Methods to reduce enterotoxemia and restore the microbiome

Abstract

To provide methods for decreasing dysbiosis, restoring the microbiome, and / or increasing recovery of a healthy microbiome (e.g., following a dysbiosis inducing event), by administering pharmaceutical compositions to a subject.SOLUTION: Provided is a method for decreasing dysbiosis in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more purified bacterial strains to decrease dysbiosis in the subject.SELECTED DRAWING: Figure 44

Description

[Technical Field]

[0001] Related applications This application claims the benefits under Section 119(e) of U.S. Patent Act, relating to U.S. Provisional Application No. 62 / 765,165 filed on 17 August 2018, U.S. Provisional Application No. 62 / 724,185 filed on 29 August 2018, U.S. Provisional Application No. 62 / 815,395 filed on 8 March 2019, U.S. Provisional Application No. 62 / 829,513 filed on 4 April 2019, and U.S. Provisional Application No. 62 / 829,959 filed on 5 April 2019. The entire contents of each of these referenced applications are incorporated herein by reference.

[0002] Field of Invention This disclosure relates to methods for reducing enterotoxemia, restoring the microbiome, and / or increasing the recovery of the microbiome (e.g., after an enterotoxemia-induced event) by administering a pharmaceutical composition to a subject. Also provided are methods for protecting and / or establishing the microbiome of a subject by administering a pharmaceutical composition to a subject. [Background technology]

[0003] The human gut microbiome consists of tens of trillions of bacteria from over 1,000 identified species. An individual's microbiome is as unique as a fingerprint, and even among close relatives, there is considerable diversity. The majority of species in the human microbiome are commensal organisms, neither harming nor injuring their host. Many bacterial species residing in the human gut, including members of the taxonomic groups Lactobacillus, Firmicutes, and Bacteriodetes, are symbiotic organisms that perform functions beneficial to humans, such as the metabolism of food by-products into absorbable nutrients. However, pathogenic bacterial species, such as strains of Escherichia coli, can also reside in the human gut and, if excessively concentrated in the human microbiome, can cause disease. Therefore, maintaining a balance of bacterial species within the human microbiome is crucial for overall human health.

[0004] Disruptions in the abundance of bacterial species within the human microbiome, decreased bacterial diversity, and altered bacterial functional capacity are signs of enterotoxosis, which can result from events such as infection, recurrent and inadequate antibiotic treatment, and inflammation. Enterotoxosis occurs when the presence of few commensal or symbiotic bacterial species in the human microbiome leads to a proliferation of opportunistic pathogenic species. Conventional therapies to reverse enterotoxosis and restore the microbiome include fecal transplantation (FMT). While FMT can be effective in treating enterotoxosis, it lacks a standardized method for administration and carries the potential risk of in transplanting pathogens or allowing pathogen proliferation to occur before recovery is achieved. [Overview of the project]

[0005] Aspects of this disclosure provide a method for reducing enterotoxemia in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains to reduce the subject's enterotoxemia. In some embodiments, the reduction in enterotoxemia includes an increase in the amount of Bacteroides compared to the amount of Bacteroides before administration of the pharmaceutical composition. In some embodiments, the pharmaceutical composition does not contain Bacteroides.

[0006] In some embodiments, the reduction in enterotoxemia includes an increase in the abundance of Firmicutes compared to the abundance of Firmicutes before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia includes an increase in the abundance of bacterial strains belonging to Clostridium cluster IV and / or XIVa compared to the abundance of bacterial strains belonging to Clostridium cluster IV and / or XIVa compared to the abundance of bacterial strains belonging to Clostridium cluster XVII compared to the abundance of bacterial strains belonging to Clostridium cluster XVII before administration of the pharmaceutical composition.

[0007] In some embodiments, the reduction in enterotoxemia includes a decrease in the abundance of inflammation-associated microorganisms compared to the abundance of inflammation-associated microorganisms before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia includes a decrease in the abundance of Proteobacteria compared to the abundance of Proteobacteria before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia does not correlate with a proportional increase in the diversity of the target microbiome.

[0008] Aspects of this disclosure provide a method for restoring the microbiome in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains to restore the microbiome in the subject. In some embodiments, the subject has not experienced an enterotoxosis-induced event. In some embodiments, the subject does not have an infection. In some embodiments, the subject does not have a Clostridium difficile infection. In some embodiments, the subject has not been treated with antibiotics.

[0009] Aspects of this disclosure provide a method for increasing the recovery of a healthy microbiome in a subject after an enterotoxemia-induced event, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains to increase the recovery of a healthy microbiome.

[0010] In some embodiments, the enterotoxemia-induced event is treatment with one or more antibiotics. In some embodiments, the enterotoxemia-induced event is treatment with one or more antibiotics related to surgery. In some embodiments, the antibiotic is vancomycin.

[0011] In some embodiments, the enterotoxosis-inducing event is an infection. In some embodiments, the enterotoxosis-inducing event is an infection with Clostridium difficile. In some embodiments, the enterotoxosis-inducing event is a primary infection with Clostridium difficile. In some embodiments, the enterotoxosis-inducing event is a secondary or recurrent infection with Clostridium difficile. In some embodiments, the enterotoxosis-inducing event is traveler's diarrhea.

[0012] In some embodiments, microbiome recovery in the subject occurs without detectable colonization of the bacterial strain of the pharmaceutical composition. In some embodiments, microbiome recovery is increased compared to the recovery of a healthy microbiome in the absence of administration of the pharmaceutical composition. In some embodiments, microbiome recovery is increased compared to the recovery of a healthy microbiome in a subject that has received fecal matter transplantation.

[0013] Aspects of this disclosure provide methods for protecting the microbiome in a subject, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains to protect the microbiome. In some embodiments, the microbiome is protected from antibiotic treatment. In some embodiments, the microbiome is protected from attack by infectious agents. In some embodiments, the microbiome is protected from Clostridium difficile infection. In some embodiments, the microbiome is protected from secondary or recurrent Clostridium difficile infection.

[0014] Aspects of this disclosure provide a method for colonizing a target microbiome, the method comprising administering to a target a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains to colonize the microbiome. In some embodiments, at least 25% of the bacterial strains of the pharmaceutical composition colonize the target microbiome. In some embodiments, at least 50% of the bacterial strains of the pharmaceutical composition colonize the target microbiome. In some embodiments, 100% of the bacterial strains of the pharmaceutical composition colonize the target microbiome.

[0015] In some embodiments, after administration, at least 25% of the bacterial strains in the target microbiome are the bacterial strain of the pharmaceutical composition. In some embodiments, after administration, at least 50% of the bacterial strains in the target microbiome are the bacterial strain of the pharmaceutical composition.

[0016] In some embodiments, one or more bacterial strains are detected in the microbiome at least four weeks after the first dose of the pharmaceutical composition. In some embodiments, one or more bacterial strains are detected in the microbiome at least six weeks after the first dose of the pharmaceutical composition. In some embodiments, one or more bacterial strains are detected in the microbiome at least twelve weeks after the first dose of the pharmaceutical composition. In some embodiments, one or more bacterial strains are detected in the microbiome at least six months after the first dose of the pharmaceutical composition. In some embodiments, one or more bacterial strains are detected in the microbiome at least twelve months after the first dose of the pharmaceutical composition.

[0017] Aspects of this disclosure provide a method for colonizing a target microbiome, the method comprising administering an antibiotic to the target, followed by administering a therapeutically effective amount of a pharmaceutical composition containing one or more bacterial strains to colonize the microbiome. In some embodiments, each of the bacterial strains of the bacterial composition colonizes the microbiome.

[0018] Aspects of this disclosure provide a method for treating a C. difficile infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more purified bacterial strains to treat the C. difficile infection. In some embodiments, the C. difficile infection is either a primary C. difficile infection or a recurrent C. difficile infection.

[0019] Aspects of this disclosure provide a method for treating food allergies in subjects, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising one or more purified bacterial strains to treat the food allergy. In some embodiments, the composition suppresses the production of IgE antibodies. In some embodiments, the composition suppresses one or more Th2 immune responses. In some embodiments, the composition suppresses one or more mast cell function and / or mast cell degranulation. In some embodiments, the composition modulates immune responses associated with food allergies.

[0020] In some embodiments, the bacterial strains of the pharmaceutical composition colonize the microbiome over a long period of time. In some embodiments, the antibiotic is vancomycin. In some embodiments, the pharmaceutical composition is administered as a single dose. In some embodiments, the pharmaceutical composition is administered in multiple doses. In some embodiments, one or more bacterial strains include one or more Clostridium difficile inhibitory strains. In some embodiments, the pharmaceutical composition is administered to a subject in a therapeutically effective dose containing one or more bacterial strains to colonize the microbiome in order to treat graft-versus-host disease (GvHD).

[0021] In some embodiments, the pharmaceutical composition comprises one or more bacterial strains belonging to Clostridium clusters IV, XIVa, and XVII. In some embodiments, the pharmaceutical composition comprises one or more bacterial strains belonging to each of Clostridium clusters IV, XIVa, and XVII. In some embodiments, the pharmaceutical composition comprises the bacterial strain Dorea longicatena. In some embodiments, the pharmaceutical composition comprises the bacterial strain Dorea longicatena. In some embodiments, the pharmaceutical composition comprises a 16S rDNA sequence with at least 97% sequence identity to the nucleic acid sequence shown as SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises a single bacterial strain comprising a 16S rDNA sequence with at least 97% sequence identity to the nucleic acid sequence shown as SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises at least 50% bacterial strains belonging to Clostridium cluster XIVa. In some embodiments, the pharmaceutical composition comprises at least 75% bacterial strains belonging to Clostridium clusters IV and / or XIVa. In some embodiments, the pharmaceutical composition comprises Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavinofractor plautii.In some embodiments, the pharmaceutical composition comprises a purified bacterial mixture consisting of bacterial strains containing 16S rDNA sequences with at least 97% sequence identity to the sequences shown as 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, and SEQ ID NO: 8.

[0022] In some embodiments, the pharmaceutical composition comprises Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Clostridium innocuum, and Flavinofractor plautii. In some embodiments, the pharmaceutical composition comprises a purified bacterial mixture consisting of bacterial strains containing 16S rDNA sequences with at least 97% sequence identity to the nucleic acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments, the pharmaceutical composition comprises a purified bacterial mixture consisting of bacterial strains containing 16S rDNA sequences with at least 97% sequence identity to the nucleic acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8.

[0023] In some embodiments, the pharmaceutical composition comprises a purified bacterial mixture comprising Clostridium saccharogumia (Clostridium ramosum JCM 1298), Flavonifractor plautii (Pseudoflavonifractor capillosus ATCC 29799), Clostridium hathewayi (Clostridium saccharolyticum WM1), Blautia coccoides (Lachnospiraceae bacterium 6_1_63FAA), Clostridium genus (Clostridium bolteae ATCC BAA-613), cf. Clostridium genus MLG055 (Erysipelotrichaceae bacterium 2_2_44A), Clostridium indolis (Anaerostipes caccae DSM 14662), Anaerotruncus colihominis (Anaerotruncus colihominis DSM 17241), Ruminococcus genus ID8 (Lachnospiraceae bacterium 2_1_46FAA), Clostridium lavalense (Clostridium asparagiforme DSM 15981), Clostridium symbiosum (Clostridium symbiosum WAL-14163), Clostridium ramosum, Eubacterium contortum (Clostridium genus D5), Clostridium scindens (Lachnospiraceae bacterium 5_1_57FAA), Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1), Clostridium genus 316002 / 08 (Clostriales bacterium 1_7_47FAA), Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1).In some embodiments, the pharmaceutical composition consists of a purified bacterial mixture comprising Clostridium saccharogumia (Clostridium ramosum JCM 1298), Flavonifractor plautii (Pseudoflavonifractor capillosus ATCC 29799), Clostridium hathewayi (Clostridium saccharolyticum WM1), Blautia coccoides (Lachnospiraceae bacterium 6_1_63FAA), Clostridium genus (Clostridium bolteae ATCC BAA-613), cf. Clostridium genus MLG055 (Erysipelotrichaceae bacterium 2_2_44A), Clostridium indolis (Anaerostipes caccae DSM 14662), Anaerotruncus colihominis (Anaerotruncus colihominis DSM 17241), Ruminococcus genus ID8 (Lachnospiraceae bacterium 2_1_46FAA), Clostridium lavalense (Clostridium asparagiforme DSM 15981), Clostridium symbiosum (Clostridium symbiosum WAL-14163), Clostridium ramosum, Eubacterium contortum (Clostridium genus D5), Clostridium scindens (Lachnospiraceae bacterium 5_1_57FAA), Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1), Clostridium genus 316002 / 08 (Clostriales bacterium 1_7_47FAA), Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1).

[0024] In some embodiments, the pharmaceutical composition contains at least 1.6×10 9 CFU (colony forming units). In some embodiments, the pharmaceutical composition contains at least 4.0×10 9 CFU (colony forming units). In some embodiments, the pharmaceutical composition contains at least 8.0×10 9 CFU (colony forming units). In some embodiments, the pharmaceutical composition contains at least 4.0×10 10 CFU (colony forming units). In some embodiments, the pharmaceutical composition contains at least 1.1×10 11 CFU (colony forming units). In some embodiments, the pharmaceutical composition contains at least 1.1×10 17 CFU (colony forming units).

[0025] In some embodiments, the pharmaceutical composition is administered as a single dose. In some embodiments, the pharmaceutical composition is administered as multiple doses. In some embodiments, each dose includes the administration of multiple capsules. In some embodiments, each capsule contains at least 8.0×10 8 CFU (colony forming units). In some embodiments, each capsule contains at least 1.6×10 9 CFU (colony forming units).

[0026] In some embodiments, the pharmaceutical composition contains at least 1.6×10 9 CFU (colony forming units) and is administered as a single dose. In some embodiments, the pharmaceutical composition contains at least 4.0×10 10 CFU (colony forming units) and is administered as a single dose. In some embodiments, the pharmaceutical composition contains at least 8.0×10 9 CFU (colony forming units) and is administered as a single dose. In some embodiments, the pharmaceutical composition contains at least 4.0×10 10 CFU (colony forming units) and is administered as multiple doses. In some embodiments, the pharmaceutical composition contains at least 4.0×1010 It contains CFU (colony-forming units) and is administered in five doses. In some embodiments, the pharmaceutical composition contains at least 2.8 × 10 10 It contains CFU (colony-forming units) and is administered in multiple doses. In some embodiments, the pharmaceutical composition contains at least 2.8 × 10 10 It contains CFU (colony-forming units) and is administered in seven doses. In some embodiments, the pharmaceutical composition contains at least 5.6 × 10 10 It contains CFU (colony-forming units) and is administered in multiple doses. In some embodiments, the pharmaceutical composition contains at least 5.6 × 10 10 It contains CFU (colony-forming units) and is administered in 14 doses. In some embodiments, the pharmaceutical composition contains at least 1.1 × 10 11 It contains CFU (colony-forming units) and is administered in multiple doses. In some embodiments, the pharmaceutical composition contains at least 1.1 × 10⁻⁶ 11 It contains CFU (colony-forming units) and is administered in 14 doses. In some embodiments, the pharmaceutical composition contains at least 1.1 × 10 17 It contains CFU (colony-forming units) and is administered in multiple doses. In some embodiments, the pharmaceutical composition contains at least 1.1 × 10⁻⁶ 17 It contains CFU (colony-forming units) and is administered in 14 doses.

[0027] In some embodiments, multiple doses are administered daily. In some embodiments, the method includes one or more additional doses of the pharmaceutical composition. In some embodiments, one or more additional doses of the pharmaceutical composition contain fewer CFUs (colony-forming units) compared to the initial dose of the pharmaceutical composition.

[0028] In some embodiments, one or more additional doses of the pharmaceutical composition are administered daily following the initial dose. In some embodiments, one or more additional doses of the pharmaceutical composition are administered at least 6 weeks after the initial dose. In some embodiments, one or more additional doses of the pharmaceutical composition are administered at least 12 weeks after the initial dose.

[0029] In some embodiments, the pharmaceutical composition contains at least 2.1 × 10 10 Contains CFU (colony-forming units). In some embodiments, the pharmaceutical composition is administered in multiple doses. In some embodiments, the pharmaceutical composition is administered in five doses.

