WO2025076184A1

WO2025076184A1 - Methods of decreasing dysbiosis and restoring a microbiome

Info

Publication number: WO2025076184A1
Application number: PCT/US2024/049743
Authority: WO
Inventors: Rajita MENON
Original assignee: Vedanta Biosciences Inc
Current assignee: Vedanta Biosciences Inc
Priority date: 2023-10-03
Filing date: 2024-10-03
Publication date: 2025-04-10
Anticipated expiration: 2026-04-03

Abstract

Provided herein are methods and compositions for decreasing dysbiosis, restoring the microbiome, and/or increasing recovery of a healthy microbiome (e.g., following a dysbiosis inducing event), by administering compositions to a subject. Also provided are methods for protecting the microbiome of a subject and/or colonizing the microbiome of a subject by administering compositions to the subject.

Description

METHODS OF DECREASING DYSBIOSIS AND RESTORING A MICROBIOME

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/587,567, filed October 3, 2023, which is incorporated by reference herein in its entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Contract No. 75A50120C00177 awarded by the Biomedical Advanced Research and Development Authority. The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (P074570039WO00-SEQ-NTJ.xml; Size: 20,409 bytes; and Date of Creation: September 26, 2024) are herein incorporated by reference in their entirety.

FIELD

The disclosure relates to methods for decreasing dysbiosis, restoring the microbiome, and/or increasing recovery of a microbiome (e.g., following a dysbiosis inducing event), by administering bacterial compositions to a subject. Also provided are methods for protecting the microbiome of a subject and/or colonizing the microbiome of a subject by administering bacterial compositions to the subject.

BACKGROUND

Clostridium difficile infection (CDI) is typically treated with antibiotics, which perturbs the gut microbiome, thereby increasing susceptibility to recurrent CDI (rCDI). For patients who have experienced CDI recurrences, fecal microbiota transplant (FMT), and other donor-derived products have been successful in preventing further recurrences of CDI. The composition and quality attributes of these donor-derived procedures are inherently variable, they have occasionally resulted in transmission of harmful pathogens from the human donors of the feces (DeFilipp et al., 2019), the procedure requires extensive and costly donor screening (Craven et al., 2017), and the precise components of the gastrointestinal microbiota that provide resistance against C. difficile remain undetermined (Khanna et al., 2021; McDonald et al., 2018).

SUMMARY

Some aspects of the disclosure relate to methods and compositions for treating and/or preventing C. difficile infection, and/or colonizing a microbiome, by administering compositions comprising bacterial strains to subjects likely to experience therapeutic benefits from such bacterial strains. It was discovered, in a study of human subjects administered a purified bacterial composition, that (i) advanced age, (ii) high concentrations of primary bile acids, (iii) low concentrations of secondary bile acids, and (iv) low concentrations of shortchain fatty acids (SCFAs) were associated with increased frequency of recurrent Clostridium difficile infection. Without wishing to be bound by theory, it is expected that subjects with less advanced age (e.g., less than 60 years old), lower stool concentrations of primary bile acids, higher stool concentrations of secondary bile acids, and/or higher stool concentrations of SCFAs are expected to experience increased efficacy (e.g.. reduction in C. difficile recurrence risk) following administration of a purified bacterial composition.

Additionally, detection of bacterial strains of the administered composition was associated with clinical efficacy and reduced risk of C. difficile infection recurrence, and that high concentrations of primary bile acids or inflammatory cytokines predicted reduced likelihood of engraftment of bacterial strains of the administered composition, such as Flavonifractor plautii. Without wishing to be bound by theory, it is expected that subjects with lower stool concentrations of primary bile acids and/or lower serum concentrations of inflammatory cytokines are more likely to experience successful engraftment by bacterial strains of an administered composition, and consequently more likely to experienced increased efficacy (e.g.. reduction in C. difficile recurrence risk).

Accordingly, some aspects of the disclosure relate to a method of treating or preventing Clostridiodes difficile (C. difficile) infection in a subject, the method comprising administering to the subject a composition comprising a purified bacterial mixture, wherein the purified bacterial mixture comprises: (i) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 1; (ii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 2; (iii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 3; (iv) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 4; (v) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 5; (vi) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 6; (vii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 7; and (viii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 8, wherein: (a) the subject is younger than 60 years old; and/or (b) a fecal sample of the subject obtained prior to administration of the composition comprises a lower concentration of a primary bile acid, relative to a reference stool concentration of the primary bile acid; and/or (c) a fecal sample of the subject obtained prior to administration of the composition comprises a higher concentration of a secondary bile acid, relative to a reference stool concentration of the secondary bile acid; and/or (d) a fecal sample of the subject obtained prior to administration of the composition comprises a higher concentration of a short-chain fatty acid (SCFA), relative to a reference stool concentration of the SCFA; and/or (e) a serum sample of the subject obtained prior to administration of the composition comprises a lower concentration of one or more inflammatory cytokines, relative to a reference serum concentration of the inflammatory cytokine; and/or (f) the subject has received vancomycin within two weeks prior to administration of the composition; and/or (g) the subject has not received an antibiotic other than vancomycin within two weeks prior to administration of the composition.

In some embodiments, the subject is younger than 60 years old. In some embodiments, the subject is 55 years old or younger, 50 years old or younger, 45 years old or younger, or 40 years old or younger.

In some embodiments, the fecal sample of the subject comprises a lower concentration of the primary bile acid, relative to the reference stool sample concentration of the primary bile acid. In some embodiments, the primary bile acid is glycocholic acid (GCA), taurocholic acid (TCA), or taurochenodeoxy cholic acid (TCDCA). In some embodiments, the primary bile acid concentration in the subject’s fecal sample is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the reference stool sample concentration of the primary bile acid.

In some embodiments, the fecal sample of the subject comprises a higher concentration of a secondary bile acid, relative to a reference stool sample concentration of the secondary bile acid. In some embodiments, the secondary bile acid is deoxy cholic acid (DCA), isoallolithocholic acid (isoalloLCA), lithocholic acid (LCA), tauroursodeoxycholic acid (TUDCA), or ursodeoxycholic acid (UDCA). In some embodiments, the secondary bile acid concentration in the fecal sample of the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the reference stool sample concentration of the secondary bile acid.

In some embodiments, a fecal sample of the subject comprises a higher concentration of a short-chain fatty acid (SCFA), relative to a reference stool sample concentration of the SCFA. In some embodiments, the SCFA is hexanoate, isovalerate, or butyrate. In some embodiments, the SCFA concentration in the fecal sample of the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the reference stool sample concentration of the SCFA.

In some embodiments, the serum sample of the subject comprises a lower concentration of one or more inflammatory cytokines, relative to a reference serum concentration of the inflammatory cytokine. In some embodiments, the inflammatory cytokine is CXCL9, eotaxin-2, IL-27, MIP-ip, SCF, SDF-la, or SDF-lb.

Some aspects relate to a method of colonizing a gut microbiome in a subject, the method comprising administering to the subject a composition comprising a purified bacterial mixture, wherein the purified bacterial mixture comprises: (i) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 1; (ii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 2; (iii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 3; (iv) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 4; (v) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 5; (vi) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 6; (vii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 7; and (viii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 8, wherein: (a) the subject is younger than 60 years old; and/or (b) a fecal sample of the subject obtained prior to administration of the composition comprises a lower concentration of a primary bile acid, relative to a reference stool sample concentration of the primary bile acid; and/or (c) a serum sample of the subject obtained prior to administration of the composition comprises a lower concentration of an inflammatory cytokine, relative to a reference serum sample concentration of the inflammatory cytokine; and/or (d) the subject has been administered vancomycin within two weeks prior to administration of the composition; and/or (e) the subject has not received an antibiotic other than vancomycin within two weeks prior to administration of the composition.

In some embodiments, the fecal sample of the subject comprises a lower concentration of a primary bile acid, relative to a reference stool sample concentration of the primary bile acid. In some embodiments, the primary bile acid is cholic acid, chenodeoxy cholic acid, or glycochenodeoxy cholic acid. In some embodiments, the primary bile acid concentration in the subject’s fecal sample is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the reference fecal sample concentration of the primary bile acid.

In some embodiments, the serum sample of the subject comprises a lower concentration of an inflammatory cytokine, relative to a reference serum sample concentration of the inflammatory cytokine. In some embodiments, the inflammatory cytokine is IL-6, IL- 10, IL- 18, IL-22, MCP-2, MIP-10, and/or TNF-a. In some embodiments, the inflammatory cytokine concentration in the serum sample of the subject is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the reference serum sample concentration of the inflammatory cytokine.

In some embodiments, one or more of the bacterial strains produce a short chain fatty acid (SCFA). In some embodiments, one or more of the bacterial strains produce acetate, butyrate, and/or valerate. In some embodiments, one or more of the bacterial strains produce a secondary bile acid. In some embodiments, one or more of the bacterial strains produce ursodeoxycholic acid (UDCA).

In some embodiments, the bacterial strains are lyophilized. In some embodiments, the bacterial strains are spray-dried. In some embodiments, one or more of the bacterial strains are in spore form. In some embodiments, each of the bacterial strains is in spore form. In some embodiments, one or more of the bacterial strains are in vegetative form. In some embodiments, each of the bacterial strains is in vegetative form.

In some embodiments, the composition is a pharmaceutical composition comprising the bacterial strains and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for oral delivery. In some embodiments, the pharmaceutical composition is formulated for rectal delivery. In some embodiments, the pharmaceutical composition is formulated for delivery to the intestine. In some embodiments, the pharmaceutical composition is formulated for delivery to the colon. In some embodiments, the pharmaceutical composition comprises one or more enteric polymers. In some embodiments, the pharmaceutical composition is in a capsule. In some embodiments, the capsule comprises IxlO⁷ to IxlO¹⁰ colony-forming units (CFUs) of each of the bacterial strains.

In some embodiments, the composition is present in a food product comprising the bacterial strains and a nutrient.

In some embodiments, the subject has a pathogenic infection. In some embodiments, the composition is administered in a therapeutically effective amount to treat a pathogenic infection in the subject. In some embodiments, the pathogenic infection is a C. difficile infection. In some embodiments, the pathogenic infection is a recurrent C. difficile infection.

In some embodiments, the composition is administered in one dose. In some embodiments, the composition is administered in multiple doses. In some embodiments, each dose of the composition comprises administration of multiple capsules. In some embodiments, the subject is administered vancomycin prior to administration of the composition. In some embodiments, the composition induces the proliferation and/or accumulation of regulatory T (Treg) cells in the subject. In some embodiments, the subject is a human.

These and other aspects of the disclosure, as well as various embodiments thereof, will become more apparent in reference to the drawings and detailed description of the disclosure.

Each of the elements of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the elements of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1C show the dosing, monitoring, and endpoints of a double-blind placebo- controlled Phase 2 clinical trial in which an 8-strain bacterial mixture (VE303) was administered to subjects at risk of recurrent C. difficile infection (CD I). FIG. 1A shows the timeline of antibiotic administration and VE303 dosing (weeks 0-2), monitoring through primary endpoint (% of subjects with C. difficile recurrence by week 8), and safety monitoring (through week 24). FIG. IB shows recurrence-free probability in subjects treated with placebo, low-dose VE303, or high-dose VE303. FIG. 1C shows data collected from serum and stool samples at screening and weeks 0, 1, 2, 4, and 8. Subjects receiving the high- dose of VE303 had a significantly higher recurrence-free probability compared to placebo subjects. See, Dsouza & Menon, et al., Cell Host & Microbe (2022)30.4: 583-598; Louie et al., JAMA. 2023. 329(16): 1356- 1366.

FIGs. 2A-2D show bacterial strain colonization, and association of colonization and recurrence-free probability. FIG. 2A shows the number of distinct bacterial strains detected in each treatment group over time. FIG. 2B shows the relative abundance of administered bacterial strains (% of total bacteria in stool) over time. FIG. 2C shows the recurrence-free probability over time in subjects that were colonized by 5-8 strains of the composition, or 0-4 strains, with colonization by 5-8 strains being associated with lower CDI recurrence (log rank test, p = 0.08). FIG. 2D shows that in subjects dosed with VE303, detection of VE303-08 (colonization > 0) at the end of dosing was associated with a lower CDI recurrence rate and greater recurrence-free probability (adjusted p = 0.08, log rank test).

FIGs. 3A-3G show predictions of response to VE303 and VE303 strain colonization using serum cytokine concentrations, stool bile acid concentrations, and clinical factors (e.g., antibiotic exposure) at screening. FIG. 3A shows the association between measured cytokine and bile acid concentrations in serum and stool, respectively, or other clinical factors, and colonization by strains of VE303. FIG. 3B shows log mean exposure over 1 month to each bacterial strain of VE303, with subjects treated with vancomycin for the qualifying CDI episode experiencing increased exposure to bacterial strains of VE303. FIG. 3C shows increased diversity in subjects whose qualifying CDI episode was treated with vancomycin, compared to fidaxomicin. FIGs. 3D-3F show random Forest (RF) model importance plots predicting on-study CDI recurrence based on the indicated variables including age, screening concentrations of the indicated cytokines (FIG. 3D), bile acids (BAs) (FIGs. 3D and 3E), and the indicated short chain fatty acids (SCFAs) (FIGs. 3D and 3F). FIG. 3G shows RF model importance plots predicting colonization by VE303 strains based on the indicated variables including serum cytokine concentrations, and stool BA and SCFA concentrations. In FIGs. 3D-3G, variable importance is defined as the drop in predictive power when a variable is held out of the model. Bar color indicates the fraction of model iterations (out of 20) wherein the variable was found to be important. Vertical legend indicates association with recurrence. TCDCA = taurochenodeoxycholic acid; ECA = lithocholic acid; DCA = deoxycholic acid; AlloisoECA or IsoalloECA = isoallolithocholic acid; CDCA = chenodeoxycholic acid; CA = cholic acid; DHLCA = dehydrolithocholic acid; GUDCA = glycoursodeoxycholic acid; TUDCA = tauroursodeoxycholic acid; and GCDCA= glycochenodeoxycholic acid.

FIGs. 4A-4E show association of bile acid (BA) and short-chain fatty acid (SCFA) concentrations and on-study recurrence of CDI. FIG. 4A shows distributions of the indicated BAs (FIGs. 4A and 4B) and SCFAs (FIGs. 4A and 4C) at screening in recurrent vs nonrecurrent subjects. The box-and-whisker plots depict the median, interquartile range (IQR, at the top and bottom of the boxes), and reasonable extreme values at 1.5 X IQR in the dataset (where the vertical lines end). FIG. 4D shows a volcano plot representing the results of a linear mixed effects (LME) model to relate bacterial species dynamics through day 14 with CDI recurrence or non-recurrence of disease in the VE303-dosed groups. Species in the right half of the plot are associated with non-recurrence, and those in the left half are associated with recurrence; species above the red dashed line have model-adjusted p-values < 0.2. Bacterial strains present in VE303 are indicated. FIG. 4E shows significant relationships (p- adjust<0.05, LME model) between bacterial strains of VE303 and production of the indicated short-chain fatty acids (SCFAs) and secondary bile acids (BA). GCA = glycocholic acid; TCDCA = taurochenodeoxycholic acid; TCA = taurocholic acid; AlloisoLCA or isoalloLCA = isoallolithocholic acid; LCA = lithocholic acid; and UDCA = ursodeoxycholic acid.

FIGs. 5A-5E show colonization of VE303 strains over time across treatment groups and association with clinical efficacy. FIG. 5A shows detection of VE303 strains. FIG. 5B shows total relative abundance of VE303 strains as a proportion of a subject’s total microbiota in stool at the indicated timepoints for subjects, excluding post- antibiotic samples. The box-and-whisker plots depict the median, interquartile range (IQR), and reasonable extreme values at 1.5x IQR in the dataset. FIG. 5C shows line plots that indicate the median (solid line) and mean (dashed line) relative abundance per strain across subjects at the indicated collection timepoints. Abundance of individual VE303 strains is shown in each row, with treatment group ordered by column. Whiskers represent the standard error in the mean, computed across subjects at each timepoint. The dashed line indicates the sensitivity of the marker panel assay [0.01% relative abundance per strain per subject]; colonization levels below this value may not be detected. FIG. 5D shows the fraction of subjects with VE303 strains detected in any sample over the 2 weeks of VE303 dosing. FIG. 5E shows VE303-08 strain colonization at the end of dosing was associated with a lower CDI recurrence rate and greater recurrence-free probability in subjects dosed with VE303. Kaplan-Meier curves are shown for high vs low colonized subjects (adjusted p=0.08, log-rank test). FIGs. 6A-6C show a clinical study design and summary of data collection. FIG. 6A shows clinical study design. Subjects were treated with VE303 or Placebo after being randomized 1:1:1 into treatment groups. CDI recurrence was evaluated at week 8, with additional safety follow-up through week 24. FIG. 6B shows metagenomic, metabolomic, and immune datasets examined. FIG. 6C shows a summary of sample collection (total samples, samples from recurrent subjects, samples from non-recurrent subjects) for VE303- dosed groups up to day 14.

FIGs. 7A-7I show detection of VE303 strains in all enrolled subjects over time in study VE303-002. Stacked-bar charts showing detection of individual VE303 strains over time, presented for each subject in the VE303 high-dose (FIGs. 7A-7C), VE303 low-dose (FIGs. 7D-7F), and placebo groups (FIGs. 7G-7I). Data are shown for all analyzed samples.

FIGs. 8A-8C show a random forest classification model schematic and performance. FIG. 8A is a schematic of a random forest model used to predict features of CDI recurrence and VE303 colonization. Subjects were classified into recurrent/non-recurrent and VE303 colonized/non-colonized using a combination of continuous and categorical independent variables including metagenomics, immunological and metabolite datasets, and patient-level metadata such as demographics and medical history. FIGs. 8B and 8C show random forest classification across different data modalities is used to predict the colonization status of VE303 strains across all dosed subjects (FIG. 8B) and on-study CDI recurrence status (FIG. 8C). Leave-one-out cross-validation was performed to assess the model performance. AUC was computed for all models; an AUC of <0.5 (dashed line) indicates random classification or unreliable model performance. Number labels on each graph indicate the total number of samples available per timepoint and dataset; brackets convey class sizes for colonized/non- colonized (FIG. 8B) and recurrent/non-recurrent classes (FIG. 8C).

FIG. 9 shows a brick plot showing detection of individual VE303 strains at day 14 in subjects dosed with VE303.

FIG. 10 shows predictors of early strain colonization at baseline. Shown is a random forest summary of important variables predicting VE303 strain colonization based on screening concentrations of serum cytokines, SCFAs, Bas, microbial, and demographic variables. Variable importance is defined as the drop in predictive power when a variable is held out of the model; the heatmap shows the frequency with which a variable was identified as important across all model iterations. The vertical legend indicates whether the variable was associated with strain colonization, non-colonization, or was not a significant predictor (NS). FIGs. 11A-11D show the effect of antibiotic pretreatment and residual vancomycin concentration on VE303 colonization. FIG. 11A shows the mean abundance per strain through day 28. FIG. 11B shows a change in diversity from screening to day 1 in VE303- dosed subjects treated with vancomycin QID or fidaxomicin as standard-of-care (SoC) antibiotic treatment for their qualifying CDI episode. The box-and-whisker plots depict the median, IQR, and reasonable extreme values at 1.5x IQR in the dataset. FIG. 11C shows log- transformed vancomycin concentrations over time in subjects given vancomycin for their qualifying CDI episode across VE303-dosed groups. The thick line represents the median concentration across subjects. Vancomycin dynamics in placebo recipients was consistent with those in the VE303-dosed groups (data not shown). FIG. 11D shows the correlation between log-transformed mean vancomycin concentrations (with a limit-of-detection value of 1) and log-transformed mean relative abundance (with a pseudocount of 0.01% added to each value) for each VE303 strain at day 7 and day 14. Subjects given vancomycin for the qualifying CDI episode in VE303-dosed groups are included. The shaded region indicates the 95% confidence interval for predictions from a linear model.

FIG. 12 shows the longer standard-of-care (SoC) antibiotic treatment immediately prior to VE303 dosing improves VE303 strain colonization. Correlation between SoC antibiotics treatment length and log-transformed mean relative abundance per subject (with a pseudocount of 0.01% added to each value) for each VE303 strain during days 7-28. Organisms with a significant positive relationship between exposure and treatment length are shown (adjusted p < 0.1, LME model, Table E2-3). In each plot, the shaded region indicates the 95% confidence interval for predictions from a linear model.

FIGs. 13A-13D show microbial, metabolite, and immune profiles at screening predict response to VE303. Variables that predict on-study CDI recurrence in random forest models based on screening datasets for stool bacterial species (FIG. 13A), serum cytokines (FIG. 13B), stool SCFAs (FIG. 13C), and BAs (FIG. 13D). Importance is defined as the drop in predictive power when a variable is held out of the model. Bar color indicates the fraction of model iterations (out of 100) wherein the variable was found to be important. The vertical legend indicates the association with CDI recurrence or non-recurrence.

FIG. 14 shows a Random Forest model summary for features linked to recurrence. Random Forest model results are shown for microbial, metabolic, and immune features predicting CDI recurrence (at screening) or associated with CDI recurrence (post- screening) in subjects treated with VE303. The heatmap represents the frequency with which the feature was selected to be important across all model iterations. Recurrence-related features and response-related features are shown.

FIGs. 15A-15C show that increased age and inflammation at the time of screening predicts recurrence. FIG. 15A shows the concentration of cytokines is predictive of on-study recurrence at screening in subjects enrolled in the VE303-dosed groups. FIG. 15B shows box-and- violin plots showing the age of subjects enrolled in the VE303-dosed groups grouped by on-study CDI outcomes. FIG. 15C shows correlations between MIP-lb concentrations at baseline vs subject age. The shaded region indicates the 95% confidence interval for predictions from a linear model. The box-and-whisker plots depict the median, IQR, (at the top and bottom of the boxes) and reasonable extreme values at 1.5x IQR.

FIGs. 16A-16C show recovery of endogenous microbiota and associations with CDI recurrence. FIG. 16A shows line plots of species alpha diversity represented by the Shannon Index over time across treatment groups over time. The thick lines represent the median diversity across subjects at each timepoint and the error bars represent the median absolute deviation. Stars show significant differences between treatment groups at the indicated timepoints (p<0.05; LME model). FIG. 16B shows a volcano plot representing the results of the LME model to relate bacterial species dynamics through day 14 with CDI recurrence or non-recurrence in the VE303-dosed groups. Species in the right half of the plot are associated with non-recurrence and those in the left half are associated with recurrence; species above the horizontal dashed line have model-adjusted p-values <0.2. FIG. 16C shows an effect-size bar plot for all species with unadjusted p-value <0.05. Bars are colored according to the taxonomic class of each organism. Species associated with non-recurrence include VE303- 08, -02, and -01, whereas species associated with recurrence include putative pathogenic organisms from the phylum Proteobacteria.

FIGs. 17A-17E show associations of endogenous taxa at species, genus, family, order and class level with CDI recurrence and VE303 abundance in study VE303-002. Effect-size bar plots for all organisms that have significant associations with CDI recurrence or non- recurrence (unadjusted p-value <0.05) at species (FIG. 17A), genus (FIG. 17B), family (FIG. 17C), order (FIG. 17D), and class level (FIG. 17E). Taxa in the right half of each plot are associated with non-recurrence; those in the left half are associated with recurrence, and bars are colored according to taxonomic class. Vertical annotations on the right indicate significant model correlations with total VE303 abundance up to day 14; blue boxes indicate a positive correlation; and red boxes indicate a negative correlation with VE303. Significance level of correlations with VE303 are indicated by stars. FIGs. 18A and 18B show short-chain fatty acid (SCFA) and bile acid (BA) associations with endogenous bacteria and clinical outcome in dosed subjects. FIG. 18A shows log-transformed concentrations (ng/mg stool) of individual primary BA, secondary BA, and SCFA found to be associated with clinical outcome in Random Forest models up to day 14. The mean metabolite concentration per subject is plotted. Box colors indicate associations with recurrence or non-recurrence. The box-and- whisker plots depict the median, IQR (at the top and bottom of the boxes), and reasonable extreme values at 1.5x IQR of the log-transformed concentrations. FIG. 18B shows stool SCFA and BA concentrations correlated with endogenous bacterial taxa. All significant associations (adjusted p<0.05, LME) between endogenous taxa and VE303 strains, and measured stool metabolites are shown. Blue boxes indicate positive correlations, and red boxes indicate negative correlations between taxon-metabolite pairs. VE303 organisms significantly associated with metabolite concentrations are highlighted in red.

FIGs. 19A-19C show a concentration of primary bile acids, secondary bile acids, and short-chain fatty acids over time and association with VE303 organisms. Log-transformed total stool concentrations (ng/mg) of primary BA (FIG. 19A), secondary BA (FIG. 19B), and SCFA (FIG. 19C) over time. The blue boxes indicate the concentrations for subjects who did not have an on-study CDI recurrence. The red boxes indicate the concentrations for subjects who did have an on-study CDI recurrence. The primary BAs include CA, CDCA, TCA, TCDCA, GCA, GCDCA. The secondary BAs include DCA, LCA, 3-oxoLCA, TLCA, TDCA, GLCA, GDCA, UDCA, TUDCA, GUDCA, and Isoallo- + IsoLCA. The box-and- whisker plots depict the median, IQR (at the top and bottom of the boxes), and reasonable extreme values at 1.5x IQR of the log-transformed metabolite concentrations at each timepoint.

DETAILED DESCRIPTION

Methods and uses of compositions

Some aspects relate to methods of treating and/or preventing C. difficile infection by administering compositions comprising bacterial strains to subjects that (i) are 60 years old or younger; (ii) have lower stool concentrations of primary bile acids; (iii) have higher stool concentrations of secondary bile acids; and/or (iv) have higher stool concentrations of shortchain fatty acids (SCFAs). These characteristics were discovered to be associated with increased responsiveness to the therapeutic effects of a bacterial composition in a clinical trial, with younger subjects, for example, being less likely to experience recurrence of C. difficile infection than older subjects following administration of such a composition. Similarly, reduced likelihood of C. difficile infection recurrence was associated with lower levels of primary bile acids, higher levels of secondary bile acids, and higher levels of SCFAs in stool at the time of screening. Without wishing to be bound by theory, the inventors posit that such characteristics (younger age, lower primary bile acid levels, higher secondary bile acid levels, and/or higher SCFA levels) may be used to identify subjects who are likely to benefit from administration of a bacterial composition. Any of the compositions described herein may be administered to a subject in a therapeutically effective amount or a dose of a therapeutically effective amount to treat or prevent C. difficile infection.

