was higher than that of mice without FMT (99% vs 95%, p = 0.0243, two-way ANOVA). On the other hand, for AIEC-infected mice, the body weight of mice with or without FMT treatment on the 14th day did not show significant differences (p = 0.98, two-way ANOVA). Comparison of the body weight recovery on the 14th day after FMT found that the body weight of K12-infected mice recovered to 97% of the initial body weight, while the body weight of mice infected with AIEC only recovered to 91% (p = 0.048, two-way ANOVA).

[0310] For colon length, in mice infected with AIEC, there was no significant difference in colon length between groups before and after FMT (p = 0.88, Kruska-Wallis test, using multiple comparisons). In contrast, the colon length of K12-infected mice increased significantly after FMT (mean colon length ± SEM 6.80 ± 0.12 mm before FMT vs 8.02 ± 0.27 mm after FMT, p = 0.0042, unpaired t test).

[0311] For histological scores, after FMT, the improvement of histological scores in mice infected with AIEC was less than that in mice infected with K12 (2.0vs 3.0, p=0.086, Mann-Whitney test), indicating that the colitis in mice infected with AIEC recovered less. These results indicate that FMT improves the DSS-induced colitis in AIEC-negative (K12-infected) mice, but its efficacy is impaired in the presence of AIEC.

[0312] Absence of beneficial bacteria in AIEC-infected mice despite FMT treatment

[0313] The mucosal and fecal microbiota of the above-mentioned AIEC-infected and K12-infected mice before and after FMT were analyzed. The AIEC present in the mouse intestine caused a decrease in the bacterial diversity of the mouse fecal microbiota. FMT treatment increased the α diversity of K12-infected mice but not AIEC-infected mice. LEfSe analysis revealed several classifications that were successfully transplanted into K12 mice but failed to colonize in AIEC-infected mice. Most of these classifications belong to the Firmicutes family, which is similar to the characteristics of the mucosal microbiota of IBD patients, indicating that AIEC is a driving factor for dysregulation in the mouse mucosa. These data show the accidental role of AIEC in the pathogenesis of IBD. These classifications are listed in Table 11.

[0314] The presence of AIEC may hamper FMT efficacy. Preventing colonization of potentially beneficial commensal organisms may be one mechanism by which AIEC may affect FMT efficacy.

[0315] Example 6 Obesity

[0316] method

[0317] An open-label human study of FMT for obesity and weight manipulation

[0318] An open-label clinical trial of fecal microbiota transplantation in obese subjects was conducted (NCT03789461). Patients aged 18-75 years with a body mass index of ≥28 kg / m 2 and <45kg / m 2 The subjects with a body mass index (BMI) of , and informed consent were obtained. During this study, the subjects received intensive FMT for a total of 20 days. During the treatment period, the subjects received 5 days of FMT per week (5 days, and 2 days of rest). In the same period, the subjects also received diet and lifestyle advice.

[0319] FMT: Stool was obtained from volunteers from the general population including spouses or partners, first-degree relatives, other relatives, friends, and other people known or unknown to the recipient. Volunteers were screened by questionnaires and laboratory tests before stool donation. A series of laboratory tests and interviews for infectious diseases were performed. Stool from qualified donors was used in this study. Before receiving FMT, the subjects received 5 days of antibiotics consisting of the following: vancomycin 500 mg 3 times a day; metronidazole 500 mg 3 times a day; and amoxicillin 500 mg 3 times a day to enhance the transplantation of microbiota from FMT. The subjects then received FMT for 20 days. 100-200 ml of FMT solution was infused into the patient by standard procedures in hospital or outpatient settings, including esophago-gastro-duodenoscopy (OGD), sigmoidoscopy, or enema.

[0320] 1. By OGD: 100-200 ml of FMT solution is infused into the distal duodenum or jejunum over 2-3 minutes by OGD. After infusion, the subject is monitored for 1 hour before leaving.

[0321] 2. Through sigmoidoscopy: 100-200 ml of FMT solution is infused into the distal colon over 2-3 minutes through sigmoidoscopy. After infusion, the subject is monitored for 1 hour before leaving.