[0030] In some embodiments, the pharmaceutical composition is administered for five consecutive days. In some embodiments, the pharmaceutical composition is administered as two high doses followed by three low doses. In some embodiments, the high dose is 8.0 × 10⁻⁶. 9 CFU (colony-forming units) is used in some embodiments. In some embodiments, the low dose is 1.6 × 10⁻⁶. 9 It is a CFU (colony-forming unit). In some embodiments, the pharmaceutical composition is 8.0 × 10 9 Two doses of CFU (colony-forming units), followed by 1.6 × 10 9 It is administered as three doses of CFU (colony-forming units). In some embodiments, antibiotics are not administered before administration of the pharmaceutical composition. In some embodiments, vancomycin is not administered before administration of the pharmaceutical composition.

[0031] In some embodiments, the method further includes administering an antibiotic to the target before administering the pharmaceutical composition. In some embodiments, the antibiotic is vancomycin, metronidazole, fidaxomicin, or ridinirazole. In some embodiments, the antibiotic is vancomycin. In some embodiments, vancomycin is administered at a dose of 500 mg per day. In some embodiments, vancomycin is administered in four doses of 125 mg per day. In some embodiments, vancomycin is administered at a dose of 250 mg per day. In some embodiments, vancomycin is administered in two doses of 125 mg per day. In some embodiments, vancomycin is administered at a dose of 125 mg per day.

[0032] In some embodiments, vancomycin is administered for five consecutive days. In some embodiments, vancomycin is administered for three consecutive days. In some embodiments, vancomycin is administered for one day. In some embodiments, vancomycin is administered on the day immediately preceding the administration of the pharmaceutical composition.

[0033] In some embodiments, 250 mg of vancomycin is administered two days before the administration of the pharmaceutical composition, and the method includes a drug-free day before the administration of the pharmaceutical composition. In some embodiments, 250 mg of vancomycin is administered for three consecutive days immediately before the administration of the pharmaceutical composition.

[0034] In some embodiments, 500 mg of vancomycin is administered for five consecutive days immediately prior to the administration day of the pharmaceutical composition. In some embodiments, 500 mg of vancomycin is administered for five consecutive days up to two days prior to the administration day of the pharmaceutical composition, and the method includes a drug-free day one day prior to the administration day of the pharmaceutical composition.

[0035] In some embodiments, one or more bacterial strains are freeze-dried. In some embodiments, one or more bacterial strains are spray-dried. In some embodiments, one or more bacterial strains are spore-forming. In some embodiments, each of the one or more bacterial strains is spore-forming. In some embodiments, one or more bacterial strains are vegetative-forming. In some embodiments, each of the one or more bacterial strains is vegetative-forming.

[0036] In some embodiments, the pharmaceutical composition further comprises one or more enteric-coated polymers. In some embodiments, administration is orally. In some embodiments, the pharmaceutical composition is a formulation 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 intestines. In some embodiments, the pharmaceutical composition is formulated for delivery to the colon.

[0037] Aspects of this disclosure provide methods for reducing the level of primary bile acids, the methods comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the primary bile acid is glycochenodeoxycholic acid, glycocholic acid, or taurocholic acid. In some embodiments, the level of primary bile acids is reduced to 1 / 10 to 1 / 100,000. In some embodiments, the subject has a Clostridium difficile infection, and optionally, the Clostridium difficile infection is recurrent. In some embodiments, the methods further include administering an antibiotic to the subject prior to the administration of the pharmaceutical composition according to any of the methods described herein.

[0038] Aspects of this disclosure provide methods for increasing levels of secondary bile acids, the methods comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the secondary bile acid is deoxycholic acid, lithocholic acid, or ursodeoxycholic acid. In some embodiments, the level of secondary bile acid increases by 10 to 1,000 times. In some embodiments, the subject has a Clostridium difficile infection, and optionally, the Clostridium difficile infection is recurrent. In some embodiments, the subject has a disease characterized by elevated levels of primary bile acids or decreased levels of secondary bile acids. In some embodiments, the disease characterized by elevated levels of primary bile acids or decreased levels of secondary bile acids is selected from the group consisting of IBD, IBS, pathogen infections, food allergies, metabolic diseases, and cardiovascular diseases. In some embodiments, the methods further include administering an antibiotic to the subject before administering the pharmaceutical composition according to any of the methods described herein.

[0039] Aspects of this disclosure provide methods for increasing short-chain fatty acid levels, the methods comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the short-chain fatty acid is an acetate, propionate, butyrate, or valerate. In some embodiments, the short-chain fatty acid level increases by 2 to 500 times. In some embodiments, the subject has a Clostridium difficile infection, optionally, the Clostridium difficile infection is recurrent. In some embodiments, the subject has a disease characterized by decreased levels of short-chain fatty amino acids. In some embodiments, the disease characterized by decreased levels of short-chain fatty amino acids is selected from the group consisting of IBD, IBS, pathogen infection, food allergies, metabolic diseases, and cardiovascular diseases. In some embodiments, the methods further include administering an antibiotic to the subject before administering the pharmaceutical composition according to any of the methods described herein.

[0040] Aspects of this disclosure provide a method for evaluating the colonization of one or more bacterial strains of a bacterial composition in a target microbiome. In some embodiments, the method includes isolating nucleic acids from a sample of the target microbiome, sequencing the isolated nucleic acids to obtain multiple nucleotide sequences of the isolated nucleic acids, and comparing the multiple nucleotide sequences with multiple genomic markers for each bacterial strain of the bacterial composition. In some embodiments, if the genomic markers of the bacterial strain are present in the multiple nucleotide sequences, the bacterial strain colonizes the microbiome. In some embodiments, if one or more of the bacterial strains of the bacterial composition are not present in the multiple nucleotide sequences, the method further includes administering one or more additional doses of the bacterial composition to the target.

[0041] In some embodiments, the subject was previously administered one or more doses of the bacterial composition. In some embodiments, the microbiome sample is a fecal sample obtained from the subject. In some embodiments, sequencing is DNA sequencing.

[0042] In some embodiments, the multiple genome markers contain a nucleotide sequence of between 200 and 1000 nucleotides for each bacterial strain in the bacterial composition. In some embodiments, the multiple genome markers contain approximately 50 nucleotides.

[0043] In some embodiments, the bacterial composition comprises a purified bacterial mixture consisting of bacterial strains containing 16S rDNA sequences with at least 97% sequence identity to the nucleic acid sequences shown as 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, and SEQ ID NO: 8. In some embodiments, the bacterial composition comprises Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavinofractor plautii.

[0044] In some embodiments, if the genomic marker for the bacterial strain is not present in multiple nucleic acid sequences, the method includes administering one or more additional doses of the bacterial composition to the target.

[0045] Aspects of this disclosure provide a method for evaluating the colonization of one or more bacterial strains of a bacterial composition in a target microbiome, the method comprising isolating nucleic acids from a sample of the target microbiome and determining the presence of at least one bacterial strain of the bacterial composition by amplifying the nucleotide sequence of a genomic marker for at least one bacterial strain in the isolated nucleic acid. In some embodiments, if a genomic marker for a bacterial strain is present in the amplified nucleotide sequence, the bacterial strain is colonized in the microbiome.

[0046] In some embodiments, amplification involves performing one or more quantitative polymerase chain reactions (qPCR). In some embodiments, the qPCR is performed using one or more pairs of primers, each pair of primers comprising a forward primer and a reverse primer for amplifying the nucleotide sequence of a bacterial strain's genomic marker.

[0047] In some embodiments, the method includes a forward primer and a reverse primer for amplifying the nucleotide sequence of a bacterial strain's genomic marker. In some embodiments, the primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes the forward primer shown in SEQ ID NO: 9 and the reverse primer shown in SEQ ID NO: 10. In some embodiments, the primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes the forward primer shown in SEQ ID NO: 12 and the reverse primer shown in SEQ ID NO: 13. In some embodiments, the primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes the forward primer shown in SEQ ID NO: 15 and the reverse primer shown in SEQ ID NO: 16. In some embodiments, the primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes the forward primer shown in SEQ ID NO: 18 and the reverse primer shown in SEQ ID NO: 19. In some embodiments, the primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes the forward primer shown in SEQ ID NO: 21 and the reverse primer shown in SEQ ID NO: 22. In some embodiments, a primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes a forward primer shown in SEQ ID NO: 24 and a reverse primer shown in SEQ ID NO: 25. In some embodiments, a primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes a forward primer shown in SEQ ID NO: 27 and a reverse primer shown in SEQ ID NO: 28. In some embodiments, a primer pair for amplifying the nucleotide sequence of a bacterial strain's genomic marker includes a forward primer shown in SEQ ID NO: 30 and a reverse primer shown in SEQ ID NO: 31.

[0048] In some embodiments, the qPCR reaction further includes a DNA probe. In some embodiments, the DNA probe includes a fluorophore and at least one quencher. In some embodiments, the DNA probe includes the sequences shown in SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, and / or SEQ ID NO: 32.

[0049] In some embodiments, if a genomic marker for the bacterial strain is not present in the amplified nucleotide sequence, the method further comprises administering one or more additional doses of the bacterial composition to the target.

[0050] Each limitation of the present invention may encompass various embodiments of the present invention. Therefore, it is expected that limitations of the present invention involving any one element or combination of elements may be included in each aspect of the present invention. In its application, the present invention is not limited to the structural and arrangement details of the components shown in the following description or illustrated in the drawings. Other embodiments of the present invention are possible and can be practiced or carried out in various ways.

[0051] The attached drawings are not intended to be scaled to actual size. These drawings are illustrative and not essential to enabling this disclosure. For clarity, not all components are labeled in all drawings. [Brief explanation of the drawing]