Some aspects relate to methods of colonizing a microbiome by administering compositions comprising bacterial strains to subjects that (i) have lower stool concentrations of primary bile acids; and/or (ii) have lower serum concentration of cytokines associated with inflammation. These characteristics were discovered to be associated with engraftment of bacterial strains of a bacterial composition in a clinical trial, with subjects having lower serum concentrations of inflammatory cytokines, for example, being more likely to be colonized by Strain 8 of the bacterial composition. Colonization by this strain was predictive of freedom from C. difficile infection recurrence (z.e., subjects colonized by this strain did not experience C. difficile infection recurrence during the study). Without wishing to be bound by theory, the inventors posit that characteristics associated with colonization of bacterial strains (lower primary bile acid levels in stool, and/or lower inflammatory cytokine levels in serum) may be used to identify subjects who are more likely to be colonized by bacterial strains of a composition, and consequently less likely to experience C. difficile infection recurrence. Any of the compositions described herein may be administered to a subject in a therapeutically effective amount or a dose of a therapeutically effective amount to colonize a microbiome.

The terms “treat” or “treatment” refer to reducing or alleviating one or more of the symptoms associated with a disease or disorder (e.g., C. difficile infection). In some embodiments, the therapeutically effective amount is an amount sufficient to treat C. difficile infection. The terms “prevent” or “prevention” encompass prophylactic administration and may reduce the incidence or likelihood of experiencing a disease or disorder (e.g., C. difficile infection). In some embodiments, the therapeutically effective amount is an amount sufficient to reduce the incidence or likelihood of experiencing C. difficile infection.

As used herein, the term “therapeutically effective amount” may be used interchangeably with the term “effective amount.” A therapeutically effective amount or an effective amount of a composition, such as a pharmaceutical composition, as described herein, is any amount that results in a desired response or outcome in a subject, such as those described herein. In some embodiments, the therapeutically effective amount is an amount sufficient to treat or prevent C. difficile infection. In some embodiments, the therapeutically effective amount is an amount sufficient to colonize the microbiome. It should be appreciated that the term “effective amount,” in reference to a composition comprising bacterial strains, may be expressed as the number of bacteria or colony-forming units (CFUs) to be administered. It should further be appreciated that the bacteria can multiply once administered. Thus, administration of even a relatively small amount of bacteria may have therapeutic effects.

As used herein, “subject,” “individual,” and “patient” are used interchangeably, and refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, humans, non-human primates or murine, bovine, equine, canine or feline species. In some embodiments, the subject is a human. In some embodiments, the human subject is a neonatal subject, a pediatric subject, an adolescent subject, an adult subject, or a geriatric subject.

Some aspects relate to combinations of purified bacterial strains. Some embodiments relate to compositions comprising a purified bacterial mixture comprising two or more bacterial strains. In some embodiments, the composition is a combination product. “Combination product,” as used herein, has the definition specified in 21 CFR 3.2(e). At the time of filing the instant specification, “combination product” defined under 21 CFR 3.2(e) includes: (1) A product comprised of two or more regulated components, z.e., drug/device, biologic/device, drug/biologic, or drug/device/biologic, that are physically, chemically, or otherwise combined or mixed and produced as a single entity; (2) Two or more separate products packaged together in a single package or as a unit and comprised of drug and device products, device and biological products, or biological and drug products; (3) A drug, device, or biological product packaged separately that according to its investigational plan or proposed labeling is intended for use only with an approved individually specified drug, device, or biological product where both are required to achieve the intended use, indication, or effect and where upon approval of the proposed product the labeling of the approved product would need to be changed, e.g., to reflect a change in intended use, dosage form, strength, route of administration, or significant change in dose; or (4) Any investigational drug, device, or biological product packaged separately that according to its proposed labeling is for use only with another individually specified investigational drug, device, or biological product where both are required to achieve the intended use, indication, or effect. Some embodiments relate to methods of administering a composition comprising a combination of purified bacterial strains. Some embodiments relate to methods of administering a combination of purified bacterial strains to a subject. Some embodiments relate to kits comprising a combination of purified bacterial strains. The skilled artisan will appreciate that because bacterial strains may colonize the gastrointestinal tract of a subject and exert a therapeutic effect through their presence in the gastrointestinal tract, it is not necessary that all bacterial strains administered to the subject be present in a single dosage form (e.g., a single capsule comprising all bacterial strains of the combination).

Bacterial strains of a combination may be included in separate individual dosage forms, in arbitrary combinations in two or more dosage forms, or all bacterial strains may be included in a single dosage form. When administered as separate dosage forms, the administration form and route may be the same or different for each dosage form. Dosage forms may be administered simultaneously, sequentially, and/or separately. In some embodiments, simultaneous administration of a plurality of dosage forms occurs when all dosage forms of the plurality are administered within a period of 1, 2, 3, 4, or 5 minute(s). In some embodiments, sequential administration of a plurality of dosage forms occurs when all dosage forms of the plurality are administered in series. In some embodiments, separate administration of a plurality of dosage forms occurs when no two dosage forms of the plurality are administered within a period of 1, 2, 3, 4, or 5 minute(s).

Uses in subjects of younger age, having reduced primary bile acid or cytokine levels, and/or increased secondary bile acid or short-chain fatty acid levels

Some aspects relate to administering a bacterial composition to a subject below a certain age. As described in the Examples, advanced age was associated with C. difficile infection recurrence, and therefore younger subjects are expected to be more responsive to the therapeutic effects of the bacterial compositions described herein. In some embodiments, a composition is administered to a subject that is 60 years old or younger. In some embodiments, the subject is no more than 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 years old. In some embodiments, the subject is 55 years old or younger. In some embodiments, the subject is 50 years old or younger. In some embodiments, the subject is 45 years old or younger. In some embodiments, the subject is 40 years old or younger. In some embodiments, the subject is between 4-17, 18-20, 20-25, 25- 30, 30-35, 35-40, 40-45, 45-50, 50-55, or 55-60 years old. In some embodiments, the subject is between 4-60, 18-60, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, or 55-60 years old. In some embodiments, the subject is between 18-55, 18-50, 18-45, 18-40, 18-35, 18-30, 18- 25, or 18-20 years old.

Some aspects relate to administering a bacterial composition to a subject having a low stool concentration of one or more primary bile acids. As described in the Examples, lower primary bile acid concentrations in stool were associated with reduced probability of C. difficile infection recurrence, and subjects having such low stool concentrations of primary bile acid are expected to be more responsive to the therapeutic effects of the bacterial compositions described herein. Similarly, lower stool concentrations of primary bile acids were associated with a higher probability of engraftment by bacterial strains of administered compositions, such as Flavonifractor plautii, and subjects in which this strain engrafted did not experience or had reduced incidence of C. difficile infection recurrence. Thus, administration to subjects with lower stool concentrations of one or more primary bile acids is expected to increase the likelihood of engraftment by bacterial strains.

In some embodiments, a composition is administered to a subject having a primary bile acid stool concentration that is lower than a reference stool concentration of the primary bile acid. In some embodiments, a reference stool concentration of the primary bile acid is an average stool concentration of the primary bile acid. Any suitable method may be used to determine the average stool concentration of the primary bile acid. In some embodiments, the average stool concentration of the primary bile acid is determined by comparison to a reference database. An average concentration may be calculated using an entire set of concentration data available from a database. An average concentration may be calculated using a subset of a database. For example, an average concentration may be calculated using only data obtained from individuals of the same or similar age and/or sex of the subject. Other criteria for filtering samples of a database may include history of C. difficile infection, history of inflammatory bowel disease (IBD), diet, weight, cholecystectomy, surgical history, medication history, immunocompromised status, and/or family history of any of the preceding factors.

In some embodiments, a subject's stool concentration of the primary bile acid is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less than the reference stool concentration of the primary bile acid. In some embodiments, the average stool concentration of the primary bile acid is calculated as the mean of concentrations. In some embodiments, the average stool concentration of the primary bile acid is calculated as the median of concentrations.

The reference stool concentration of a primary bile acid may be a concentration measured in one or more previous fecal samples of the subject. In some embodiments, a composition is administered to a subject having a primary bile acid stool concentration that is lower than a primary bile acid stool concentration of a previously measured stool sample from the subject. In some embodiments, the previously measured stool sample was obtained during a C. difficile infection. In some embodiments, the previously measured stool sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after detection of C. difficile infection. In some embodiments, the previously measured stool sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before detection of C. difficile infection. In some embodiments, at the time of administration of a bacterial composition, the subject has a stool concentration of the primary bile acid that is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less than the primary bile acid concentration in a previously measured stool sample of the subject.

In some embodiments, a composition is administered to a subject whose fecal sample has a primary bile acid stool concentration that is lower than an average primary bile acid stool concentration of previously measured stool samples from the subject. In some embodiments, a subject's stool concentration of the primary bile acid is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less than the average stool concentration of the primary bile acid in the subject. An average stool concentration of the subject may be determined using any number of stool samples. In some embodiments, an average stool concentration of a primary bile acid is calculated from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 stool samples. In some embodiments, an average stool concentration of a primary bile acid is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days apart. In some embodiments, an average stool concentration of a primary bile acid is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart. In some embodiments, an average stool concentration of a primary bile acid is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, an average stool concentration of a primary bile acid is calculated from at least two stool samples collected daily. In some embodiments, an average stool concentration of a primary bile acid is calculated from at least two stool samples collected weekly. In some embodiments, an average stool concentration of a primary bile acid is calculated from at least two stool samples collected monthly. In some embodiments, the subject did not have a Clostridium difficile infection during collection of their stool samples. In some embodiments, the average stool concentration of the primary bile acid is calculated as the mean of concentrations. In some embodiments, the average stool concentration of the primary bile acid is calculated as the median of concentrations.

In some embodiments, the primary bile acid is cholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), or taurochenodeoxycholic acid (TCDCA). In some embodiments, the primary bile acid is taurochenodeoxycholic acid (TCDCA). In some embodiments, the primary bile acid is glycocholic acid (GCA). In some embodiments, the primary bile acid is taurocholic acid (TCA).

Some aspects relate to administering a bacterial composition to a subject having an elevated stool concentration of one or more secondary bile acids. As described in the Examples, higher secondary bile acid concentrations in stool were associated with reduced probability of C. difficile infection recurrence, and subjects having such elevated stool concentrations of secondary bile acids are expected to be more responsive to the therapeutic effects of bacterial compositions. In some embodiments, a reference stool concentration of the secondary bile acid is an average stool concentration of the secondary bile acid. In some embodiments, a composition is administered to a subject having a secondary bile acid stool concentration that is higher than a reference stool concentration of the secondary bile acid. Any suitable method may be used to determine the average stool concentration of the secondary bile acid. In some embodiments, the average stool concentration of the secondary bile acid is determined by comparison to a reference database. An average concentration may be calculated using an entire set of concentration data available from a database. An average concentration may be calculated using a subset of a database. For example, an average concentration may be calculated using only data obtained from individuals of the same or similar age and/or sex of the subject. Other criteria for filtering samples of a database may include history of C. difficile infection, history of inflammatory bowel disease (IBD), diet, weight, cholecystectomy, surgical history, medication history, immunocompromised status, and/or family history of any of the preceding factors.

In some embodiments, a subject's stool concentration of the secondary bile acid is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the reference stool concentration of the secondary bile acid. In some embodiments, the average stool concentration of the secondary bile acid is calculated as the mean of concentrations. In some embodiments, the average stool concentration of the secondary bile acid is calculated as the median of concentrations.

The reference stool concentration of a secondary bile acid may be a concentration measured in one or more previous fecal samples of the subject. In some embodiments, a composition is administered to a subject having a secondary bile acid stool concentration that is higher than a secondary bile acid stool concentration of a previously measured stool sample from the subject. In some embodiments, the previously measured stool sample was obtained during a C. difficile infection. In some embodiments, the previously measured stool sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after detection of C. difficile infection. In some embodiments, the previously measured stool sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before detection of C. difficile infection. In some embodiments, at the time of administration of a bacterial composition, the subject has a stool concentration of the secondary bile acid that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the secondary bile acid concentration in a previously measured stool sample of the subject.

In some embodiments, a composition is administered to a subject whose fecal sample has a secondary bile acid stool concentration that is higher than an average secondary bile acid stool concentration of previously measured stool samples from the subject. In some embodiments, a subject's stool concentration of the secondary bile acid is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the average stool concentration of the secondary bile acid in the subject. An average stool concentration of the subject may be determined using any number of stool samples. In some embodiments, an average stool concentration of a secondary bile acid is calculated from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 stool samples. In some embodiments, an average stool concentration of a secondary bile acid is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days apart. In some embodiments, an average stool concentration of a secondary bile acid is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart. In some embodiments, an average stool concentration of a secondary bile acid is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, an average stool concentration of a secondary bile acid is calculated from at least two stool samples collected daily. In some embodiments, an average stool concentration of a secondary bile acid is calculated from at least two stool samples collected weekly. In some embodiments, an average stool concentration of a secondary bile acid is calculated from at least two stool samples collected monthly. In some embodiments, the subject did not have a Clostridium difficile infection during collection of their stool samples. In some embodiments, the average stool concentration of the secondary bile acid is calculated as the mean of concentrations. In some embodiments, the average stool concentration of the secondary bile acid is calculated as the median of concentrations.

In some embodiments, the secondary bile acid is deoxycholic acid (DCA), dehydrolithocholic acid (DHLCA), glycodeoxy cholic acid (GDC A), glycolithocholic (GLCA), glycoursodeoxycholic acid (GUDCA), isoallolithocholic acid (IsoalloLCA), isolithocholic acid (IsoLCA), lithocholic acid (LCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or 3-oxolithocholic acid (3-oxoLCA). In some embodiments, the secondary bile acid is deoxy cholic acid (DCA) or lithocholic acid (LCA). In some embodiments, the secondary bile acid is DCA. In some embodiments, the secondary bile acid is LCA. In some embodiments, the secondary bile acid is isoallolithocholic acid (isoalloLCA). In some embodiments, the secondary bile acid is tauroursodeoxycholic acid (TUDCA). In some embodiments, the secondary bile acid is ursodeoxycholic acid (UDCA).

Some aspects relate to administering a bacterial composition to a subject having an elevated stool concentration of one or more short-chain fatty acids (SCFAs). As described in the Examples, higher SCFA concentrations in stool were associated with reduced probability of C. difficile infection recurrence, and subjects having such elevated stool concentrations of SCFAs are expected to be more responsive to the therapeutic effects of bacterial compositions. In some embodiments, a composition is administered to a subject having a SCFA stool concentration that is higher than a reference stool concentration of the SCFA. In some embodiments, a reference stool concentration of the SCFA is an average stool concentration of the SCFA. Any suitable method may be used to determine the average stool concentration of the SCFA. In some embodiments, the average stool concentration of the SCFA is determined by comparison to a reference database. An average concentration may be calculated using an entire set of concentration data available from a database. An average concentration may be calculated using a subset of a database. For example, an average concentration may be calculated using only data obtained from individuals of the same or similar age and/or sex of the subject. Other criteria for filtering samples of a database may include history of C. difficile infection, history of inflammatory bowel disease (IBD), diet, weight, cholecystectomy, surgical history, medication history, immunocompromised status, and/or family history of any of the preceding factors.

In some embodiments, a subject's stool concentration of the SCFA is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the reference stool concentration of the SCFA. In some embodiments, the average stool concentration of the SCFA is calculated as the mean of concentrations. In some embodiments, the average stool concentration of the SCFA is calculated as the median of concentrations.

The reference stool concentration of an SCFA may be a concentration measured in one or more previous fecal samples of the subject. In some embodiments, a composition is administered to a subject having a SCFA stool concentration that is higher than a SCFA stool concentration of a previously measured stool sample from the subject. In some embodiments, the previously measured stool sample was obtained during a C. difficile infection. In some embodiments, the previously measured stool sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after detection of C. difficile infection. In some embodiments, the previously measured stool sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before detection of C. difficile infection. In some embodiments, at the time of administration of a bacterial composition, the subject has a stool concentration of the SCFA that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the SCFA concentration in a previously measured stool sample of the subject.

In some embodiments, a composition is administered to a subject whose fecal sample has a SCFA stool concentration that is higher than an average SCFA stool concentration of previously measured stool samples from the subject. In some embodiments, a subject's stool concentration of the SCFA is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the average stool concentration of the SCFA in the subject. An average stool concentration of the subject may be determined using any number of stool samples. In some embodiments, an average stool concentration of a SCFA is calculated from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 stool samples. In some embodiments, an average stool concentration of a SCFA is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days apart. In some embodiments, an average stool concentration of a SCFA is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart. In some embodiments, an average stool concentration of a SCFA is calculated from at least two stool samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, an average stool concentration of a SCFA is calculated from at least two stool samples collected daily. In some embodiments, an average stool concentration of a SCFA is calculated from at least two stool samples collected weekly. In some embodiments, an average stool concentration of a SCFA is calculated from at least two stool samples collected monthly. In some embodiments, the subject did not have a Clostridium difficile infection during collection of their stool samples. In some embodiments, the average stool concentration of the SCFA is calculated as the mean of concentrations. In some embodiments, the average stool concentration of the SCFA is calculated as the median of concentrations.

In some embodiments, the SCFA is acetate, butyrate, formate, hexanoate (caproate), isobutyrate, isovalerate, propionate, succinate, valerate, or 2-methylbutyrate. In some embodiments, the SCFA is hexanoate, isovalerate, or butyrate. In some embodiments, the SCFA is hexanoate. In some embodiments, the SCFA is isovalerate. In some embodiments, the SCFA is butyrate.

Some aspects relate to administering a bacterial composition to a subject having a low serum concentration of one or more cytokines. As described in the Examples, lower serum concentrations of inflammatory cytokines, or cytokines associated with inflammation, were associated with higher probability of engraftment by bacterial strains of administered compositions, such as Flavonifractor plautii, and subjects in which this strain engrafted did not experience C. difficile infection recurrence. Thus, administration to subjects with lower serum concentrations of such cytokines is expected to increase the likelihood of engraftment by bacterial strains, and consequently reduce the likelihood of C. difficile infection recurrence.

In some embodiments, a composition is administered to a subject having a serum cytokine concentration that is lower than a reference serum concentration of the cytokine. In some embodiments, the cytokine is an inflammatory cytokine. An “inflammatory cytokine,” as used herein, refers to a cytokine that induces one or more inflammatory responses. Inflammatory responses include, without limitation, redness, swelling, heat, pain, loss of tissue function, increased vascular permeability, leukocyte recruitment, and leukocyte infiltration. In some embodiments, the cytokine is associated with inflammation. A cytokine “associated with inflammation,” as used herein, refers to a cytokine having elevated serum concentration in a subject experiencing inflammation. Those of ordinary skill in the art will understand that while IL- 10, for example, has anti-inflammatory effects, it is produced during an inflammatory response and plays a role in balancing inflammation to limit tissue damage and maintain homeostasis.

In some embodiments, a reference serum concentration of the cytokine is an average serum concentration of the cytokine. Any suitable method may be used to determine the average serum concentration of the cytokine. In some embodiments, the average serum concentration of the cytokine is determined by comparison to a reference database. An average concentration may be calculated using an entire set of concentration data available from a database. An average concentration may be calculated using a subset of a database. For example, an average concentration may be calculated using only data obtained from individuals of the same or similar age and/or sex of the subject. Other criteria for filtering samples of a database may include history of C. difficile infection, history of inflammatory bowel disease (IBD), diet, weight, cholecystectomy, surgical history, medication history, immunocompromised status, and/or family history of any of the preceding factors.

In some embodiments, a subject's serum concentration of the cytokine is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less than the reference serum concentration of the cytokine. In some embodiments, the average serum concentration of the cytokine is calculated as the mean of concentrations. In some embodiments, the average serum concentration of the cytokine is calculated as the median of concentrations.

The reference serum concentration of a cytokine may be a concentration measured in one or more previous serum samples of the subject. In some embodiments, a composition is administered to a subject having a serum cytokine concentration that is lower than a serum cytokine concentration of a previously measured serum sample from the subject. In some embodiments, the previously measured serum sample was obtained during a C. difficile infection. In some embodiments, the previously measured serum sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after detection of C. difficile infection. In some embodiments, the previously measured serum sample was obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before detection of C. difficile infection. In some embodiments, at the time of administration of a bacterial composition, the subject has a serum concentration of the cytokine that is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less than the cytokine concentration in a previously measured serum sample of the subject.

In some embodiments, a composition is a serum cytokine concentration that is lower than an average serum cytokine concentration of previously measured serum samples from the subject. In some embodiments, a subject's serum concentration of the cytokine is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less than the average serum concentration of the cytokine in the subject. An average serum concentration of the subject may be determined using any number of serum samples. In some embodiments, an average serum concentration of a cytokine is calculated from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 serum samples. In some embodiments, an average serum concentration of a cytokine is calculated from at least two serum samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days apart. In some embodiments, an average serum concentration of a cytokine is calculated from at least two serum samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart. In some embodiments, an average serum concentration of a cytokine is calculated from at least two serum samples collected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, an average serum concentration of a cytokine is calculated from at least two serum samples collected daily. In some embodiments, an average serum concentration of a cytokine is calculated from at least two serum samples collected weekly. In some embodiments, an average serum concentration of a cytokine is calculated from at least two serum samples collected monthly. In some embodiments, the subject did not have a Clostridium difficile infection during collection of their serum samples. In some embodiments, the average serum concentration of the cytokine is calculated as the mean of concentrations. In some embodiments, the average serum concentration of the cytokine is calculated as the median of concentrations.

In some embodiments, the cytokine is CXCL9, eotaxin-2, IL-6, IL- 10, IL- 18, IL-22, IL-27, MCP-2, MIP-1 , MIP-10, SCF, SDF-la, SDF-lb, or TNF-a. In some embodiments, the cytokine is eotaxin-2. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is IL-22. In some embodiments, the cytokine is IL-27. In some embodiments, the cytokine is MCP-2. In some embodiments, the cytokine is MIP-10. In some embodiments, the cytokine is MIP-10. In some embodiments, the cytokine is SCF. In some embodiments, the cytokine is SDF-la. In some embodiments, the cytokine is SDF-lb. In some embodiments, the cytokine is TNF-a.

Uses for treating or preventing C. difficile infection

Some aspects relate to methods of treating and/or preventing C. difficile infection. As used herein, methods of treating C. difficile infection involve relieving or alleviating at least one symptom associated with C. difficile infection. In some embodiments, the subject has a C. difficile infection. In some embodiments, the subject has a primary (first instance of) C. difficile infection. In some embodiments, the subject has a recurrent C. difficile infection. In some embodiments, the subject does not have a C. difficile infection at the time a composition is administered. In some embodiments, the subject has not previously had a C. difficile infection. In some embodiments, the bacterial strains of the compositions provided herein can treat and/or prevent C. difficile infection because of synergy between the bacterial strains.

In some embodiments, the therapeutically effective amount is an amount sufficient to restore the microbiome following a state of disease. In some embodiments, the therapeutically effective amount is an amount sufficient to reduce or eliminate at least one symptom associated with pathogenic infection, or reduce the severity of at least one symptom associated with pathogenic infection. In some embodiments, the therapeutically effective amount is an amount sufficient to reduce or eliminate at least one symptom associated with pathogenic infection, or reduce the severity of at least one symptom associated with pathogenic infection.

It should be appreciated that the term “effective amount,” in reference to a composition comprising bacterial strains, may be expressed as the number of bacteria or CFUs to be administered. It should further be appreciated that the bacteria can multiply once administered. Thus, administration of even a relatively small amount of bacteria may have therapeutic effects.

In some embodiments, the specific combination of one or more bacterial strains of the compositions described herein provides a synergistic effect that promotes treating and/or preventing pathogen infection and/or reducing the risk and/or occurrence of pathogen infection. In some embodiments, the specific combination of one or more bacterial strains of the compositions described herein provides a synergistic effect that promotes treating and/or preventing C. difficile infection and/or reducing the risk and/or recurrence of C. difficile infection. In some embodiments, the specific combination of one or more bacterial strains of the compositions described herein provides a synergistic effect that promotes treating and/or preventing C. difficile infection in a subject. In some embodiments, the synergistic effect is provided by the capacity of the combination to metabolize specific nutrients. In some embodiments, the synergistic effect is provided by the capacity of the combination to provide specific metabolites to the environment. Such specific metabolites may suppress growth of the pathogen and/or stimulate growth of non-pathogens.

In some embodiments, the synergistic effect is provided by the capacity of the combination to provide short-chain fatty acids to the environment. In some embodiments, the synergistic effect is provided by the capacity of the combination to provide specific shortchain fatty acids to the environment. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce butyrate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce acetate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce lactate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce propionate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce succinate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce multiple metabolites. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce multiple short-chain fatty acids. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce both butyrate and acetate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce both butyrate and lactate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce both butyrate and propionate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce both butyrate and succinate. In some embodiments, the synergistic effect is provided by the capacity of the combination to produce butyrate, acetate, and additional short-chain fatty acids.

In some embodiments, the specific combination of two or more bacterial strains of the compositions provided herein is superior in the use of nutrients and in grafting when compared to other strains (e.g., pathogens), thereby protecting and or restoring the microbiome, for instance through suppressing the growth of the pathogen. In some embodiments, the specific combination of one or more bacterial strains of the compositions provided herein induces an immune response in the subject that promotes colonizing the microbiome of the subject with one of or of the bacterial strains of the compositions. In some embodiments, the specific combination of one or more bacterial strains of the compositions provided herein induces an immune response in the subject that reduces symptoms of C. difficile infection or risk of C. difficile infection recurrence.

Uses for colonizing a microbiome

Some aspects relate to methods of colonizing a microbiome, which comprise administering a composition comprising two or more bacterial strains. For example, a composition, pharmaceutical composition, or food product as described herein may be administered to a subject. The composition may be administered in an amount sufficient for one or more bacterial strains of the composition to colonize the microbiome of a subject. In some embodiments, the composition is administered in a therapeutically effective amount for treating a pathogenic infection in a subject. In some embodiments, the method further comprises administering an antibiotic to the subject prior to administration of the composition. In some embodiments, the method further comprises administering vancomycin to the subject prior to administration of any of the compositions described herein.