[0322] 3. Enema: 100-150 ml of FMT solution was given by enema, either by self-administration or with the help of the research team. Subjects were instructed to retain the enema for 20-30 minutes. 4 mg of loperamide was given before each enema to increase retention of the FMT solution.

[0323] Fecal DNA isolation and metagenomic sequencing

[0324] Using DNeasy blood and tissue kit (QIAGEN), extract the fecal sample DNA collected from the donor according to the manufacturer's scheme, and further purify.By first constructing a paired end library with an insertion size of 350bp according to the manufacturer's instructions (illumina), and sequencing on the NovaSeq Illumina sequencer to carry out metagenomic sequencing.Using MetaPhlan2, the community composition is calculated using default parameters.In the R program package vegan, the bacterial classification summary, the sparseness analysis of microbial diversity, and the composition difference (the difference value indicated by the unweighted UniFrac distance) are calculated.

[0325] Discover

[0326] The weight changes of obese subjects when reaching the 8th week were analyzed. 5 consecutive subjects received FMT from 3 different donors. The fecal bacteria groups of these donors were characterized by metagenomic sequencing. Donor 15 and donor 16 have 2 and 3 species with a relative abundance of>0.1 that are beneficial to weight loss (Table 12); and 2 species with a relative abundance of ≤0.01% that are unfavorable to weight loss (Table 13). In contrast, donor 8 does not have these favorable bacteria possessed by donors 15 and 16, and the relative abundance of 2 unfavorable species is>0.01% (Table 12 and Table 13). Based on these characteristics, donor 8 is considered to be a donor that is unfavorable to weight loss, while donors 15 and 16 are considered to be donors that are beneficial to weight loss. At the 8th week, the subjects who received FMT from donors 15 and 16 had more weight loss than the recipients of donor 8 (Table 14).

[0327] Example 7 Obesity and lipid metabolism

[0328] Experimental models for obesity and lipid metabolism

[0329] Bacteria and culture medium

[0330] Waldersartella Sutterella wadsworthensis ) Culture conditions

[0331] Waldersartella (SW) was obtained from DSMZ, lipid medium without glucose, supplemented with different combinations of syrups corresponding to a sucrose concentration of 2%; sodium lactate (Chempur Poland) 7.41 g / L; sodium acetate (Chempur Poland) 7 g / L; and 0.2% yeast extract (BD Bioscences USA). The starting pH of all media was 7.0. The cultures were incubated at 37°C in a microaerophilic atmosphere consisting of 5.9% oxygen, 7.2% carbon dioxide, 3.6% hydrogen, and 83.3% nitrogen. The culture medium was obtained from Microbiology bv(9200JB Drachten,Netherlands) The atmosphere was generated by an atmosphere generation system. Minced meat carbohydrate medium (CM) was purchased from BD. Cultures were grown at 37°C without shaking under anaerobic conditions (Coy Laboratory Products, 75% N2, 20% CO2, 5% H2). Plates were evaluated twice a week for up to 1 month. Any bacterial isolates that were considered Gram-negative and oxygen-sensitive (by means of failed subclones in room air) were identified by 16S rRNA gene sequencing and sequence searches on NCBI BLAST.

[0332] Roseburia intestinalis (Roseburia intestinalis) Culture conditions

[0333] Roseburia intestinalis (RI) was purchased from the DSMZ Collection, and DSM-13018 medium was prepared and maintained anaerobically using an anaerobic apparatus. Isolates were regularly maintained by culturing in 7 ± 5 ml aliquots of M2GSC medium at 37 °C for 16–18 h. Substrate utilization and hydrolysis. The medium consisted of (in 100 ml) 1 g casein, 0 ± 2.5 g yeast extract, 0 ± 4 g NaHCO3, 0 ± 1 g cysteine, 0 ± 0.45 g K2HPO4, 0 ± 0.45 g KH2PO4, 0 ± 09 g NaCl, 0 ± 009 g MgSO4, 0 ± 009 g CaCl2, 0 ± 1 mg resazurin, 1 mg hemin, 1 μg biotin, 1 μg cobalamin, 3 μg p-aminobenzoic acid, 5 μg folic acid, and 15 μg pyridoxamine. Polyacidic glaucoma Mitsuokella multacida ) Culture conditions