[0052] [Figure 1] This disclosure presents charts illustrating the various treatment cohorts. [Figure 2A]This data shows the abundance of bacterial strains belonging to Clostridium clusters IV and XIVa. It shows the abundance of bacterial taxa belonging to Clostridium clusters IV and XIVa after fecal transplantation (FMT) (see van Nood, et al., N.Engl.J.Med. (2013) 368:407-415). "Pre-rCDI" refers to recurrent C. difficile infection before FMT transplantation, and "Post-rCDI" refers to recurrent C. difficile infection after FMT transplantation. [Figure 2B] This section shows data indicating the abundance of bacterial strains belonging to Clostridium clusters IV and XIVa. It shows the abundance of bacterial strains belonging to Clostridium clusters IV, XIVa, and XVII after administration of composition VE303. For each time point, data is presented from left to right as follows: vancomycin ("Vanco"), index / cohort 1, cohort 2, cohort 3, cohort 4, and cohort 5. [Figure 3A] A graph showing the recovery of the microbiome in subjects after vancomycin treatment is presented. The relative abundance of bacterial phyla before and after FMT introduction is shown (see Smilie, et al., Cell Host Microbe (2018) 23(2):229-240). [Figure 3B] A graph showing the recovery of the microbiome in subjects after treatment with vancomycin is presented. The relative abundance of bacterial phyla is shown before (baseline), after administration of vancomycin, and after administration of composition VE303. [Figure 3C] A graph showing the recovery of the microbiome in subjects after vancomycin treatment is presented. The abundance of Bacteriodetes in each cohort ("Coh") or vancomycin control ("Vanco") is shown, either less than one week after administration of composition VE303 or more than one week after administration of composition VE303. [Figure 3D]A graph showing the recovery of the microbiome in subjects after vancomycin treatment is presented. The abundance of Proteobacteria (Figure 3D) in each cohort ("Coh") or vancomycin control ("Vanco") is shown, either less than one week after administration of composition VE303 or more than one week after administration of composition VE303. [Figure 4A] This paper illustrates the dynamics of the microbiome after administration of vancomycin and after administration of composition VE303. The microbial communities of all cohorts are shown at baseline, after administration of vancomycin, less than one week after administration of composition VE303 (recovery), and more than one week after administration of composition VE303. [Figure 4B] This paper illustrates the dynamics of the microbiome after administration of vancomycin and after administration of composition VE303. The microbial communities of Cohort 5 are shown at baseline, after administration of vancomycin, less than one week after administration of composition VE303 (recovery), and more than one week after administration of composition VE303. [Figure 4C] This paper illustrates the dynamics of the microbiome after administration of vancomycin and after administration of composition VE303. It shows the changes in bacterial species present in the microbiome at baseline, after administration of vancomycin, less than one week after administration of composition VE303 (recovery), and more than one week after administration of composition VE303 (recovery). [Figure 5] This is a detailed species analysis highlighting the presence of Proteobacteria and other pathogens immediately after vancomycin administration. [Figure 6-1] A graph is presented showing colonization data, which represents the number of bacterial strains of composition VE303 detected in the microbiome of all target populations. ND: Data not collected. Vanco: 0 CFU, Cohort 1: 1.6 × 10⁹ CFU (1 day), Cohort 2: 4.0 × 10⁹ CFU (1 day), Cohort 3: 8.0 × 10⁹ CFU (1 day), Cohort 4: 4.0 × 10¹⁰ CFU (5 days), Cohort 5: 1.1 × 10¹¹ CFU (14 days). [Figure 6-2]A graph is presented showing colonization data, which represents the number of bacterial strains of composition VE303 detected in the microbiome of all target populations. ND: Data not collected. Vanco: 0 CFU, Cohort 1: 1.6 × 10⁹ CFU (1 day), Cohort 2: 4.0 × 10⁹ CFU (1 day), Cohort 3: 8.0 × 10⁹ CFU (1 day), Cohort 4: 4.0 × 10¹⁰ CFU (5 days), Cohort 5: 1.1 × 10¹¹ CFU (14 days). [Figure 6-3] A graph is presented showing colonization data, which represents the number of bacterial strains of composition VE303 detected in the microbiome of all target populations. ND: Data not collected. Vanco: 0 CFU, Cohort 1: 1.6 × 10⁹ CFU (1 day), Cohort 2: 4.0 × 10⁹ CFU (1 day), Cohort 3: 8.0 × 10⁹ CFU (1 day), Cohort 4: 4.0 × 10¹⁰ CFU (5 days), Cohort 5: 1.1 × 10¹¹ CFU (14 days). [Figure 6-4] A graph is presented showing colonization data, which represents the number of bacterial strains of composition VE303 detected in the microbiome of all target populations. ND: Data not collected. Vanco: 0 CFU, Cohort 1: 1.6 × 10⁹ CFU (1 day), Cohort 2: 4.0 × 10⁹ CFU (1 day), Cohort 3: 8.0 × 10⁹ CFU (1 day), Cohort 4: 4.0 × 10¹⁰ CFU (5 days), Cohort 5: 1.1 × 10¹¹ CFU (14 days). [Figure 7A-1] A graph is presented showing the number of bacterial strains of composition VE303 detected in the microbiome of each target group. The graph shows the number of bacterial strains of composition VE303 detected in the microbiome of subjects (A-N) in Cohort 4 and Cohort 5. [Figure 7A-2] A graph is presented showing the number of bacterial strains of composition VE303 detected in the microbiome of each target group. The graph shows the number of bacterial strains of composition VE303 detected in the microbiome of subjects (A-N) in Cohort 4 and Cohort 5. [Figure 7B]A graph is presented showing the number of bacterial strains of composition VE303 detected in the microbiome of each target group. The graph shows the number of bacterial strains of composition VE303 detected in the microbiome of control cohort subjects (O-S) that did not receive vancomycin. For each time point, from top to bottom, the bacterial strains of composition VE303 are shown: VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. Shaded areas indicated by "*" indicate the time of vancomycin administration. Areas indicated by "#" indicate the time of composition VE303 administration. [Figure 8] This section summarizes the bacterial strains of composition VE303 detected in the total number of subjects' microbiomes one week after administration of composition VE303 (for example, in cohort 3, strain VE303-01 was found in 2 out of 3 subjects). An asterisk (*) indicates that the strains detected in each subject are after subtracting background levels. [Figure 9A] This shows the relative abundance and durability of bacterial strains of composition VE303 in the microbiome of each individual subject in each cohort. This also shows the overall abundance of each bacterial strain of composition VE303 in each individual subject in each cohort. For each subject, from top to bottom, the bacterial strains of composition VE303 are shown: VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. [Figure 9B-1] This shows the relative abundance and durability of bacterial strains of composition VE303 in the microbiome of each individual subject in each cohort. The total abundance of bacterial strains of composition VE303 over time per cohort and a summary table are also shown. [Figure 9B-2] This shows the relative abundance and durability of bacterial strains of composition VE303 in the microbiome of each individual subject in each cohort. The total abundance of bacterial strains of composition VE303 over time per cohort and a summary table are also shown. [Figure 9C] This shows the relative abundance and durability of bacterial strains of composition VE303 in the microbiome of each individual subject in each cohort. Durable colonization is observed (see also Figure 6). [Figure 10-1]This shows the relative abundance of each bacterial strain of composition VE303 in the microbiome of each cohort over time. [Figure 10-2] This shows the relative abundance of each bacterial strain of composition VE303 in the microbiome of each cohort over time. [Figure 11A] This shows the relative abundance of the VE303 bacterial strain in cohorts 4 and 5. A plot showing the relative abundance of the VE303 bacterial strain in cohorts 4 and 5 over time is presented. [Figure 11B] This shows the relative abundance of bacterial strains of composition VE303 in subjects of Cohorts 4 and 5. A summary table of the relative abundance of bacterial strains of composition VE303 over approximately 4 weeks is also shown. [Figure 12] This shows the diversity of the microbiome over time at baseline, after administration of vancomycin ("post-Vanco"), less than one week after administration of composition VE303, and more than one week after administration of composition VE303. For each treatment day, the data are shown from left to right as vancomycin only ("Vanco"), Cohort 4, and Cohort 5. [Figure 13A] A rationally defined consortium of human bacterial strains associated with rCDI is shown. Figure 13A shows a clustered heatmap of top bacterial genera associated with recurrent Clostridium difficile (rCDI) recovery after fecal microbiota transplantation (FMT). Feces from healthy donors and rCDI patients were collected in collaboration with Leiden University Medical Center (LUMC) and the Netherlands Donor Feces Bank (NDFB). The response to FMT is associated with the transfer of Clostridium clusters IV and XIVa. [Figure 13B] This shows a reasonably defined consortium of human bacterial strains of rCDI. Kaplan-Meier plots are shown illustrating the survival of mice administered with VE303 composition, human FMT, or vancomycin. For comparison, mice administered with phosphate-buffered saline (PBS) or negative live biological agents (negative LBP) died from Clostridium difficile infection. [Figure 14-1] This shows a Phase 1a / b dose-escalation trial in normal, healthy volunteers. Various cohorts were treated according to the chart in Figure 1. Fecal samples were collected over a long period at the indicated times for each subject and analyzed by Illumina shotgun metagenomics sequencing. The first shaded area (left) represents daily administration of vancomycin. The second shaded area (right) represents daily administration of VE303. Each data point represents a fecal collection. [Figure 14-2] This shows a Phase 1a / b dose-escalation trial in normal, healthy volunteers. Various cohorts were treated according to the chart in Figure 1. Fecal samples were collected over a long period at the indicated times for each subject and analyzed by Illumina shotgun metagenomics sequencing. The first shaded area (left) represents daily administration of vancomycin. The second shaded area (right) represents daily administration of VE303. Each data point represents a fecal collection. [Figure 15] The pharmacodynamics of VE303 in normal, healthy volunteers are shown. Changes in the absolute abundance of each bacterial phylum are based on the mass and DNA yield of extracted stool samples. All data points are displayed in the upper panel, and a subset of data points are displayed in the lower panel. Treatment with vancomycin significantly reduced bacterial biomass, but increased Proteobacteria DNA in some subjects. [Figure 16] This chart shows the changes in the absolute abundance of Firmicutes, Bacteroidetes, and Proteobacteria at the time of selection. The arrows indicate the recovery trend in VE303-related factors within one week after vancomycin treatment. [Figure 17A] This shows the pharmacokinetics (PK) of VE303 in normal, healthy volunteers. It also shows the number of VE303 strains detected over time in each normal, healthy volunteer group. [Figure 17B] This chart shows the pharmacokinetics (PK) of VE303 in normal, healthy volunteers. The percentage of subjects in which each strain was established is shown, with values ​​greater than the median highlighted. All eight VE303 strains were detected in nearly all subjects in cohorts 4 and 5. [Figure 17C]This shows the pharmacokinetics (PK) of VE303 in normal, healthy volunteers. For all data points (upper panel) and subsets of data points (lower panel), the total abundance of VE303 in each subject over time is shown. [Figure 17D] This chart shows the pharmacokinetics (PK) of VE303 in normal, healthy volunteers. The median abundance per VE303 strain is shown, with values ​​greater than the median highlighted. [Figure 18-1] This shows the pharmacodynamics (PD) of VE303 in normal, healthy volunteers. Changes in the total relative abundance per subject of the highest-abundant Firmicutes, Bacteroidetes, and Proteobacteria are shown after vancomycin (post-vancomycin), up to 1 week after recovery (less than 1 week after recovery), or more than 1 week after recovery (more than 1 week after recovery). Spontaneous recovery after vancomycin monotherapy is compared to recovery in the presence of VE303 (Cohorts 4 and 5). An asterisk (*) indicates a Kruskal-Wallis value with BH-adjusted p<0.05. [Figure 18-2] This shows the pharmacodynamics (PD) of VE303 in normal, healthy volunteers. Changes in the total relative abundance per subject of the highest-abundant Firmicutes, Bacteroidetes, and Proteobacteria are shown after vancomycin (post-vancomycin), up to 1 week after recovery (less than 1 week after recovery), or more than 1 week after recovery (more than 1 week after recovery). Spontaneous recovery after vancomycin monotherapy is compared to recovery in the presence of VE303 (Cohorts 4 and 5). An asterisk (*) indicates a Kruskal-Wallis value with BH-adjusted p<0.05. [Figure 18-3]This shows the pharmacodynamics (PD) of VE303 in normal, healthy volunteers. Changes in the total relative abundance per subject of the highest-abundant Firmicutes, Bacteroidetes, and Proteobacteria are shown after vancomycin (post-vancomycin), up to 1 week after recovery (less than 1 week after recovery), or more than 1 week after recovery (more than 1 week after recovery). Spontaneous recovery after vancomycin monotherapy is compared to recovery in the presence of VE303 (Cohorts 4 and 5). An asterisk (*) indicates a Kruskal-Wallis value with BH-adjusted p<0.05. [Figure 19A] Figure 19A shows the number and abundance of bacterial strains of composition VE303 detected in the microbiomes of individual subjects. [Figure 19B] Figure 19B shows the percentage abundance of each VE303 strain detected in the microbiome of subjects treated with vancomycin alone and subjects treated with vancomycin followed by VE303. For each time point, from top to bottom, the bacterial strains of composition VE303 are shown: VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. Shaded areas marked with "*" indicate the time of vancomycin administration. Areas marked with "#" indicate the time of composition VE303 administration. [Figure 20] Figures illustrating various additional treatment cohorts of this disclosure are presented. [Figure 21-1] Graphs are presented showing the number of bacterial strains of composition VE303 detected in the microbiomes of individual subjects. These graphs show the number of bacterial strains of composition VE303 detected in the microbiomes of subjects in cohorts 1-6 and 8. For each time point, from top to bottom, the bacterial strains of composition VE303 are shown: VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. "B" indicates the number of VE303 composition strains detected at baseline, and "V" indicates the number of VE303 composition strains detected during vancomycin treatment. [Figure 21-2] Graphs are presented showing the number of bacterial strains of composition VE303 detected in the microbiomes of individual subjects. These graphs show the number of bacterial strains of composition VE303 detected in the microbiomes of subjects in cohorts 1-6 and 8. For each time point, from top to bottom, the bacterial strains of composition VE303 are shown: VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. "B" indicates the number of VE303 composition strains detected at baseline, and "V" indicates the number of VE303 composition strains detected during vancomycin treatment. [Figure 22-1] A graph is presented showing colonization data, represented by the number of bacterial strains of composition VE303 detected in the microbiome of all target populations in cohorts 1-6 and cohort 8. Samples were collected at the indicated time points up to week 24. [Figure 22-2] A graph is presented showing colonization data, represented by the number of bacterial strains of composition VE303 detected in the microbiome of all target populations in cohorts 1-6 and cohort 8. Samples were collected at the indicated time points up to week 24. [Figure 22-3] A graph is presented showing colonization data, represented by the number of bacterial strains of composition VE303 detected in the microbiome of all target populations in cohorts 1-6 and cohort 8. Samples were collected at the indicated time points up to week 24. [Figure 23] This shows the overall abundance and durability of each bacterial strain of composition VE303 in the microbiome of each individual subject in each cohort. For each subject, the bacterial strains of composition VE303, VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8, are shown from top to bottom. [Figure 24-1] This shows the relative abundance of each bacterial strain of composition VE303 in the microbiome of each of the cohorts 1-6 and cohort 8 over time. [Figure 24-2] This shows the relative abundance of each bacterial strain of composition VE303 in the microbiome of each of the cohorts 1-6 and cohort 8 over time. [Figure 24-3]This shows the relative abundance of each bacterial strain of composition VE303 in the microbiome of each of the cohorts 1-6 and cohort 8 over time. [Figure 24-4] This shows the relative abundance of each bacterial strain of composition VE303 in the microbiome of each of the cohorts 1-6 and cohort 8 over time. [Figure 25] This chart shows the relative abundance of bacterial phyla before (baseline) administration of vancomycin, after administration, and after administration of composition VE303. "B" indicates the number of VE303 composition strains detected at baseline, and "V" indicates the number of VE303 composition strains detected during vancomycin treatment. [Figure 26-1] This shows the quantification of bacterial phyla before (baseline) administration of vancomycin, after administration, and after administration of composition VE303. "B" indicates the amount of each bacterial phylum detected at baseline, "V" indicates the amount of each bacterial phylum detected during vancomycin treatment, and "R" indicates the amount of each bacterial phylum detected during recovery from vancomycin treatment. [Figure 26-2] This shows the quantification of bacterial phyla before (baseline) administration of vancomycin, after administration, and after administration of composition VE303. "B" indicates the amount of each bacterial phylum detected at baseline, "V" indicates the amount of each bacterial phylum detected during vancomycin treatment, and "R" indicates the amount of each bacterial phylum detected during recovery from vancomycin treatment. [Figure 27] This shows the time-series abundance of Clostridium cluster IV and XIVa bacteria after vancomycin treatment in cohorts 4, 5, and 8. The Kruskal-Wallis test was used to determine the p-values: "ns" indicates a p-value greater than 0.05, "*" indicates a p-value of approximately 0.05, and "**" indicates a p-value less than 0.001. [Figure 28] This example illustrates how a gut disrupted by antibiotics can be restored to health through treatment with VE303. [Figure 29]This study shows that the restoration of short-chain fatty acids (SCFAs) and secondary bile acids is associated with recovery in patients with Clostridium difficile infection (CDI) (Seekatz et al., Anaerobe 2018, Oct, 53:64-73). [Figure 30-1] This shows the decrease in primary conjugated bile acids (BAs) after treatment with VE303. Various primary bile acids (glycohenodeoxycholic acid, glycoholic acid, taurochenodeoxycholic acid, and taurocholic acid) from subjects in cohorts 1–5 were quantified from samples taken at the indicated time points. "B" indicates the amount of each primary conjugated BA detected at baseline, "V" indicates the amount of each primary conjugated BA detected during vancomycin treatment, and "R" indicates the amount of each primary conjugated BA detected during recovery from vancomycin treatment. "*" indicates an interesting trend in which VE303 administration is associated with rapid depletion of primary BAs. [Figure 30-2] This shows the decrease in primary conjugated bile acids (BAs) after treatment with VE303. Various primary bile acids (glycohenodeoxycholic acid, glycoholic acid, taurochenodeoxycholic acid, and taurocholic acid) from subjects in cohorts 1–5 were quantified from samples taken at the indicated time points. "B" indicates the amount of each primary conjugated BA detected at baseline, "V" indicates the amount of each primary conjugated BA detected during vancomycin treatment, and "R" indicates the amount of each primary conjugated BA detected during recovery from vancomycin treatment. "*" indicates an interesting trend in which VE303 administration is associated with rapid depletion of primary BAs. [Figure 31]This shows the recovery of secondary uncoupled bile acids (BAs) after treatment with VE303. Various secondary bile acids (deoxycholic acid, lithocholic acid, and ursodeoxycholic acid) from subjects in cohorts 1–5 were quantified from samples taken at the indicated time points. "B" indicates the amount of each secondary uncoupled BA detected at baseline, "V" indicates the amount of each secondary uncoupled BA detected during vancomycin treatment, and "R" indicates the amount of each secondary uncoupled BA detected during recovery from vancomycin treatment. "*" indicates an interesting trend in which VE303 administration is associated with rapid recovery of secondary BAs. [Figure 32] This shows a decrease in primary conjugated BAs and a recovery of secondary unconjugated BAs after treatment with VE303. Various primary and secondary BAs (glycohenodeoxycholic acid, glycoholic acid, and taurocholic acid; deoxycholic acid, lithocholic acid, and ursodeoxycholic acid) from subjects in Cohorts 4 and 5 were quantified from samples taken at the indicated time points. "B" indicates the amount of each primary or secondary BA detected at baseline, "V" indicates the amount of each primary or secondary BA detected during vancomycin treatment, and "R" indicates the amount of each primary or secondary BA detected during recovery from vancomycin treatment. "*" indicates an interesting trend in which VE303 administration is associated with rapid depletion of primary BAs or recovery of secondary BAs. [Figure 33-1] The study shows the reduction and recovery of BA after antibiotic treatment and administration of VE303. Various primary and secondary BAs (chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, and ursodeoxycholic acid) from subjects in Cohorts 4 and 5 were quantified from samples taken at the indicated time points. [Figure 33-2] The study shows the reduction and recovery of BA after antibiotic treatment and administration of VE303. Various primary and secondary BAs (chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, and ursodeoxycholic acid) from subjects in Cohorts 4 and 5 were quantified from samples taken at the indicated time points. [Figure 34A] The results of a linear mixed-effects model analysis on the association between VE303 treatment and bile acid production are presented. VE303 is strongly associated with the restoration of secondary bile acids and the reduction of primary bile acids. [Figure 34B] The results of a linear mixed-effects model analysis on the association between VE303 treatment and bile acid production are presented. VE303 is strongly associated with the restoration of secondary bile acids and the reduction of primary bile acids. [Figure 35-1] This document outlines the characteristics of the top 20 bacterial strains important for the metabolism of primary and secondary bile acids. The VE303 bacterial strain is important for the metabolism of both primary and secondary bile acids. [Figure 35-2] This document outlines the characteristics of the top 20 bacterial strains important for the metabolism of primary and secondary bile acids. The VE303 bacterial strain is important for the metabolism of both primary and secondary bile acids. [Figure 35-3] This document outlines the characteristics of the top 20 bacterial strains important for the metabolism of primary and secondary bile acids. The VE303 bacterial strain is important for the metabolism of both primary and secondary bile acids. [Figure 35-4] This document outlines the characteristics of the top 20 bacterial strains important for the metabolism of primary and secondary bile acids. The VE303 bacterial strain is important for the metabolism of both primary and secondary bile acids. [Figure 36A-1] This shows the VE303 species and commensal microorganisms that are important for bile acid (BA) recovery. VE303 group A strains (VE303-01, VE303-02, VE303-03, VE303-05, VE303-07, VE303-8) are negatively associated with primary BA recovery and positively associated with secondary BA recovery. VE303 group B strains (VE303-04, VE303-06) are positively associated with secondary BA recovery. [Figure 36A-2] This shows the VE303 species and commensal microorganisms that are important for bile acid (BA) recovery. VE303 group A strains (VE303-01, VE303-02, VE303-03, VE303-05, VE303-07, VE303-8) are negatively associated with primary BA recovery and positively associated with secondary BA recovery. VE303 group B strains (VE303-04, VE303-06) are positively associated with secondary BA recovery. [Figure 36B]This shows the VE303 species and commensal microorganisms that are important for bile acid (BA) recovery. VE303 group A strains (VE303-01, VE303-02, VE303-03, VE303-05, VE303-07, VE303-8) are negatively associated with primary BA recovery and positively associated with secondary BA recovery. VE303 group B strains (VE303-04, VE303-06) are positively associated with secondary BA recovery. [Figure 37] This shows the recovery of short-chain fatty acids (SCFAs) after antibiotic treatment and VE303 administration. Various SCFAs (acetate, propionate, butyrate, and valerate) from subjects in cohorts 1–5 were quantified from samples taken at the indicated time points. "B" indicates the amount of each SCFA detected at baseline, "V" indicates the amount of each SCFA detected during vancomycin treatment, and "R" indicates the amount of each SCFA detected during recovery from vancomycin treatment. "*" indicates an interesting trend where VE303 administration is associated with a rapid recovery of SCFAs. [Figure 38] This shows the recovery of short-chain fatty acids (SCFAs) after antibiotic treatment and VE303 administration. Various SCFAs (acetate, propionate, and butyrate) from subjects in Cohorts 4 and 5 were quantified from samples taken at the indicated time points. "B" indicates the amount of each SCFA detected at baseline, "V" indicates the amount of each SCFA detected during vancomycin treatment, and "R" indicates the amount of each SCFA detected during recovery from vancomycin treatment. "*" indicates an interesting trend where VE303 administration is associated with rapid SCFA recovery. [Figure 39A] The results of a linear mixed-effects model analysis on the association between VE303 treatment and short-chain fatty acid (SCFA) recovery are presented. VE303 is strongly associated with the recovery of acetate, butyrate, and propionate. [Figure 39B] The results of a linear mixed-effects model analysis on the association between VE303 treatment and short-chain fatty acid (SCFA) recovery are presented. VE303 is strongly associated with the recovery of acetate, butyrate, and propionate. [Figure 40-1]This report identifies the top 20 bacterial strains important for the recovery of short-chain fatty acids (SCFAs) after antibiotic treatment. The VE303 bacterial strain is particularly important for SCFA recovery. [Figure 40-2] This report identifies the top 20 bacterial strains important for the recovery of short-chain fatty acids (SCFAs) after antibiotic treatment. The VE303 bacterial strain is particularly important for SCFA recovery. [Figure 40-3] This report identifies the top 20 bacterial strains important for the recovery of short-chain fatty acids (SCFAs) after antibiotic treatment. The VE303 bacterial strain is particularly important for SCFA recovery. [Figure 41A-1] This shows the VE303 species and commensal microorganisms that are important for short-chain fatty acid (SCFA) recovery. VE303 group A