In some embodiments, the two or more of the bacterial strains of the compositions provided herein colonize or recolonize the gastrointestinal tract or parts thereof (e.g., the colon or the cecum) of the subject. Such colonization may also be referred to as grafting or engraftment. In some embodiments, two or more of the bacterial strains of the compositions recolonize the intestinal tract (e.g., the colon or the cecum) of the subject after the naturally present microbiome has been partially or completely removed, e.g., due to administration of an antibiotic. In some embodiments, the two or more of the bacterial strains of the compositions recolonize the intestinal tract (e.g., the colon or the cecum) of the subject after the naturally present microbiome has been partially or completely removed by antibiotic (e.g., vancomycin) treatment. In some embodiments, the two or more of the bacterial strains of the compositions colonize a dysbiotic gastrointestinal tract (e.g., a gastrointestinal tract that has undergone antibiotic treatment). In some embodiments, all of the bacterial strains of the composition colonize the gastrointestinal tract. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of the bacterial strains of the compositions colonize the gastrointestinal tract. In some embodiments, all of the bacterial strains of the compositions colonize a dysbiotic gastrointestinal tract. In some embodiments, multiple doses of the bacterial compositions are administered to allow for all of the bacterial strains of the composition colonize the gastrointestinal tract. In some embodiments, multiple doses of the bacterial compositions are administered to allow for all of the bacterial strains of the compositions colonize a dysbiotic gastrointestinal tract. In some embodiments, colonization of the microbiome using the compositions and methods provide for an increase in the abundance of bacterial species beneficial to the microbiome.

In some embodiments, colonization of a microbiome with the bacterial strains of the compositions described herein results in a healthy microbiome. As used herein, a “healthy microbiome,” refers to a microbiome from a subject who does not have overt disease (e.g., a healthy subject). Although the microbial composition of healthy microbiomes can vary widely, several trends have emerged which characterize healthy microbiomes. For example, the gastrointestinal microbiome may perform a number of metabolic and/or other molecular functions, including the metabolism of carbohydrates, lipids, and other nutrients which are performed by healthy microbiomes, regardless of the specific species composition. In some instances, the metabolic and molecular functions carried out by a healthy microbiome cannot be performed by the host subject, resulting in a symbiotic host-microbial relationship. Additionally, healthy microbiomes tend to be resilient to external (e.g., dietary or pharmaceutical) and/or internal (e.g., age, disease- state, stress, inflammation) changes in the subject. The resilience of a healthy microbiome can also be characterized by the ability and the rate at which a healthy state is restored after occurrence of a perturbation. Alternatively, or in addition, a healthy microbiome may be characterized by a high (e.g., greater than 75%) relative abundance of bacterial species from the phylum Firmicutes and genus Bacteroides relative to species from the phylum Proteobacteria. In some embodiments, administration of the compositions described herein reduces the risk of C. difficile infection (CDI) in a subject. In some embodiments, administration of the compositions described herein prevents CDI in a subject. In some embodiments, administration of the compositions reduces the risk of recurrent C. difficile infection (rCDI) in a subject. rCDI is CDI that occurs more than once in the same subject. rCDI is associated with reduced abundance of SCFAs, increased abundance of primary bile acids, and reduced abundance of secondary bile acids. In some embodiments, administration of the compositions described herein prevents rCDI in a subject.

In some embodiments, administration of the compositions described herein results in a decrease in the abundance of microorganisms associated with recurrence of C. difficile infection. Microorganisms associated with recurrence of C. difficile infection are described herein in Example 1 and include, for example, Lactobacillus harbinensis, Campylobacter concisus, Lactobacillus rhamnosus, Clostridia bacterium UC5.1.1F8, Veillonella sp. oral taxon 158, Lactobacillus paracasei, Blautia massiliensis, Bacteroides caccae CAG.21, Enterobacter cloacae. Additional microorganisms associated with recurrence of C. difficile infection include those of the genera Campylobacter, Kluyvera, and Veillonella, those of the family Campylobacteraceae, those of the order Campylobacterales, and those of the class Epsilonproteobacteria.

In some embodiments, administration of the compositions described herein results in a decrease in abundance of Proteobacteria in the subject (or microbiome thereof) by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10⁴-fold, 10⁵- fold or more, as compared to the abundance of Proteobacteria in the subject (or microbiome thereof) prior to administering the compositions. In some embodiments, the abundance of Proteobacteria in the subject prior to the administration of the compositions was higher because of treatment with an antibiotic. In some embodiments, the abundance of Proteobacteria in the subject prior to the administration of the compositions was higher because of treatment with an antibiotic. In some embodiments, administration of the compositions described herein results in a decrease in abundance of Proteobacteria in the subject (or microbiome thereof) by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10⁴-fold, 10⁵-fold or more, as compared to the abundance of Proteobacteria in another subject (e.g., a reference subject) (or microbiome thereof) who did not receive the compositions.

In some embodiments, administration of the compositions described herein results in a decrease in abundance of Proteobacteria in the subject (or microbiome thereof) by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, as compared to the abundance of Proteobacteria in the subject (or microbiome thereof) prior to administering the compositions. In some embodiments, administration of the compositions described herein results in decrease in the abundance of Proteobacteria in the subject (or microbiome thereof) by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150% or more, as compared to the abundance of Proteobacteria in a subject (e.g., a reference subject) (or microbiome thereof) who did not receive the compositions.

In some embodiments, the one or more bacterial strains of the compositions colonize the microbiome because they can “outgrow” other bacterial strains (e.g., pathogens such as C. difficile'). In some embodiments, the subject has been treated with an antibiotic resulting in a removal of most of the microbiome, providing a “clean slate” environment for both the one or more bacterial strains of compositions and any other bacterial strains (e.g., pathogens, strains associated with inflammation or undesired immune responses). Thus, without being limited to a specific mechanism, if a pathogen and bacterial strains of the compositions provided herein are both present in the intestinal tract (e.g., the colon or the cecum), the bacterial strains of compositions provided herein grow faster (e.g., have a shorter doubling time) than the pathogen, thereby preventing the pathogen from accumulating in the intestinal tract (e.g., the colon or the cecum) and allowing the bacterial strains of the compositions to colonize. In some embodiments, the faster growth results because the bacterial strains of the compositions provided herein are better at grafting in the intestinal tract (e.g., the colon or the cecum). In some embodiments, the faster growth results because the bacterial strains of the compositions provided herein are better at metabolizing nutrients present in the intestinal tract (e.g., the colon or the cecum). In some embodiments, the compositions of bacterial strains provided herein prevent or inhibit production of bacterial toxins by an infectious agent, or prevent or inhibit the cytopathic or cytotoxic effects of such toxins. In some embodiments, the bacterial strains of the compositions provided herein can treat pathogenic infections, because of the synergy between the bacterial strains. Thus, without being limiting, in some embodiments, the combination of the bacterial strains of the compositions provided herein act synergistically because the combination of the strains is particularly well- suited to use nutrients in the intestinal tract (e.g., the colon or the cecum), or instance through metabolic interactions, and/or because the combination is superior in grafting (e.g., by providing a favorable microenvironment). In some embodiments, the bacterial strains of the compositions described herein are able to colonize specific niches in the intestinal tract (e.g., the colon or the cecum). In some embodiments, the bacterial strains of the compositions described herein are able to colonize specific niches in the intestinal tract (e.g., the colon or the cecum) that became available after antibiotic treatment.

In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the bacterial strains of the composition colonize the microbiome of the subject. In some embodiments, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7- 10, 8-10, 9-10, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 of the bacterial strains of the composition colonize the microbiome of the subject. In some embodiments, 1 to 3 of the bacterial strains of the composition colonize the microbiome of the subject. In some embodiments, 1 to 8 of the bacterial strains of the composition colonize the microbiome of the subject. In some embodiments, 3 to 8 of the bacterial strains of the composition colonize the microbiome of the subject. In some embodiments, Clostridium bolteae colonizes the microbiome of the subject. In some embodiments, Anaerotruncus colihominis colonizes the microbiome of the subject. In some embodiments, Sellimonas intestinalis colonizes the microbiome of the subject. In some embodiments, Clostridium symbiosum colonizes the microbiome of the subject. In some embodiments, Blautia producta colonizes the microbiome of the subject. In some embodiments, Dorea longicatena colonizes the microbiome of the subject. In some embodiments, Clostridium innocuum colonizes the microbiome of the subject. In some embodiments, Flavonifractor plautii colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 1 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 2 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 3 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 4 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 5 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 6 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 7 colonizes the microbiome of the subject. In some embodiments, a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 8 colonizes the microbiome of the subject. The extent of colonization of any of the bacterial strains may be determined, for example by detecting the presence of one or more bacterial strains and/or by quantifying the abundance of the one or more bacterial strains. In some embodiments, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,

28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,

44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,

60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the bacterial strains of the compositions colonize the microbiome of the subject. In some embodiments, at least 25% of the bacterial strains of the compositions colonize the microbiome of the subject. In some embodiments, at least 50% of the bacterial strains of the compositions colonize the microbiome of the subject. In some embodiments, 100% of the bacterial strains of the compositions colonize the microbiome of the subject. In some embodiments, the percentage of the bacterial strains of the compositions that colonize the microbiome of the subject is increased by administering additional doses of the compositions.

Uses for metabolite modulation

Some aspects of the compositions and methods described herein decrease the abundance of primary bile acids, increase the abundance of secondary bile acids, and/or promote conversion of primary bile acids into secondary bile acids. Bile acids are steroid acids that allow the digestion of dietary fats and oils by acting as surfactants that turn the fats and oils into micelles. Bile acids also act as hormones utilizing the farnesoid X receptor and GBPAR1. Primary bile acids are synthesized in the liver from cholesterol and a conjugated with either taurine or glycine prior to secretion. When the primary bile acids are secreted into the lumen of the intestine, bacteria partially dehydroxylate and remove the glycine or taurine groups, forming secondary bile acids.

Some Clostridia strains, such as those of the compositions and methods provided herein, effectively metabolize primary bile acids to secondary bile acids and also produce substantial amounts of SCFAs. The metabolic activities of these and other bacterial strains are described, for instance, in PCT Publication No. WO 2020/037271, which is incorporated herein by reference in its entirety. See, e.g., Example 5 of PCT Publication No. WO 2020/037271, demonstrating the ability of Clostridium bolteae, Anaerotruncus colihominis, Drancourtella massiliensis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and F lav onifr actor plautii to increase levels of secondary bile acids in a subject when administered after antibiotic treatment to clear other gut-resident microflora (e.g., FIGs. 32-33). See, also, Example 6 of PCT Publication No. WO 2020/037271, demonstrating the ability of the same bacterial strains to produce SCFAs such as acetate, propionate, and butyrate, when administered to subjects under similar conditions (e.g., FIGs. 37-38).

Non-limiting examples of primary bile acids are cholic acid (CA), chenodeoxy cholic acid (CDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), and taurochenodeoxycholic acid (TCDCA). Non-limiting examples of secondary bile acids are deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), and tauroursodeoxycholic acid (TUDCA).

Several diseases and disorders, such as inflammatory bowel disease (e.g., ulcerative colitis), cancer (e.g., colorectal carcinoma), and pathogenic organism infections (e.g., Clostridium difficile infection), are associated with increased primary bile acids and reduced secondary bile acids. The primary bile acids are reduced, and the secondary bile acids are increased following fecal matter transplant (FMT) (Seekatz, et al., Anaerobe (2018) 53: 64- 73). In some embodiments, administration of the bacterial strains or a composition as described herein reduces primary bile acids and/or increases secondary bile acids.

In some embodiments, the levels of primary bile acids are reduced by 10-fold to 100,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of primary bile acids are reduced by 2-fold to 1,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of primary bile acids are reduced by 10- fold to 1,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of primary bile acids are reduced 20-fold to 10,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of primary bile acids are reduced by 2- fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 10,000-fold, 20,000-fold, 30,000-fold, 40,000-fold, 50,000- fold, 60,000-fold, 70,000-fold, 80,000-fold, 90,000-fold, or 100,000-fold following administration of the bacterial strains or any of the compositions described herein.

In some embodiments, the levels of secondary bile acids are increased by 2-fold to 10,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of secondary bile acids are increased by 10-fold to 10,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of secondary bile acids are increased by 10-fold to 1,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of secondary bile acids are increased by 20-fold to 100-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, the levels of secondary bile acids are increased by 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000- fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, or 1,000-fold following administration of the bacterial strains or any of the compositions described herein.

Some embodiments of the compositions and methods described herein increase production of regulatory metabolites, such as short chain fatty acids (e.g., in the gastrointestinal tract of the subject), in a subject. In some embodiments, the methods involve administering to a subject one or more compositions containing bacterial strains that produce short chain fatty acids. SCFAs are abundant in healthy subjects and decreased in subjects having particular diseases and disorders.

SCFA produced in the gastrointestinal tract are thought to function as signaling molecules between the gut microbiota and the host organism, with the SCFA playing a role in local, intermediary and peripheral metabolism of the host. See, e.g., Morrison, et al. Gut Microbes (2016) 7(3): 189-200.

Short chain fatty acids (SCFAs) are fatty acids containing six or fewer carbon atoms. They are produced when dietary fiber is fermented in the intestine. They are primarily absorbed in the portal vein following lipid digestion. SCFAs can affect the production of lipids, energy, and vitamins, as well as playing a critical role in maintaining intestinal epithelial cell membrane integrity. Examples of SCFA include, without limitation, formic acid, acetic acid, butyric acid, isobutyric acid, valeric acid, or isovaleric acid.

In some embodiments, SCFAs are increased by 2-fold to 10,000-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, SCFAs are increased by 10-fold to 500-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, SCFAs are increased by 2-fold to 250-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, SCFAs are increased by 100-fold to 500-fold following administration of the bacterial strains or any of the compositions described herein. In some embodiments, SCFAs are increased by 2-fold, 5-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300- fold, 400-fold, or 500-fold following administration of the bacterial strains or any of the compositions described herein.

Uses for Treg induction

In some embodiments, the compositions provided herein induce the proliferation and/or accumulation of regulatory T cells in the subject. As will be evident to one of ordinary skill in the art, regulatory T cells, also referred to as “Tregs,” are a subset of T lymphocytes that are generally thought to suppress an abnormal or excessive immune response and play a role in immune tolerance. Regulatory T cells may be identified based expression of the markers Foxp3 and CD4 (Foxp3+ CD4+). The term regulatory T cells may also include Foxp3-negative regulatory T cells that are IL-10-producing CD4-positive T cells.

In some embodiments, the therapeutically effective amount is an amount sufficient to induce the proliferation and/or accumulation of Tregs in the subject (or in a sample obtained from a subject) by at least 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 150-fold, 200-fold, 500-fold or more, as compared to the amount of Tregs in a subject (e.g. , a subject having or at risk of IBD or GvHD) that has not received any of the compositions described herein or as compared to a fecal sample from the same subject that was collected prior to administration of any of the compositions.

As used herein, the phrase “induces proliferation and/or accumulation of regulatory T cells” refers to an effect of inducing the differentiation of immature T cells into regulatory T cells, which differentiation leads to the proliferation and/or the accumulation of regulatory T cells. Further, the meaning of “induces proliferation and/or accumulation of regulatory T cells” includes in vivo effects, in vitro effects, and ex vivo effects. In some embodiments, the proliferation and/or accumulation of regulatory T cells may be assessed by detecting and/or quantifying the number of cells that express markers of regulatory T cells (e.g., Foxp3 and CD4), for example by flow cytometry. In some embodiments, the proliferation and/or accumulation of regulatory T cells may be assessed by determining the activity of the regulatory T cells, such as the production of cytokines (e.g., IL-10).

In some embodiments, administration of the compositions described herein results in an increase in the proliferation and/or accumulation of regulatory T cells (e.g., total Tregs or a specific subset of Treg) by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10⁴-fold, 10⁵-fold or more, as compared to the quantity of regulatory T cells in the subject (or particular site in the subject) prior to administration of the compositions. In some embodiments, administration of the compositions described herein results in an increase in the proliferation and/or accumulation of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10⁴- fold, 10⁵-fold or more, as compared to the quantity of regulatory T cells in another subject (e.g., a reference subject) who did not receive the compositions.

In some embodiments, administration of the compositions described herein results in an increase in the proliferation and/or accumulation of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150% or more, as compared to the quantity of regulatory T cells in the subject (or particular site in the subject) prior to administration of the compositions. In some embodiments, administration of the compositions described herein results in an increase in the proliferation and/or accumulation of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150% or more, as compared to the quantity of regulatory T cells in another subject (e.g., a reference subject) who did not receive the compositions.

In some embodiments, administration of the compositions described herein results in an increase in the activity of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) at a particular site (e.g., the gastrointestinal tract) in the subject. In some embodiments, administration of the compositions described herein results in an increase in the activity of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10⁴-fold, 10⁵-fold or more, as compared to the activity of regulatory T cells in the subject (or particular site in the subject) prior to administration of the compositions. In some embodiments, administration of the compositions described herein results in an increase in the activity of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10⁴-fold, 10⁵-fold or more, as compared to the activity of regulatory T cells in another subject (e.g., a reference subject) who did not receive the compositions.

In some embodiments, administration of the compositions described herein results in an increase in the activity of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150% or more, as compared to the activity of regulatory T cells in the subject (or particular site in the subject) prior to administration of the compositions. In some embodiments, administration of the compositions described herein results in an increase in the activity of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150% or more, as compared to the activity of regulatory T cells in another subject (e.g., a reference subject) who did not receive the compositions.

The abundance of regulatory T cells (e.g., total Tregs or a specific subset of Tregs) can be assessed by any method known in the art, for example by detecting a cellular marker indicative of regulatory T cells (e.g., FoxP3), assessing a direct or indirect activity of regulatory T cells, and/or by measuring the production of one or more cytokines produced by regulatory T cells (e.g., IL-10).

Uses with antibiotics

In some embodiments, administration of multiple doses of the compositions described herein provides enhanced colonization (engraftment) of one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of a single dose of the composition results in the same or a similar level of engraftment (e.g., total bacteria) as administration of multiple doses of the composition, however the engraftment may be dominated by one bacterial strain or only a subset of the bacterial strains of the compositions.

Dosage regimes

Any of the methods described herein may further comprise administering vancomycin to the subject prior to administration of the compositions described herein. In some embodiments, the subject was previously administered vancomycin prior to administration of the compositions described herein. In some embodiments, administration of any of the compositions described herein is not preceded by administration of an antibiotic other than vancomycin. Vancomycin administration has been found to alter the composition of human gut microbiota. See, e.g., Reijnders et al. Cell Metabolism (2016) 24(1): 63-72. Without wishing to be bound by any particular theory, it is thought that administration of vancomycin may aid engraftment of the bacterial strain(s) of the compositions described herein, for example by removing other microbes present in the gastrointestinal tract.

In some embodiments, the vancomycin is administered at a dose (a single dose or multiple doses) in a sufficient amount to allow for colonization of one or more of the bacterial strains of the compositions described herein. In some embodiments, the vancomycin is administered to the subject once, as a single dose. In some embodiments, the vancomycin is administered to the subject in multiple doses. In some embodiments, the vancomycin is administered to the subject in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more doses. The multiple doses of vancomycin may be administered to the subject at regular intervals prior to administering any of the compositions described herein. In some embodiments, each of the multiple doses of vancomycin are administered on consecutive days e.g., first dose on day 1, second dose of day 2, third dose on day 3, etc.). In some embodiments, the vancomycin is administered to the subject for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive days. In some embodiments, vancomycin is administered to the subject for one day. In some embodiments, vancomycin is administered to the subject each day for three consecutive days. In some embodiments, vancomycin is administered to the subject each day for five consecutive days. In some embodiments, the vancomycin is administered to the subject each day for seven consecutive days. In any of the embodiments described herein, a subject may be administered one or more doses of a first antibiotic followed by one or more doses of a second antibiotic.

In some embodiments, a single dose of any of the compositions described herein, or the first dose in a treatment regimen of multiple doses, is administered, the same day as the administration of the final dose of vancomycin. In some embodiments, a single dose of any of the compositions described herein, or the first dose in a treatment regimen of multiple doses, is administered, immediately following the administration of the final dose of vancomycin. In some embodiments, a single dose of any of the compositions described herein, or the first dose in a treatment regimen of multiple doses, is administered, the day after administration of the final dose of vancomycin. In some embodiments, a single dose of any of the compositions described herein, or the first dose in a treatment regimen of multiple doses, is administered, two days after administration of the final dose of vancomycin. In some embodiments, the methods provided herein allow for a wash out day between the final dose of vancomycin and the first dose of the composition. In some embodiments, a single dose of any of the compositions described herein, or the first dose in a treatment regimen of multiple doses, is administered, three days, four days, five days, six days, ten days or more, after administration of the final dose of vancomycin. In some embodiments, the methods provided herein allow for multiple wash out days between the final dose of vancomycin and the first dose of the composition. In some embodiments, the methods provided herein allow for two wash out days between the final dose of vancomycin and the first dose of the composition.

Each dose of the vancomycin may be the same amount of vancomycin or may be a different amount of vancomycin. In some embodiments, the vancomycin is administered in an amount sufficient to allow for colonization of one or more of the bacterial strains of the compositions described herein. In some embodiments, the subject is administered between about 50 mg and 1 g, 100 mg and 750 mg, 100 mg and 500 mg, 200 mg and 750 mg, 200 mg and 500 mg, 300 mg and 750 mg, 300 mg and 500 mg, 100 mg and 400 mg, 100 mg and 300 mg, 100 mg and 200 mg, 200 mg and 400 mg, 200 mg and 300 mg, or 450 mg to 550 mg vancomycin per day. As will be appreciated by one of skill in the art, the total amount of vancomycin administered to the subject per day may be administered in a single dose or between multiple doses, which in sum results in the total amount of vancomycin per day.

In some embodiments, the subject is administered about 500 mg vancomycin per day prior to administration of any of the compositions described herein. In some embodiments, 500 mg vancomycin per day is administered in a single dose (e.g., 500 mg). In some embodiments, 500 mg vancomycin per day is administered in multiple doses (e.g., 2, 3, 4, 5 or more), which in sum results in 500 mg vancomycin per day. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day. In some embodiments, 500 mg vancomycin is administered to the subject for one day. In some embodiments, 500 mg vancomycin is administered to the subject per day for two days. In some embodiments, 500 mg vancomycin is administered to the subject per day for three days. In some embodiments, 500 mg vancomycin is administered to the subject per day for four days. In some embodiments, 500 mg vancomycin is administered to the subject per day for five days. In some embodiments, 500 mg vancomycin is administered to the subject per day for six days. In some embodiments, 500 mg vancomycin is administered to the subject per day for seven days. In some embodiments, 500 mg vancomycin is administered to the subject per day for eight days. In some embodiments, 500 mg vancomycin is administered to the subject per day for nine days. In some embodiments, 500 mg vancomycin is administered to the subject per day for ten days.

In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for one day. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for two days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for three days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for four days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for five days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for six days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for seven days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for eight days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for nine days. In some embodiments, 500 mg vancomycin is administered in 4 doses of 125 mg vancomycin per day for ten days.

In some embodiments, the vancomycin is administered according to a pulse tapered- regimen. See e.g., Sirbu et al., Clinical Infectious Diseases (2017) 65: 1396-1399.

In some embodiments, the vancomycin is administered to the subject at least 1, 2, 3, 4, 5, 6, 7 days or more prior to administration of the compositions described herein. In some embodiments, administration of vancomycin is terminated at least one day e.g., 1, 2, 3, 4, 5, or more) prior to administration of any of the compositions described herein. In some embodiments, administration of vancomycin is terminated two days prior to administration of any of the compositions described herein.

In some embodiments, additional antibiotics are administered in combination with the vancomycin regimes provided herein.

It should be appreciated, in some embodiments, that any of the vancomycin doses or administration regimens may be combined with any of the composition doses or administration regimens provided herein.

In some embodiments, the disclosure provides methods comprising administering one or more antibiotics to the subject and subsequently administering any of the bacterial compositions to the subject once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, at least 14 times or more. In some embodiments, the disclosure provides methods comprising administering one or more antibiotics to the subject and subsequently administering any of the bacterial compositions described herein to the subject in multiple doses at a regular interval, such as every 2 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or more. In some embodiments, one dose of any of the compositions described herein is administered and a second dose of the composition is administered the following day (e.g., the consecutive day). In some embodiments, one dose of any of the compositions described herein is administered and each of the additional doses of the composition are administered on consecutive days (e.g., first dose on day 1, second dose on day 2, third dose on day 3, etc.).

In some embodiments, the subject is treated by multiple cycles, each of which may involve a period of administering multiple doses of any of the compositions described herein, followed by a period during which the compositions are not administered. In some embodiments, each cycle further involves administering antibiotics prior to administering any of the compositions described herein. In some embodiments, the subject is treated by multiple cycles, each of which may involve a period of administering multiple doses of any of the compositions described herein for at least 7 days, followed by a period during which the compositions are not administered. In some embodiments, the subject is treated by multiple cycles, each of which may involve a period of administering multiple doses of any of the compositions described herein for at least 14 days, followed by a period during which the compositions are not administered. In some embodiments, a cycle further comprises determining whether the subject is colonized by one or more of the bacterial strains of the composition, and administering the composition if the one or more of the bacteria of the composition are not detected. In some embodiments, the cycle is repeated after a period of time, such as after 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In one aspect, the disclosure provides methods comprising administering one or more antibiotics to the subject and subsequently administering any of the bacterial compositions as multiple daily doses of the compositions. In some embodiments, the compositions are administered on a daily basis for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

In some embodiments, the compositions are administered on a daily basis for a period of time, followed by a period of time during which the compositions are not administered. In some embodiments, the methods may involve administering the compositions to the subject on a daily basis for a second period of time (e.g., a cycle), which may be followed a second period of time during which the compositions are not administered.

In one aspect the disclosure provides methods comprising the administration of an antibiotic (e.g., vancomycin) followed by the administration of a composition provided herein, wherein the administration of an antibiotic (e.g., vancomycin) is followed by the administration of a single dose or multiple doses of the composition. In some embodiments, administration of an antibiotic (e.g., vancomycin) followed by the administration of a single dose or multiple doses of the composition results in an increase in the abundance of bacterial strains of the compositions in the microbiome of the subject (engraftment) compared to the administration of a composition without the administration of the antibiotic. In some embodiments, administration of an antibiotic (e.g., vancomycin) followed by the administration of a single dose or multiple doses of the composition results in an increase in the duration of the colonization of bacterial strains of the composition in the microbiome of the subject (e.g., up to 6 months) compared to the administration of a composition without the administration of the antibiotic.

In some embodiments, administration of an antibiotic (e.g., vancomycin) followed by the administration of a single dose or multiple doses of the composition results in an increase in the rate of engraftment of the initial amount of the bacterial strains of the composition in the microbiome of the subject by between ten- to one hundred- fold (e.g., within the first 48 hours) compared to the administration of a composition without the administration of the antibiotic.