[0334] Polyacidopsis luminilis (MM) was kept frozen at -20°C as glycerol stocks. L-10 liquid or solid (1% w / v agar) medium containing maltose and glucose (0.1% w / v), or a modified Scott and Dehority medium containing 10% (v / v) rumen fluid, 0.2% (w / v) glucose, 0.2% (w / v) cellobiose and 0.3% (v / v) starch [1] was used for cultivation. The cultures were grown in Hungate tubes containing 5 ml of medium under anaerobic and static conditions in an atmosphere consisting of CO2 / H2 (90:10) at a temperature of 37°C or at 39°C in 100% CO2.

[0335] Mouse Model

[0336] Eight-week-old male C57BL / 6 mice (average initial weight 20 g) were purchased from the Chinese University of Hong Kong and adapted to the animal facility environment for two weeks before the experiment. In all experiments, male mice were used. All mice were kept under a strict 24-hour light-dark cycle with light hours from 6 a.m. to 6 p.m. Each experimental group consisted of two cages per group to control cage effects (n=5 / cage).

[0337] The mice were divided into 5 groups, namely SW group, MM group, RI group, medium group and combination (combination of SW, MM and RI) group. The mice were given 1*10 9 doses of 3 single bacteria or combinations (combination of 3 bacteria, each accounting for 33.33%). Body weight was measured every 5 days. The study design is shown in Fig.11 middle.

[0338] antibiotic

[0339] For antibiotic treatment, mice were given a combination of kanamycin (0.4 mg / mL), gentamicin (0.035 mg / mL), colistin (850 U / mL), metronidazole (0.215 mg / mL), and vancomycin (0.045 mg / mL) in their drinking water for two weeks as described previously.

[0340] The samples were immediately transferred to Hungate anaerobic culture tubes in the animal facility and the supernatant and bacteria were administered to the mice by oral gavage. On the scheduled days, feces were collected at the beginning of the dark phase and immediately snap frozen and transferred for storage at 80°C until further processing.

[0341] Lipid properties

[0342] 20 days after the 6th administration, mice were killed by cervical dislocation for blood collection. Blood samples were collected in non-heparinized tubes and centrifuged at 1,600 × g for 10 min at 4 ° C. The levels of serum TG (GPO-PAP determination; catalog number A110-1), TC (GPO-PAP determination; catalog number A111-1), LDL-C (catalog number A113-1) and HDL-C (catalog number A112-1) were determined by Fisher Multiskan FC multifunctional detection microplate reader using a kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The levels of TG and TC were determined at 510nm and expressed as mmol / L. The levels of LDL-C and HDL-C were determined at 546nm and expressed as mmol / L.

[0343] Discover

[0344] Administration of S. waldensis (SW) significantly reduced body weight gain (p < 0.001; two-way repeated measures ANOVA), whereas administration of R. intestinalis (RI) showed a trend toward reduced body weight gain. The effect was particularly pronounced after day 15 (after 3 oral gavages) ( Fig.12 The percentage of body weight gain on day 20 and day 45 compared to day 0 was calculated for each mouse. The percentage of body weight gain on day 20 and day 45 was significantly lower in mice given SW and RI compared to those given medium ( Fig.13). Mean food intake was calculated as g food intake / mouse day. After administration of the combination (combination of SW, MM and RI), Pleurotus polyacicola (MM), RI and SW, mean food intake showed a decreasing trend, in contrast to administration of the medium alone, which showed an increasing trend. The effect of reducing food intake was most pronounced in RI and SW ( Fig.14 The average food efficiency was calculated as the weight gain (g) / total food intake (g) in the 5 days for each mouse. The average food efficiency of mice given combination, MM, RI, and SW after day 15 was reduced compared to that given medium ( Fig.15 ). The effects on lipid metabolism were studied. Administration of SW alone reduced LDL-C, total cholesterol (TC), and triglycerides (TG). Administration of RI alone reduced LDL-C and TC. Administration of the combination reduced LDL-C and TC. All results were compared with the control given medium on day 45 ( Fig.16 ).