strains (VE303-01, VE303-02, VE303-06, VE303-07) are positively associated with hexanoate recovery, while VE303 group B strains (VE303-03, VE303-04, VE303-08) are positively associated with propionate. [Figure 41A-2] This shows the VE303 species and commensal microorganisms that are important for short-chain fatty acid (SCFA) recovery. VE303 group A strains (VE303-01, VE303-02, VE303-06, VE303-07) are positively associated with hexanoate recovery, while VE303 group B strains (VE303-03, VE303-04, VE303-08) are positively associated with propionate. [Figure 41B] This shows the VE303 species and commensal microorganisms that are important for short-chain fatty acid (SCFA) recovery. VE303 group A strains (VE303-01, VE303-02, VE303-06, VE303-07) are positively associated with hexanoate recovery, while VE303 group B strains (VE303-03, VE303-04, VE303-08) are positively associated with propionate. [Figure 42]This study demonstrates that VE303 promotes early recovery of the microbiome after vancomycin administration. Mean relative abundances (+ / - SEM) of Bacteroidetes (left panel) and Proteobacteria (right panel) in healthy volunteers (HV) treated with vancomycin alone (daily for 5 days) ("Vanco") or VE303 for 14 days ("Cohort 5"). The "Vanco" time point includes samples collected within 24 hours of vancomycin administration. The "Early Recovery" time point includes samples collected within the first 7 days of recovery. The "Late Recovery" time point includes samples collected after the first 7 days of recovery. [Figure 43] This study demonstrates that the VE303 consortium and strain 6 (Dorea longicatena) reduced Clostridium difficile (C. difficile) growth in in vitro competitive experiments. VE303 without Dorea longicatena was less effective in suppressing VE303 growth. C. difficile cultures were cultured in the presence of Clostridium bifermentans (positive control), VE303, Dorea longicatena, VE303 without Dorea longicatena, or in the absence of one or more competing strains (C. difficile only). The amount of C. difficile is presented as a percentage of the control (C. difficile only). [Figure 44] This disclosure presents various charts illustrating different treatment cohorts. [Figure 45A] This shows the establishment of VE303. It indicates the total number of bacterial strains of composition VE303 detected in the microbiome of the target population. [Figure 45B]This shows VE303 retention. The top row of Figure 45B contains all data points, and the bottom row magnifies the points below the dashed line in the top row. Vancomycin ("Vanco"): 0 CFU, Cohort 1: 1.6 × 10⁹ CFU (1 day), Cohort 2: 4.0 × 10⁹ CFU (1 day), Cohort 3: 8.0 × 10⁹ CFU (1 day), Cohort 4: 4.0 × 10¹⁰ CFU (5 days), Cohort 5: 1.1 × 10¹¹ CFU (14 days), Cohort 6: 1.7 × 10¹¹ CFU (21 days). D6 is day 6 of VE303 treatment, D6-D11 is days 6-11, and D0-20 is days 0-20. The median (+ / - interquartile range and 95% confidence interval) is plotted for each cohort. The VE303 administration dates are indicated by arrows for each cohort. [Figure 46A] This diagram shows the pharmacodynamics of the microbiome after VE303 administration. It shows the relative abundance of bacterial phyla at baseline (left side of the dashed box), after vancomycin administration (dashed box), and during recovery after vancomycin administration (right side of the dashed box). Each bar corresponds to the relative abundance of bacteria at a single time point in a single subject, and is arranged in order of increasing time. D6 is day 6 of VE303 treatment, D6-D11 is days 6-11, and D0-20 is days 0-20. [Figure 46B] The pharmacodynamics of the microbiome after VE303 administration are shown. The microbiome index (MI) at different treatment times is shown (see Equation 4 in Example 11). "Baseline" corresponds to the community at the start of treatment, "Vanco" corresponds to days 4–6, "Early recovery" corresponds to days 7–13 of the study (or one week after vancomycin treatment with or without VE303), "Late recovery" corresponds to days 14 or longer, "Early, without vanco" corresponds to days 1–7 (or one week after VE303 for cohort 6 only), and "Late, without vanco" corresponds to days 8 or longer for cohort 6 only. MI is plotted on a log10 scale. [Figure 46C]This figure shows the pharmacodynamics of the microbiome after VE303 administration. The percentage abundance of Bacteroidetes (left panel) and Proteobacteria (right panel) in each cohort or in the vancomycin control group at the "early recovery" point for each cohort is shown. Figure 46D shows the microbiome response (measured by MI) to escalating VE303 doses at the "early recovery" point. [Figure 46D] This shows the pharmacodynamics of the microbiome after VE303 administration. It also shows the response of the microbiome to gradually increasing doses of VE303 at the "early recovery" point (measured by MI). [Figure 46E] This report presents the pharmacodynamics of the microbiome in response to VE303 administration. It shows the microbiome response (measured by MI) to escalating VE303 doses at the "early recovery" and "late recovery" time points. Mean + / - standard error is shown. [Figure 47A] This chart shows the bile acid pharmacodynamics of VE303 administration. It shows the relative abundance of bile acids measured in the stool of healthy volunteers at baseline, after vancomycin administration (in the dashed box), or during the recovery period after vancomycin administration. Each bar corresponds to the relative bile acid abundance at a single time point in one subject. "Vanco" is vancomycin, "BA" is bile acid, D6 is day 6 of VE303 treatment, D6-D11 is days 6-11, and D0-20 is days 0-20. [Figure 47B] The bile acid pharmacodynamics of VE303 administration are shown. Bile acid index (BAI) (see Equation 3.5 in Example 11). "Baseline" is the population at baseline, "Vanco" is days 4–6, "Early recovery" corresponds to days 7–13 of the study (or one week after vancomycin treatment with or without VE303 administration), "Late recovery" corresponds to days 14 or longer, "Early, without vanco" corresponds to days 1–7 of the study (or one week after VE303 for cohort 6 only), and "Late, without vanco" corresponds to study days 8 or longer for cohort 6 only. The most recent date of sample collection for each cohort is shown in the lower right corner of each panel. [Figure 47C-1]This shows the bile acid pharmacodynamics of VE303 administration. It shows the decrease and recovery of bile acid (BA) at baseline and after antibiotic treatment and VE303 administration. For each time point, from left to right, various primary and secondary BAs (chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, and ursodeoxycholic acid) from subjects in the "vanco" cohort, cohort 4, and cohort 5 were quantified from samples taken at the indicated time. [Figure 47C-2] This shows the bile acid pharmacodynamics of VE303 administration. It shows the decrease and recovery of bile acid (BA) at baseline and after antibiotic treatment and VE303 administration. For each time point, from left to right, various primary and secondary BAs (chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, and ursodeoxycholic acid) from subjects in the "vanco" cohort, cohort 4, and cohort 5 were quantified from samples taken at the indicated time. [Figure 47D-1] This paper presents the bile acid pharmacodynamics of VE303 administration. Predicted measured bile acid abundances are shown as a function of measured microbial abundances, as predicted by random forest regression. 100 different random forest regressions were created by randomly selecting one sample per patient. This process was repeated 30 times to accommodate various random seed initializations. Microorganisms with sorted importance p-values ​​less than or equal to 0.05 in at least 50% of the 30 × 100 iterations were evaluated by cumulative local effects (ALE) analysis. A linear model was fitted to the resulting ALE plots to qualitatively determine the positive / negative contribution of each microorganism to each measured bile acid. [Figure 47D-2]This paper presents the bile acid pharmacodynamics of VE303 administration. Predicted measured bile acid abundances are shown as a function of measured microbial abundances, as predicted by random forest regression. 100 different random forest regressions were created by randomly selecting one sample per patient. This process was repeated 30 times to accommodate various random seed initializations. Microorganisms with sorted importance p-values ​​less than or equal to 0.05 in at least 50% of the 30 × 100 iterations were evaluated by cumulative local effects (ALE) analysis. A linear model was fitted to the resulting ALE plots to qualitatively determine the positive / negative contribution of each microorganism to each measured bile acid. [Figure 47E] This paper presents the bile acid pharmacodynamics of VE303 administration. Predicted measured bile acid abundances are shown as a function of measured microbial abundances, as predicted by random forest regression. 100 different random forest regressions were created by randomly selecting one sample per patient. This process was repeated 30 times to accommodate various random seed initializations. Microorganisms with sorted importance p-values ​​less than or equal to 0.05 in at least 50% of the 30 × 100 iterations were evaluated by cumulative local effects (ALE) analysis. A linear model was fitted to the resulting ALE plots to qualitatively determine the positive / negative contribution of each microorganism to each measured bile acid. [Figure 48A] This shows the pharmacodynamics of short-chain fatty acids (SCFAs) after VE303 administration. SCFA concentrations are shown as measured in the stool of healthy volunteers at baseline, after vancomycin administration, and during recovery after vancomycin administration. "B" represents baseline, "V" represents vancomycin administration, and "R" represents recovery. [Figure 48B] This paper presents the pharmacodynamics of short-chain fatty acids (SCFAs) after VE303 administration. It shows the SCFA concentrations (mean + / - sem) in stool samples over time in subjects who received vancomycin alone, compared with subjects who received multiple doses of VE303 (Cohorts 4 and 5). [Figure 48C]This paper presents the pharmacodynamics of short-chain fatty acids (SCFAs) administered with VE303. Predicted and measured SCFA abundances are shown as a function of measured microbial abundances. 100 different random forest regressions were created by randomly selecting one sample per patient. This process was repeated 30 times to accommodate various random seed initializations. Microorganisms with sorted importance p-values ​​less than or equal to 0.05 in at least 50% of the 30 × 100 iterations were evaluated by cumulative local effects (ALE) analysis. A linear model was fitted to the resulting ALE plots to qualitatively determine the positive / negative contribution of each microorganism to the measured SCFAs. [Figure 48D-1] This paper presents the pharmacodynamics of short-chain fatty acids (SCFAs) administered with VE303. Predicted and measured SCFA abundances are shown as a function of measured microbial abundances. 100 different random forest regressions were created by randomly selecting one sample per patient. This process was repeated 30 times to accommodate various random seed initializations. Microorganisms with sorted importance p-values ​​less than or equal to 0.05 in at least 50% of the 30 × 100 iterations were evaluated by cumulative local effects (ALE) analysis. A linear model was fitted to the resulting ALE plots to qualitatively determine the positive / negative contribution of each microorganism to the measured SCFAs. [Figure 48D-2] This paper presents the pharmacodynamics of short-chain fatty acids (SCFAs) administered with VE303. Predicted and measured SCFA abundances are shown as a function of measured microbial abundances. 100 different random forest regressions were created by randomly selecting one sample per patient. This process was repeated 30 times to accommodate various random seed initializations. Microorganisms with sorted importance p-values ​​less than or equal to 0.05 in at least 50% of the 30 × 100 iterations were evaluated by cumulative local effects (ALE) analysis. A linear model was fitted to the resulting ALE plots to qualitatively determine the positive / negative contribution of each microorganism to the measured SCFAs. [Figure 49-1]This shows a volunteer-based Phase 1a / b dose-escalation clinical trial. Various cohorts were treated according to the chart in Figure 44. Fecal samples were collected at the indicated times for each subject and analyzed by Illumina shotgun metagenomics sequencing. The first shaded area (left) shows daily administration of vancomycin. The second shaded area (right) shows daily administration of VE303. Each data point represents a fecal collection. "Vanco" refers to vancomycin. [Figure 49-2] This shows a volunteer-based Phase 1a / b dose-escalation clinical trial. Various cohorts were treated according to the chart in Figure 44. Fecal samples were collected at the indicated times for each subject and analyzed by Illumina shotgun metagenomics sequencing. The first shaded area (left) shows daily administration of vancomycin. The second shaded area (right) shows daily administration of VE303. Each data point represents a fecal collection. "Vanco" refers to vancomycin. [Figure 50A] A graph is presented showing the number of bacterial strains of composition VE303 detected in individual target microbiomes. The graph shows the number of bacterial strains of composition VE303 detected in the target microbiome of the cohort that received only vancomycin ("Vanco"). [Figure 50B-1] A graph is presented showing the number of bacterial strains of composition VE303 detected in each target microbiome. The graph shows the number of bacterial strains of composition VE303 detected in the target microbiomes of Cohorts 1-3. [Figure 50B-2] A graph is presented showing the number of bacterial strains of composition VE303 detected in each target microbiome. The graph shows the number of bacterial strains of composition VE303 detected in the target microbiomes of Cohorts 1-3. [Figure 50C-1] A graph is presented showing the number of bacterial strains of composition VE303 detected in each target microbiome. The graph shows the number of bacterial strains of composition VE303 detected in the target microbiomes of Cohort 4 and Cohort 5. [Figure 50C-2]A graph is presented showing the number of bacterial strains of composition VE303 detected in each target microbiome. The graph shows the number of bacterial strains of composition VE303 detected in the target microbiomes of Cohort 4 and Cohort 5. [Figure 50D] A graph is presented showing the number of bacterial strains of composition VE303 detected in each target microbiome. The graph shows the number of bacterial strains of composition VE303 detected in the target microbiome of Cohort 6. For each column at each time point, from top to bottom, the bacterial strains of composition VE303 are shown: VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. [Figure 51A] Figure 51A shows the depth and coverage of the VE303 marker panel using the detection method described in Example 11. The mean VE303 marker depth for each cohort is shown in Figure 51A. [Figure 51B] Figure 51B shows the coverage of VE303 markers (percentage of detected VE303 markers) for each cohort. Each data point represents the average depth or coverage of VE303 strains detected in the target fecal sample metagenomics across all time points. The maximum number of detected strains is 8. [Figure 52A] A graph showing the abundance of bacterial strains of composition VE303 detected in individual target microbiomes is presented. Figure 52A shows the abundance of bacterial strains of composition VE303 detected in the target microbiome of the cohort that received only vancomycin ("Vanco"). [Figure 52B-1] Figure 52B shows the abundance of bacterial strains of composition VE303 detected in the target microbiomes of cohorts 1-3. [Figure 52B-2] Figure 52B shows the abundance of bacterial strains of composition VE303 detected in the target microbiomes of cohorts 1-3. [Figure 52C] Figure 52C shows the abundance of bacterial strains of composition VE303 detected in the target microbiome of Cohort 4. [Figure 52D] Figure 52D shows the abundance of bacterial strains of composition VE303 detected in the target microbiome of Cohort 5. [Figure 52E] Figure 52E shows the abundance of bacterial strains of composition VE303 detected in the target microbiome of Cohort 6. For each column at each time point, from top to bottom, are shown the bacterial strains of composition VE303-1, VE303-2, VE303-3, VE303-4, VE303-5, VE303-6, VE303-7, and VE303-8. In Figures 52C-52E, the top row contains all data points, and the bottom row magnifies the points below the dashed line in the top row. [Figure 53A] This shows the species richness and diversity of the target fecal microbiome. The species richness is evaluated by the number of bacterial species detected in the microbiome of each cohort. [Figure 53B] This shows the species richness and diversity of the stool microbiome of the target group. The bacterial species diversity index (Shannon) for the microbiome of each cohort is shown. [Figure 53C] This shows the species richness and diversity of the target fecal microbiome. It displays the bacterial species diversity (Inv Simpson) in the microbiome of each cohort. "Shannon" is the Shannon diversity index, and "Inv Simpson" is the reciprocal of the Simpson diversity index. [Figure 54A] This shows the absolute bacterial abundance. It shows the quantification of bacterial DNA for each indicated phylum before vancomycin administration (baseline), after vancomycin administration, and after administration of composition VE303. The top row contains all data points, and the bottom row magnifies the points below the dashed line in the top row. "B" indicates the amount of each phylum detected at baseline, "V" indicates the amount of each phylum detected during vancomycin treatment, and "R" indicates the amount of each phylum detected during recovery from vancomycin treatment. [Figure 54B] This shows the absolute bacterial abundance. It shows the total abundance of VE303 strains in fecal samples from the indicated cohort. The top row contains all data points, and the bottom row magnifies the points below the dashed line in the top row. "B" indicates the amount of each bacterial phylum detected at baseline, "V" indicates the amount of each bacterial phylum detected during vancomycin treatment, and "R" indicates the amount of each bacterial phylum detected during recovery from vancomycin treatment. [Figure 55-1] This shows the relative abundance of each of the indicated bacterial classes based on individual samples at different time points within each of the indicated cohorts. [Figure 55-2] This shows the relative abundance of each of the indicated bacterial classes based on individual samples at different time points within each of the indicated cohorts. [Figure 56] The pharmacodynamics (PD) of the microbiome in each cohort are shown. The relative abundances of the most abundant Firmicutes (top row), Bacteroidetes (middle row), and Proteobacteria (bottom row) are shown at baseline, after vancomycin administration ("Vanco"), up to 1 week after recovery ("early recovery"), or more than 1 week after recovery ("late recovery"). For each time point, the cohorts are, from left to right, vancomycin alone ("vanco"), cohort 4, and cohort 5. Spontaneous recovery after vancomycin alone is compared to recovery in the presence of VE303 (cohorts 4 and 5). An asterisk (*) indicates a BH-corrected Kruskal-Wallis p<0.05. [Figure 57A-1] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-2]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-3] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-4]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-5] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-6]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-7] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-8]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-9] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57A-10]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-1] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-2]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-3] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-4]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-5] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-6]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-7] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-8]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-9] This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 57B-10]This panel shows bacterial strains associated with the recovery of conjugated primary bile acids ("conjugated 1°BA": glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid), unconjugated primary bile acids ("nonconjugated 1°BA": cholic acid, chenodeoxycholic acid), secondary bile acids ("secondary BA": deoxycholic acid, lithocholic acid, ursodeoxycholic acid), and conjugated secondary bile acids ("conjugated 2°BA": glycodeoxycholic acid, glycoursodeoxycholic acid). Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria that were found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded dots indicate the frequency of statistical significance calculated against the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have either a positive or negative impact on bile acid levels. [Figure 58A-1] This heatmap shows the identity percentage of the VE303 bacterial strain open reading frame (ORF) (or control reference genome ORF) for known bile acid uncoupling, epimerization, and dihydroxylation genes. [Figure 58A-2] This heatmap shows the identity percentage of the VE303 bacterial strain open reading frame (ORF) (or control reference genome ORF) for known bile acid uncoupling, epimerization, and dihydroxylation genes. [Figure 58B-1] This heatmap shows the identity percentage of the VE303 bacterial strain open reading frame (ORF) (or control reference genome ORF) for known bile acid uncoupling, epimerization, and dihydroxylation genes. [Figure 58B-2] This heatmap shows the identity percentage of the VE303 bacterial strain open reading frame (ORF) (or control reference genome ORF) for known bile acid uncoupling, epimerization, and dihydroxylation genes. [Figure 59A-1]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-2] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-3]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-4] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-5]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-6] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-7]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59A-8] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-1]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-2] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-3]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-4] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-5]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-6] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-7]This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 59B-8] This panel shows bacterial strains associated with the recovery of short-chain fatty acids (SCFAs: acetate, butyrate, propionate, hexanoate, isobutyrate, isovalerate, valerate, and 2-methylbutyrate) after antibiotic treatment. Bacterial strains were identified based on sorted importance analysis and ranked according to the increase in mean squared predictive error when sorted. Each panel shows bacteria found to be significant (sorted p-value less than 0.05) in at least one replicate. Shaded points indicate the frequency of statistical significance calculated relative to the total number of random forest replicates. Each of the VE303 strains considered important is indicated by an arrow. The species identified by this analysis may have a positive or negative impact on SCFA abundance. [Figure 60] This document provides a summary of exemplary methods described herein for defining the pharmacokinetics (PK) and pharmacodynamics (PD) of live biological products (LBPs). The genomic sequences and unique marker sequences of each LBP consortium member may be identified, bacterial strains may be used to colonize the target, and the effects of the LBP on the target's symbiotic microbiome may be observed during LBP development. Short-chain fatty acids (SCFAs) and bile acids (BAs) in feces may be useful in characterizing the PD of LBPs designed for the treatment of specific indications. [Figure 61]This model illustrates the relationship between the microbiome and metabolites and VE303 administration. It shows a simplified model of interactions between symbiotic microorganisms, BA, SCFA, and intestinal epithelium in the gut of healthy humans (left panel), under conditions of Clostridium difficile infection (center panel), and in healthy humans after VE303 administration resulting in restoration of microbiome, bile acid, and SCFA homeostasis (right panel). [Figure 62] This diagram shows a schematic of the development of an assay to detect the presence of bacterial strains of a bacterial composition in a target microbiome. The upper schematic shows the identification of genomic markers specific to each bacterial strain in the bacterial composition. The lower schematic shows the detection of specific genomic markers in DNA sequences obtained from a target microbiome sample to determine the presence of bacterial strains in the target microbiome. [Figure 63] This paper describes the optimization of an assay for detecting the presence of bacterial strains of a bacterial composition in the target microbiome. [Figure 64] This shows qPCR amplification of DNA isolated from the VE303 strain of bacteria. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 65] This shows qPCR amplification of DNA isolated from two VE303 strain bacteria. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 66] This shows qPCR amplification of DNA isolated from three VE303 bacterial strains. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 67]This shows qPCR amplification of DNA isolated from four VE303 bacterial strains. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 68] This shows qPCR amplification of DNA isolated from VE303 strain 5 bacteria. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 69] This shows qPCR amplification of DNA isolated from VE303 strain 6 bacteria. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 70] This shows qPCR amplification of DNA isolated from VE303 strain 7 bacteria. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 71] This shows qPCR amplification of DNA isolated from eight VE303 bacterial strains. CT is the number of qPCR cycles in which the amount of amplified DNA is detectable above the background. ng / rxn is the amount of DNA in nanograms present at the start of each qPCR reaction. The slope of the line correlates with the efficiency of qPCR amplification. [Figure 72A] The number of VE303 strains detected in fecal samples from individuals within the cohort at the indicated time point after administration of the bacterial composition is shown. The results are shown using 25 PCR cycles. [Figure 72B] The number of VE303 strains detected in fecal samples from individuals within the cohort at the indicated time point after administration of the bacterial composition is shown. The results are shown using 30 PCR cycles. [Figure 72C] The number of VE303 strains detected in fecal samples from individuals within the cohort at the indicated time point after administration of the bacterial composition is shown. The results are shown using 35 PCR cycles. [Figure 73A] This shows the number of qPCR-positive samples for each strain of composition VE303 detected in fecal samples from individuals within the cohort at the indicated time after administration of the bacterial composition. The results are shown using 25 PCR cycles. [Figure 73B] This shows the number of qPCR-positive samples for each strain of composition VE303 detected in fecal samples from individuals within the cohort at the indicated time after administration of the bacterial composition. The results are shown using 30 PCR cycles. [Figure 73C] This shows the number of qPCR-positive samples for each strain of composition VE303 detected in fecal samples from individuals within the cohort at the indicated time after administration of the bacterial composition. The results are shown using 35 PCR cycles. [Modes for carrying out the invention]