In some embodiments, administration of an antibiotic (e.g., vancomycin) followed by the administration of a single dose or multiple doses of the composition results in a greater number (amount) of subjects having all of the bacterial strains of the composition present in their microbiome as compared to compared to the administration of a composition without the administration of the antibiotic.

In some embodiments, administration of an antibiotic (e.g., vancomycin) followed by the administration of multiple doses of the composition results in a higher abundance of the bacterial strains of the composition in the microbiome of the subject as compared to the administration of a single dose of the composition. In some embodiments, the disclosure provides methods comprising the administration of a composition provided herein, wherein the administration of multiple doses of the composition results in higher abundance of the bacterial strains of the composition in the microbiome of the subject compared to the administration of a single dose of the composition. In some embodiments, administration of an antibiotic (e.g., vancomycin) followed by the administration of multiple doses of the composition results in a greater number (amount) of subjects having all of the bacterial strains of the composition present in their microbiome as compared to the administration of a single dose of the composition. In some embodiments, the disclosure provides methods comprising the administration of a composition provided herein, wherein the administration of multiple doses of the results in a greater number (amount) of subject having all of the bacterial strains of the composition in their microbiome as compared to the administration of a single dose of the composition.

A physician, veterinarian or other trained practitioner, can start doses of the composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect (e.g., colonization of a microbiome, treatment of pathogenic (e.g., C. difficile) infection) is achieved. In general, effective doses of the compositions described herein, for the prophylactic treatment of groups of people as described herein vary depending upon many different factors, including routes of administration, physiological state of the subject, whether the subject is human or an animal, other medications administered, and the therapeutic effect desired. Dosages need to be titrated to optimize safety and efficacy. In some embodiments, the dosing regimen entails oral administration of a dose of any of the compositions described herein. In some embodiments, the dosing regimen entails oral administration of a single dose of any of the compositions described herein. In some embodiments, the dosing regimen entails oral administration of multiple doses of any of the compositions described herein. In some embodiments, any of the compositions described herein are administered the subject once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, or more. In some embodiments, any of the compositions described herein are administered the subject in multiple doses at a regular interval, such as every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or more. In some embodiments, one dose of any of the compositions described herein is administered and a second dose of the composition is administered the following day (e.g., consecutive day). In some embodiments, one dose of any of the compositions described herein is administered and each of the additional doses of the composition are administered on consecutive days (e.g., first dose on day 1, second dose of day 2, third dose on day 3, etc.). In some embodiments, the disclosure relates to methods comprising administration of multiple doses of the compositions. In some embodiments, the disclosure relates to methods comprising administration of antibiotic (e.g., vancomycin) followed by multiple doses of the compositions. In some embodiments, administration of multiple doses of the compositions described herein provides enhanced colonization (engraftment) of one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides enhanced recovery of one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides increased abundance of one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides an increase in the number of subjects that were colonized with of all of bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides enhanced durability of colonization with one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides durable colonization of some or all of the bacterial strains of the compositions described herein over an extended period of time as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides durable colonization (e.g., up to 6 months) of one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. In some embodiments, administration of multiple doses of the compositions described herein provides durable colonization (e.g., up to 6 months or longer) of some or all of the bacterial strains of the compositions as compared to administration of a single dose of the composition. It should further be appreciated that administration of multiple doses may result in a combination of the results described. Thus, for example, in some embodiments, administration of multiple doses of the compositions described herein provides enhanced colonization (engraftment) and increased rate of recovery of one or more bacterial strains of the compositions as compared to administration of a single dose of the composition. Dosage amounts

Dosages of the active ingredients in the compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired pharmaceutical response for a particular subject, composition, and mode of administration, without being toxic or having an adverse effect on the subject. The selected dosage level depends upon a variety of factors including the activity of the particular compositions employed, the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health, and prior medical history of the subject being treated, and like factors.

The compositions, including the compositions disclosed herein, include compositions that contain selected bacterial strains. The amount of bacteria, including the amount of bacteria of each of the bacterial strains, in the compositions, including compositions, may be expressed in weight, number of bacteria and/or CFUs (colony-forming units). In some embodiments, the compositions, including compositions, comprise about 10, about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about IO¹⁰, about 10¹¹, about 10¹², about 10¹³ or more of each of the bacterial strains per dosage amount. In some embodiments, the compositions, including compositions, comprise about 10, about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about IO¹⁰, about 10¹¹, about 10¹², about 10¹³ or more total bacteria per dosage amount. It should further be appreciated that bacteria of each of the bacterial strains may be present in different amounts. Thus, for instance, as a non-limiting example, the composition may include 10³ of bacteria A, 10⁴ of bacteria B and 10⁶ of bacteria C. In some embodiments, the compositions, including compositions, comprise about 10, about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about IO¹⁰, about 10¹¹, about 10¹², about 10¹³ or more CFUs of each of the bacterial strains per dosage amount. In some embodiments, the compositions, including compositions, comprise about 10¹, about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about IO¹⁰, about 10¹¹, about 10¹², about 10¹³ or more CFUs in total for all of the bacterial strains combined per dosage amount. As discussed above, bacteria of each of the bacterial strains may be present in different amounts. In some embodiments, the compositions, including compositions, contain about 10'⁷, about 10'⁶, about 10'⁵, about 10'⁴, about 10'³, about 10'², about 10'¹ or more grams of bacteria of each of the bacterial strains in the composition per dosage amount. In some embodiments, the compositions, including compositions, contain about 10'⁷, about 10'⁶, about 10'⁵, about 10'⁴, about 10'³, about 10'², about 10'¹ or more grams of bacteria in total for all of the bacterial strains combined per dosage amount. In some embodiments, the compositions, including compositions, comprise about 10⁹ CFUs in total for all of the bacterial strains combined per dosage amount. In some embodiments, the compositions, including compositions, comprise about IO¹⁰ CFUs in total for all of the bacterial strains combined per dosage amount.

In some embodiments, the dosage amount is one administration device (e.g., one table, pill or capsule). In some embodiments, the dosage amount is the amount administered at one time, which may be in the form of more than one administration device (e.g., more than one table, pill or capsule). In some embodiments, the dosage amount is the amount that is administered in a particular period (e.g., one day or one week).

As described herein, any of the compositions described herein may be administered once, as a single dose. In some embodiments, the compositions described herein are administered in multiple doses. In some embodiments, each dose is administered in the form of one or more capsules. In some embodiments, each dose comprises administration of multiple capsules. In some embodiments, each dose is administered in the form of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more capsules. In some embodiments, each dose is administered in the form of 5 capsules. In some embodiments, each dose is administered in the form of 10 capsules.

In some embodiments, each capsule contains between 10 and 10¹³, between 10² and 10¹³, between 10³ and 10¹³, between 10⁴ and 10¹³, between 10⁵ and 10¹³, between 10⁶ and

10¹³, between 10⁷ and 10¹³, between 10⁸ and 10¹³, between 10⁹ and 10¹³, between IO¹⁰ and

10¹³, between 10¹¹ and 10¹³, between 10¹² and 10¹³, between 10 and 10¹², between 10² and 10¹², between 10³ and 10¹², between 10⁴ and 10¹², between 10⁵ and 10¹², between 10⁶ and

10¹², between 10⁷ and 10¹², between 10⁸ and 10¹², between 10⁹ and 10¹², between IO¹⁰ and

10¹², between 10¹¹ and 10¹², between 10 and 10¹¹, between 10² and 10¹¹, between 10³ and

10¹³, between 10⁴ and 10¹³, between 10⁵ and 10¹³, between 10⁶ and 10¹³, between 10⁷ and

10¹¹, between 10⁸ and 10¹¹, between 10⁹ and 10¹¹, between IO¹⁰ and 10¹¹, between 10 and

IO¹⁰, between 10² and IO¹⁰, between 10³ and IO¹⁰, between 10⁴ and IO¹⁰, between 10⁵ and

IO¹⁰, between 10⁶ and IO¹⁰, between 10⁷ and IO¹⁰, between 10⁸ and IO¹⁰, between 10⁹ and

IO¹⁰, between 10 and 10⁹, between 10² and 10⁹, between 10³ and 10⁹, between 10⁴ and 10⁹, between 10⁵ and 10⁹, between 10⁶ and 10⁹, between 10⁷ and 10⁹, between 10⁸ and 10⁹, between 10 and 10⁸, between 10² and 10⁸, between 10³ and 10⁸, between 10⁴ and 10⁸, between 10⁵ and 10⁸, between 10⁶ and 10⁸, between 10⁷ and 10⁸, between 10 and 10⁷, between 10² and 10⁷, between 10³ and 10⁷, between 10⁴ and 10⁷, between 10⁵ and 10⁷, between 10⁶ and 10⁷, between 10 and 10⁶, between 10² and 10⁶, between IO³ and 10⁶, between 10⁴ and 10⁶, between 10⁵ and 10⁶, between 10 and 10⁵, between 10² and 10⁵, between IO³ and 10⁵, between 10⁴ and 10⁵, between 10 and 10⁴, between 10² and 10⁴, between IO³ and 10⁴, between 10 and IO³, between 10² and IO³, or between 10 and 10² of each of the bacterial strains per capsule.

In some embodiments, each capsule contains between 10 and IO¹³, between 10² and IO¹³, between IO³ and IO¹³, between 10⁴ and IO¹³, between 10⁵ and IO¹³, between 10⁶ and

IO¹³, between 10⁷ and IO¹³, between IO⁸ and IO¹³, between IO⁹ and IO¹³, between IO¹⁰ and

IO¹³, between IO¹¹ and IO¹³, between 10¹² and IO¹³, between 10 and 10¹², between 10² and 10¹², between IO³ and 10¹², between 10⁴ and 10¹², between 10⁵ and 10¹², between 10⁶ and

10¹², between 10⁷ and 10¹², between IO⁸ and 10¹², between IO⁹ and 10¹², between IO¹⁰ and

10¹², between IO¹¹ and 10¹², between 10 and IO¹¹, between 10² and IO¹¹, between IO³ and

10¹³, between 10⁴ and IO¹³, between 10⁵ and IO¹³, between 10⁶ and IO¹³, between 10⁷ and

IO¹¹, between IO⁸ and IO¹¹, between IO⁹ and IO¹¹, between IO¹⁰ and IO¹¹, between 10 and

IO¹⁰, between 10² and IO¹⁰, between IO³ and IO¹⁰, between 10⁴ and IO¹⁰, between 10⁵ and

IO¹⁰, between 10⁶ and IO¹⁰, between 10⁷ and IO¹⁰, between IO⁸ and IO¹⁰, between IO⁹ and

IO¹⁰, between 10 and IO⁹, between 10² and IO⁹, between IO³ and IO⁹, between 10⁴ and IO⁹, between 10⁵ and IO⁹, between 10⁶ and IO⁹, between 10⁷ and IO⁹, between IO⁸ and IO⁹, between 10 and IO⁸, between 10² and IO⁸, between IO³ and IO⁸, between 10⁴ and IO⁸, between 10⁵ and IO⁸, between 10⁶ and IO⁸, between 10⁷ and IO⁸, between 10 and 10⁷, between 10² and 10⁷, between IO³ and 10⁷, between 10⁴ and 10⁷, between 10⁵ and 10⁷, between 10⁶ and 10⁷, between 10 and 10⁶, between 10² and 10⁶, between IO³ and 10⁶, between 10⁴ and 10⁶, between 10⁵ and 10⁶, between 10 and 10⁵, between 10² and 10⁵, between IO³ and 10⁵, between 10⁴ and 10⁵, between 10 and 10⁴, between 10² and 10⁴, between IO³ and 10⁴, between 10 and IO³, between 10² and IO³, or between 10 and 10² total bacteria per capsule. In some embodiments, each capsule contains between 10⁷ and IO⁹, between 10⁷ and IO⁸, or between IO⁸ and IO⁹ total bacteria. In some embodiments, each capsule contains about 1.0 x 10⁷, 2.0 x 10⁷, 3.0 x 10⁷, 4.0 x 10⁷, 5.0 x 10⁷, 6.0 x 10⁷, 7.0 x

10⁷, 8.0 x 10⁷, 9.0 x 10⁷, 1.0 x IO⁸, 2.0 x IO⁸, 3.0 x IO⁸, 4.0 x IO⁸, 5.0 x IO⁸, 6.0 x IO⁸, 7.0 x

10⁸, 8.0 x IO⁸, 9.0 x IO⁸, 1.0 x IO⁹, 1.1 x IO⁹, 1.2 x IO⁹, 1.3 x IO⁹, 1.4 x IO⁹, 1.5 x IO⁹, 1.6 x

10⁹, 1.7 x IO⁹, 1.8 x IO⁹, 1.9 x IO⁹, 2.0 x IO⁹, 2.1 x IO⁹, 2.2 x IO⁹, 2.3 x IO⁹, 2.4 x IO⁹, 2.5 x

IO⁹, 2.6 x IO⁹, 2.7 x IO⁹, 2.8 x IO⁹, 2.9 x IO⁹, 3.0 x IO⁹, 3.1 x IO⁹, 3.2 x IO⁹, 3.3 x IO⁹, 3.4 x

IO⁹, 3.5 x IO⁹, 3.6 x IO⁹, 3.7 x IO⁹, 3.8 x IO⁹, 3.9 x IO⁹, 4.0 x IO⁹, 4.1 x IO⁹, 4.2 x IO⁹, 4.3 x IO⁹, 4.4 X IO⁹, 4.5 X IO⁹, 4.6 x IO⁹, 4.7 x IO⁹, 4.8 x IO⁹, 4.9 x IO⁹, 5.0 x IO⁹ total bacteria. In some embodiments, each capsule contains about IxlO⁹ total bacteria.

10¹¹, between IO⁸ and IO¹¹, between IO⁹ and IO¹¹, between IO¹⁰ and IO¹¹, between 10 and

10¹⁰, between 10 and IO⁹, between 10² and IO⁹, between IO³ and IO⁹, between 10⁴ and IO⁹, between 10⁵ and IO⁹, between 10⁶ and IO⁹, between 10⁷ and IO⁹, between IO⁸ and IO⁹, between 10 and IO⁸, between 10² and IO⁸, between IO³ and IO⁸, between 10⁴ and IO⁸, between 10⁵ and IO⁸, between 10⁶ and IO⁸, between 10⁷ and IO⁸, between 10 and 10⁷, between 10² and 10⁷, between IO³ and 10⁷, between 10⁴ and 10⁷, between 10⁵ and 10⁷, between 10⁶ and 10⁷, between 10 and 10⁶, between 10² and 10⁶, between IO³ and 10⁶, between 10⁴ and 10⁶, between 10⁵ and 10⁶, between 10 and 10⁵, between 10² and 10⁵, between IO³ and 10⁵, between 10⁴ and 10⁵, between 10 and 10⁴, between 10² and 10⁴, between IO³ and 10⁴, between 10 and IO³, between 10² and IO³, or between 10 and 10² of each bacterial strain per capsule.

In some embodiments, the compositions contain between 10 and IO¹³, between 10² and IO¹³, between IO³ and IO¹³, between 10⁴ and IO¹³, between 10⁵ and IO¹³, between 10⁶ and

IO¹³, between IO¹¹ and IO¹³, between 10¹² and IO¹³, between 10 and 10¹², between 10² and

10¹², between IO³ and 10¹², between 10⁴ and 10¹², between 10⁵ and 10¹², between 10⁶ and

IO¹⁰, between 10⁶ and IO¹⁰, between 10⁷ and IO¹⁰, between IO⁸ and IO¹⁰, between IO⁹ and 10¹⁰, between 10 and 10⁹, between 10² and IO⁹, between IO³ and IO⁹, between 10⁴ and IO⁹, between 10⁵ and IO⁹, between 10⁶ and IO⁹, between 10⁷ and IO⁹, between IO⁸ and IO⁹, between 10 and IO⁸, between 10² and IO⁸, between IO³ and IO⁸, between 10⁴ and IO⁸, between 10⁵ and IO⁸, between 10⁶ and IO⁸, between 10⁷ and IO⁸, between 10 and 10⁷, between 10² and 10⁷, between IO³ and 10⁷, between 10⁴ and 10⁷, between 10⁵ and 10⁷, between 10⁶ and 10⁷, between 10 and 10⁶, between 10² and 10⁶, between IO³ and 10⁶, between 10⁴ and 10⁶, between 10⁵ and 10⁶, between 10 and 10⁵, between 10² and 10⁵, between IO³ and 10⁵, between 10⁴ and 10⁵, between 10 and 10⁴, between 10² and 10⁴, between IO³ and 10⁴, between 10 and IO³, between 10² and IO³, or between 10 and 10² CFUs of each of the bacterial strains per dosage amount. In some embodiments, the compositions contain between 10 and IO¹³, between 10² and IO¹³, between IO³ and IO¹³, between 10⁴ and IO¹³, between 10⁵ and IO¹³, between 10⁶ and IO¹³, between 10⁷ and IO¹³, between IO⁸ and IO¹³, between IO⁹ and IO¹³, between IO¹⁰ and IO¹³, between IO¹¹ and IO¹³, between 10¹² and IO¹³, between 10 and 10¹², between 10² and 10¹², between IO³ and 10¹², between 10⁴ and 10¹², between 10⁵ and 10¹², between 10⁶ and 10¹², between 10⁷ and 10¹², between IO⁸ and

10¹², between IO⁹ and 10¹², between IO¹⁰ and 10¹², between IO¹¹ and 10¹², between 10 and

10¹¹, between 10² and IO¹¹, between IO³ and IO¹³, between 10⁴ and IO¹³, between 10⁵ and

10¹³, between 10⁶ and IO¹³, between 10⁷ and IO¹¹, between IO⁸ and IO¹¹, between IO⁹ and

IO¹¹, between IO¹⁰ and IO¹¹, between 10 and IO¹⁰, between 10² and IO¹⁰, between IO³ and

IO¹⁰, between 10⁴ and IO¹⁰, between 10⁵ and IO¹⁰, between 10⁶ and IO¹⁰, between 10⁷ and

IO¹⁰, between IO⁸ and IO¹⁰, between IO⁹ and IO¹⁰, between 10 and IO⁹, between 10² and IO⁹, between IO³ and IO⁹, between 10⁴ and IO⁹, between 10⁵ and IO⁹, between 10⁶ and IO⁹, between 10⁷ and IO⁹, between IO⁸ and IO⁹, between 10 and IO⁸, between 10² and IO⁸, between IO³ and IO⁸, between 10⁴ and IO⁸, between 10⁵ and IO⁸, between 10⁶ and IO⁸, between 10⁷ and IO⁸, between 10 and 10⁷, between 10² and 10⁷, between IO³ and 10⁷, between 10⁴ and 10⁷, between 10⁵ and 10⁷, between 10⁶ and 10⁷, between 10 and 10⁶, between 10² and 10⁶, between IO³ and 10⁶, between 10⁴ and 10⁶, between 10⁵ and 10⁶, between 10 and 10⁵, between 10² and 10⁵, between IO³ and 10⁵, between 10⁴ and 10⁵, between 10 and 10⁴, between 10² and 10⁴, between IO³ and 10⁴, between 10 and IO³, between 10² and IO³, or between 10 and 10² total CFUs per dosage amount.

In some embodiments, the compositions contain at least about 1.0 x IO⁸, 1.1 x IO⁸, 1.2 x IO⁸, 1.3 x IO⁸, 1.4 x IO⁸, 1.5 x IO⁸, 1.6 x IO⁸, 1.7 x IO⁸, 1.8 x IO⁸, 1.9 x IO⁸, 2.0 x IO⁸, 2.1 x

IO⁸, 2.2 x IO⁸, 2.3 x IO⁸, 2.4 x IO⁸, 2.5 x IO⁸, 2.6 x IO⁸, 2.7 x IO⁸, 2.8 x IO⁸, 2.9 x IO⁸, 3.0 x

IO⁸, 3.1 x IO⁸, 3.2 x IO⁸, 3.3 x IO⁸, 3.4 x IO⁸, 3.5 x IO⁸, 3.6 x IO⁸, 3.7 x IO⁸, 3.8 x IO⁸, 3.9 x IO⁸, 4.0 X IO⁸, 4.1 X IO⁸, 4.2 x IO⁸, 4.3 x IO⁸, 4.4 x IO⁸, 4.5 x IO⁸, 4.6 x IO⁸, 4.7 x IO⁸, 4.8 x

IO⁸, 4.9 x IO⁸, 5.0 x IO⁸, 5.1 x IO⁸, 5.2 x IO⁸, 5.3 x IO⁸, 5.4 x IO⁸, 5.5 x IO⁸, 5.6 x IO⁸, 5.7 x

IO⁸, 5.8 x IO⁸, 5.9 x IO⁸, 6.0 x IO⁸, 6.1 x IO⁸, 6.2 x IO⁸, 6.3 x IO⁸, 6.4 x IO⁸, 6.5 x IO⁸, 6.6 x

IO⁸, 6.7 x IO⁸, 6.8 x IO⁸, 6.9 x IO⁸, 7.0 x IO⁸, 7.1 x IO⁸, 7.2 x IO⁸, 7.3 x IO⁸, 7.4 x IO⁸, 7.5 x

IO⁸, 7.6 x IO⁸, 7.7 x IO⁸, 7.8 x IO⁸, 7.9 x IO⁸, 8.0 x IO⁸, 8.1 x IO⁸, 8.2 x IO⁸, 8.3 x IO⁸, 8.4 x

IO⁸, 8.5 x IO⁸, 8.6 x IO⁸, 8.7 x IO⁸, 8.8 x IO⁸, 8.9 x IO⁸, 9.0 x IO⁸, 9.1 x IO⁸, 9.2 x IO⁸, 9.3 x

10⁸, 9.4 x IO⁸, 9.5 x IO⁸, 9.6 x IO⁸, 9.7 x IO⁸, 9.8 x IO⁸, 9.9 x IO⁸, 1.0 x IO⁹, 1.1 x IO⁹, 1.2 x

10⁹, 1.3 x IO⁹, 1.4 x IO⁹, 1.5 x IO⁹, 1.6 x IO⁹, 1.7 x IO⁹, 1.8 x IO⁹, 1.9 x 10⁹, 2.0 x IO⁹, 2.1 x

IO⁹, 2.2 x IO⁹, 2.3 x IO⁹, 2.4 x IO⁹, 2.5 x IO⁹, 2.6 x IO⁹, 2.7 x IO⁹, 2.8 x IO⁹, 2.9 x IO⁹, 3.0 x

IO⁹, 3.1 x IO⁹, 3.2 x IO⁹, 3.3 x IO⁹, 3.4 x IO⁹, 3.5 x IO⁹, 3.6 x IO⁹, 3.7 x IO⁹, 3.8 x IO⁹, 3.9 x

IO⁹, 4.0 x IO⁹, 4.1 x IO⁹, 4.2 x IO⁹, 4.3 x IO⁹, 4.4 x IO⁹, 4.5 x IO⁹, 4.6 x IO⁹, 4.7 x IO⁹, 4.8 x

IO⁹, 4.9 x IO⁹, 5.0 x IO⁹, 5.1 x IO⁹, 5.2 x IO⁹, 5.3 x IO⁹, 5.4 x IO⁹, 5.5 x IO⁹, 5.6 x IO⁹, 5.7 x

IO⁹, 5.8 x IO⁹, 5.9 x IO⁹, 6.0 x IO⁹, 6.1 x IO⁹, 6.2 x IO⁹, 6.3 x IO⁹, 6.4 x IO⁹, 6.5 x IO⁹, 6.6 x

IO⁹, 6.7 x IO⁹, 6.8 x IO⁹, 6.9 x IO⁹, 7.0 x IO⁹, 7.1 x IO⁹, 7.2 x IO⁹, 7.3 x IO⁹, 7.4 x IO⁹, 7.5 x

IO⁹, 7.6 x IO⁹, 7.7 x IO⁹, 7.8 x IO⁹, 7.9 x IO⁹, 8.0 x IO⁹, 8.1 x IO⁹, 8.2 x IO⁹, 8.3 x IO⁹, 8.4 x

IO⁹, 8.5 x IO⁹, 8.6 x IO⁹, 8.7 x IO⁹, 8.8 x IO⁹, 8.9 x IO⁹, 9.0 x IO⁹, 9.1 x IO⁹, 9.2 x IO⁹, 9.3 x

IO⁹, 9.4 x IO⁹, 9.5 x IO⁹, 9.6 x IO⁹, 9.7 x IO⁹, 9.8 x IO⁹, 9.9 x IO⁹, 1.0 x IO¹⁰, 1.1 x IO¹⁰, 1.2 x IO¹⁰, 1.3 x IO¹⁰, 1.4 x IO¹⁰, 1.5 x IO¹⁰, 1.6 x IO¹⁰, 1.7 x IO¹⁰, 1.8 x IO¹⁰, 1.9 x IO¹⁰, 2.0 x IO¹⁰, .1 x IO¹⁰, 2.2 x IO¹⁰, 2.3 x IO¹⁰, 2.4 x IO¹⁰, 2.5 x IO¹⁰, 2.6 x IO¹⁰, 2.7 x IO¹⁰, 2.8 x IO¹⁰, 2.9 x

IO¹⁰, 3.0 x IO¹⁰, 3.1 x IO¹⁰, 3.2 x IO¹⁰, 3.3 x IO¹⁰, 3.4 x IO¹⁰, 3.5 x IO¹⁰, 3.6 x IO¹⁰, 3.7 x IO¹⁰,

3.8 x IO¹⁰, 3.9 x IO¹⁰, 4.0 x IO¹⁰, 4.1 x IO¹⁰, 4.2 x IO¹⁰, 4.3 x IO¹⁰, 4.4 x IO¹⁰, 4.5 x IO¹⁰, 4.6 x

IO¹⁰, 4.7 x IO¹⁰, 4.8 x IO¹⁰, 4.9 x IO¹⁰, 5.0 x IO¹⁰, 5.1 x IO¹⁰, 5.2 x IO¹⁰, 5.3 x IO¹⁰, 5.4 x IO¹⁰,

5.5 x IO¹⁰, 5.6 x IO¹⁰, 5.7 x IO¹⁰, 5.8 x IO¹⁰, 5.9 x IO¹⁰, 6.0 x IO¹⁰, 6.1 x IO¹⁰, 6.2 x IO¹⁰, 6.3 x

IO¹⁰, 6.4 x IO¹⁰, 6.5 x IO¹⁰, 6.6 x IO¹⁰, 6.7 x IO¹⁰, 6.8 x IO¹⁰, 6.9 x IO¹⁰, 7.0 x IO¹⁰, 7.1 x IO¹⁰, .2 x IO¹⁰, 7.3 x IO¹⁰, 7.4 x IO¹⁰, 7.5 x IO¹⁰, 7.6 x IO¹⁰, 7.7 x IO¹⁰, 7.8 x IO¹⁰, 7.9 x IO¹⁰, 8.0 x

IO¹⁰, 8.1 x IO¹⁰, 8.2 x IO¹⁰, 8.3 x IO¹⁰, 8.4 x IO¹⁰, 8.5 x IO¹⁰, 8.6 x IO¹⁰, 8.7 x IO¹⁰, 8.8 x IO¹⁰,

8.9 x IO¹⁰, 9.0 x IO¹⁰, 9.1 x IO¹⁰, 9.2 x IO¹⁰, 9.3 x IO¹⁰, 9.4 x IO¹⁰, 9.5 x IO¹⁰, 9.6 x IO¹⁰, 9.7 x

10¹⁰, 9.8 x IO¹⁰, 9.9 x IO¹⁰, 1.0 x IO¹¹, 1.1 x IO¹¹, 1.2 x IO¹¹, 1.3 x IO¹¹, 1.4 x IO¹¹, 1.5 x IO¹¹,

1.6 x IO¹¹, 1.7 x IO¹¹, 1.8 x IO¹¹, 1.9 x 10ⁿ, 2.0 x IO¹¹, 2.1 x IO¹¹, 2.2 x IO¹¹, 2.3 x IO¹¹, 2.4 x

10¹¹, 2.5 x IO¹¹, 2.6 x IO¹¹, 2.7 x IO¹¹, 2.8 x IO¹¹, 2.9 x IO¹¹, 3.0 x IO¹¹, 3.1 x IO¹¹, 3.2 x IO¹¹,

3.3 x IO¹¹, 3.4 x IO¹¹, 3.5 x IO¹¹, 3.6 x IO¹¹, 3.7 x IO¹¹, 3.8 x IO¹¹, 3.9 x IO¹¹, 4.0 x IO¹¹, 4.1 x

IO¹¹, 4.2 x IO¹¹, 4.3 x IO¹¹, 4.4 x IO¹¹, 4.5 x IO¹¹, 4.6 x IO¹¹, 4.7 x IO¹¹, 4.8 x IO¹¹, 4.9 x IO¹¹,

5.0 x IO¹¹, 5.1 x IO¹¹, 5.2 x IO¹¹, 5.3 x IO¹¹, 5.4 x IO¹¹, 5.5 x IO¹¹, 5.6 x IO¹¹, 5.7 x IO¹¹, 5.8 x

IO¹¹, 5.9 x IO¹¹, 6.0 x IO¹¹, 6.1 x IO¹¹, 6.2 x IO¹¹, 6.3 x IO¹¹, 6.4 x IO¹¹, 6.5 x IO¹¹, 6.6 x IO¹¹, 6.7 x IO¹¹, 6.8 x IO¹¹, 6.9 x IO¹¹, 7.0 x IO¹¹, 7.1 x IO¹¹, 7.2 x IO¹¹, 7.3 x IO¹¹, 7.4 x IO¹¹, 7.5 x IO¹¹, 7.6 x IO¹¹, 7.7 x IO¹¹, 7.8 x IO¹¹, 7.9 x IO¹¹, 8.0 x IO¹¹, 8.1 x IO¹¹, 8.2 x IO¹¹, 8.3 x IO¹¹, 8.4 x IO¹¹, 8.5 x IO¹¹, 8.6 x IO¹¹, 8.7 x IO¹¹, 8.8 x IO¹¹, 8.9 x IO¹¹, 9.0 x IO¹¹, 9.1 x IO¹¹, 9.2 x IO¹¹, 9.3 x IO¹¹, 9.4 x IO¹¹, 9.5 x IO¹¹, 9.6 x IO¹¹, 9.7 x IO¹¹, 9.8 x IO¹¹, 9.9 x IO¹¹, or 1.0 x 10¹² total CFUs.