[0345] Abbreviations sw: S. walderii; ri - enteric rosella; mm: Pseudomonas aeruginosa; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; TG: triglycerides; TC: total cholesterol; OGTT: oral glucose tolerance test; ITT: insulin tolerance test.

[0346] Example 8 Carbapenem-resistant Enterobacteriaceae

[0347] method

[0348] Clinical trials of FMT for eradication of CRE in humans

[0349] Patients aged ≥18 years with two or more stool or rectal swabs positive for CRE at least one week apart and who had not received antimicrobial therapy for at least 48 hours before FMT infusion were enrolled in the clinical trial (NCT03479710). Patients with active CRE or VRE infection requiring antimicrobial therapy, pregnancy, active gastrointestinal infection or inflammatory conditions, recent intra-abdominal surgery, short bowel syndrome, or use of medications that alter gastrointestinal motility were excluded. CRE was defined as the presence of any Enterobacteriaceae that was resistant to any carbapenem. A total of 3 subjects with CRE, the donor, and 4 healthy family member subjects were enrolled and baseline stool samples were obtained for microbiome analysis. The sample collection timeline for donors and recipients is shown in Fig.17 middle.

[0350] In this study, patients received 2 FMTs using frozen donor stool samples. In 2-3 minutes, 100 ml of FMT solution (original stool 50 g) in 0.9% sterile saline was infused into the distal duodenum or jejunum via esophago-gastroduodenoscope (OGD). Fecal samples were collected from patients in advance before and after FMT. For these 2 FMTs, recipients received FMTs from the same single donor.

[0351] Feces for FMT infusion were obtained from donors recruited to the Stool Biobank for the Faculty of Medicine, The Chinese University of Hong Kong. Donors were volunteers from the general population including spouses or partners, first-degree relatives, other relatives, friends, and other people known or unknown to the potential patient. Donors were required to meet a series of eligibility criteria and pass laboratory tests for screening infectious diseases including CRE and vancomycin-resistant enterococci (VRE).

[0352] Fecal DNA extraction

[0353] Approximately 100 mg of fecal sample was pre-washed with 1 ml of ddH2O and pelleted by centrifugation at 13,000 × g for 1 min. The fecal pellet was resuspended in 800 μL TE buffer (pH 7.5) supplemented with 1.6 μL 2-mercaptoethanol and 500 U lyticase (Sigma) and incubated at 37°C for 60 min. The sample was then centrifuged at 13,000 × g for 2 min, and the supernatant was discarded. After pretreatment, the fecal pellet was subsequently used Fecal DNA was extracted from the pellet using the RSC PureFood GMO and Authentication kit (Promega) according to the manufacturer's instructions. Briefly, 1 ml of CTAB buffer was added to the fecal pellet and vortexed for 30 seconds, and then the sample was heated at 95°C for 5 minutes. Afterwards, the sample was thoroughly vortexed with the beads at maximum speed for 15 minutes. Then 40 μl of proteinase K and 20 μl of RNase A were added to the sample, and the mixture was incubated at 70°C for 10 minutes. The supernatant was then obtained by centrifugation at 13,000 × g for 5 minutes and then added to The RSC machine was used for DNA extraction. The extracted fecal DNA was subjected to 16S rDNA sequencing and metagenomic sequencing.

[0354] Metagenomic sequencing and analysis

[0355] Qualified fecal DNA was cut into fragments, and sequencing libraries were prepared by end repair, adding A to the tail, purification, and PCR amplification. The fecal DNA library was deeply sequenced on the Illumina Hiseqxten PE150 platform by the Beijing Genomics Institute (BGI), and an average of 4800 ± 5.3 million reads (12G data) were generated for each sample.