[0053] Methods for reducing enterotoxosis in subjects receiving a pharmaceutical composition containing one or more purified bacterial strains are provided herein. Methods for restoring the microbiome in subjects receiving a pharmaceutical composition containing one or more purified bacterial strains are provided herein. Methods for increasing the recovery of the microbiome in subjects receiving a pharmaceutical composition containing one or more purified bacterial strains are also provided herein. Methods for protecting the microbiome of subjects receiving a pharmaceutical composition containing one or more purified bacterial strains are also provided herein. Methods for establishing bacteria in the microbiome of subjects receiving a pharmaceutical composition containing one or more purified bacterial strains are also provided herein.

[0054] The present invention is not limited in its application to the structural and arrangement details of the components shown in the following description or illustrated in the drawings. Other embodiments of the present invention are possible and can be practiced or carried out in various ways. Furthermore, the expressions and terms used herein are for illustrative purposes only and should not be considered limiting. The use herein of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof means to include the items listed below and their equivalents, as well as additional items.

[0055] Aspects of this disclosure relate to a method for reducing enterotoxemia in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising one or more purified bacterial strains to the subject. In some embodiments, the pharmaceutical composition is administered to reduce enterotoxemia of the subject's intestinal microbiota. In some embodiments, the method further comprises administering one or more additional doses or amounts of the pharmaceutical composition described herein. In some embodiments, the method further comprises administering vancomycin to the subject prior to the administration of the pharmaceutical composition.

[0056] As used herein, “enterotoxemia” refers to an imbalance of the microbiome within or on the surface of an object. The microbiome, for example, in mammalian objects, is present on the skin, in the gastrointestinal tract (i.e., the intestines), in the oral cavity, and in the vaginal canal of female objects, and includes bacteria, archaea, protists, fungi, and viruses. In some cases, species present in the microbiome benefit the object by performing useful or necessary functions, such as assisting in the digestion of food in the object's intestinal tract, protecting the body from the invasion of pathogenic microorganisms, and promoting immune progression. Organisms in the microbiome that perform these functions are sometimes referred to as symbiotic or commensal organisms because they are present in the object without causing harm and, in some cases, actually benefit the host. In enterotoxemia, a variety of diseases and / or disorders can result from the disruption or damage of the object's normal microbiome. Enterotoxemia can result, for example, the loss of beneficial species, loss of microbial diversity, an increase in pathogenic organisms (one or more), and / or changes in metabolic capacity. In some embodiments, species that normally dominate the microbiome become few in number (e.g., commensal or symbiotic species), and species that normally exist in small numbers (e.g., opportunistic species) become dominant. See also Petersen et al., "Defining dysbiosis and its influence on host immunity and disease." Cell Microbiol 2014, July 16(7), 1024-1033. It should be understood that the compositions and methods of this disclosure may reduce enterotoxosis in various ways in some embodiments. For example, in some embodiments, the compositions and methods result in an increase in the abundance of beneficial bacterial species in the microbiome. In some embodiments, the compositions and methods result in a decrease in the abundance of pathogenic bacterial species. However, it should also be understood that the compositions and methods provided herein do not necessarily reduce all characteristics of enterotoxosis. For example, in some embodiments, the compositions and methods result in an increase in the abundance of beneficial bacterial species in the microbiome, but without simultaneously increasing the diversity of the microbiome. This disclosure relates to a method for reducing enterotoxemia in the subject.As used herein, “reduction of enterotoxemia” refers to the restoration of the composition and homeostasis of the microbial community.

[0057] Disruption of the microbiome can allow pathogens from within or from other sources to colonize, overcrowde, and / or cause disease in the subject. Enterotoxosis is associated with many diseases and / or disorders, including Clostridium difficile infection, cancer, inflammatory bowel disease (IBD), obesity, colitis, chronic fatigue syndrome, periodontitis, and bacterial vaginosis.

[0058] Enterotoxemia can be detected and / or monitored by a variety of methods, including stool tests (e.g., identification and / or quantification of microbial populations, enzyme assays, metabolite assays, immune function tests), hydrogen / methane breath tests, and many others.

[0059] In some embodiments, a reduction in enterotoxemia is accompanied by a change (increase or decrease) in the abundance of one or more bacterial populations. Bacterial abundances (e.g., bacteria belonging to a particular phylum), including the abundance of a specific species or strain of bacteria and the abundance of a bacterial population, can be assessed using any method known in the art. Generally, bacterial abundances may be assessed directly or indirectly. Examples of methods for directly assessing bacterial abundance in a sample (e.g., a microbiome or a sample thereof) include identifying and quantifying bacterial strains in a fecal sample from a subject. Examples of methods for indirectly assessing bacterial abundance in a sample (e.g., a microbiome or a sample thereof) include sequencing of nucleic acid samples (e.g., 16S rRNA genes of a given bacterial species or other bacterial genes obtained from a fecal or biopsy sample), and detection and quantification of metabolites associated with specific bacteria in a fecal sample from a subject (e.g., phospholipid fatty acid metabolism, microbial biomass carbon analysis).

[0060] The abundance of one or more bacterial populations in a sample from a subject may be compared to the abundance of bacterial populations in samples from the same subject obtained at a different time (e.g., previously or later). Alternatively, the abundance of one or more bacterial populations in a sample from a subject may be compared to the abundance of bacterial populations in samples from a different subject (e.g., a reference subject).

[0061] In some embodiments, enterotoxosis of the microbiome of interest (e.g., the gastrointestinal microbiome) is characterized by an increased abundance of microorganisms associated with inflammation and / or disease. In some embodiments, enterotoxosis is characterized by an increased abundance of Proteobacteria. In some embodiments, the increased abundance of microorganisms associated with inflammation and / or disease is compared to the abundance of microorganisms associated with inflammation before exposure to an event referred to as an enterotoxosis-induced event, as described herein.

[0062] In some embodiments, enterotoxosis of the target microbiome (e.g., gastrointestinal microbiome) is characterized by a decrease in the abundance of microorganisms that are thought to provide one or more beneficial effects to the target. In some embodiments, enterotoxosis of the target microbiome (e.g., gastrointestinal microbiome) is characterized by a decrease in the abundance of bacteria of the phylum Bacteroidetes. In some embodiments, enterotoxosis of the target microbiome (e.g., gastrointestinal microbiome) is characterized by a decrease in the abundance of bacteria of the phylum Firmicutes. In some embodiments, enterotoxosis of the target microbiome (e.g., gastrointestinal microbiome) is characterized by a decrease in the abundance of bacteria belonging to Clostridium cluster IV and / or XIVa. In some embodiments, enterotoxosis of the target microbiome (e.g., gastrointestinal microbiome) is characterized by a decrease in the abundance of bacteria belonging to Clostridium cluster XVII. In some embodiments, the decrease in the abundance of beneficial microorganisms is compared to the abundance of microorganisms associated with inflammation prior to exposure to an event referred to as an enterotoxosis-inducing event, as described herein.

[0063] In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria of the phylum Bacteroidetes (e.g., bacteria of the genus Bacteroides) compared to the abundance of Bacteroides in the subject (or its microbiome) before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria of the phylum Bacteroidetes (e.g., bacteria of the genus Bacteroides) compared to the abundance of Bacteroides in the subject (e.g., reference subject) (or its microbiome) that did not receive the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of one or more bacterial species belonging to the genus Bacteroides. In some embodiments, the reduction in enterotoxemia leads to an increase in the overall abundance of bacterial species belonging to the genus Bacteroides.

[0064] In some embodiments, the methods described herein increase the abundance of Bacteroides. Interestingly, in some embodiments, the pharmaceutical compositions described herein do not contain Bacteroides species. While we do not wish to be bound by any particular theory, in some embodiments, the pharmaceutical compositions may promote the establishment and / or proliferation of Bacteroides species, for example, by providing an environment favorable for the engraftment and / or proliferation of Bacteroides species.

[0065] In some embodiments, administration of the pharmaceutical compositions described herein results in an increase in the abundance of Bacteroidetes bacteria in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, and 10 times compared to the abundance of Bacteroidetes bacteria in the subject (or its microbiome) before administration of the pharmaceutical compositions. 4 double, 10 5The amount increases by a factor of two or more. In some embodiments, the abundance of Bacteroidetes bacteria in a subject before administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein increases the abundance of Bacteroidetes bacteria in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, 10 times, 10 times, 30 times, 40 times, 50 times, 10 times, 10 times, 10 4 double, 10 5 The amount increases twofold or more. In some embodiments, the abundance of Bacteroidetes bacteria in the reference sample was lower due to antibiotic treatment.