In some embodiments, the composition comprises about 1 x 10⁹ total CFUs. In some embodiments, the composition comprises about 1 x 10⁹ total CFUs and is administered as a single dose. In some embodiments, the composition comprises about 1 x 10⁹ total CFUs and is administered as multiple (e.g., 2, 3, 4, 5, or more) doses. In some embodiments, the composition comprises about 1 x 10⁹ total CFUs and is administered as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more doses. In some embodiments, each of the multiple doses are administered at regular intervals. In some embodiments, each of the multiple doses are on consecutive days (e.g., first dose on day 1, second dose of day 2, third dose on day 3, etc.).

In some embodiments, the composition comprises about 1 x IO¹⁰ total CFUs. In some embodiments, the composition comprises about 1 x IO¹⁰ total CFUs and is administered as a single dose. In some embodiments, the composition comprises about 1 x IO¹⁰ total CFUs and is administered as multiple (e.g., 2, 3, 4, 5, or more) doses. In some embodiments, the composition comprises about 1 x IO¹⁰ total CFUs and is administered as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more doses. In some embodiments, each of the multiple doses are administered at regular intervals. In some embodiments, each of the multiple doses are on consecutive days (e.g., first dose on day 1, second dose of day 2, third dose on day 3, etc.).

In some embodiments, the composition comprises about 5 x 10⁹ total CFUs. In some embodiments, the composition comprises about 5 x 10⁹ total CFUs and is administered as a single dose. In some embodiments, the composition comprises about 5 x 10⁹ total CFUs and is administered as multiple (e.g., 2, 3, 4, 5, or more) doses. In some embodiments, the composition comprises about 5 x 10⁹ total CFUs and is administered as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more doses. In some embodiments, each of the multiple doses are administered at regular intervals. In some embodiments, each of the multiple doses are on consecutive days (e.g., first dose on day 1, second dose of day 2, third dose on day 3, etc.).

As described herein, any of the compositions described herein may be administered to a subject in one dose or in multiple doses (e.g., an initial administration), which may be followed by one or more additional doses of any of the compositions described herein. In some embodiments, any of composition described herein may be administered to a subject in one dose or in multiple doses in an initial administration, followed by one or more additional doses of a composition comprising the same one or more bacterial strains as the composition of the initial administration. In some embodiments, any of the compositions described herein may be administered to a subject in one dose or in multiple doses in an initial administration, followed by one or more additional doses of a composition comprising more total bacteria (colony-forming units) relative to the initial administration of the composition. In some embodiments, any of the compositions described herein may be administered to a subject in one dose or in multiple doses in an initial administration, followed by one or more additional doses of a composition comprising fewer total bacteria (colony-forming units) relative to the initial administration of the composition. In some embodiments, the initial administration includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more doses of any of the compositions described herein. In some embodiments, the additional administration includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more doses of any of the compositions described herein. In some embodiments, the initial administration comprises two doses of any of the compositions and the additional administration comprises three doses of any of the compositions described herein.

In some embodiments, any of the compositions described herein may be administered to a subject in one dose or in multiple doses in an initial administration, followed by one or more additional doses of a composition comprising fewer total bacteria (colony-forming units) relative to the initial administration of the composition.

Bacterial compositions

Aspects of methods described herein comprise administering to the subject a composition comprising a purified bacterial mixture comprising one or more bacterial strains selected from the group consisting of Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and F lav onifr actor plautii. Some aspects of the disclosure relate to methods for treating and/or preventing neutropenia in a subject comprising administering to the subject a composition comprising a purified bacterial mixture comprising one or more bacterial strains comprising 16S rDNA sequences having at least 97% sequence identity with the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-8. Bacterial strains

Some aspects of the disclosure relate to compositions comprising mixtures of bacterial strains. Some embodiments of the bacterial strains are described for instance in PCT Publication No. WO 2017/218680, which is incorporated herein by reference in its entirety.

In some embodiments of the compositions provided herein, the composition includes one or more of the bacterial strains provided in Table 1. In some embodiments of the compositions provided herein, the composition includes one or more bacterial strains belonging to the following species: (i) Clostridium bolteae (Lachnoclostridiu bolteae, Enterocloster bolteae), (ii) Anaerotruncus colihominis, (iii) Sellimonas intestinalis (Drancourtella massilienses, Ruminococcus torques, Eubacterium f issicalena), (iv) Clostridium symbiosum (Lachnoclostridium symbiosum), (v) Blautia producta (Blautia spOOl 304935), (vi) Dorea longicatena, (vii) Clostridium innocuum (Erysipelotrichaceae innocuum, Eubacterium innocuum, Absiella innocuum, Longicatena innocuum, Erysipelotrichaceae bacterium), and (viii) Flavonifractor plautii (Clostridium orbiscindens, Subdolinogranulum spp). In some embodiments, the compositions described herein comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 bacterial strains (e.g., purified bacterial strains) listed in Table 1.

As will be appreciated by one of ordinary skill in the art, a bacterial strain may be closely related to one or more bacterial species. Alternatively or in addition, a bacterial strain may be referred to by one or more bacterial species names, based on changing nomenclature and phylogenetic classification. In some embodiments, the composition includes Clostridium bolteae. In some embodiments, the bacterial strain referred to as Clostridium bolteae and having a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 1 may also be referred to, for example, as Lachnoclostridium bolteae or Enterocloster bolteae. In some embodiments, the composition includes Anaerotruncus colihominis. In some embodiments, the bacterial strain referred to as Anaerotruncus colihominis has a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the composition includes Sellimonas intestinalis. In some embodiments, the bacterial strain referred to as Sellimonas intestinalis and having a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 3 may also be referred to, for example, as Drancourtella massilienses, Eubacterium fissicatena, or Ruminococcus torques. In some embodiments, the composition includes Clostridium symbiosum. In some embodiments, the bacterial strain referred to as Clostridium symbiosum and having a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 4 may also be referred to, for example, as Lachnoclostridium symbiosum. In some embodiments, the composition includes Blautia producta. In some embodiments, the bacterial strain referred to as Blautia producta and having a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 5 may also be referred to, for example, as Blautia sp001304935. In some embodiments, the composition includes Dorea longicatena. In some embodiments, the bacterial strain referred to as Dorea longicatena has a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the composition includes Clostridium innocuum. In some embodiments, the bacterial strain referred to as Clostridium innocuum and having a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 7 may also be referred to, for example, as Erysipelotrichaceae bacterium, Erysipelotrichaceae innocuum, Eubacterium innocuum, Absiella innocuum, and Longicatena innocuum. In some embodiments, the composition includes Flavonifractor plautii. In some embodiments, the bacterial strain referred to as Flavonifractor plautii and having a 16S rDNA sequence comprising the nucleic acid sequence of SEQ ID NO: 8 may also be referred to, for example, as Subdolinogranulum spp. Or Clostridium orbiscindens .

In some aspects, the composition comprises a purified bacterial mixture comprising one or more bacterial strains selected from the group consisting of Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavonifractor plautii. In some aspects, the composition comprises a purified bacterial mixture consisting of one or more bacterial strains selected from the group consisting of Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavonifractor plautii.

In some aspects, the composition comprises a purified bacterial mixture comprising Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavonifractor plautii. In some aspects, the composition comprises a purified bacterial mixture consisting of Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavonifractor plautii.

Identifying bacteria by their sequences furthermore allows for the identification of additional bacterial strains that are identical or highly similar to the exemplified bacteria. For instance, the 16s rRNA sequences of bacterial strains were used to identify the closest relative (based on percent identity) through whole genome sequencing and by comparing these sequences with 16S databases (Table 1). In addition, based on whole genome sequencing (WGS) and comparing of the whole genome to whole genome (WG) databases, the bacterial strains having 16S rRNA sequences provided by SEQ ID NOs: 1-8 are most closely related to the following bacterial species: Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavonifractor plautii (see, e.g., Table 1). Thus, in one aspect it should be appreciated that each row of Table 1, the bacterial strains are highly similar and/or are identical. In some embodiments, in context of the instant disclosure the names of bacterial strains within a row of Table 1 can be used interchangeably.

Bacterial strains of species listed in Table 1 are available to those of ordinary skill in the art, from repositories such as the American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). ATCC is a repository for reference cultures and biological materials, including microorganisms, distributing cultures to scientists worldwide. Similarly, DSMZ is an active collection of biological resources including microorganisms, from which catalogued strains are available.

Aspects of the disclosure relate to bacterial strains with 16S rDNA sequences that have homology or identity to a nucleic acid sequence of any one of the sequences of the bacterial strains or species described herein. In some embodiments, the bacterial strain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity relative to any of the strains or bacterial species described herein over a specified region of nucleic acid or amino acid sequence or over the entire nucleic acid or amino acid sequence. It would be appreciated by one of skill in the art that the terms “homology” or “percent homology,” may be used interchangeably with “identity” or “percent identity,” respectively. In the context of two or more nucleic acid sequences or amino acid sequences, the terms homology or identity refer to a measure of similarity between two or more sequences or portion(s) thereof. The homology or identity may exist over a region of a sequence that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length. In some embodiments, the homology or identity exists over the length the 16S rRNA or 16S rDNA sequence, or a portion thereof.

In some embodiments, the compositions include one or more bacterial strains, wherein the one or more bacterial strains comprising 16S rDNA sequences having at least 97% identity with nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, or SEQ ID NO:8. In some embodiments, the compositions include one or more bacterial strains, wherein the bacterial strains comprise 16S rDNA sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or up to 100% identity with nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

In some embodiments, the compositions consist of bacterial strains comprising 16S rDNA sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or up to 100% identity with nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

Additionally, or alternatively, two or more sequences may be assessed for the identity between the sequences. The terms “identical” or “percent identity” in the context of two or more nucleic acids or amino acid sequences, refer to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity) over a specified region of a nucleic acid or amino acid sequence or over an entire sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length. In some embodiments, the identity exists over the length the 16S rRNA or 16S rDNA sequence.

In some embodiments, the compositions include one or more bacterial strains, wherein the one or more bacterial strains comprising 16S rDNA sequences having at least 97% sequence identity with nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, the compositions include one or more bacterial strains, wherein the bacterial strains comprise 16S rDNA sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or up to 100% sequence identity with nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

In some embodiments, the compositions consist of bacterial strains comprising 16S rDNA sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or up to 100% sequence identity with nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

Additionally, or alternatively, two or more sequences may be assessed for the alignment between the sequences. The terms “alignment” or “percent alignment” in the context of two or more nucleic acids or amino acid sequences, refer to two or more sequences or subsequences that are the same. Two sequences are “substantially aligned” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical) over a specified region of the nucleic acid or amino acid sequence or over the entire sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the alignment exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length. In some embodiments, the identity exists over the length the 16S rRNA or 16S rDNA sequence.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. Methods of alignment of sequences for comparison are well known in the art. See, e.g., by the local homology or identity algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology or identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. (1970) 48:443, by the search for similarity method of Pearson and Lipman. Proc. Natl. Acad. Sci. USA (1998) 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group. Madison. WI), or by manual alignment and visual inspection (see. E.g., Brent el al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. (1977) 25:3389-3402, and Altschul et al., J. Mol. Biol. (1990) 215:403-410, respectively.

It should be appreciated that the terms “bacteria,” “bacterial strain,” and “bacterial strains” as used herein are interchangeable.

Sources of bacterial strains

In some embodiments of the compositions provided herein, one or more of the bacterial strains are human-derived bacteria, meaning the one or more bacterial strains were obtained from or identified from a human or a sample therefrom (e.g., a human donor). In some embodiments of the compositions provided herein, all of the bacterial strains are human-derived bacteria. In some embodiments of the compositions provided herein, the bacterial strains are derived from more than one human donor.

The bacterial strains used in the compositions provided herein generally are isolated from the microbiome of healthy individuals. In some embodiments, the compositions include strains originating from a single individual. In some embodiments, the compositions include strains originating from multiple individuals. In some embodiments, the bacterial strains are obtained from multiple individuals, isolated, and grown up individually. The bacterial compositions that are grown up individually may subsequently be combined to provide the compositions of the disclosure. It should be appreciated that the origin of the bacterial strains of the compositions provided herein is not limited to the human microbiome from a healthy individual. In some embodiments, the bacterial strains originate from a human with a microbiome in dysbiosis. In some embodiments, the bacterial strains originate from nonhuman animals or the environment (e.g., soil or surface water). In some embodiments, the combinations of bacterial strains provided herein originate from multiple sources (e.g., human and non-human animals).

In some embodiments of the compositions provided herein, the composition includes one or more anaerobic bacteria. In some embodiments of the compositions provided herein, the composition includes only anaerobic bacteria. In some embodiments of the compositions provided herein, the composition includes one or more facultative anaerobic bacteria. In some embodiments of the compositions provided herein, the composition includes only facultative anaerobic bacteria. In some embodiments of the compositions provided herein, the composition includes one or more obligate anaerobic bacteria. In some embodiments of the compositions provided herein, the composition includes only obligate anaerobic bacteria. In some embodiments of the compositions provided herein, one or more of the bacterial strains is a spore-former. In some embodiments of the compositions provided herein, one or more of the bacterial strains is in spore form. In some embodiments of the compositions provided herein, one or more of the bacterial strains is a non-spore former.

In some embodiments, the compositions described herein comprise spore forming and non-spore forming bacterial strains. In some embodiments, the compositions described herein comprise spore-forming bacterial strains. In some embodiments, the compositions described herein comprise only spore-forming bacterial strains. In some embodiments, the compositions described herein comprise only non-spore forming bacterial strains. The spore-forming bacteria can be in spore form (z.e., as spores) or in vegetative form (z.e., as vegetative cells). In spore form, bacteria are generally more resistant to environmental conditions, such as heat, acid, radiation, oxygen, chemicals, and antibiotics. In contrast, in the vegetative state or actively growing state, bacteria are more susceptible to such environmental conditions, compared to in the spore form. In general, bacterial spores are able to germinate from the spore form into a vegetative/actively growing state, under appropriate conditions. For instance, bacteria in spore format may germinate when they are introduced in the intestine.

In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of the bacterial strains in the composition is a spore former. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of the bacterial strains in the composition is in spore form. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of the bacterial strains in the composition is a non-spore former. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of the bacterial strains in the composition is in vegetative form (as discussed above, spore forming bacteria can also be in vegetative form). In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) of the bacterial strains in the composition is in spore form and at least one (e.g., 1, 2, 3, 4, 5, or more) of the bacterial strains in the composition is in vegetative form. In some embodiments, at least one bacterial strain is considered able to form spores (i.e., a spore-former) but is present in the composition in vegetative form. In some embodiments, at least one bacterial strain that is considered able to form spores is present in the composition both in spore form and in vegetative form.

It is envisioned that the bacterial strains of the compositions provided herein are alive and will be alive when they reach the target area (e.g., the intestines). Bacterial spores are considered to be alive in this regard. In some embodiments, bacteria that are administered as spores may germinate in the target area (e.g., the intestines). It should further be appreciated that not all of the bacteria are alive and the compositions can include a percentage (e.g., by weight) that is not alive. In addition, in some embodiments, the compositions include bacterial strains that are not alive when administered or at the time when the composition reaches the target area (e.g., the intestines). It is envisioned that non-living bacteria may still be useful by providing some nutrients and metabolites for the other bacterial strains in the composition.

In any of the compositions provided herein, in some embodiments, the bacterial strains are purified. In any of the compositions provided herein, in some embodiments, the bacterial strains are isolated. Any of the bacterial strains described herein may be isolated and/or purified, for example, from a source such as a culture or a microbiota sample (e.g., fecal matter). The bacterial strains used in the compositions provided herein generally are isolated from the microbiome of healthy individuals. However, bacterial strains can also be isolated from individuals that are considered not to be healthy. In some embodiments, the compositions include strains originating from multiple individuals. As used herein, the term “isolated” refers to bacteria that have been separated from one or more undesired component, such as another bacterium or bacterial strain, one or more component of a growth medium, and/or one or more component of a sample, such as a fecal sample. In some embodiments, the bacteria are substantially isolated from a source such that other components of the source are not detected. As also used herein, the term “purified” refers to a bacterial strain or composition comprising such that has been separated from one or more components, such as contaminants. In some embodiments, the bacterial strain is substantially free of contaminants. In some embodiments, one or more bacterial strains of a composition may be independently purified from one or more other bacteria produced and/or present in a culture or a sample containing the bacterial strain. In some embodiments, a bacterial strain is isolated or purified from a sample and then cultured under the appropriate conditions for bacterial replication, e.g., under anaerobic culture conditions. The bacteria that are grown under appropriate conditions for bacterial replication can subsequently be isolated/purified from the culture in which it is grown.

Preparation of bacterial strains

In some embodiments, one of or more of the bacterial strains of the compositions have been spray-dried. The process of spray-drying refers to production of a dry powder from a liquid comprising bacterial compositions. (See e.g., Ledet el al., Spray-Drying of Pharmaceuticals in “Lyophilized Biologies and Vaccines” pages 273-194, Springer). In general, the process involves rapidly drying the bacterial compositions with a hot gas. A bacterial strain may be combined with a pharmaceutical excipient prior to combining it with the other bacterial strains or multiple spray-dried bacterial strains may be combined while in spray-dried form, and the mixture of bacterial strains, once combined, may be subsequently combined with a pharmaceutical excipient.

Any of the compositions described herein, including the pharmaceutical compositions and food products comprising bacterial strains, the bacterial strains may be in any form, for example in an aqueous form, such as a solution or a suspension, embedded in a semi-solid form, in a powdered form, or freeze-dried form. In some embodiments, the composition or the bacterial strains are lyophilized. In some embodiments, a subset of the bacterial strains is lyophilized. Suitable methods of lyophilizing compositions, specifically compositions comprising bacteria, are described, for example, in U.S. Patent No. 3,261,761; U.S. Patent No. 4,205,132; and PCT Publication Nos. WO 2014/029578 and WO 2012/098358, herein incorporated by reference in their entirety. The bacteria may be lyophilized as a combination and/or the bacteria may be lyophilized separately and combined prior to administration. A bacterial strain may be combined with a pharmaceutical excipient prior to combining it with the other bacterial strain(s) or multiple lyophilized bacteria may be combined while in lyophilized form and the mixture of bacteria, once combined may subsequently be combined with a pharmaceutical excipient. In some embodiments, the bacterial strain is a lyophilized cake. In some embodiments, the composition comprising the one or more bacterial strains is a lyophilized cake.

The bacterial strains can be manufactured using any suitable fermentation technique. In some embodiments, the bacteria are propagated or manufactured using anaerobic fermenters, which can support the rapid growth of anaerobic bacterial species. The anaerobic fermenters may be, for example, stirred tank reactors or disposable wave bioreactors. Culture media such as BL media and EG media, or similar versions of these media devoid of animal components, can be used to support the growth of the bacterial species. The bacterial product can be purified and concentrated from the fermentation broth by traditional techniques, such as centrifugation and filtration, and can optionally be dried and lyophilized by suitable methods.

Pharmaceutical compositions

In some embodiments, the live bacterial product may be formulated for administration as a pharmaceutical composition. The term “pharmaceutical composition” as used herein means a product that results from the mixing or combining of at least one active ingredient, such as any of the bacterial strains described herein, and one or more inactive ingredients, which may include one or more pharmaceutically acceptable excipients. Excipients

An “acceptable” excipient refers to an excipient that must be compatible with the active ingredient and not deleterious to the subject to which it is administered. In some embodiments, the pharmaceutically acceptable excipient is selected based on the intended route of administration of the composition, for example a composition for oral or nasal administration may comprise a different pharmaceutically acceptable excipient than a composition for rectal administration. Examples of excipients include sterile water, physiological saline, solvent, a base material, an emulsifier, a suspending agent, a surfactant, a stabilizer, a flavoring agent, an aromatic, an excipient, a vehicle, a preservative, a binder, a diluent, a tonicity adjusting agent, a soothing agent, a bulking agent, a disintegrating agent, a buffer agent, a coating agent, a lubricant, a colorant, a sweetener, a thickening agent, and a solubilizer.

Pharmaceutical compositions can be prepared in accordance with methods well known and routinely practiced in the art (see e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co. 20th ed. 2000). The pharmaceutical compositions described herein may further comprise any carriers or stabilizers in the form of a lyophilized formulation or an aqueous solution. Acceptable excipients, carriers, or stabilizers may include, for example, buffers, antioxidants, preservatives, polymers, chelating reagents, and/or surfactants. Pharmaceutical compositions are preferably manufactured under GMP conditions. The pharmaceutical compositions can be used orally, nasally or parenterally, for instance, in the form of capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewables, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, cataplasms, transdermal absorption systems, lotions, inhalations, aerosols, injections, suppositories, and the like.

Delivery compositions

In some embodiments, the compositions comprising bacterial strains are formulated for oral delivery. In some embodiments, the compositions comprising bacterial strains are formulated for delivery to the intestines (e.g., the small intestine and/or the colon). In some embodiments, the composition comprising bacterial strains may be formulated with an enteric coating that increases the survival of the bacteria through the harsh environment in the stomach. The enteric coating is one which resists the action of gastric juices in the stomach so that the bacteria of the composition therein will pass through the stomach and into the intestines. The enteric coating may readily dissolve when in contact with intestinal fluids, so that the bacteria enclosed in the coating will be released in the intestinal tract. Enteric coatings may consist of polymers and copolymers well known in the art, such as commercially available EUDRAGIT (Evonik Industries). (See e.g., Zhang, AAPS PharmSciTech (2016) 17 (1), 56-67).

The compositions comprising bacterial strains may also be formulated for rectal delivery to the intestine (e.g., the colon (large intestine)). Thus, in some embodiments, compositions comprising bacterial strains may be formulated for delivery by suppository, colonoscopy, endoscopy, sigmoidoscopy, or enema. A pharmaceutical preparation or formulation and particularly a pharmaceutical preparation for oral administration, may include an additional component that enables efficient delivery of the compositions of the disclosure to the intestine (e.g., the colon). A variety of pharmaceutical preparations that allow for the delivery of the compositions to the intestine (e.g., the colon) can be used. Examples thereof include pH sensitive compositions, more specifically, buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach. When a pH sensitive composition is used for formulating the pharmaceutical preparation, the pH sensitive composition is preferably a polymer whose pH threshold of the decomposition of the composition is between about 6.8 and about 7.5. Such a numeric value range is a range in which the pH shifts toward the alkaline side at a distal portion of the stomach, and hence is a suitable range for use in the delivery to the colon. It should further be appreciated that each part of the intestine (e.g., the duodenum, jejunum, ileum, cecum, colon and rectum), has different biochemical and chemical environment. For instance, parts of the intestines have different pHs, allowing for targeted delivery by compositions that have a specific pH sensitivity. Thus, the compositions provided herein may be formulated for delivery to the intestine or specific parts of the intestine (e.g., the duodenum, jejunum, ileum, cecum, colon and rectum) by providing formulations with the appropriate pH sensitivity. (See, e.g., Villena et al., Int J Pharm (2015) 487 (1-2): 314-9).