[0356] The raw sequences were screened and quality trimmed by Trimmomatic v0.36 18 as follows: 1) trimming using a 4:8 quality sliding window; 2) trimming the sequence to remove 20 bases from the beginning and more than 220 bases from the end; 3) removing sequences less than 150 bp in length. Human host contamination reads were then filtered out using default parameters by Kneaddata (https: / / bitbucket.org / biobakery / kneaddata / wiki / Home, reference database: GRCh38 p12) to generate clean reads.

[0357] Bacterial taxonomy and functional characterization were performed in humann2 v0.11.1 19, with the following processes: taxonomic identification using branch-specific biomarkers 20 by MetaPhlAn2, annotation of species pangenomes using the ChocoPhlAn database by Bowtie2 21, translation searches of unmapped reads against the UniRef90 universal protein reference database 23 by DIAMOND 22, and pathway collections from the resulting gene lists using the Metacyc database 24. Gene family and pathway abundance files for all samples were merged and then normalized to relative abundance.

[0358] The taxonomic identity of fungi was determined from the fecal DNA metagenomic dataset using Kraken2 v2.0.7-beta. The complete NCBI fungal and viral RefSeq database was constructed from NCBI using the Jellyfish program by counting unique 31-mers in the reference library for each k-mer in the reads mapped to the lowest common ancestor of all reference genomes with an exact k-mer match 25. Each query was then sorted into the taxonomy with the highest total match of matching k-mers by pruning the general taxonomic tree to which the mapped genomes belonged.

[0359] Statistical analysis

[0360] The abundance data of bacteria, viruses, and fungi were entered into R 3.3.5. The richness, diversity, and rarefaction were calculated using the phyloseq package. The principal component analysis and principal coordinate analysis (PCoA) based on the Bray-Curtis dissimilarity matrix of the microbial community structure were calculated using the vegan R package. Heatmaps were performed using the ggplot R package.

[0361] Discover

[0362] The relative abundance of Ruminococcus sp_5_1_39BFAA, Collinsella tanakaei, and Eubacterium sicaeum was undetectable before FMT and increased to levels similar to or even higher than those of the donors after FMT ( Fig.18 ). LEfSe analysis also showed that these bacteria were significantly increased in the post-FMT samples compared to the pre-FMT samples, with LDA scores > 2.0 ( Fig. 20 ). These represent bacteria that are beneficial for CRE decolonization (Table 15).

[0363] To answer the question of whether FMT affects the gut microbiome, deep metagenomic sequencing of fungal enrichment was performed, and sequence reads were then aligned to a reference database using Kraken2 to identify fungal taxa. The alpha diversity of the patient's gut fungal community after FMT showed extensive fluctuations over time ( Fig. 20 a), Fungal communities in samples after FMT showed reduced differences from those from their corresponding donors ( Fig. 20 b), indicating a possible transplantation of donor fungi in the recipient or restoration of the recipient fungal community to a healthy state. Further discriminant analysis of the fecal microbiome before and after FMT by LEfSe showed that Xylonales, Wallemiales, and Ceraceosorales were differentially enriched genera in the feces after FMT ( Fig. 20 c, LDA effect size >2, p<0.05). More detailed species-level characterization revealed taxonomic differences in each recipient after FMT, such that CRE patients were transplanted with 22 fungal species, including Daedalea quercina, Pisolithus tinctorius, and Aspergillus arachidicola ( Fig. 20 d, Table 16). Although the transferred microbiota after FMT is composed mainly of bacteria, some fungal transplants may also be present, albeit in smaller numbers.

[0364] All patents, patent applications, and other publications cited in this application, including GenBank Accession Numbers and other sequence identification numbers, are incorporated herein by reference in their entirety for all purposes.