[0066] In some embodiments, administration of the pharmaceutical compositions described herein results in an increase of at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, and 10 times compared to the amount of Bacteroides in the subject (or its microbiome) before administration of the pharmaceutical compositions. 4 double, 10 5 The amount increases by a factor of two or more. In some embodiments, the bacterial abundance of Bacteroides in a subject prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein increases the abundance of Bacteroides in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, 10 times, 10 times, 30 times, 40 times, 50 times, 100 times, 1000 times, 10 times, 30 times, 30 times, 40 times, 50 times, 100 times, 10 times, 10 times, 30 times, 30 times, 1000 times, 1 4 double, 10 5The amount doubles or more. In some embodiments, the bacterial abundance of Bacteroides in the reference sample was lower due to antibiotic treatment.

[0067] In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of Bacteroidetes bacteria in a subject (or its microbiome) 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 compared to the abundance of Bacteroidetes bacteria in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, the abundance of Bacteroidetes bacteria in subjects prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of bacteria of the phylum Bacteroidetes in a subject (or its microbiome) 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 compared to the abundance of Bacteroides in a subject (e.g., a reference subject) (or its microbiome) that did not receive the pharmaceutical compositions. In some embodiments, the abundance of Bacteroidetes bacteria in the reference sample was lower due to antibiotic treatment.

[0068] In some embodiments, administration of the pharmaceutical compositions described herein increases the amount of Bacteroides in a subject (or its microbiome) 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 compared to the amount of Bacteroides in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, the amount of Bacteroides bacteria in the subjects prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical compositions described herein increases the amount of Bacteroides in a subject (or its microbiome) 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 compared to the amount of Bacteroides in a subject (or its microbiome) that did not receive the composition (e.g., a reference subject) or its microbiome. In some embodiments, the bacterial abundance of Bacteroides in the reference sample was lower due to antibiotic treatment.

[0069] In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of Firmicutes compared to the abundance of Firmicutes in the subject (or its microbiome) before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of Firmicutes compared to the abundance of Firmicutes in the subject (e.g., a reference subject) (or its microbiome) that did not receive the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of one or more bacterial species belonging to the phylum Firmicutes. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of all bacterial species belonging to the phylum Firmicutes. It should be understood that even if the pharmaceutical composition does not contain any bacterial species belonging to the phylum Firmicutes, an increase in the abundance of bacterial species belonging to the phylum Firmicutes may occur. If the pharmaceutical composition contains bacterial species belonging to the phylum Firmicutes, the increase in abundance may include both bacterial species belonging to the phylum Firmicutes that were present in the bacterial composition and bacterial species belonging to the phylum Firmicutes that were not present in the composition.

[0070] In some embodiments, administration of the pharmaceutical compositions described herein results in an increase in the amount of Firmicutes in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, and 10 times compared to the amount of Firmicutes in the subject (or its microbiome) before administration of the pharmaceutical compositions. 4 double, 10 5The amount increases by a factor of two or more. In some embodiments, the abundance of Firmicutes bacteria in a subject prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein resulted in an increase of 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, 10 times, 10 times, 30 times, 40 times, 50 times, 10 4 double, 10 5 The amount increases twofold or more. In some embodiments, the abundance of Firmicutes bacteria in the reference sample was lower due to antibiotic treatment.

[0071] In some embodiments, administration of the pharmaceutical compositions described herein increases the amount of Firmicutes in a subject (or its microbiome) 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 compared to the amount of Firmicutes in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, the abundance of Firmicutes bacteria in subjects prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of Firmicutes in a subject (or its microbiome) 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 compared to the abundance of Firmicutes in a subject (or its microbiome) that did not receive the composition (e.g., a reference subject) or its microbiome. In some embodiments, the abundance of Firmicutes bacteria in the reference sample was lower due to antibiotic treatment.

[0072] In some embodiments, according to the methods provided herein, administration of the pharmaceutical compositions described herein increases the abundance of bacterial species of the phylum Firmicutes and increases the abundance of bacterial species of the phylum Bacteroidetes.

[0073] In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria belonging to Clostridium cluster IV and / or XIVa compared to the abundance of bacteria belonging to Clostridium cluster IV and / or XIVa in the subject (or its microbiome) before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria belonging to Clostridium cluster IV, XIVa, and / or XVII compared to the abundance of bacteria belonging to Clostridium cluster IV, XIVa, and / or XVII in the subject (or its microbiome) before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria belonging to cluster IV and / or XIVa compared to the abundance of bacteria belonging to cluster IV and / or XIVa in the subject (e.g., a reference subject) (or its microbiome) that did not receive the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of one or more bacterial species belonging to Clostridium cluster IV and / or XIVa. In some embodiments, the reduction in enterotoxemia leads to an increase in the overall abundance of bacterial species belonging to Clostridium cluster IV and / or XIVa. In some embodiments, the abundance of bacteria belonging to Clostridium cluster IV, XIVa, and / or XVII in subjects before administration was lower due to antibiotic treatment.

[0074] In some embodiments, administration of the pharmaceutical compositions described herein results in an increase in the abundance of bacterial strains belonging to Clostridium cluster IV and / or XIVa in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, and 10 times, compared to the abundance of Clostridium cluster IV and / or XIVa in the subject (or its microbiome) before administration of the pharmaceutical compositions. 4 double, 10 5The amount increases by a factor of two or more. In some embodiments, the amount of Clostridium cluster IV and / or XIVa in a subject prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein increases the amount of Clostridium cluster IV and / or XIVa in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, 10 times, 10 times, 30 times, 40 times, 50 times, 10 4 double, 10 5 It doubles or increases by more than two.

[0075] In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of Clostridium cluster IV and / or XIVa in a subject (or its microbiome) 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 compared to the abundance of Clostridium cluster IV and / or XIVa in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, the amount of Clostridium cluster IV and / or XIVa present in subjects prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of Clostridium cluster IV and / or XIVa in a subject (or its microbiome) 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 compared to the abundance of Clostridium cluster IV and / or XIVa in a subject (e.g., a reference subject) (or its microbiome) that did not receive the composition.

[0076] In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria belonging to Clostridium cluster XVII compared to the abundance of bacteria belonging to Clostridium cluster XVII in the subject (or its microbiome) before administration of the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of bacteria belonging to Clostridium cluster XVII compared to the abundance of bacteria belonging to Clostridium cluster XVII in the subject (e.g., a reference subject) (or its microbiome) that did not receive the pharmaceutical composition. In some embodiments, the reduction in enterotoxemia leads to an increase in the abundance of one or more bacterial species belonging to Clostridium cluster XVII. In some embodiments, the reduction in enterotoxemia leads to an increase in the overall abundance of bacterial species belonging to Clostridium cluster XVII.

[0077] In some embodiments, administration of the pharmaceutical compositions described herein results in an increase in the abundance of bacterial strains belonging to Clostridium cluster XVII in the subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, and 10 times compared to the abundance of Clostridium cluster XVII in the subject (or its microbiome) before administration of the pharmaceutical compositions. 4 double, 10 5The amount increases by a factor of two or more. In some embodiments, the abundance of Clostridium cluster XVII in a subject prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein increases the abundance of bacterial strains belonging to Clostridium cluster XVII in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, 10 times, 10 times, 30 times, 40 times, 50 times, 10 4 double, 10 5 It doubles or increases by more than two.

[0078] In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of Clostridium cluster XVII in a subject (or its microbiome) 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 compared to the abundance of Clostridium cluster XVII in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, the amount of Clostridium cluster XVII present in subjects prior to administration of the pharmaceutical composition was lower due to antibiotic treatment. In some embodiments, administration of the pharmaceutical compositions described herein increases the abundance of Clostridium cluster XVII in a subject (or its microbiome) 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 compared to the abundance of Clostridium cluster XVII in a subject (e.g., a reference subject) (or its microbiome) that did not receive the composition.

[0079] In some embodiments, administration of the pharmaceutical compositions described herein reduces the abundance of microorganisms associated with inflammation. In some embodiments, administration of the pharmaceutical compositions described herein reduces the abundance of one or more microorganisms associated with inflammation. As used herein, the term “microorganisms associated with inflammation” refers to microorganisms that induce an inflammatory response upon colonization or infection of a subject. Microorganisms associated with inflammation are described, for example, in Zechner: “Inflammatory disease caused by intestinal pathobionts,” Current Opinion in Microbiology (2017) 35:64-69, and in numerous publications describing the role of microorganisms in IBD (see, for example, Hoffmann et al., ISME J. 2016 Feb;10(2):460-477). In some embodiments, microorganisms associated with inflammation induce acute inflammation characterized, for example, by the presence of inflammatory cytokines and / or infiltration of inflammatory immune cells into the site of colonization or infection. In some embodiments, microorganisms associated with inflammation induce chronic inflammation. In some embodiments, the microorganisms associated with inflammation are Proteobacteria. In some embodiments, a reduction in enterotoxemia leads to a decrease in the abundance of one or more bacterial species associated with inflammation. In some embodiments, a reduction in enterotoxemia leads to a decrease in the overall abundance of bacterial species associated with inflammation. In some embodiments, the abundance of bacterial species associated with inflammation (e.g., Proteobacteria) in the subject before administration increases due to antibiotic treatment.

[0080] In some embodiments, administration of the pharmaceutical compositions described herein results in an increase in the amount of inflammation-associated microorganisms in a subject (or its microbiome) by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1000 times, and 10 times compared to the amount of inflammation-associated microorganisms in the subject (or its microbiome) before administration of the pharmaceutical compositions.4 double, 10 5 The amount decreases by a factor of two or more. In some embodiments, the abundance of inflammation-associated microorganisms in a subject prior to administration of the pharmaceutical composition was higher due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein reduces the abundance of inflammation-associated microorganisms in a subject (or its microbiome) by at least 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 100, 1 / 1000, 1 / 1000, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 1000, 1 / 1000. 4 1 / 10 5 It decreases to one-tenth or less.

[0081] In some embodiments, administration of the pharmaceutical compositions described herein reduces the abundance of inflammation-associated microorganisms in a subject (or its microbiome) 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% compared to the abundance of inflammation-associated microorganisms in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, the abundance of inflammation-associated microorganisms in the subject before administration of the pharmaceutical compositions was higher due to antibiotic treatment.

[0082] In some embodiments, administration of the pharmaceutical composition reduces the amount of inflammation-associated microorganisms in a subject (or its microbiome) 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, compared to the amount of inflammation-associated microorganisms in a subject (or its microbiome) that did not receive the pharmaceutical composition.

[0083] In some embodiments, administration of the pharmaceutical composition described herein reduces the abundance of Proteobacteria. In some embodiments, administration of the pharmaceutical composition described herein reduces the abundance of one or more bacterial strains belonging to the phylum Proteobacteria. Proteobacteria is a phylum of Gram-negative bacteria that includes many pathogenic bacteria, such as Escherichia coli, Salmonella, Campylobacter, and Pseudomonas. In some embodiments, the reduction in enterotoxemia reduces the abundance of one or more bacterial species belonging to the phylum Proteobacteria. In some embodiments, the reduction in enterotoxemia reduces the overall abundance of bacterial species belonging to the phylum Proteobacteria.

[0084] In some embodiments, administration of the pharmaceutical compositions described herein reduces the amount of Proteobacteria in a subject (or its microbiome) by at least 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 1000, and 1 / 1000, respectively, compared to the amount of Proteobacteria in the subject (or its microbiome) before administration of the pharmaceutical compositions. 4 1 / 105 The amount is reduced to 1 / 1 or less. In some embodiments, the amount of Proteobacteria in the subject before administration of the pharmaceutical composition was higher due to antibiotic treatment. In some embodiments, the amount of Proteobacteria in the subject before administration of the pharmaceutical composition was higher due to antibiotic treatment. In some embodiments, administration of the pharmaceutical composition described herein reduces the amount of Proteobacteria in the subject (or its microbiome) by at least 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 1000, 1 / 10 4 1 / 10 5 It decreases to one-tenth or less.

[0085] In some embodiments, administration of the pharmaceutical compositions described herein reduces the amount of Proteobacteria in a subject (or its microbiome) 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% compared to the amount of Proteobacteria in the subject (or its microbiome) before administration of the pharmaceutical compositions. In some embodiments, administration of the pharmaceutical compositions described herein reduces the amount of Proteobacteria in a subject (or its microbiome) 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 compared to the amount of Proteobacteria in a subject (or its microbiome) that did not receive the composition (e.g., a reference subject) (or its microbiome).

[0086] In some embodiments, administration of the pharmaceutical composition described herein, according to the method provided herein, increases the abundance of bacterial species of the phylum Firmicutes, increases the abundance of bacterial species of the phylum Bacteroidetes, and decreases the abundance of bacterial species of the phylum Proteobacteria.

[0087] In some embodiments, according to the methods provided herein, administration of the pharmaceutical compositions described herein increases the abundance of bacterial species of the phylum Firmicutes and increases the abundance of bacterial species of the phylum Bacteroidetes.

[0088] In some embodiments, administration of the pharmaceutical composition described herein, according to the method provided herein, increases the abundance of bacterial species of the phylum Firmicutes and decreases the abundance of bacterial species of the phylum Proteobacteria.

[0089] In some embodiments, according to the methods provided herein, administration of the pharmaceutical compositions described herein increases the abundance of bacterial species of the phylum Bacteroidetes and decreases the abundance of bacterial species of the phylum Proteobacteria.

[0090] In some embodiments, the reduction in enterotoxemia does not correlate with a proportional increase in the diversity of the target microbiome. While microbiome diversity is generally associated with a healthy microbiome, it has been unexpectedly found herein that the compositions and methods provided herein can restore the strength of the microbiome without restoring diversity. Although not limited to a specific mechanism, it is believed that the microbiome is restored by the composition of the pharmaceutical compositions provided herein. For example, a limited number of Clostridium cluster XIVa bacterial strains in a composition can take the role of a larger group of Clostridium cluster XIVa strains found in an untreated healthy microbiome. Thus, a microbiome treated with the compositions provided herein and according to the methods provided herein may exhibit all the characteristics of a healthy microbiome, but may not have the high diversity that is sometimes associated with a healthy microbiome.

[0091] Aspects of this disclosure relate to a method for restoring a target microbiome, comprising administering a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains. In some embodiments, the method further comprises administering one or more additional doses of the pharmaceutical composition described herein. In some embodiments, the method further comprises administering an antibiotic to the target prior to the administration of the pharmaceutical composition. In some embodiments, the method further comprises administering vancomycin to the target prior to the administration of the pharmaceutical composition.

[0092] As used herein, “microbiome restoration” refers to the re-establishment or recovery of the relative abundance and / or microbial diversity of the bacterial population of the subject’s gastrointestinal microbiome. In some embodiments, the subject’s microbiome is restored to a healthy subject microbiome (healthy microbiome). In some embodiments, the subject’s microbiome is restored to a previous microbiome of the same subject. In some embodiments, the subject whose microbiome is being restored may have an inflammatory condition such as irritable bowel syndrome, ulcerative colitis, or Crohn’s disease. In some embodiments, the subject whose microbiome is being restored may have allergies (e.g., food allergies). In some embodiments, the subject’s microbiome is restored to the same subject’s microbiome prior to the inflammatory condition. Therefore, it should be understood that the microbiome requiring restoration may not have experienced an enterotoxosis-inducing event. Following the methods provided herein, in some embodiments, the subject requiring microbiome restoration may not have experienced an enterotoxosis-inducing event. In some embodiments, the subject requiring microbiome restoration does not have a Clostridium difficile infection. In some embodiments, the subject requiring microbiome restoration has not been treated with antibiotics.

[0093] Microbiota requiring restoration are characterized, for example, by the presence of identifying features associated with damaged microbiota. Generally, microbiota can be characterized based on the presence or absence of bacteria or populations of specific bacterial species. For example, damaged microbiota may be overcrowded with pathogens and / or have a reduced presence of commensal bacteria. Damaged microbiota can also be characterized by their biological function. For example, damaged microbiota may be unable to maintain the mucosal barrier and healthy immune function, and / or may be unable to fight infection and healthy microbiota.