Also within the scope of the disclosure are pharmaceutical compositions for administration by additional or alternative routes. In some embodiments, the pharmaceutical compositions are formulated for sublingual administration. In some embodiments, the pharmaceutical compositions are formulated for administration by injection.

In some embodiments, a pharmaceutical composition may include an additional component that enables efficient delivery of the compositions of the disclosure to a desired site, such as the gastrointestinal tract (e.g., the colon). In some embodiments, the pharmaceutical composition includes an adjuvant associated with providing a benefit in the treatment of allergy. In some embodiments, the pharmaceutical composition includes one or more components of an oral immunotherapeutic, an epicutaneous immunotherapeutic, or a sublingual immunotherapeutic.

Another embodiment of a pharmaceutical preparation useful for delivery of the compositions to the intestine (e.g., the colon) is one that ensures the delivery to the colon by delaying the release of the contents (e.g., the bacterial strains) by approximately 3 to 5 hours, which corresponds to the small intestinal transit time. In one embodiment of a pharmaceutical preparation for delayed release, a hydrogel is used as a shell. The hydrogel is hydrated and swells upon contact with gastrointestinal fluid, with the result that the contents are effectively released (released predominantly in the colon). Delayed release dosage units include drugcontaining compositions having a material which coats or selectively coats a drug or active ingredient to be administered. Examples of such a selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and/or enzyme degradable polymers. A wide variety of coating materials for efficiently delaying the release is available and includes, for example, cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

Additional examples of pharmaceutical compositions that allow for the delivery to the intestine (e.g., the colon) include bioadhesive compositions which specifically adhere to the colonic mucosal membrane (for example, a polymer described in the specification of US Patent No. 6,368,586) and compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.

Another example of a system enabling the delivery to the intestine (e.g., the colon) is a system of delivering a composition to the colon by pressure change in such a way that the contents are released by utilizing pressure change caused by generation of gas in bacterial fermentation at a distal portion of the stomach. Such a system is not particularly limited, and a more specific example thereof is a capsule which has contents dispersed in a suppository base and which is coated with a hydrophobic polymer (for example, ethyl cellulose).

A further example of a system enabling the delivery of a composition to the intestine (e.g., the colon), is a composition that includes a coating that can be removed by an enzyme present in the gut (e.g., the colon), such as, for example, a carbohydrate hydrolase or a carbohydrate reductase. Such a system is not particularly limited, and more specific examples thereof include systems which use food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

The compositions provided herein can also be delivered to specific target areas, such as the intestine, by delivery through an orifice (e.g., a nasal tube) or through surgery. In addition, the compositions provided herein that are formulated for delivery to a specific area (e.g., the cecum or the colon), may be administered by a tube (e.g., directly into the small intestine). Combining mechanical delivery methods such as tubes with chemical delivery methods such as pH specific coatings, allow for the delivery of the compositions provided herein to a desired target area (e.g., the cecum or the colon).

The pharmaceutical compositions comprising bacterial strains are in pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., the prophylactic or therapeutic effect). In some embodiments, the dosage form of the composition is a tablet, pill, capsule, powder, granules, solution, or suppository. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition comprises bacterial strains and is formulated such that the bacteria, or a portion thereof, remain viable after passage through the stomach of the subject. In some embodiments, the pharmaceutical composition is formulated for rectal administration, e.g., as a suppository. In some embodiments, the pharmaceutical composition is formulated for delivery to the intestine or a specific area of the intestine (e.g., the colon) by providing an appropriate coating (e.g., a pH specific coating, a coating that can be degraded by target area specific enzymes, or a coating that can bind to receptors that are present in a target area).

Food products

Some aspects of the present disclosure provide food products comprising any of the compositions provided herein and a nutrient. Also within the scope of the present disclosure are food products comprising any of the bacterial strains described herein and a nutrient. Food products are, in general, intended for the consumption by a human or an animal. Any of the bacterial strains described herein may be formulated as a food product. In some embodiments, the bacterial strains are formulated as a food product in spore form. In some embodiments, the bacterial strains are formulated as a food product in vegetative form. In some embodiments, the food product comprises both vegetative bacteria and bacteria in spore form. The compositions disclosed herein can be used in a food or beverage, such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.

Non-limiting examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products such as Western confectionery products including biscuits, cookies, and the like, Japanese confectionery products including steamed bean-jam buns, soft adzuki-bean jellies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.

Examples of food products which may be formulated to contain any of the bacterial strains described herein include, without limitation, a beverage, a drink, a bar, a snack, a dairy product, a confectionery product, a cereal product, a ready-to-eat product, a nutritional formula, such as a nutritional supplementary formulation, a food or beverage additive.

Food products containing bacterial strains described herein may be produced using methods known in the art and may contain the same amount of bacteria (e.g., by weight, amount or CFU) as the compositions provided herein. Selection of an appropriate amount of bacteria in the food product may depend on various factors, including for example, the serving size of the food product, the frequency of consumption of the food product, the specific bacterial strains contained in the food product, the amount of water in the food product, and/or additional conditions for survival of the bacteria in the food product. Table 1: Examples of bacterial species of the compositions disclosed herein.

SEQUENCES

Strain 1 Clostridium bolteae 16S ribosomal RNA coding sequence (16S rDNA)

ATGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTAAA ATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGATAACCTGCCTCACAC TGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGTACCGCATGGTACGGTGTGAAAAACTC CGGTGGTGTGAGATGGATCCGCGTCTGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGC C GAC C T GAGAGGGT GAC CGGCCACATT GGGAC T GAGAC AC GGC C C AAAC T C C T AC GGGAGGC AGC AGT GGGGAAT ATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATC AGC AGGGAAGAAAAT GAC GGT AC C T GAC T AAGAAGC C C C GGC T AAC T AC GT GC C AGC AGC C GC GGT AAT AC GT AG GGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCGAAGCAAGTCTGAAGTGAAAACCCAG GGCTCAACCCTGGGACTGCTTTGGAAACTGTTTTGCTAGAGTGTCGGAGAGGTAAGTGGAATTCCTAGTGTAGCG GT GAAAT GC GT AGAT AT T AGGAGGAAC AC C AGT GGC GAAGGC GGC T T AC T GGAC GAT AAC T GAC GT T GAGGC T C G AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGGGGG CAAAGCCCTTCGGTGCCGTCGCAAACGCAGTAAGCATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAA GGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTC TTGACATCCTCTTGACCGGCGTGTAACGGCGCCTTCCCTTCGGGGCAGGAGAGACAGGTGGTGCATGGTTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTAGTAGCCAGCAGGTAGAG C T GGGC AC T C T AGGGAGAC T GC C AGGGAT AAC C T GGAGGAAGGT GGGGAT GAC GT C AAAT C AT C AT GC C C C T T AT GATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCAAGACAGTGATGTGGAGCAAATCCCAAAAA TAACGTCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAAT GTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGCAACGCCCGAAGTCAG TGACCCAACTCGCAAGAGAGGGAGCTGCCGAAGGCGGGGCAGGTAACTGGGGTGAAGTCGTAACAAGGTAGCCGT ATCGGAAGGTGCGGCTGGATCACCTCCTTT (SEQ ID NO : 1 )

Strain 2 Anaerotruncus colihominis 16S ribosomal RNA coding sequence (16S rDNA)

C AAAGAGT T T GAT C C T GGC T C AGGAC GAAC GC T GGC GGC GC GC C T AAC AC AT GC AAGT C GAAC GGAGC T T AC GT T TTGAAGTTTTCGGATGGATGAATGTAAGCTTAGTGGCGGACGGGTGAGTAACACGTGAGCAACCTGCCTTTCAGA GGGGGATAACAGCCGGAAACGGCTGCTAATACCGCATGATGTTGCGGGGGCACATGCCCCTGCAACCAAAGGAGC AATCCGCTGAAAGATGGGCTCGCGTCCGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCGGTAG C C GGAC T GAGAGGT T GAAC GGCCACATT GGGAC T GAGAC AC GGC C C AGAC T C C T AC GGGAGGC AGC AGT GGGGGA T AT T GC AC AAT GGGC GAAAGC C T GAT GC AGC GAC GC C GC GT GAGGGAAGAC GGT C T T C GGAT T GT AAAC C T C T GT CTTTGGGGAAGAAAATGACGGTACCCAAAGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA GGGAGCAAGCGTTGTCCGGAATTACTGGGTGTAAAGGGAGCGTAGGCGGGATGGCAAGTAGAATGTTAAATCCAT CGGCTCAACCGGTGGCTGCGTTCTAAACTGCCGTTCTTGAGTGAAGTAGAGGCAGGCGGAATTCCTAGTGTAGCG GT GAAAT GC GT AGAT AT T AGGAGGAAC AC C AGT GGC GAAGGC GGC C T GC T GGGC T T T AAC T GAC GC T GAGGC T C G AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATTACTAGGTGTGGGGGGA CTGACCCCTTCCGTGCCGCAGTTAACACAATAAGTAATCCACCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAA GGAATTGACGGGGGCCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTC TTGACATCGGATGCATAGCCTAGAGATAGGTGAAGCCCTTCGGGGCATCCAGACAGGTGGTGCATGGTTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATTAGTTGCTACGCAAGAGCAC T C T AAT GAGAC T GC C GT T GAC AAAAC GGAGGAAGGT GGGGAT GAC GT C AAAT CATCATGCCCCTTAT GAC C T GGG CTACACACGTACTACAATGGCACTAAAACAGAGGGCGGCGACACCGCGAGGTGAAGCGAATCCCGAAAAAGTGTC TCAGTTCAGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGG TGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCGGTAACACCCGAAGCCAGTAGCCTA ACCGCAAGGGGGGCGCTGTCGAAGGTGGGATTGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGG TGCGGCTGGATCACCTCCTTT (SEQ ID NO : 2 )

Strain 3 Sellimonas intestinalis 16S ribosomal RNA coding sequence (16S rDNA)

ACGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGTTT T C AGAAT C T T C GGAGGAAGAGGAC AGT GAC T GAGC GGC GGAC GGGT GAGT AAC GC GT GGGC AAC CTGCCTCATAC AGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGGACCGCATGGTGTAGTGTGAAAAACTC CGGTGGTATGAGATGGACCCGCGTCTGATTAGGTAGTTGGTGGGGTAAAGGCCTACCAAGCCGACGATCAGTAGC C GAC C T GAGAGGGT GAC CGGCCACATT GGGAC T GAGAC AC GGC C C AAAC T C C T AC GGGAGGC AGC AGT GGGGAAT ATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATTTCGGTATGTAAACTTCTATC AGCAGGGAAGAAGATGACGGTACCTGAGTAAGAAGCACCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTAT GGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGATAGGCAAGTCTGGAGTGAAAACCCAG GGC T CAAC C C T GGGAC T GC T T T GGAAAC T GCAGAT C T GGAGT GC C GGAGAGGTAAGC GGAAT T C C T AGT GT AGC G GTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTGACTGACGTTGAGGCTCG AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGACTACTAGGTGTCGGTGTG CAAAGCACATCGGTGCCGCAGCAAACGCAATAAGTAGTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAA GGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGTC TTGACATCCGGATGACGGGCGAGTAATGTCGCCGTCCCTTCGGGGCATCCGAGACAGGTGGTGCATGGTTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTCAGTAGCCAGCATATAAGG TGGGCACTCT GGAGAGAC TGC C AGGGAGAAC C T GGAGGAAGGT GGGGAT GAC GT C AAAT CATCATGCCCCTTATG GCCAGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGAGGGTGACCTGGAGCGAATCCCAAAAAT AACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATG CCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGCCAGT GACCCAACCTTAGAGGAGGGAGCTGTCGAAGGCGGGACGGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTAT CGGAAGGTGCGGCTGGATCACCTCCTTT (SEQ ID NO : 3 )

Strain 4 Clostridium symbiosum 16S ribosomal RNA coding sequence (16S rDNA)

AT GAGAGT T TGATCCTGGCTCAGGAT GAAC GCTGGCGGCGTGCC T AAC AC AT GC AAGT C GAAC GAAGC GAT T T AA CGGAAGTTTTCGGATGGAAGTTGGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTGTAC TGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTC CGGTGGTACAAGATGGACCCGCGTCTGATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGC C GAC C T GAGAGGGT GAC CGGCCACATT GGGAC T GAGAC AC GGC C C AAAC T C C T AC GGGAGGC AGC AGT GGGGAAT ATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATC AGC AGGGAAGAAAAT GAC GGT AC C T GAC T AAGAAGC C C C GGC T AAC T AC GT GC C AGC AGC C GC GGT AAT AC GT AG GGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTAAAGCAAGTCTGAAGTGAAAGCCCGC GGCTCAACTGCGGGACTGCTTTGGAAACTGTTTAACTGGAGTGTCGGAGAGGTAAGTGGAATTCCTAGTGTAGCG GT GAAAT GC GT AGAT AT T AGGAGGAAC AC C AGT GGC GAAGGC GAC T T AC T GGAC GAT AAC T GAC GT T GAGGC T C G AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAG CAAAGCTCTTCGGTGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAA GGAAT T GAC GGGGAC C C GC AC AAGC GGT GGAGC AT GT GGT T T AAT T C GAAGC AAC GC GAAGAAC C T T AC C AGGT C TTGACATCGATCCGACGGGGGAGTAACGTCCCCTTCCCTTCGGGGCGGAGAAGACAGGTGGTGCATGGTTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTCTAAGTAGCCAGCGGTTCGGC C GGGAAC T C T T GGGAGAC TGC C AGGGAT AAC C T GGAGGAAGGT GGGGAT GAC GT C AAAT CATCATGCCCCTTATG ATCTGGGCTACACACGTGCTACAATGGCGTAAACAAAGAGAAGCAAGACCGCGAGGTGGAGCAAATCTCAAAAAT AAC GT C T C AGT T C GGAC T GCAGGC T GC AAC T C GC C T GC AC GAAGC T GGAAT C GC T AGT AAT C GC GAAT CAGAAT G TCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGT GACCCAACCGCAAGGAGGGAGCTGCCGAAGGCGGGACCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATC GGAAGGTGCGGCTGGATCACCTCCTTT (SEQ ID NO : 4 )

Strain 5 Blautia producta 16S ribosomal RNA coding sequence (16S rDNA)

T C AGAGAGT T TGATCCTGGCTCAGGAT GAAC GCTGGCGGCGTGCT T AAC AC AT GC AAGT C GAGC GAAGC AC T T AA GTGGATCTCTTCGGATTGAAACTTATTTGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATA CAGGGGGATAACAGTTAGAAATGGCTGCTAATACCGCATAAGCGCACAGGACCGCATGGTCTGGTGTGAAAAACT CCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAG C C GGC C T GAGAGGGT GAAC GGCCACATT GGGAC T GAGAC AC GGC C C AGAC T C C T AC GGGAGGC AGC AGT GGGGAA TATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTAT C AGC AGGGAAGAAAAT GAC GGT AC C T GAC T AAGAAGC C C C GGC T AAC T AC GT GC C AGC AGC C GC GGT AAT AC GT A GGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTG GGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTAGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGC GGTGAAATGCGTAGATATTAGGAGGAACATCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTC GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGTG GCAAAGCCATTCGGTGCCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAA AGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGT CTTGACATCCCTCTGACCGGCCCGTAACGGGGCCTTCCCTTCGGGGCAGAGGAGACAGGTGGTGCATGGTTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTAGCCAGCAGGTGAA GC T GGGC AC T C T AGGGAGAC T GC C GGGGAT AAC C C GGAGGAAGGC GGGGAC GAC GT C AAAT C AT C AT GC C C C T T A TGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGACAGCGATGTTGAGCAAATCCCAAAA ATAACGTCCTAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAA TGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCA GTGACCCAACCTTACAGGAGGGAGCTGCCGAAGGCGGGACCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGT ATCGGAAGGTGCGGCTGGATCACCTCCTTT (SEQ ID NO : 5 )

Strain 6 Dorea longicatena 16S ribosomal RNA coding sequence (16S rDNA)

AAC GAGAGT T TGATCCTGGCTCAGGAT GAAC GCTGGCGGCGTGCT T AAC AC AT GC AAGT C GAGC GAAGC AC T T T G GAAAGATTCTTCGGATGATTTCCTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATA CAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGACCACGGTACCGCATGGTACAGTGGTAAAAACT CCGGTGGTATGAGATGGACCCGCGTCTGATTAGGTAGTTGGTGGGGTAACGGCCTACCAAGCCGACGATCAGTAG C C GAC C T GAGAGGGT GAC CGGCCACATT GGGAC T GAGAC AC GGC C C AGAC T C C T AC GGGAGGC AGC AGT GGGGAA TATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAAGGATGAAGTATTTCGGTATGTAAACTTCTAT C AGC AGGGAAGAAAAT GAC GGT AC C T GAC T AAGAAGC C C C GGC T AAC T AC GT GC C AGC AGC C GC GGT AAT AC GT A GGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCACGGCAAGCCAGATGTGAAAGCCCG GGGCTCAACCCCGGGACTGCATTTGGAACTGCTGAGCTAGAGTGTCGGAGAGGCAAGTGGAATTCCTAGTGTAGC GGT GAAAT GC GT AGAT AT T AGGAGGAAC AC C AGT GGC GAAGGC GGCTTGCT GGAC GAT GAC T GAC GT T GAGGC T C GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGACTGCTAGGTGTCGGGTG GCAAAGCCATTCGGTGCCGCAGCTAACGCAATAAGCAGTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAA AGGAAT T GAC GGGGAC C C GC AC AAGC GGT GGAGC AT GT GGT T T AAT T C GAAGC AAC GC GAAGAAC C T T AC C T GAT CTTGACATCCCGATGACCGCTTCGTAATGGAAGCTTTTCTTCGGAACATCGGTGACAGGTGGTGCATGGTTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCAGTAGCCAGCAGGTTAA GCTGGGCACTCT GGAGAGAC TGC C AGGGAT AAC C T GGAGGAAGGT GGGGAT GAC GT C AAAT CATCATGCCCCTTA TGACCAGGGCTACACACGTGCTACAATGGCGTAAACAAAGAGAAGCGAACTCGCGAGGGTAAGCAAATCTCAAAA ATAACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCAGATCAGAA TGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCA GTGACCCAACCGTAAGGAGGGAGCTGCCGAAGGTGGGACCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTA TCGGAAGGTGCGGCTGGATCACCTCCTTT (SEQ ID NO : 6)

Strain 7 Clostridium innocuum 16S ribosomal RNA coding sequence (16S rDNA)

AT GGAGAGT T TGATCCTGGCTCAGGAT GAAC GCTGGCGGCATGCCTAATACAT GC AAGT C GAAC GAAGT T T C GAG GAAGCTTGCTTCCAAAGAGACTTAGTGGCGAACGGGTGAGTAACACGTAGGTAACCTGCCCATGTGTCCGGGATA AC T GC T GGAAAC GGT AGC T AAAAC C GGAT AGGT AT AC AGAGC GC AT GC T C AGT AT AT T AAAGC GC C C AT C AAGGC GTGAACATGGATGGACCTGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCCCACCAAGGCGATGATGCGTAGCCG GC C T GAGAGGGT AAAC GGCCACATT GGGAC T GAGAC AC GGC C C AAAC T C C T AC GGGAGGC AGC AGT AGGGAAT T T TCGTCAATGGGGGAAACCCTGAACGAGCAATGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGTTGT AAGT GAAGAAC GGC T C AT AGAGGAAAT GC T AT GGGAGT GAC GGT AGC T T AC C AGAAAGC C AC GGC T AAC T AC GT G CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATCATTGGGCGTAAAGGGTGCGTAGGTGGCGTA CTAAGTCTGTAGTAAAAGGCAATGGCTCAACCATTGTAAGCTATGGAAACTGGTATGCTGGAGTGCAGAAGAGGG CGATGGAATTCCATGTGTAGCGGTAAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGTCGCCTGGT CTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAATAGGATTAGATACCCTAGTAGTCCACGCCGTAAACG ATGAGAACTAAGTGTTGGAGGAATTCAGTGCTGCAGTTAACGCAATAAGTTCTCCGCCTGGGGAGTATGCACGCA AGTGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAA GAAC C T T AC CAGGC C T T GAC AT GGAAAC AAAT AC C C TAGAGATAGGGGGAT AAT TAT GGAT C AC AC AGGT GGT GC ATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCGCATGTTACC AGC AT C AAGT T GGGGAC T CAT GC GAGAC T GC C GGT GAC AAAC C GGAGGAAGGT GGGGAT GAC GT C AAAT CAT CAT GC C C C T T AT GGC C T GGGC T AC AC AC GT AC T AC AAT GGC GAC C AC AAAGAGC AGC GAC AC AGT GAT GT GAAGC GAA TCTCATAAAGGTCGTCTCAGTTCGGATTGAAGTCTGCAACTCGACTTCATGAAGTCGGAATCGCTAGTAATCGCA GATCAGCATGCTGCGGTGAATACGTTCTCGGGCCTTGTACACACCGCCCGTCAAACCATGGGAGTCAGTAATACC

CGAAGCCGGTGGCATAACCGCAAGGAGTGAGCCGTCGAAGGTAGGACCGATGACTGGGGTTAAGTCGTAACAAGG

TATCCCTACGGGAACGTGGGGATGGATCACCTCCTTT (SEQ ID NO : 7 )

Strain 8 Flavonifractor plautii 16S ribosomal RNA coding sequence (16S rDNA)

TATTGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGGTGCTCA TGACGGAGGATTCGTCCAACGGATTGAGTTACCTAGTGGCGGACGGGTGAGTAACGCGTGAGGAACCTGCCTTGG AGAGGGGAAT AAC AC T C C GAAAGGAGT GC T AAT AC C GC AT GAT GC AGT T GGGT C GC AT GGC T C T GAC T GC C AAAG ATTTATCGCTCTGAGATGGCCTCGCGTCTGATTAGCTAGTAGGCGGGGTAACGGCCCACCTAGGCGACGATCAGT AGC C GGAC T GAGAGGT T GAC CGGCCACATT GGGAC T GAGAC AC GGC C C AGAC T C C T AC GGGAGGC AGC AGT GGGG AATATTGGGCAATGGGCGCAAGCCTGACCCAGCAACGCCGCGTGAAGGAAGAAGGCTTTCGGGTTGTAAACTTCT TTTGTCGGGGACGAAACAAATGACGGTACCCGACGAATAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGTGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGCGTGTAGGCGGGATTGCAAGTCAGATGTGAAA ACTGGGGGCTCAACCTCCAGCCTGCATTTGAAACTGTAGTTCTTGAGTGCTGGAGAGGCAATCGGAATTCCGTGT GTAGCGGTGAAATGCGTAGATATACGGAGGAACACCAGTGGCGAAGGCGGATTGCTGGACAGTAACTGACGCTGA GGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGGATACTAGGTGTG GGGGGTCTGACCCCCTCCGTGCCGCAGTTAACACAATAAGTATCCCACCTGGGGAGTACGATCGCAAGGTTGAAA CTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTAC CAGGGCTTGACATCCCACTAACGAAGCAGAGATGCATTAGGTGCCCTTCGGGGAAAGTGGAGACAGGTGGTGCAT GGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCTAC GC AAGAGC AC T C T AGC GAGAC T GC C GT T GAC AAAAC GGAGGAAGGT GGGGAC GAC GT C AAAT CATCATGCCCCTT ATGTCCTGGGCCACACACGTACTACAATGGTGGTTAACAGAGGGAGGCAATACCGCGAGGTGGAGCAAATCCCTA AAAGC C AT C C C AGT T C GGAT T GC AGGC T GAAAC C C GC C T GT AT GAAGT T GGAAT C GC T AGT AAT C GC GGAT C AGC ATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTCGGGAACACCCGAAGTC CGTAGCCTAACCGCAAGGAGGGCGCGGCCGAAGGTGGGTTCGATAATTGGGGTGAAGTCGTAACAAGGTAGCCGT ATCGGAAGGTGCGGCTGGATCACCTCCTTT (SEQ ID NO : 8 )

EQUIVALENTS AND SCOPE

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in some embodiments, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in some embodiments, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. Each possibility represents a separate embodiment of the present invention.

It should be understood that, unless clearly indicated to the contrary, the disclosure of numerical values and ranges of numerical values in the specification includes both i) the exact value(s) or range specified, and ii) values that are “about” the value(s) or ranges specified (e.g., values or ranges falling within a reasonable range (e.g., about 10% similar)) as would be understood by a person of ordinary skill in the art.

It should also be understood that, unless clearly indicated to the contrary, in any methods disclosed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are disclosed.

This disclosed compositions and methods are not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosed compositions and methods are capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

EXAMPLES

Example 1: Biological Features of Response to VE303, a Defined Bacterial Consortium, in Patients with Clostridioides difficile Infection (CDI): Results of the Phase 2 CONSORTIUM Study

Background

Fecal transplants and other donor-derived treatments promote a gut environment resistant to CDI, but these treatments have inherently variable quality attributes, are difficult to scale, and can transfer emerging pathogens. VE303 is a defined consortium of 8 purified, clonal Clostridia strains, developed for the prevention of recurrent CDI (rCDI), produced by fermentation from cell banks, and thus overcoming the limitations of donor-derived treatments. In a Phase 1 study of healthy volunteers, VE303 had a favorable safety profile with dose-dependent strain colonization.

In the CONSORTIUM Phase 2 Study (NCT03788434), high-dose VE303 was well tolerated, reduced the odds of rCDI by >80% compared with placebo, and led to both robust colonization of VE303 strains and early restoration of the native microbiota. Louie et al., JAMA. 2023. 239(6): 1356-1366. VE303 organisms successfully colonized the efficacious high-dose group and were significantly associated with nonrecurrence. Multi-omic modeling identified subject antibiotic history, baseline stool metabolites, and serum cytokines as predictors of both on-study CDI recurrence and VE303 colonization. VE303 potentiated early recovery of the host microbiome and metabolites following antibiotic treatment for CDI, which is considered important to prevent future CDI recurrences. These results support that VE303 promotes efficacy in rCDI through multiple mechanisms.

Methods

This was a double-blind, placebo-controlled phase 2 trial (FIG. 1A). After completion of antibiotic treatment for a lab-confirmed CDI episode, 79 subjects were randomized to groups for administration of low-dose VE303, high-dose VE303, or placebo, and dosed orally once daily for 14 days. Subjects were followed for 24 weeks to monitor safety and rCDI episodes. Samples were collected during dosing and at weeks 4 and 8 for metagenomic, metabolomic, and immune analysis. Random forest classification models were used to identify variables that predicted strain colonization and response to VE303 administration.