[0365] Table 1a “Favourable” bacteria present in FMT donors used to induce weight loss

[0366]

[0367] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0368] Table 1b The most "unfavorable" bacteria in donors for inducing weight loss using FMT

[0369]

[0370]

[0371] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0372] Table 2. “Favorable” fungi in donors for inducing weight loss using FMT

[0373]

[0374] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0375] Table 3a Fungi in the receptors associated with successful weight loss

[0376]

[0377] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0378] Table 3b Fungi associated with weight loss failure in the receptors

[0379]

[0380] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0381] #To improve the efficacy of weight loss interventions, the relative abundance of receptors should be kept below these values

[0382] Table 4 Bacteria in donors that are “favorable” for FMT treatment of CRE

[0383]

[0384]

[0385] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0386] Table 5 Fungi in donors that are “favorable” for FMT treatment of CRE

[0387]

[0388]

[0389] *Relative abundance was calculated as the number of all sequences belonging to a taxon divided by the number of all sequences detected in the sample

[0390] Table 6a Bacteria in donors that are “favorable” for FMT in the treatment of acute graft-versus-host disease

[0391]

[0392] Table 6b Maximum abundance of bacteria in donors that are “unfavorable” for the use of FMT for aGvHD

[0393]

[0394] Table 7a Fungi in donors that are “favorable” for the use of FMT to treat aGvHD

[0395]

[0396]

[0397] Table 7b “Unfavorable” fungi in donors for FMT treatment of aGvHD

[0398]

[0399] Table 8a Overrepresented viral classes in ulcerative colitis

[0400]

[0401] Table 8b Virus categories underrepresented in UC

[0402]

[0403]

[0404] Table 9a Viruses overrepresented in enterotype 2 compared to enterotype 1

[0405]

[0406] Table 9b Viruses underrepresented in enterotype 2 compared to enterotype 1

[0407]

[0408]

[0409] Table 10a Overrepresented bacterial taxa in UC

[0410]

[0411] Table 10b Bacterial taxa underrepresented in UC

[0412] Classification (family / genus / species) LDA effect size Proteobacteria (phylum) 5.8313 Actinomycetes (phylum) 4.7581 Fusobacteria (phylum) 4.5352 Oxalobacter 5.6387 Fusobacterium 4.5406 Cellulomonas(family) 4.4426 Fusobacterium 4.5392 Marseilles 5.6450 Escherichia coli 4.7503 Cellulomonas(genus) 4.4122

[0413] Table 11 Bacterial supplementation that can improve the efficacy of FMT in Crohn's disease patients with AIEC

[0414]

[0415]

[0416] *Sort by order of importance

[0417] Table 12 Relative abundance (%) of favorable fecal samples from D8, D15 and D16.

[0418]

[0419] Table 13 Relative abundance (%) of adverse fecal samples from D8, D15 and D16.

[0420]

[0421] Table 14 Characteristics and weight loss of FMT recipients after receiving FMT on D8, D15 and D16

[0422] Receptor Study No. FB001 FB002 FB003 FB004 FB005 gender M M F F F Fecal donor D8 D8 D8 D16 D15 Original weight 113.4 127.1 95.5 111.6 98.3 Weight loss at week 4 -2.4％ 0.3％ -0.5％ -2.4％ -1.8％ Weight loss at week 6 -2.4％ -1.3％ -2.4％ -3.3％ NA Weight loss at week 8 -2.8％ 1.0％ -1.9％ -3.8％ -5.5％

[0423] Table 15 Bacteria in donors that are "favorable" for the use of FMT to treat CRE

[0424]

[0425] Table 16 Fungi in donors that are “favorable” for FMT treatment of CRE

[0426]

[0427]

Claims

1. Use of a bacterium comprising each of Roseburia intestinalis, Sutterella wadsworthensis and Mitsuokella multacida in the preparation of a formulation for weight loss in a subject.

2. Use of bacteria comprising Roseburia intestinalis, Sutterella wadsworthensis and Mitsuokella multacida in the preparation of a formulation for reducing total cholesterol and low-density lipoprotein cholesterol in a subject.

3. The use according to claim 1 or 2, wherein the preparation is a preparation administered by fecal microbiota transplantation (FMT).

4. The use according to claim 3, wherein the FMT comprises oral ingestion of a composition derived from donor fecal material.

Images