[0094] In some embodiments, restoring the target microbiome results in a microbiome substantially similar to a healthy microbiome (e.g., a healthy target microbiome). In some embodiments, restoring the target microbiome results in a microbiome that is more similar to a healthy microbiome than to a damaged microbiome. In some embodiments, restoring the target microbiome results in the restoration or increase of the presence of one or more populations of bacteria or a particular bacterial species. In some embodiments, restoring the target microbiome results in the recovery or decrease of the presence of one or more populations of bacteria or a particular bacterial species. In some embodiments, restoring the target microbiome results in the improvement of one or more functions of the microbiome. In some embodiments, restoring the target microbiome results in the deterioration of one or more functions of the microbiome.

[0095] In some embodiments, the pharmaceutical compositions described herein can restore the microbiome without restoring its complete diversity. While microbiome diversity is generally associated with a healthy microbiome, it has been unexpectedly found herein that the compositions and methods provided herein can restore the intensity of the microbiome without restoring diversity (e.g., as defined by the Shannon index). Without wishing to limit ourselves to any particular theory, the microbiome is restored by the composition of specific bacterial strains administered in the composition and by the treatment methods and drug regimens provided herein. For example, a limited number of bacterial strains belonging to the Clostridium cluster XIVa in the composition may play the role of a larger group of XIVa strains found in an untreated healthy microbiome. Thus, a microbiome treated with the compositions described herein may exhibit the distinctive characteristics of a healthy microbiome (including the presence of Bacteroidetes and Firmicutes and the absence of Proteobacteria), but may not possess the high diversity typically associated with a healthy microbiome.

[0096] As used herein, “healthy microbiome” refers to the microbiome from an object without overt disease (e.g., a healthy object). While the microbial composition of a healthy microbiome can vary considerably, several tendencies characterizing a healthy microbiome have been identified. 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 carried out by a healthy microbiome, regardless of the composition of specific species. In some cases, the metabolic and molecular functions performed by a healthy microbiome are not those that the host object can perform, resulting in a symbiotic host-microbe relationship. In addition, a healthy microbiome tends to be resilient to external (e.g., diet or medication) and / or internal (e.g., age, disease state, stress, inflammation) changes in the object. The resilience of a healthy microbiome can also be characterized by its ability and speed to restore a healthy state after a disturbance. Alternatively, or in addition, a healthy microbiome may be characterized by a higher relative abundance (e.g., over 75%) of bacterial species from the phylum Firmicutes and the genus Bacteroides compared to species from the phylum Proteobacteria.

[0097] In some embodiments, subjects with restored or healthy microbiomes exhibit increased abundance of bacteria from the phylum Firmicutes and / or genus Bacteroides compared to bacteria from the phylum Proteobacteria.

[0098] In some embodiments, subjects with restored or healthy microbiomes exhibit increased abundance of bacteria from the Firmicutes and / or Bacteroidetes phyla compared to bacteria from the Proteobacteria phylum.

[0099] In some embodiments, the subjects have not received or experienced an enterotoxosis-inducing event. In some embodiments, the subjects have not received or experienced an enterotoxosis-inducing event one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, twelve months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or more prior to administration of the pharmaceutical composition described herein. In some embodiments, the subjects do not have an infection. In some embodiments, the subjects have not had an infection one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, twelve months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or more prior to administration of the pharmaceutical composition described herein. In some embodiments, the subjects do not have a Clostridium difficile infection. In some embodiments, the subject has not had a Clostridium difficile infection one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, twelve months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or more prior to administration of the pharmaceutical composition described herein. In some embodiments, the subject has not been treated with antibiotics. In some embodiments, the subject has not been treated with antibiotics one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, twelve weeks, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or more prior to administration of the pharmaceutical composition described herein. In some embodiments, the subject has not been treated with vancomycin. In some embodiments, the subject has not been treated with vancomycin one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, twelve weeks, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or more prior to administration of the pharmaceutical composition described herein.

[0100] Aspects of this disclosure relate to methods for increasing the recovery of a healthy microbiome in a subject after an enterotoxemia-induced event, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains. In some embodiments, the methods further comprise administering one or more additional doses of the pharmaceutical composition described herein. In some embodiments, the methods further comprise administering an antibiotic to the subject prior to administering the pharmaceutical composition. In some embodiments, the methods further comprise administering vancomycin to the subject prior to administering the pharmaceutical composition.

[0101] As used herein, the term “increasing the recovery” of a healthy microbiome refers to an increase in the rate of recovery or the overall degree of recovery (e.g., the microbiome of the subject is more similar to a healthy microbiome after administration of the pharmaceutical composition). In some embodiments, increasing the recovery of a healthy microbiome refers to an increase in the rate of recovery. In some embodiments, recovery of a healthy microbiome refers to an increase in the overall degree of recovery (e.g., the microbiome of the subject is more similar to a healthy microbiome after administration of the pharmaceutical composition). In some embodiments, “increasing the recovery” of a healthy microbiome refers to an increase in both the rate of recovery and the overall degree of recovery. In some embodiments, after administration of any of the pharmaceutical compositions described herein, a healthy microbiome is recovered more rapidly in the subject compared to the recovery of a healthy microbiome in a subject that did not receive the pharmaceutical composition. In some embodiments, after administration of any of the pharmaceutical compositions described herein, a healthy microbiome is recovered more rapidly in the subject compared to the recovery of a healthy microbiome in a subject that received a fecal matter transplant. In some embodiments, the increased recovery of a healthy microbiome occurs at least 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4, 5, or 6 weeks earlier than the recovery of a healthy microbiome in subjects that were not administered the pharmaceutical composition. In some embodiments, the increased recovery of a healthy microbiome occurs at least 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4, 5, or 6 weeks earlier than the recovery of a healthy microbiome in subjects that did not receive fecal matter transplantation.

[0102] In some embodiments, increasing the recovery of a healthy microbiome refers to an increase in the recovery rate. In some embodiments, the recovery of a healthy microbiome occurs within 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks from the administration of the pharmaceutical composition described herein. In some embodiments, the recovery of a healthy microbiome occurs 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 100 times faster, or more, compared to subjects not treated with the pharmaceutical composition according to the methods provided herein.

[0103] In some embodiments, increasing the recovery of a healthy microbiome refers to an increase in the overall degree of recovery. In some embodiments, the recovery of a healthy microbiome is 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 better compared to subjects not treated with the pharmaceutical composition according to the methods provided herein.

[0104] In some embodiments, recovery occurs without the detection of colonization of one or more bacterial strains of the pharmaceutical composition. In some embodiments, recovery of a healthy microbiome is associated with colonization of one or more bacterial strains of the pharmaceutical composition.

[0105] In some embodiments of the methods provided herein, the subject is experiencing an enterotoxosis-inducing event. In some embodiments of the methods provided herein, the subject is experiencing an enterotoxosis-inducing event prior to administration of a single or multiple dose of the pharmaceutical composition provided herein. As used herein, the term “enterotoxosis-inducing event” refers to one or more events that disrupt the microbiome of the subject. In some embodiments, the subject who has experienced an enterotoxosis-inducing event requires administration of one of the pharmaceutical compositions described herein. In some embodiments, the subject who has experienced an enterotoxosis-inducing event is administered one of the pharmaceutical compositions described herein to restore or recover the microbiome to a healthy microbiome. In some embodiments, the microbiome of the subject has one or more characteristics (identifying features) of a damaged microbiome after the enterotoxosis-inducing event. Examples of enterotoxosis-inducing events include exposure to antibiotics (e.g., treatment with vancomycin), infection, Clostridium difficile infection, misuse of alcohol, surgery, treatment with chemotherapeutic agents, treatment with immunosuppressants, traveler’s diarrhea, and inappropriate diet.

[0106] In addition, it is recognized that the microbiome can be modulated by exposure to any of the various classes of therapeutic agents, and that members of the gut microbiome metabolize structurally diverse drug molecules (see Zimmermann et al. Nature (2019) 570:462-471). In some embodiments, enterotoxosis-induced events involve exposure to one or more therapeutic agents that can be metabolized by the microbiome.

[0107] Examples of drug molecules that can be metabolized by the microbiome include steroids and hormone agonists (e.g., norethindrone acetate, levonorgestrel, megestrol acetate, betamethasone acetate, dexamethasone, drospirenone, norgestimate, finasteride), hormone antagonists (e.g., cyproterone acetate, bromocriptine mesylate, naloxone, mifepristone, bicalutamide), antidepressants and drugs associated with mental health (e.g., virazodone, fluoxetine, olanzapine, thiothixene, fluvoxamine maleate, paroxetine), Sulpiride, metitepine maleate, bupropion, ziprasidone mesylate, pimozide, fluoxetine, periciazine, paliperidone), antiviral agents (e.g., famciclovir, ritonavir, zidovudine (azt), nevirapine, amantadine), antiallergic / antihistamines (e.g., roxatidine acetate, clemastine fumarate, cetirizine, nizatidine, diphenylpyraline, tranilast), antidiarrheal agents (e.g., racecadotril, trimebutine maleate, loperamide), immunosuppressants (e.g., mycophenolate mofetil, deflazacort, Tacrolimus), antihypertensive drugs (e.g., olmesatran medoxomil, diltiazem flufenadine, telmisaratan, quinapril, trandolapril, benazepril, irbesartan, losartan, valsartan, enalapril maleate, benzthiazide, doxazosin mesylate, papaverine, lofexidine, verampimil, penbutrol sulfate, ramipril, carvedilol, nitrendipine), diabetes medications (e.g., linagliptin, trazamide, nateglinide, glipizide, gliclazide, Repaglinide), antipruritics (e.g., diflorasone diacetate, betamethasone valerate), cholesterol medications (e.g., fenofibrate, bezafibrate, ezetimibe, mevastatin, lovastatin), nonsteroidal anti-inflammatory drugs (e.g., fanprofazone, pranoprofen, oxaprozin, etodolac, ketrolactromethamine, colchicine, naproxen, celecoxib, diacetamate), muscle relaxants (e.g., carisoprodol, oxybutynin chloride, metaxalone, naphtopidil, sumatriptan succinate),Orphenadrine citrate, cremisole, trihexyphnidyl, alfuzosin, alprenolol, camylofin, chlormezanone, carbapentan citrate, phenazopyridine), antifungal agents (e.g., griseoulvin, mebendazole, voriconazole, fluconazole, itraconazole), sedatives (e.g., ramelteon, zaleplon, clonidine) (e.g., eszopiclone), central nervous system drugs (e.g., riluzole, galantamine, ergotamine tartrate, nicergoline, etopropazine, methylphenidate, methosiximide, oxcarbazepine, memantine, biperiden, methyserzide maleate, etozolamide, phenytoin sodium, idebenone, entacapone), anticoagulants (e.g., clopidrogrel sulfate) Sulfonate, sulfinpyrazone, warfarin, dipyridamole, anagrelide, trimetazidine, naphronyl oxalate), blood pressure raising drugs (e.g., digitoxin, digoxin, sotalol, acecainide), respiratory disease drugs (e.g., fenpiride), antiparasitic drugs (e.g., primaquine phosphate, mefloquine, rebamisole, artemisinin), chemotherapeutic agents (e.g., dasatinib, procarbine, cyclophosphamide, anastrozole, imiatinib, paclitaxel, capecitabine, melphalan), muscle tonics / enhancing agents (e.g., ergonovine maleate, neostigmine bromide), drugs associated with gastric / mucosal protection (e.g., rebamisole, Examples include pyrophosphate (e.g., metoprolol tartrate, ranitidine, pantoprazole, tenatoprazole), intestinal disorder medications (e.g., budesonide, dicyclomine, sulfasalazine, bisacodyl), uric acid inhibitors (e.g., febuxostate, indomethacin), acid fasteners (e.g., cimetidine, benzbromarone, omeprazole), erectile dysfunction medications (e.g., tadalafil, sildenafil citrate), analgesics (e.g., noscapine, rizatriptan benzoate), natural products (e.g., vinpocetine), skin disease medications (e.g., methoxsaline), immunostimulants (e.g., pyrotimod), antibiotics, and reproductive disorder medications (e.g., danazol).

[0108] In some embodiments, the subject has experienced or is undergone an enterotoxemia-inducing event one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten months, eleven months, twelve months, or more prior to administration of the pharmaceutical composition described herein.

[0109] The administration of antibiotics is associated with gastroenterotoxemia because the killing of symbiotic and / or commensal bacteria can lead to an overpopulation of opportunistic bacteria, causing disease. In some embodiments, antibiotics are administered to treat or prevent a bacterial infection or suspected bacterial infection. In some embodiments, antibiotics are administered in connection with surgery on the subject. In some embodiments, antibiotics are administered to the subject before (prophylactically) or after surgical procedures.

[0110] Non-exclusive examples of antibiotics that can induce enterotoxemia include cephalosporin antibiotics, cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cephamandol, cefoxitin, cefprodil, ceftoviprole, clindamycin, ceftriaxone, cefotaxime, cefazolin, cefoperazone, cefuroxime, cefmetazole, fluoroquinolones, ciprofloxacin, levaquin, floxin, techin, avelox, norflox, tetracycline, minocycline, and oxytetracycline. (Doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, benzylpenicillin, carbenicillin, vancomycin, and methicillin), ertapenem, doripenem, imipenem / cilastatin, meropenem, clavulanic acid, tazobactam, piperacillin, ceftriaxone, cefotaxime, cefazolin, fluoroquinolone, imipenem, meropenem, metronidazole, fidaxomyxin, lydinirazole, trimethoprim / sulfamextoxazole, ceftobioprol, cephthaloline, dalbavancin, da Ptomycin, Fusidic acid, Linezolid, Mupirocin, Omadacycline, Oritabancin, Tedizolid, Terabancin, Tigecycline, Ceftazidime, Cefepime, Ceftolozane / Tazobactam, Piperacillin / Tazobactam, Ticarcillin / Clavulanate, Streptogramin, Daptomycin, Amikacin, Kanamycin, Neomycin, Netirimycin, Tobramycin, Paromycin, Spectinomycin, Geldanamycin, Helbimycin, Rifamixin, Loracarbef, Cefurodoxil, Cefaldine, Cefadrine, Cefapillin Cephalexin, cefotetan, cefmetazole, cefonisid, cefprodil, cefuroxime, cefiximin, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, ceftarolinfosamil, teicoplanin, terbancin, dalbavancin, oritabancin, lincomycin, daptomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin,Fidaxomicin, azetrenoma, furazolidone, nitrofurantoin, posizolid, radezolid, trezolid, azurocillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, piperacillin, temocillin, ticalcillin, bacitracin, colistin, polymyxin B, enoxacin, gatifloxacin, gemifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, naldixic acid, norfloxacin, ofloxacin, trovafloxacin, glepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sul Examples include phadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizol, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, sulfoamindochrysoidine, demeclocycline, metacycline, minocycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifubutin, rifapentin, arsphenamine, fosfomycin, fusidic acid, mupirocin, platensimycin, quinupristin / dalfopristin, thianphenicol, tigecycline, and tinidazole.

[0111] In some embodiments, the enterotoxemia-induced event is treated with antibiotics. In some embodiments, the enterotoxemia-induced event is treatment with antibiotics associated with surgery. In some embodiments, the antibiotic is vancomycin.

[0112] In some embodiments, the enterotoxosis-induced event is an infection, such as an infection caused by a virus, bacteria, fungus, yeast, parasite, or a combination thereof. In some embodiments, the subject is administered one or more therapeutic agents (e.g., antivirals, antibiotics, antifungals, antiparasitic agents, etc.) to treat the infection. In some embodiments, the enterotoxosis-induced event is a Clostridium difficile infection. Antibiotics typically used to treat Clostridium difficile infections include metronidazole, vancomycin, and fidaxomicin.

[0113] In some embodiments, the enterotoxosis-induced event is a gastrointestinal infection. In some embodiments, the subject is experiencing a gastrointestinal infection caused by, for example, Clostridium perfringens, Staphylococcus, Enterococcus faecium, Roseburia hominis, Raecalibacterium prausnitzii, Clostridioides difficile, Escherichia coli, Salmonella typhii, Vibrio cholerae, Shigella flexneri, Campylobacter, Peptostreptococcus, Yersinia, or Proteobacteria. In some embodiments, the enterotoxosis-induced event is traveler's diarrhea. Generally, traveler's diarrhea is a gastrointestinal disorder characterized by diarrhea, abdominal colic, nausea, vomiting, and / or fever, caused by exposure to an infectious pathogen during travel (e.g., typically eating contaminated food or drinking contaminated water).