Results

VE303 strains colonized rapidly in VE303 recipients by day 14, with significantly greater colonization in VE303-dosed subjects vs placebo, and in high-dose vs low-dose (total VE303, VE303 -03, -08) (FIGs. 2A and 2B). Regardless of the dose received, colonization by at least 5 strains, or VE303-08, at the end of dosing were associated with greater recurrence-free probability (FIGs. 2C and 2D).

Reduced colonization was predicted by high screening levels of primary bile acids (BAs) in stool, high inflammatory cytokines in serum, advanced age, and prior nonvancomycin antibiotic exposure at screening (FIGs. 3A and 3G). Increased colonization was predicted by vancomycin, rather than fidaxomicin, treatment for the qualifying CDI episode (FIGs. 3A-3C and 3G). Without wishing to be bound by theory, the increased colonization observed in subjects treated with vancomycin may be due to better clearance of a niche for bacterial strains of VE303.

On-study CDI recurrence was predicted by higher primary bile acid (BA) and inflammatory cytokine levels at screening, as well as increased age and prior antibiotic exposure (FIG. 3D-3F). By day 14, subjects that exhibited on-study CDI recurrence had elevated primary BA levels, and reduced levels of secondary BAs and SCFAs (FIGs. 4A-4C) By contrast, non-recurrence was predicted by lower primary BA levels, and higher secondary BA and SCFA levels at screening (FIG. 3D). In particular, subjects that did not experience recurrence had lower levels of primary BAs deoxycholic acid (DC A), taurochenodeoxy cholic acid (TCDCA), and taurocholic acid (TCA), higher levels of secondary BAs isoallolithocholic acid (isoalloLCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and higher levels of SCFAs butyrate, hexanoate, and isovalerate (FIGs. 4A-4C).

VE303-08 (F 'lav onifr actor plautii), VE303-02 (Anaerotruncus colihominis). and VE303-01 (Clostridium bolleae) were significantly associated with nonrecurrence (see, FIG. 4D, p-adjust<0.25, linear mixed effects [EME] model). Furthermore, multiple VE303 strains were positively correlated with gut metabolites including the SCFAs valerate, acetate and butyrate, and the secondary BA ursodeoxycholic acid (UDCA) (FIG. 4E, p-adjust<0.05, EME), which are thought to confer colonization resistance against C. difficile and inhibit spore germination. Conclusions

In subjects at high risk of rCDI, BA concentrations at screening (high primary BAs, low secondary BAs, and/or low SCFAs) were predictive of on-study CDI recurrence. Furthermore, high levels of primary BAs and inflammatory cytokines at the time of screening predicted low VE303 colonization.

Example 2: Colonization and Effect of VE303, a Defined Bacterial Consortium, in Patients with Clostridioides difficile Infection (CDI): Results of the Phase 2 CONSORTIUM Study

Donor-derived fecal microbiota treatments are efficacious in preventing recurrent Clostridioides difficile Infection (rCDI), but they have inherently variable quality attributes, are difficult to scale, and harbor the risk of pathogen transfer. VE303 is a rationally defined consortium of 8 purified, clonal bacterial strains . In the Phase 2 CONSORTIUM Study, high-dose VE303 was well tolerated and reduced the odds of rCDI by >80% compared with placebo. VE303 organisms robustly colonized the gut in the high-dose group and were among the top taxa associated with non-recurrence. Multi-omic modeling identified subject antibiotic history, baseline stool metabolites, and serum cytokines as predictors of both on- study CDI recurrence and VE303 colonization. VE303 potentiated early recovery of the host microbiome and metabolites following antibiotic treatment for CDI, which is considered important to prevent CDI recurrences. These results support that VE303 promotes efficacy in rCDI through multiple mechanisms.

In the United States, Clostridioides difficile infection (CDI) affects approximately 500,000 people and causes approximately 30,000 deaths annually¹. CDI is typically treated with antibiotics, which perturb the gut microbiome, thereby increasing susceptibility to recurrent CDI (rCDI). Fecal microbiota transplantation (FMT) and other donor-derived formulations have been successful in preventing rCDI^{2 4} ; two products manufactured from human fecal matter were recently approved by the US Food and Drug Administration. However, the composition and quality attributes of these treatments are inherently variable due to donor differences and natural fluctuations in microbiome composition within a donor over time. Furthermore, donor stools require extensive and costly screening to mitigate the risk of pathogen transfer^{4 7}. In contrast, defined bacterial consortia are produced by fermentation from cell banks and thus have a standardized composition and active ingredient dose, reproducible quality attributes, and no risk of pathogen transfer from donors. VE303 is a defined consortium of 8 bacterial strains developed for prevention of rCDI. In a Phase 1 study of healthy volunteers, VE303 had a favorable safety profile with dose-dependent strain colonization⁸. Subsequently, VE303 was evaluated in the Phase 2 CONSORTIUM study (NCT03788434) for the prevention of rCDI. Subjects given the high dose of VE303 had a significantly improved clinical response compared with placebo recipients, and VE303 strain detection was associated with higher recurrence-free probability⁹.

Following antibiotic administration for CDI, microbiota-based treatments aim to restore microbiome diversity and function, to provide colonization resistance to C. difficile. Here recipient microbiome composition, fecal metabolites, and host immune function were profiled to identify factors that predicted VE303 strain colonization and clinical response to treatment. VE303 strains rapidly colonized subjects with CDI in a dose-dependent manner. Increased total VE303 colonization and colonization of organisms VE303-01, -02, -03, and - 08 were linked to non-recurrence of CDI, establishing a direct connection between exposure and clinical response. VE303 facilitated recovery of the recipient’s microbiome following standard-of-care (SoC) antibiotic treatment for CDI. VE303 organisms were negatively correlated with recurrence-associated pathobionts and positively correlated with response- associated Clostridia species, short-chain fatty acids (SCFAs), and the secondary bile acid (BA) ursodeoxycholic acid (UDCA). Furthermore, high baseline levels of fecal primary BAs and peripheral inflammatory cytokines predicted both on-study CDI recurrence and lower VE303 colonization, suggesting that a more disrupted microbiome and increased inflammation may influence outcomes when treating patients at risk of rCDI.

Following antibiotic administration for CDI, microbiota-based treatments aim to restore microbiome diversity and function, to provide colonization resistance to C. difficile. Here, recipient microbiome composition, fecal metabolites, and host immune function were analyzed to identify factors that predicted VE303 strain colonization and clinical response to treatment. VE303 strains rapidly colonized subjects with CDI in a dose-dependent manner. Increased total VE303 colonization and colonization of organisms VE303-01, -02, -03, and - 08 were linked to non-recurrence of CDI, establishing a direct connection between exposure and clinical response. VE303 facilitated recovery of the recipient’s microbiome following standard-of-care (SoC) antibiotic treatment for CDI. VE303 organisms were negatively correlated with recurrence-associated pathobionts and positively correlated with response- associated Clostridia species, short-chain fatty acids (SCFAs), and the secondary bile acid (BA) ursodeoxycholic acid (UDCA). Furthermore, high baseline levels of fecal primary BAs and peripheral inflammatory cytokines predicted both on-study CDI recurrence and lower VE303 colonization, suggesting that a more disrupted microbiome and increased inflammation may influence outcomes when treating patients at risk of rCDI.

Results

The CONSORTIUM Study was a randomized, double-blind, placebo-controlled, dose-finding study in individuals at high risk of rCDI. After completing a course of SoC antibiotics for a laboratory-confirmed CDI episode, eligible subjects were randomized 1:1:1 to receive VE303 high dose (HD) (8xl0⁹ colony-forming units [CFU] daily, cumulative dose l.lxlO¹¹ CFU); VE303 low dose (LD) (1.6xl0⁹ CFU daily, cumulative dose 2.2xlO¹⁰ CFU); or placebo for 14 days (FIG. 6A)⁹. The primary efficacy endpoint was the on-study recurrence rate at week 8. Subjects were followed for 24 weeks to monitor secondary endpoints, including safety, rCDI episodes, and gut microbiota composition. VE303 significantly reduced the risk of rCDI, with a CDI recurrence in 4/29 (13.8%) subjects in the VE303 high-dose group, 10/27 (37%) in the VE303 low-dose group, compared to 10/22 (45%) subjects in the placebo group⁹. Stool and/or serum samples were collected at screening, after SoC antibiotics but before start of VE303 or placebo (day 1), and on study days 7, 14, 28, 56, and 168 (FIGs. 6B, 6C).

Antibiotic treatment of a previous CDI episode is an important risk factor for recurrence^{10 11}, providing a favorable environment for C. difficile growth and toxin production by altering the intestinal microbiota and metabolite composition¹². In The CONSORTIUM Study, most recipients of VE303 HD or ED who experienced recurrences did so early after SoC antibiotics, by day 14, or otherwise experienced sustained cure through week 24⁹. These results indicate that administration of VE303 within this 2- week window after completion of antibiotic treatment for the qualifying CDI episode is useful for reducing the chance of CDI recurrence.

1.1 VE303 strains colonize robustly and predict greater recurrence-free probability

VE303 colonization was quantified in fecal samples (FIGs. 6B, 6C) as previously described^{8 9}. VE303 strains were largely undetected in the placebo group at screening and after the start of dosing, showing high specificity of the strain detection method⁸. The relative abundance of most strains was significantly increased (/? <0.05 ) in both dosed groups vs the placebo group by day 14, indicating dose sufficiency for VE303 strain colonization (increased in HD vs placebo: VE303-01, -02, -03, -04, -05, -07, and -08; increased in ED vs placebo: VE303-01, -03, -04, -07, and -08; see, FIGs. 5A-5E, FIGs. 7A-7I, Table E2-1). A median exposure of 0.1% to 1.2% relative abundance per strain per subject was observed during the month post-dosing for VE303 strains -01, -03, -04, -05, -07, and -08 in the HD and LD groups; strains VE303-02 and -06 showed low levels of colonization in the present study, despite previously having been shown to effectively colonize healthy volunteers⁰.

The number and abundance of VE303 strains increased rapidly in VE303 recipients during the 2 weeks of dosing (FIGs. 5A-5C), with a plateau in strain detection by day 28 and significantly higher colonization in the HD group vs the LD group in terms of both the number of VE303 strains (median 5.5 vs 3; p-adjust=0.02, Wilcoxon day 14) and total VE303 relative abundance (median 5.2% vs 0.8%; p-adjust=0.09, Wilcoxon day 14). Long-term colonization was achieved for a subset of strains, with a median of 2.5 (LD) and 3 (HD) VE303 strains detected on day 168. Strains were generally more prevalent in the VE303 HD vs VE303 LD group during the first 2 weeks after study start (FIG. 5D); in particular, on day 14, strains VE303-03 and VE303-08 were elevated in the VE303 HD group versus the VE303 LD group (p-adjust=0.05, Table E2-1).

The observed differences between HD and LD suggested that by day 14, VE303 colonization protected against subsequent CDI recurrence. “High” colonized subjects had a greater recurrence-free probability when >5 strains colonized, regardless of the dose received⁹. Next, the effect of colonization of specific consortium strains on predicted outcomes was tested using a Cox proportional hazard model (Table E2-2, Methods). Consistent with the dose-exposure relationship described above, colonization of strain VE303-08 was associated (un-adjusted p=0.01; adjusted p=0.08, FIG. 5E, Table E2-2), whereas colonization of strain VE303-03 trended higher (un-adjusted =0.06; adjusted p=0.26, Table E2-2) with greater recurrence-free probability. Overall, these data demonstrate the existence of a positive dose-colonization-response relationship and suggest that superior individual and collective strain colonization soon after SoC antibiotics were contributed to VE303 efficacy.

1.2 Factors affecting VE303 colonization and. on-study CDI recurrence

An understanding of the ecological factors and subject characteristics that predict VE303 colonization and CDI recurrence can yield insights into mechanism(s) of action, support biomarker design to monitor trial outcomes, and inform patient selection in future studies. Random forest models were used to infer the relationship between CDI recurrence or VE303 strain colonization status with ecological and other data collected during the study, including stool BAs, SCFAs, calprotectin, and vancomycin measurements; serum cytokine concentrations; stool microbiota community abundances; and subject-level metadata variables relating to demographics, antibiotic use, and medical history (FIGs. 8A-8C).

1.21 Baseline gut microbial ecology and subject matter attributes influence VE303 strain colonization

Although overall colonization in the VE303-dosed groups was both effective and durable (FIGs. 5A-5E), variability was observed in the patterns of individual strain detection (FIG. 5D). To understand the basis of this heterogeneity, colonization was modeled status as a function of baseline (z.e., prior to SoC antibiotics) ecological and subject- level data. Several strains were not detected in VE303 recipients by day 7 but appeared to colonize during the second week of dosing (FIGs. 5A-5E, FIGs. 7A-7I), potentially reflecting early abundances below the limit of strain detection. Therefore, a strain was considered to have colonized a subject if detected at 2 of 3 available timepoints from day 7 to day 28; subjects were grouped into binary “colonized” and “not-colonized” categories for the classification model. Model performance was assessed using the area under the receiver operating characteristic (AUC) curve (FIGs. 8A-8C); models with median AUC<0.5 over 10 random seed iterations with 5- fold cross-validation were considered non-informative. The remaining models showed adequate performance, with median AUCs ranging between 0.5 and 0.9 (FIG. 8B).

The ability of individual VE303 strains to colonize was predicted by a diverse set of microbial, metabolic, and immune features at screening (FIG. 10). Strains VE303-02, -03, and -08 colonized better in subjects with a high pre- antibiotic abundance of Firmicutes (including Clostridium, Lachnoclostridiu and Lachnospiraceae species), suggesting higher potential for VE303 strain colonization in subjects with a native gut environment conducive to Clostridia. Cytokine profiles likewise associated with colonization of strains VE303-02, - 03 and -08; lower colonization was predicted by increased concentrations of TGF-|32, CXCL10, and MCP-2. Low strain VE303-08 colonization was also predicted by higher primary BAs chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), whereas an increase in taurochenodeoxycholic acid (TCDCA) predicted low strain VE303-02 colonization.

Strain colonization was also predicted by subject antibiotic history, age, and body mass index (BMI). Colonization of VE303-02, -03, -05 and -08 was decreased in older subjects and in subjects with higher BMI. Colonization of most strains was improved by higher exposure (N days administered) to SoC antibiotics immediately before dosing with VE303, and with use of vancomycin for the qualifying CDI episode. However, colonization of strains VE303-03, -05, and -08 was reduced in subjects with higher cumulative exposure (N days administered) to any antibiotics during the month prior to study start. No models were found to be informative for strain VE303-06.

About 72% of subjects in our study (n=56) were treated with vancomycin four times daily (QID) and 21% received fidaxomicin (n= 16) for the qualifying CDI episode. The remaining subjects received metronidazole (n=3) or a non-QID vancomycin regimen (72=3). To further investigate the role of antibiotics in facilitating colonization, VE303 strain abundance was examined in subjects receiving different SoC antibiotic regimens across treatment groups (FIG. 11A). Consistent with the random forest results, strains VE303-02, - 03, -05, -07 and -08 colonized better in subjects that received more days of on-study antibiotic treatment (Table E2-3, FIG. 12). Vancomycin treatment boosted colonization, with significantly higher abundance of strains VE303-01, -02, -03, -07, -08, and total VE303, both during the first 2 weeks and over the long-term (to day 56) among VE303 recipients treated with vancomycin versus fidaxomicin (Table E2-3, FIG. 11A). Vancomycin also achieved significantly better niche clearance (measured as change in diversity) compared with fidaxomicin by day 1, consistent with the expected spectra of activity of these antibiotics (FIG. 11B). Stool vancomycin concentrations peaked at day 1, decayed by 1.5 to 2 logs over the first week of VE303 dosing, and were undetectable in most subjects by day 14 (FIG. 11C); similar vancomycin decay dynamics were seen in placebo recipients (data not shown). During this period, negative correlations between VE303 relative abundance and vancomycin levels were observed for all strains except VE303-06 (p<0.0001 LME; VE303-06 model was unreliable due to low number of subjects colonized, Table E2-3, FIG. 11D). This observation is consistent with prior studies indicating susceptibility of the VE303 strains to vancomycin, with minimum inhibitory concentration values below the concentrations achieved in the stool following vancomycin QID dosing¹³ (Table E2-4). Overall, these findings indicate that a broad but brief perturbation of the gut microbiota with vancomycin can improve VE303 colonization, even with partial strain inhibition during antibiotic decay in the first week of dosing.

1.2.2 Microbial, metabolomic, and. immunological predictors of on-study recurrence

In models predicting rCDI, subjects’ clinical outcomes were defined as recurrent or non-recurrent based on a CDI diagnosis through week 8. Datasets collected at screening, day 1, day 7, and day 14; screening models can identify features that predispose subjects to a recurrent phenotype prior to the start of dosing, whereas models using later timepoints elucidate on-study associations with recurrence. A resampling approach was used to handle class imbalance due to the small number of recurrences in the dosed groups^{14 16} (FIGs. 5A- 5E, Methods). Models with median AUC<0.5 over 100 resampling iterations with 5-fold cross validation were considered non-informative (FIGs. 8A-8C).

Screening levels of stool metabolites (SCFA and BAs), serum cytokines, and stool microbiome datasets showed modest performance for predicting on-study CDI recurrence, with median AUCs ranging between 0.55 and 0.7 (FIG. 8C). Individual features found to be important predictors of recurrence in each model are summarized in FIGs. 13-13D and FIG. 14. Among the clinical variables tested, age and prolonged antibiotic exposure proved detrimental to patient outcomes. Advanced age has been connected with worse CDI outcome^{17 19} , consistent with model predictions (FIGs. 13A-13D, FIGs. 15A-15C). On-study recurrence was also predicted by increased antibiotic exposure during the month prior to study start (N days administered and N courses of antibiotics). Among the resident microbes, increased levels of Veillonella parvula, Veillonella rogosae, Citrobacter freundii, Morganella morganii, and Klebsiella michiganensis were observed in the screening stool of subjects who went on to recur (FIG. 13A). Several metabolic markers were also associated with outcomes; higher levels of primary BAs TCDCA, taurocholic acid (TCA), and glycocholic acid (GCA) (FIG. 13C), and lower levels of SCFAs hexanoate, isovalerate, and butyrate (FIG. 13D) predicted recurrence.

In addition to quantifying metabolic and metagenomic profiles of individual stool samples, measured cytokine concentrations were measured in serum samples as a measure of host immune activation. Concentrations of Eotaxin-2, MIP-ip, SCF, and CXCL9 were increased in subjects who went on to recur, whereas SDF-la/b was depleted (FIGs. 15A- 15C). Interestingly, subject age was positively correlated with the inflammatory cytokines CXCL9 and MIP-ip, indicating higher inflammation in older subjects (FIGs. 15A-15C). Both CDI recurrence and low VE303 colonization were predicted by increased age; expression of TGF-|32; glycine- or taurine-conjugated forms of the primary BAs CA and CDCA; and increased exposure to any antibiotic during the month prior to screening.

1.3 Recovery of host microbiota and metabolites with VE303 dosing

Antibiotic treatment for CDI can lower species diversity and exacerbate gut microbial imbalance, with a low abundance of taxa from the bacterial classes Clostridia and Bacteroidia, and a high abundance of Gammaproteobacteria^20,21. This is accompanied by a reduction in bacterial metabolites such as SCFAs and secondary BAs, and an over- abundance of primary BAs^22,23. The restoration of healthy gut microbiota and metabolites is important to facilitate colonization resistance to C. difficile and alleviate infection. The abundance of primary BAs after antibiotic treatment in patients with CDI can trigger C. difficile spore germination and permit outgrowth of vegetative cells^24,25, whereas recovery of the secondary BA pool abates C. difficile spore germination and growth^{25 27}. Similarly, gut microbe- produced SCFAs enhance colonization resistance against C. difficile and protect against C. t/z//zcz7c- induced colitis^23,28. VE303 promotes recovery of the microbiota community and metabolites following vancomycin exposure in healthy volunteers⁸. Here, the effects of VE303 dosing on recovery of the resident bacterial community, and stool SCFA and BA concentrations in subjects with CDI, were examined.

1.3.1 Among bacterial taxa, VE303 organisms were among the top predictors of nonrecurrence

Metagenomics sequencing and analysis of stool samples was conducted to characterize the microbiome profiles of subjects in the CONSORTIUM Study. Species alpha diversity was calculated across treatment and clinical response categories using the Shannon Index as a global measure of microbiome recovery. Higher diversity was associated with nonrecurrence in all treatment groups, but that the relative diversity change in cured versus recurrent subjects was highest for the VE303 HD group⁹. Over time, a reduction in diversity after SoC antibiotic treatment for the qualifying CDI episode, followed by recovery in all treatment groups, was observed (FIG. 16A). However, diversity at day 14 and diversity increase during the first 2 weeks was significantly higher after VE303 HD than with VE303 LD or placebo (p<0.05, LME, FIG. 16A), indicating expedited microbiome recovery in VE303 HD recipients.

Relative abundance of bacteria at species and higher taxonomic level were estimated from quality-filtered metagenomic reads²⁹ (Methods), analyzing bacterial associations with rCDI in the VE303-dosed groups up to day 14. Most species were not significantly associated with outcome, with a false discovery rate (FDR) adjusted p-value >0.2 (LME), potentially due to the reduced number of samples and the unbalanced nature of the dataset n= 14 with recurrence, zz=42 without recurrence). However, Flavonifractor sp. VE303-08 and Anaerotruncus sp. VE303-02 were significantly associated with non-recurrence, with FDR adjusted -valuc <0.2; Lachnoclostridium sp. VE303-01 and Bifidobacterium longum were also associated with non-recurrence (un-adjusted p-value <0.05) (FIG. 16B). Consistent with the above and with the recurrence-free probability analysis presented in FIG. 16C Flavonifractor sp. VE303-08, Lachnoclostridium sp. VE303-01, and the total number of detected VE303 strains were important features associated with non-recurrence in the random forest model at day 14 (FIG. 14). Broadly, species associated with non-recurrence belonged to class Clostridia, Bacteroidia, or Actinobacteria; those associated with recurrence included Negativicutes, Proteobacteria, and Bacilli (FIG. 13B, FIG. 14, FIGs. 17A-17E). These observations are consistent with prior work showing that the presence and recovery of commensal organisms such as Clostridia, Bifidobacterium and Faecalibacterium spp. are associated with protection against recurrence of CDI^{30 32}, whereas resurgence of pathobionts such as Enterobacteriaceae, Veillonella, and Klebsiella are linked to CDI recurrence and exacerbation of antibiotic-associated diarrhea^{33 36}. Importantly, VE303 was significantly negatively correlated (FDR adjusted p-value <0.05) with recurrence-associated Veillonella, Klebsiella, and Citrobacter species, and positively correlated with response-associated taxa (FIG. 14, FIGs. 17A-17E). Together, these results suggest that VE303 can expedite restoration of gut microbial diversity and drive changes in abundance of specific bacteria to protect against CDI recurrence.

1.3.2 Metabolite associations with clinical recurrence and bacterial dynamics

Stool concentrations of BAs and SCFAs were measured over time, to determine the effect of VE303 on metabolite recovery, especially during the first 2 weeks after antibiotics. Consistent with known changes in the gut metabolome following antibiotic administration^23,37, total stool concentration of primary BAs increased from screening to day 1, with a corresponding decrease in secondary BAs and SCFAs for all subjects (FIGs. 19A- 19C). Over time and after the start of VE303 or placebo administration, the total concentration of secondary BAs and SCFAs increased as expected^38,39. No significant differences in total SCFA or BA concentrations were seen between recurrent and nonrecurrent subjects by day 14; however, individual metabolites were found to be associated with recurrence (random forest regression, FIG. 14). Specifically, the primary BAs TCDCA, TCA, and GCA were elevated in recurrent subjects, the secondary BAs UDCA, lithocholic acid (ECA), and alloisoECA and the SCFAs butyrate, hexanoate, and isovalerate were elevated in non-recurrent subjects post-screening and up to day 14 (FIG. 18A).

A small set of taxa, including VE303 organisms, were significantly associated with recurrence- or response-related metabolites during the 2 weeks post-antibiotics (FIG. 18B). VE303-02, -04, -05, -07, and -08 were positively correlated with acetate, butyrate, valerate, or propionate (FDR-adjusted p <0.05, LME), whereas the commensal species Bifidiobacterium dentium was correlated with an increased butyrate concentration. Strikingly, negative correlations were seen between acetate, butyrate, the secondary BAs LCA and UDCA, and Gammaproteobacteria species predictive of recurrence in Screening samples (FIG. 13A). In contrast, taxa positively correlated with LCA, DCA, or UDCA included VE303-05, -07, and -08, Eggerthella lenta and Ruminococcus gnavus (FIG. 18B). The latter two organisms are known to encode 3a- and 3P-HSDH enzymes, required for production of LCA and DCA from CA^{40 42}. Lastly, VE303-05 was negatively correlated with the primary BAs GCA and glycodeoxycholic acid (GDCA), whereas VE303-05, -07, and -08 were all positively correlated with UDCA (FDR- adjusted p <0.05). Overall, the observed VE303- metabolite associations are consistent with known in vitro SCFA production, and the in silico conversion potential of CA to UDCA by VE303 organisms⁸. The role of VE303 in facilitating UDCA recovery may have significance in light of the growing body of work showing inhibition of C. difficile by this compound, and its evaluation as a standalone treatment for CDI^{43 4}\

Discussion

Donor-derived treatments such as FMT and stool fractions have shown clinical promise for the prevention of rCDI, but it remains challenging to characterize their active ingredients. Maintaining these modalities requires stringent donor screening practices with revision to mitigate the risk of emergent pathogen transfer. VE303 is a rationally defined bacterial consortium of 8 bacterial strains, developed for prevention of rCDI, manufactured from clonal cell banks, improving product consistency, and eliminating the need for donor stool. VE303 colonized effectively with an acceptable safety profile in a Phase 1 doseranging study⁸ and was safe and effective in a Phase 2 study in subjects with CDI⁹. Here, VE303 colonization dynamics and changes in serum cytokines, stool metabolites, and gut microbiome are described; baseline predictors of clinical response and VE303 colonization in subjects at risk of rCDI are also identified.

The 2- to 4- week period of severe microbiome disruption after antibiotic treatment for a CDI episode is considered to be a “window of vulnerability” for subsequent recurrences^46,47. Most CDI recurrences in the CONSORTIUM Study fell within the 14-day window after completion of SoC antibiotics; early VE303 colonization and restoration of the gut microbiota likely mitigated infection in the VE303 HD subjects. Seven of the 8 VE303 strains colonized VE303-dosed vs placebo subjects, and colonization differences between the VE303 HD and LD groups at day 14 mirrored the respective efficacy profiles. Total VE303 proportion and strains VE303-03 and VE303-08 were elevated in the VE303 HD versus VE303 LD group at day 14 and were likewise associated with lower probability of recurrence. Overall, organisms VE303-01, -02, -03, -08, and the total proportion of colonizing VE303 strains, were linked with non-recurrence using time-to-event, LME, and random forest models. These results suggest that colonization by at least 5 VE303 strains contributes to clinical efficacy⁹. However, the specific colonizing strains tended to differ across subjects (FIG. 9), suggesting that protection against rCDI was conferred by the consortium overall rather than through colonization of individual strains.

Increased exposure to the VE303 strains was associated with clinical efficacy. Therefore, available ecological and subject-level data were analyzed to identify predictors of VE303 colonization. Compared with fidaxomicin, vancomycin use resulted in more effective niche clearance and increased VE303 colonization. However, high cumulative exposure to any antibiotic (days administered) in the month prior to the start of dosing predicted lower VE303 colonization, suggesting that prolonged or repeated antibiotic exposure may create an environment unfavorable for VE303 and microbiota community recovery. Moreover, residual vancomycin in the gut suppressed early VE303 colonization. Taken together, this highlights the importance of treating with VE303 for 14 consecutive days following SoC antibiotics. With variable elimination of vancomycin among individuals, continuous VE303 treatment ensures the consortium strains are present when the ecological niche and antibiotic levels are permissive for colonization.

Heterogeneity of response to VE303 was analyzed using random forest models. The presence of Veillonella, Morganella, Klebsiella, and Citrobacter species at baseline and early during dosing was linked to CDI recurrence, consistent with studies demonstrating that CDI severity may be greater in bacterial populations with pathogenic potential^20,48. Like the effect on VE303 strain colonization, the number of courses of antibiotics during the month prior to the study were identified as predicting worse patient outcomes and may have exacerbated C. t/z//zcz7c- induced microbial and metabolic dysbiosis. Increased age and elevated inflammatory cytokines at baseline predicted both lower VE303 strain colonization and higher rates of recurrence.

Gut metabolites, including secondary BAs and SCFAs, are important for providing colonization resistance against C. difficile. Consistent with prior studies, baseline primary BAs including TCA predicted CDI recurrence, whereas CA, CDCA, and TCDCA were associated with reduced VE303 colonization. Early recovery of UDCA, butyrate, and isovalerate protected against recurrence. Butyrate is known to confer colonization resistance against C. difficile^23,23 butyrate and valerate suppress C. difficile in vitro^49,50. Additionally, the primary BA TCA potentiates C. difficile spore germination²⁴, whereas UDCA is a potent inhibitor of C. difficile^54,52 and is being investigated as a treatment for CDI^44,45. Notably, VE303 organisms encode pathways needed for the conversion of CA to UDCA, have been linked to higher UDCA levels in healthy volunteers⁸, and were associated with early recovery of UDCA in patients with CDI. More broadly, recurrence-associated Veillonella, Klebsiella, and Citrobacter organisms were negatively associated with multiple key metabolites, whereas VE303 species were linked with their recovery. This suggests that CDI outcomes in this study were related to a microbe-dependent reduction in beneficial metabolites, which was countered by dosing with VE303.

These findings collectively indicate that greater exposure to VE303 strains, individually and as a consortium, contributed to the clinical response observed in the VE303 HD arm of the CONSORTIUM Study. In this study, VE303 protected against rCDI through a combination of robust colonization of VE303 strains and VE303-mediated recovery of endogenous microbiota and metabolites following SoC antibiotics. These results provide a rationale for advancing VE303 HD into Phase 3, to determine the efficacy and safety of VE303 in the prevention of rCDI in a larger cohort of subjects.

Methods

To be eligible for enrollment in the CONSORTIUM Study, subjects were required to have a laboratory-confirmed qualifying CDI episode. These subjects received SoC antibiotic treatment with one of the following for 10 to 21 days and were also required to have a clinical response, with resolution of diarrhea, to be eligible for randomization: vancomycin QID (N=56), fidaxomicin (N=16), metronidazole (N=3), or vancomycin twice daily (BID) (N=3). On the last day of SoC antibiotics or no later than the following day, subjects were randomized 1:1:1 to receive VE303 HD (8xl0⁹ CFU daily, cumulative dose l.lxlO¹¹ CFU), VE303 LD (1.6xl0⁹ CFU daily, cumulative dose 2.2x10¹⁰ CFU), or matching placebo for 14 days (FIGs. 6A-6C). Stool samples were collected longitudinally from each subject at screening and on days 1, 7, and 14 during dosing with VE303 or placebo; and subsequently on days 28, 56, and 168 (FIGs. 6A-6C). Stool samples were used to determine the prevalence and abundance of the eight VE303 consortium bacterial strains during and after treatment with VE303, to characterize the endogenous bacterial community, and to quantify stool metabolite concentrations. Serum samples were likewise collected on screening and days 14 and 56 and used to quantify cytokine concentrations. Subjects were evaluated for the primary efficacy endpoint of CDI recurrence at week 8, in an analysis that included subjects with a laboratory-confirmed on-study CDI recurrence and subjects who were treated with antibiotics for a CDI recurrence in the absence of laboratory confirmation⁹.

1.4 Metagenomics Sequencing of Subject Fecal Samples

1.4.1 Sample Processing and Stool Metagenomic Sequencing

Stool samples were collected fresh and approximately 500 mg was transferred to an OMNIgene-GUT (OMR-200) tube and resuspended in the preservation buffer according to the manufacturer’s instructions. Tubes were shipped to Diversigen, a microbiome analytics laboratory (Minneapolis, Minnesota, USA), where preserved stool suspensions were extracted and sequenced on the Illumina NovaSeq platform using standard operating procedures. Stool metagenomic sequences (or reads) were assigned to taxonomy using the k- mer-based One Codex platform (onecodex.com) with a reference database including National Center for Biotechnology Information (NCBI, USA) genomes.

1.4.2 Analytical Methods for VE303 Strain Detection and Community Characterization

VE303 strains were detected in subject stool samples using a bioinformatics assay previously described⁸. The estimated relative abundance of endogenous bacterial species in the microbial community was determined using the standard One Codex algorithm from quality-filtered metagenomic reads after removal of unclassified reads and reads that map to the human host ⁹ (onecodex.com). At higher taxonomic levels (e.g., Phylum or Class level), the relative abundance was calculated for each sample as the ratio of sequence reads assigned at the desired taxonomic level (plus all reads assigned below) to the total number of assigned reads.

1.5 Metabolite and Vancomycin Quantification in Subject Fecal Samples

1.5.1 Bile Acid and SCFA Quantification in Stool

Stool samples were collected longitudinally for the measurement of BA and SCFA concentrations. The following BAs were measured by high-performance liquid chromatography (HPLC) and compared with internal standards (Metabolon Incorporated, Durham, North Carolina, USA): chenodeoxycholic acid (CDCA), cholic acid (CA), taurocholic acid (TCA), taurochenodeoxy cholic acid (TCDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), dehydrolithocholic acid (herein 3-oxoLCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), glycodeoxycholic acid (GDCA), glycoursodeoxycholic acid (GUDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), and tauroursodeoxycholic acid (TUDCA). Isoallo- and isolithocholic acid were also measured, but their peak areas were indistinguishable from each other in the assay. These data were reported as “isoallo- + isoLCA.” Approximately 8 to 12 mg of lyophilized human stool was used for each sample. The peak area of each BA was measured against the internal standards and corrected for sample weight (dry lyophilized stool). The final concentrations were provided as ng BA/mg lyophilized feces.

Stool SCFA concentrations, including acetic acid, propionic acid, isobutyric acid, butyric acid, 2-methylbutyric acid, isovaleric acid, valeric acid, succinic acid, and hexanoic acid were determined by liquid chromatography with tandem mass spectrometry (LC- MS/MS) and compared with internal standards (Metabolon, Incorporated, Durham, North Carolina, USA). Approximately 100 mg of raw, frozen human stool was used for each sample. The peak area of each SCFA was measured against the internal standards and corrected for sample weight (wet stool). The final concentrations were provided as ng SCFA/mg feces.

1.5.2 Vancomycin Quantification in Stool

Stool vancomycin concentrations were determined by LC-MS/MS and compared with internal standards (Metabolon, Incorporated, Durham, North Carolina, USA). Approximately 200 mg to 500 mg of raw, frozen human stool was used for each sample. The final concentrations were provided as pg vancomycin/g feces.

1.6 Analysis of Peripheral Cytokines

Blood was collected into 5-mL BD Vacutainer Serum Tube (Cat: 367814, Becton Dickinson, Franklin Lakes, New Jersey, USA) and incubated at room temperature for 15 to 30 minutes prior to centrifugation for 10 minutes at 1,000 to 2,000 x g. Serum was collected and stored at -80°C until analysis. Serum samples were sent to the Forsyth Institute Multiplex Core (Cambridge, Massachusetts, USA) for analysis with Millipore Sigma Kits (Burlington, Massachusetts, USA) on a Luminex platform. HYCTA-60K-PX48, HCP2MAG62KPX236K, and TGFBMAG-64K-03 kits were used according to the manufacturer’s protocol for a total analysis of 74 analytes. 1.7 Statistical Analysis of VE303 Colonization and Host Recovery in Study Subjects

1.7.1 Models to Assess Colonization ofVE303 in High-Dose, Low-Dose and Placebo Groups over Time

To examine early (/.<?., through day 14) colonization and associations with CDI recurrence for total and individual VE303 strains across the VE303 HD, VE303 LD, and placebo groups, an LME model was used on log-transformed microbiome relative abundance data. Mixed effect models together with appropriate mathematical transformations are commonly used to conduct statistical analysis of longitudinal microbiome datasets; they allow inference of feature-phenotype associations, while accounting for multiple samples collected per subject ⁵³~⁵⁵. Treatment group, SoC antibiotic (Abx) type (vancomycin, fidaxomicin, or metronidazole) and sequencing batch were included as covariates in the model; repeated sampling per subject was handled as a random effect following the equation: VE303 ~ Treatment (HD/LD/Placebo) + Abx Type + Batch + 1 [Subject. The model was fit using early dosing timepoints (t < day 14). Model results for strain VE303-06 were unreliable due to sparsity of detection and were therefore excluded from the analysis.

To examine differences in total VE303 colonization at day 14 between the VE303- dosed and placebo groups, a Wilcoxon rank sum test was conducted on total relative abundance data, raw total VE303 proportion, and individual VE303 strain prevalence values. For individual VE303 strains, /-tests were conducted after first log-transforming the relative abundance values to meet the normality assumption. The Wilcoxon test was chosen because it assumes no underlying distribution, is robust to outlier values, and is appropriate for ordinal data such as total strain proportion. In all models, samples were excluded if collected after the onset of an on-study CDI recurrence event or after onset of on-study antibiotics, whichever occurred first.

1.7.2 Recurrence-Free Probability Analysis in High vs Low VE303 -Colonized Subjects To model the probability of recurrence-free survival as a function of exposure to

VE303, Kaplan-Meier (KM) curves were calculated for subjects with “High Colonization” and “Low Colonization” in the two VE303-dosed arms (HD and LD). The probability of recurrence-free survival was modeled as a function of individual VE303 strains, here defining the colonization groups using the relative abundance of the strain in individual subjects on day 14. Subjects with strain relative abundance above the median that was observed across all subjects were categorized as having “High” colonization, whereas those with relative abundance below the median across all subjects were categorized as having “Low” colonization. If a strain was absent in more than half of the subjects, a cut point of 0 was chosen (z.e., High Colonized = Subjects with non-zero abundance). Event-free probability was calculated as a function of colonization using a Cox proportional hazard model⁵⁶ , “A Package for Survival Analysis in R.” R package version 3.2-12); the comparative probability of recurrence in the “Low” vs “High” groups was computed over the duration of the study and represented by the hazard ratio (z.e., the exponential of the model estimate).

1.7.3 Random Forest Classification Models to Determine Features Predicting VE303 Colonization and On-study Recurrence

We used random forest classification models to identify variables linked to subjects’ on-study CDI recurrence status, and colonization with individual VE303 strains through day 28. Models were built separately for data at individual timepoints; Screening datasets were used to identify features predictive of CDI recurrence, and model results using day 1 to 14 datasets were interpreted as being features associated with CDI recurrence. Data modalities used to predict subsequent recurrence and colonization status included stool metabolites, i.e., BAs, SCFAs, and vancomycin measurements; serum cytokine concentrations; host microbiota from metagenomics stool sequencing analysis; and subject-level clinical metadata variables relating to demographics and medical history (FIG. 14). The following clinical metadata variables were included in each model: Age; BMI; number of CDI recurrences prior to and including the qualifying episode; number of times a subject was prescribed antibiotics in the month before screening; number of days a subject was exposed to any antibiotic within the month before screening; number of days of on-study antibiotic for the qualifying episode; total administered dose of on-study antibiotic for the qualifying episode; and antibiotic type (vancomycin or other i.e., fidaxomicin or metronidazole). A subset of individuals started antibiotics prior to collection of Screening samples, which could skew the measurement of microbiome and metabolite profiles. Therefore, antibiotics screening status was included as a separate variable in the random forest model.

Subjects were classified as recurrent or non-recurrent based on their CDI status through week 8. For predicting VE303 strain colonization, a strain was considered colonized if it was detected at 2 out of 3 sampled timepoints from day 7 to day 28. Subjects were grouped into binary “colonized” (C) and “not-colonized” (NC) categories for the classification model. To address the extreme class imbalance seen in the recurrence models, owing to the small number of CDI recurrences in the VE303-dosed groups, a resampling procedure was followed, whereby the training datasets for the random forest models undersampled the majority class (non-recurrent subjects) to match the minority class (recurrent subjects)^{14 l6}. Model validation was performed on 100 iterations of random subsampling of the minority class with 5-fold stratified cross-validation. For predicting colonization, a standard (without under- sampling) random forest model realized over 10 random seeds was fit, and leave-one-out cross-validation was performed to assess model performance. In all cases, feature selection was applied to reduce the feature space by removing sparse or nonsense variables⁵⁷ and computed the AUC to discern meaningful models. Any models for which the median AUC was <0.5 were not considered meaningful, as this indicated random or unreliable model performance (FIGs. 8A-8C).

1.7.4 Models to Determine Associations between Endogenous Taxa, Metabolites, Response, and VE303

The species alpha diversity, abundance of endogenous microbiota, SCFA, and BA were compared across treatment groups and by CDI recurrence status using an LME model. The model was fit using early dosing timepoints (t < day 14), and excluding all samples collected after the onset of a CDI recurrence event or after the onset of on-study antibiotics, whichever occurred first. Microbiota analysis was conducted at the species, genus, family, order, and class taxonomic levels. A prevalence threshold (taxa present in at least 20% of samples) and an abundance threshold (taxa >0.01% average abundance across subjects) were applied to exclude taxa that were too sparse or low in abundance to obtain accurate model results.

The LME model was also used to assess the associations of the microbiota and metabolite concentrations with total VE303 abundance during the early recovery (t < day 14) period. Treatment group, SoC antibiotic type (vancomycin, fidaxomicin, or metronidazole), recurrence status, and sequencing batch were included as categorical covariates in the model, with VE303 abundance included as a continuous covariate; repeated sampling per subject was handled as a random effect following the equation: Diversity /Metabolite/Microbial Taxon ~ Treatment (HD/LD/Placebo) + Abx Type + Recurrence + Batch + VE303 + 1 (Subject. Diversity differences between treatment groups per timepoint and diversity change from screening to day 14 per treatment group were assessed by fitting additional models that encoded an interaction between treatment and Visit.

1.7.5 Models to Determine the Effect of Antibiotics on VE303 Colonization

To assess the difference in VE303 colonization following SoC antibiotics of different types (vancomycin, fidaxomicin, metronidazole, or other) and duration, the log-transformed relative abundance of individual strains or total VE303 was regressed as: VE303 ~ Abx Type + 1 [Subject; and VE303 ~ Abx length + 1 [Subject. Subjects in the VE303-dosed groups were included in this analysis, and models were run to assess colonization up to day 14 and up to day 56. Finally, the correlation between individual VE303 strain abundances and vancomycin concentrations were assessed up to day 14. The log-transformed relative abundance per strain was regressed against the log-transformed vancomycin level; repeated sampling per subject was handled as a random effect: VE303 ~ Vancomycin + Treatment (HD/LD) + 1 [Subject.

In models comparing the effects of different antibiotic treatment regimens, analysis was restricted to subjects treated with vancomycin QID (n = 56) and fidaxomicin (n = 16), as these constituted 92.3% of enrolled subjects. The remaining regimens could not be evaluated statistically due to small numbers enrolled (n = 3 metronidazole, n = 3 vancomycin non-

QID).

Table E2-1 Linear Mixed Model Summary: Comparison of VE303 Strain Colonization in VE303 High Dose, VE303 Low Dose and Placebo Groups During Day 1 to Day 14 in Study VE303-002

Models were fit to examine the differences in individual VE303 strain colonization up to Day 14 between the indicated groups. A positive value for Effect Size indicates an increase in the first group relative to the second group mentioned in column 1. Table E2-2 Cox Model Summary: VE303 Colonization Associations with Recurrence- Free Survival in VE303-Dosed Groups in Study VE303-002

*logrank test. Cox inapplicable as coefficient diverges

Results of the Cox models for recurrence-free survival as a function of individual VE303 strain colonization. The log-rank p-value is shown for strain VE303-08 and VE303-06, as the Cox p-value was not evaluable. A positive value for the Effect Size indicates increased probability of recurrence-free survival in subjects in the “High” Colonized group.

Table E2-3 Linear Mixed Model Summary: VE303 Abundance Correlation with Type and Length (Days) Antibiotic Treatment for the Qualifying CDI Episode in Study VE303-002

Linear mixed models to estimate the effect of antibiotics type (vancomycin versus fidaxomicin), treatment length (N days) and residual vancomycin concentration on VE303 strain colonization in dosed subjects. A negative value for the Effect Size indicates lower VE303 colonization in fidaxomicin-treated subjects (top), or a negative relationship between the indicated covariate and VE303 colonization.

Table E2-4 Minimum Inhibitory Concentrations of Clinically Relevant Antibiotics for VE303 Organisms

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Claims

What is claimed is: CLAIMS

1. A method of treating or preventing Clostridiodes difficile ( C. difficile) infection in a subject, the method comprising administering to the subject a composition comprising a purified bacterial mixture, wherein the purified bacterial mixture comprises:

(i) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 1;

(ii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 2;

(iii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 3;

(iv) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 4;

(v) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 5;

(vi) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 6;

(vii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 7; and

(viii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 8, wherein:

(a) the subject is younger than 60 years old; and/or

(b) a fecal sample of the subject obtained prior to administration of the composition comprises a lower concentration of a primary bile acid, relative to a reference stool concentration of the primary bile acid; and/or

(c) a fecal sample of the subject obtained prior to administration of the composition comprises a higher concentration of a secondary bile acid, relative to a reference stool concentration of the secondary bile acid; and/or

(d) a fecal sample of the subject obtained prior to administration of the composition comprises a higher concentration of a short-chain fatty acid (SCFA), relative to a reference stool concentration of the SCFA; and/or (e) a serum sample of the subject obtained prior to administration of the composition comprises a lower concentration of one or more inflammatory cytokines, relative to a reference serum concentration of the inflammatory cytokine; and/or

(f) the subject has received vancomycin within two weeks prior to administration of the composition; and/or

(g) the subject has not received an antibiotic other than vancomycin within two weeks prior to administration of the composition.

2. The method of claim 1, wherein the subject is younger than 60 years old.

3. The method of claim 1 or 2, wherein the subject is 55 years old or younger, 50 years old or younger, 45 years old or younger, or 40 years old or younger.

4. The method of any one of claims 1-3, wherein the fecal sample of the subject comprises a lower concentration of the primary bile acid, relative to the reference stool sample concentration of the primary bile acid.

5. The method of any one of claims 1-4, wherein the primary bile acid is glycocholic acid (GCA), taurocholic acid (TCA), or taurochenodeoxycholic acid (TCDCA).

6. The method of any one of claims 1-5, wherein the primary bile acid concentration in the subject’s fecal sample is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the reference stool sample concentration of the primary bile acid.

7. The method of any one of claims 1-6, wherein the fecal sample of the subject comprises a higher concentration of a secondary bile acid, relative to a reference stool sample concentration of the secondary bile acid.

8. The method of any one of claims 1-7, wherein the secondary bile acid is deoxy cholic acid (DCA), isoallolithocholic acid (isoalloLCA), lithocholic acid (LCA), tauroursodeoxycholic acid (TUDCA), or ursodeoxycholic acid (UDCA).

9. The method of any one of claims 1-8, wherein the secondary bile acid concentration in the fecal sample of the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the reference stool sample concentration of the secondary bile acid.

10. The method of any one of claims 1-9, wherein a fecal sample of the subject comprises a higher concentration of a short-chain fatty acid (SCFA), relative to a reference stool sample concentration of the SCFA.

11. The method of any one of claims 1-10, wherein the SCFA is hexanoate, isovalerate, or butyrate.

12. The method of any one of claims 1-11, wherein the SCFA concentration in the fecal sample of the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, or at least 300% of the reference stool sample concentration of the SCFA.

13. The method of any one of claims 1-12, wherein the serum sample of the subject comprises a lower concentration of one or more inflammatory cytokines, relative to a reference serum concentration of the inflammatory cytokine.

14. The method of any one of claims 1-13, wherein the inflammatory cytokine is CXCL9, eotaxin-2, IL-27, MIP-ip, SCF, SDF-la, or SDF-lb.

15. A method of colonizing a gut microbiome in a subject, the method comprising administering to the subject a composition comprising a purified bacterial mixture, wherein the purified bacterial mixture comprises:

(iii) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 3; (iv) a bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to SEQ ID NO: 4;

(a) the subject is younger than 60 years old; and/or

(b) a fecal sample of the subject obtained prior to administration of the composition comprises a lower concentration of a primary bile acid, relative to a reference stool sample concentration of the primary bile acid; and/or

(c) a serum sample of the subject obtained prior to administration of the composition comprises a lower concentration of an inflammatory cytokine, relative to a reference serum sample concentration of the inflammatory cytokine; and/or

(d) the subject has been administered vancomycin within two weeks prior to administration of the composition; and/or

(e) the subject has not received an antibiotic other than vancomycin within two weeks prior to administration of the composition.

16. The method of claim 15, wherein the subject is younger than 60 years old.

17. The method of claim 16, wherein the subject is 55 years old or younger, 50 years old or younger, 45 years old or younger, or 40 years old or younger.

18. The method of any one of claims 1-17, wherein the fecal sample of the subject comprises a lower concentration of a primary bile acid, relative to a reference stool sample concentration of the primary bile acid.

19. The method of any one of claims 1-18, wherein the primary bile acid is cholic acid, chenodeoxycholic acid, or glycochenodeoxycholic acid.

20. The method of any one of claims 1-19, wherein the primary bile acid concentration in the subject’s fecal sample is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the reference fecal sample concentration of the primary bile acid.

21. The method of any one of claims 1-20, wherein the serum sample of the subject comprises a lower concentration of an inflammatory cytokine, relative to a reference serum sample concentration of the inflammatory cytokine.

22. The method of any one of claims 1-21, wherein the inflammatory cytokine is IL-6, IL- 10, IL- 18, IL-22, MCP-2, MIP-10, and/or TNF-a.

23. The method of any one of claims 1-22, wherein the inflammatory cytokine concentration in the serum sample of the subject is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the reference serum sample concentration of the inflammatory cytokine.

24. The method of any one of claims 1-23, wherein one or more of the bacterial strains produce a short chain fatty acid (SCFA).

25. The method of any one of claims 1-24, wherein one or more of the bacterial strains produce acetate, butyrate, and/or valerate.

26. The method of any one of claims 1-25, wherein one or more of the bacterial strains produce a secondary bile acid.

27. The method of any one of claims 1-26, wherein one or more of the bacterial strains produce ursodeoxycholic acid (UDCA).

28. The method of any one of claims 1-27, wherein the bacterial strains are lyophilized.

29. The method of any one of claims 1-27, wherein the bacterial strains are spray-dried.

30. The method of any one of claims 1-29, wherein one or more of the bacterial strains are in spore form.

31. The method of any one of claims 1-30, wherein each of the bacterial strains is in spore form.

32. The method of any one of claims 1-30, wherein one or more of the bacterial strains are in vegetative form.

33. The method of any one of claims 1-29 and 32, wherein each of the bacterial strains is in vegetative form.

34. The method of any one of claims 1-33, wherein the composition is a pharmaceutical composition comprising the bacterial strains and a pharmaceutically acceptable excipient.

35. The method of claim 34, wherein the pharmaceutical composition is formulated for oral delivery.

36. The method of claim 34, wherein the pharmaceutical composition is formulated for rectal delivery.

37. The method of any one of claims 34-36, wherein the pharmaceutical composition is formulated for delivery to the intestine.

38. The method of any one of claims 34-37, wherein the pharmaceutical composition is formulated for delivery to the colon.

39. The method of any one of claims 34-38, wherein the pharmaceutical composition comprises one or more enteric polymers.

40. The method of any one of claims 34-39, wherein the pharmaceutical composition is in a capsule.

41. The method of claim 40, wherein the capsule comprises IxlO⁷ to IxlO¹⁰ colonyforming units (CFUs) of each of the bacterial strains.

42. The method of any one of claims 1-33, wherein the composition is present in a food product comprising the bacterial strains and a nutrient.

43. The method of any one of claims 1-42, wherein the subject has a pathogenic infection.

44. The method of claim 43, wherein the composition is administered in a therapeutically effective amount to treat a pathogenic infection in the subject.

45. The method of claim 43 or 44, wherein the pathogenic infection is a C. difficile infection.

46. The method of any one of claims 43-45, wherein the pathogenic infection is a recurrent C. difficile infection.

47. The method of any one of claims 43-46, wherein the composition is administered in one dose.

48. The method of any one of claims 43-46, wherein the composition is administered in multiple doses.

49. The method of claim 47 or 48, wherein each dose of the composition comprises administration of multiple capsules.

50. The method of any one of claims 43-49, wherein the subject is administered vancomycin prior to administration of the composition.

51. The method of any one of claims 43-50, wherein the composition induces the proliferation and/or accumulation of regulatory T (Treg) cells in the subject.

52. The method of any one of claims 43-51, wherein the subject is a human.