[0114] In some embodiments, as described herein, the administration of one or more therapeutic agents for treating an infection, C. difficile infection, or traveler's diarrhea may contribute to gastrointestinal toxemia.

[0115] In some embodiments, the enterotoxosis-induced event is exposure to an antifungal agent. In some embodiments, the subject is administered one or more antifungal agents, for example, to treat a fungal infection. Examples of antifungal agents include, but are not limited to, clotrimazole, econazole, micronazole, fluconazole, terbinafine, ketoconazole, amphotericin, thioconazole, itraconazole, posaconazole, voriconazole, isabconazonium, casophungin, anidurafungin, micafungin, griseofulvin, flucytosine, terbinafine, nyastin, amphotericin B, and candidiasis. Examples include citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, citronellol, and citronellol.

[0116] In some embodiments, the enterotoxemia-inducing event is exposure to antiparasitic agents. In some embodiments, the subject is administered one or more antiparasitic agents, for example, to treat a parasitic infection. Examples of antiparasitic agents include, but are not limited to, nitazonxanide, melarsoprol, eflornithine, metronidazole, tinidazole, meltefosin, mebenzaole, pyrantel pamoate, thiabendazole, diethylcarbamuzin, ivermectin, niclosamide, praziquantel, albenzaole, praziquantel, albenzaole, praziquantel, rifampin, amphotericin B, fumagiline, befenium, diethylcarbamazine, nicrossamide, piperazine, pyantel, pyrvinium, flubenazole, benzyl benzoate / disulfiram, lindane, malathion, permethrin, benzyl alcohol, piperonyl butoxide / pyrethrin, spinosad, and crotamiton.

[0117] In some embodiments, the enterotoxosis-induced event is exposure to one or more immunosuppressants. Examples of immunosuppressants include, but are not limited to, prednisone, dexamethasone, hydrocortisone, methotrexate, azatipurine, mecaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mitramycin, cyclosporine, tacrolimus, evalolimus, sirolimus, rapamycin, infliximab, etanercept, adaluminab, and mycopheno. Examples include uric acid, fingolimond, myriocin, hydroxychloroquine, pexidalatinib, darolutamide, ferric maltol, glucagon, lilonacept, benralizumab, canakinumab, brodalumab, anakinra, reslizumab, ustekinumab, mepolizumab, tocilizumab, dupilumab, ixekizumab, guselkumab, secukinumab, thurirumab, tildrakizumab, basiliximab, risankizumab, siltuximab, daclizumab, and thalidomide.

[0118] In some embodiments, the enterotoxosis-induced event is exposure to one or more chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, doxorubicin, docetaxel, bleomycin, vinblastine, dacarbazine, mustine, vincristine, procarbazine, prednisolone, etoposide, cisplatin, epirubicin, cisplatin, capecitabine, folinic acid, oxaliplatin, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine, streptozotocin, dacarbazine, mitozolomid, temozolomid, thiote Examples include mitomycin, diaziquan, procarbazine, hexamethylmelamine, pemetrexed, capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nerarabine, cladribine, clofabine, pentostatin, thioguanine, mecaptopurine, vinblastine, vinorelbine, vindesine, vinflunin, paclitaxel, docetaxel, podophyllotoxin, teniposide, camptothecin, novobiocin, melbaron, acralubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, and mitoxantrone.

[0119] In some embodiments, the enterotoxemia-inducing event is bowel cleansing.

[0120] Aspects of this disclosure relate to methods for protecting a target microbiome, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains. In some embodiments, the methods further comprise administering one or more additional doses of the pharmaceutical composition described herein. In some embodiments, the methods further comprise administering an antibiotic to the target prior to administering the pharmaceutical composition. In some embodiments, the methods further comprise administering vancomycin to the target prior to administering the pharmaceutical composition.

[0121] A number of factors can influence the composition of the gastrointestinal microbiota (e.g., diversity, abundance of specific populations). The compositions described herein may be used to protect the microbiota from such factors. As used herein, the term “protecting the microbiota” means preventing or minimizing disruption to the microbiota in question. In some embodiments, the compositions described herein help maintain the normal gastrointestinal microbiota in question. In some embodiments, the compositions described herein help maintain a healthy gastrointestinal microbiota. In some embodiments, by protecting the microbiota, microbiota-mediated disorders such as antibiotic-induced adverse reactions, Clostridium difficile infection, ulcerative colitis, Crohn's disease, and irritable bowel syndrome may be treated or prevented.

[0122] In some embodiments, the microbiome is considered protected if its composition (e.g., diversity, abundance of a particular population) does not substantially change after an event. In some embodiments, the microbiome is considered protected if its composition (e.g., diversity, abundance of a particular population) does not detectably change after an event. In some embodiments, the composition (e.g., diversity, abundance of a particular population) of the gastrointestinal microbiome changes by 10% or less (9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) after an event. In some embodiments, events that may affect the composition of the microbiome are antibiotic treatment, attack by infectious agents (e.g., infection), and / or C. difficile infection.

[0123] In some embodiments, administration of the pharmaceutical compositions described herein protects the microbiome of the subject from antibiotic treatment. For example, the subject may be administered antibiotics to treat or prevent infection, according to the method provided herein, and the administration of these antibiotics (e.g., vancomycin) does not result in severe enterotoxemia. In some embodiments, administration of the pharmaceutical compositions described herein protects the microbiome of the subject from attack by infectious agents (e.g., infection). In some embodiments, administration of the pharmaceutical compositions described herein protects the microbiome of the subject from C. difficile infection. In some embodiments, the pharmaceutical compositions described herein protect the microbiome of the subject from C. difficile by increasing the recovery rate of the microbiome after administration of antibiotics to treat C. difficile infection. While we do not wish to be bound by any particular theory, it is thought that increasing the recovery rate of the microbiome after administration of antibiotics prevents C. difficile colonization (implantation) in the preferred depressions of the gastrointestinal tract. In some embodiments, the colonization of C. difficile is prevented by the colonization of one or more bacterial strains of the pharmaceutical composition or bacterial strains associated with the bacterial strains of the pharmaceutical composition in the C. difficile-preferred depressions of the gastrointestinal tract.

[0124] Aspects of this disclosure relate to methods for establishing a target microbiome, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition containing one or more purified bacterial strains. In some embodiments, the methods further comprise administering one or more additional doses or amounts of the pharmaceutical composition described herein. In some embodiments, the methods further comprise administering an antibiotic to the target before administering the pharmaceutical composition. In some embodiments, the methods further comprise administering vancomycin to the target before administering the pharmaceutical composition.

[0125] In some embodiments, one or more bacterial strains of the pharmaceutical compositions provided herein colonize or re-colonize the target gastrointestinal tract or a portion thereof (e.g., the colon or cecum). Such colonization may also be referred to as transplantation or engraftment. In some embodiments, one or more bacterial strains of the compositions colonize the target gastrointestinal tract (e.g., the colon or cecum) after the naturally occurring microbiome has been partially or completely removed, for example, due to an enterotoxin-induced event. In some embodiments, one or more bacterial strains of the compositions colonize the target gastrointestinal tract (e.g., the colon or cecum) after the naturally occurring microbiome has been partially or completely removed by antibiotic (e.g., vancomycin) treatment. In some embodiments, one or more bacterial strains of the compositions colonize the gastrointestinal tract with an imbalance in the intestinal flora (e.g., the gastrointestinal tract treated with antibiotics). In some embodiments, all of the bacterial strains of the compositions colonize the gastrointestinal tract. In some embodiments, all of the bacterial strains of the compositions colonize the gastrointestinal tract with an imbalance in the intestinal flora. In some embodiments, multiple doses of the bacterial composition are administered to allow all of the bacterial strains of the composition to colonize the gastrointestinal tract. In some embodiments, multiple doses of the bacterial composition are administered to allow all of the bacterial strains of the composition to colonize the gastrointestinal tract in a state of intestinal symbiotic imbalance.

[0126] In some embodiments, one or more bacterial strains of the pharmaceutical composition can grow faster than other bacterial strains (e.g., pathogens) and thus colonize the microbiome. In some embodiments, the subject is treated with antibiotics, resulting in the removal of a large portion of the microbiome and creating a "blank slate" environment for both one or more bacterial strains of the composition and any other bacterial strains (e.g., pathogens). Therefore, although not limited to a specific mechanism, if both pathogens and one or more bacterial strains of the composition provided herein are present in the intestinal tract (e.g., colon or cecum), one or more bacterial strains of the composition provided herein grow faster than pathogens (e.g., have a shorter doubling time), preventing the accumulation of pathogens in the intestinal tract (e.g., colon or cecum) and enabling colonization of one or more bacterial strains of the composition. In some embodiments, one or more bacterial strains of the composition provided herein are superior to transplantation in the intestinal tract (e.g., colon or cecum), resulting in faster growth. In some embodiments, one or more bacterial strains of the compositions provided herein are superior in metabolizing nutrients present in the intestinal tract (e.g., colon or cecum), resulting in faster growth. In some embodiments, the compositions of bacterial strains provided herein prevent or inhibit the production of bacterial toxins by infectious agents, 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 due to synergistic effects between the bacterial strains. For this reason, in some embodiments, but not limited to, the combination of bacterial strains of the compositions provided herein acts synergistically, because the combination of strains is particularly suitable for the use of nutrients in the intestinal tract (e.g., colon or cecum) (e.g., through metabolic interactions) and / or the combination is superior for transplantation (e.g., by providing a favorable microenvironment). In some embodiments, one or more bacterial strains of the compositions described herein can colonize specific depressions in the intestinal tract (e.g., colon or cecum).In some embodiments, one or more bacterial strains of the compositions described herein can colonize specific depressions in the intestinal tract (e.g., the colon or cecum) that become available after antibiotic treatment.

[0127] In some embodiments, the pharmaceutical composition comprises seven bacterial strains, with at least two strains colonizing the control microbiome. In some embodiments, the pharmaceutical composition comprises seven bacterial strains, with at least four strains colonizing the target microbiome. In some embodiments, the pharmaceutical composition comprises seven bacterial strains, with each of the seven bacterial strains colonizing the target microbiome.

[0128] The degree of colonization of one or more bacterial strains may be determined, for example, by detecting the presence of one or more bacterial strains and / or by quantifying the abundance of one or more bacterial strains. In some embodiments, the bacterial strains of the pharmaceutical composition may be 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%, 5 4%, 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% colonize the target microbiome. In some embodiments, at least 25% of the bacterial strains of the pharmaceutical composition colonize the target microbiome. In some embodiments, at least 50% of the bacterial strains of the pharmaceutical composition colonize the target microbiome. In some embodiments, 100% of the bacterial strains of the pharmaceutical composition colonize the target microbiome. In some embodiments, the percentage of bacterial strains of the pharmaceutical composition colonizing the target microbiome increases by administering additional doses of the pharmaceutical composition. In one embodiment, it should be understood that the method provided herein provides better colonization of the bacterial strains of the pharmaceutical composition (e.g., higher number / percentage colonization and / or higher abundance) compared to colonization of the same pharmaceutical composition administered according to different methods. For this reason, in one embodiment, colonization is better because the pharmaceutical composition is administered according to a specific dosing regimen, dose, and / or antibiotic treatment regimen provided herein.

[0129] In some embodiments, the pharmaceutical composition contains eight bacterial strains, with at least two strains colonizing the target microbiome. In some embodiments, the pharmaceutical composition contains eight bacterial strains, with at least four strains colonizing the target microbiome. In some embodiments, the pharmaceutical composition contains eight bacterial strains, with each of the eight bacterial strains colonizing the target microbiome.

[0130] The degree of colonization of the microbiome by one or more bacterial strains of the pharmaceutical composition described herein may be based on the relative abundance of the bacterial strains of the pharmaceutical composition in the microbiome. For example, 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% of the bacterial strains of the target microbiome. 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% are bacterial strains of the pharmaceutical composition. In some embodiments, at least 25% of the bacterial strains detected in the target microbiome are bacterial strains of the pharmaceutical composition. In some embodiments, at least 50% of the bacterial strains detected in the target microbiome are the bacterial strains of the pharmaceutical composition. In some embodiments, the percentage of bacterial strains of the bacterial composition in the target microbiome increases by administering an additional dose of the pharmaceutical composition.

[0131] In some embodiments, the pharmaceutical compositions described herein result in the long-term colonization of one or more bacterial strains in the composition. In some embodiments, one or more bacterial strains of the pharmaceutical compositions described herein are detected in the target microbiome for an extended period. In some embodiments, one or more bacterial strains of the pharmaceutical compositions described herein colonize the target microbiome for an extended period. In some embodiments, one or more bacterial strains of the pharmaceutical compositions described herein are detected in the target microbiome for at least one week, two weeks, three weeks, four weeks, five weeks, or six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year after administration of the pharmaceutical composition.

[0132] In one embodiment, it should be understood that the method provided herein provides better colonization (e.g., higher number / percentage colonization and / or higher abundance) and more durable colonization of bacterial strains of the pharmaceutical composition compared to colonization of the same pharmaceutical composition administered according to a different method. Therefore, in one embodiment, the colonization is better and more durable because the pharmaceutical composition is administered according to a specific dosing regimen, amount, and / or antibiotic treatment regimen provided herein.

[0133] In one embodiment, it should be understood that the method provided herein provides more durable colonization of bacterial strains of the pharmaceutical composition compared to colonization of the same pharmaceutical composition administered according to a different method. Therefore, in one embodiment, the colonization is more durable because the pharmaceutical composition is administered according to a specific dosing regimen, dosage, and / or antibiotic treatment regimen provided herein.

[0134] The methods described herein involve administering one of the pharmaceutical compositions described herein to a subject in need thereof. As used herein, “subject,” “individual,” and “patient” are interchangeable and refer to vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, human primates, non-human primates, or species of rodents, cattle, horses, dogs, or cats. In some embodiments, the subject is a human subject. In some embodiments, the human subject is a neonatal subject, a child subject, an adolescent subject, an adult subject, or an elderly subject. In some embodiments, the subject has or is at risk of having enterotoxosis, or has experienced or is at risk of experiencing an enterotoxosis-inducing event. In some embodiments, the subject has risk factors associated with enterotoxosis. In some embodiments, the subject has risk factors associated with microbiome disturbance.

[0135] Any of the compositions described herein may be administered to a subject in a therapeutically effective dose or a therapeutically effective amount. In some embodiments, the therapeutically effective dose is an amount sufficient to treat or prevent a disease or disorder. The terms “treat” or “medicate” mean to reduce or alleviate one or more of the symptoms associated with a disease or disorder (e.g., enterotoxemia).

[0136] In some embodiments, administering a therapeutically effective amount of any of the compositions described herein may prevent a disease or disorder (e.g., enterotoxemia), prevent disruption of the microbiome, and / or prevent colonization by a pathogen. The terms “prevent” or “prevent” encompass prophylactic administration and may, for example, reduce the incidence or likelihood of a disease or disorder. In some embodiments, the compositions reduce the incidence or likelihood of microbiome disruption. In some embodiments, the compositions reduce the incidence or likelihood of prevention of pathogen colonization. For example, in some embodiments, administration of the compositions provided herein results in a healthy microbiome, which provides an effect in subjects that reduces the incidence or likelihood of a disease or disorder. In some embodiments, administration of the compositions provided herein results in reduction or alleviation of one or more symptoms associated with a disease or disorder.

[0137] As used herein, the term “therapeutic effective dose” may be used interchangeably with the term “effective dose.” A therapeutic effective dose or effective dose of a composition, such as a pharmaceutical composition described herein, is any amount that produces a desired response or outcome in a subject, such as one described herein, which includes, but is not limited to, delaying the onset, halting the progression, at least one symptom of a disease or disorder (e.g., enterotoxosis), disruption of the microbiome, and / or reduction or mitigation of microbiome colonization by a pathogen. In some embodiments, a therapeutic effective dose is an amount sufficient to restore the microbiome, increase the recovery of the microbiome after an enterotoxosis-induced event, protect the microbiome, and / or colonize the microbiome of the subject. In some embodiments, a therapeutic effective dose is an amount sufficient to treat primary and / or secondary (recurrent) Clostridium difficile infection. In some embodiments, a therapeutic effective dose is an amount sufficient to treat or suppress a food allergy.

[0138] It should be understood that the term "effective dose" in relation to compositions containing bacterial strains may be expressed as the number of bacteria or CFUs administered. Furthermore, it should be understood that bacteria can grow once administered. Therefore, even relatively small doses of bacteria may have a therapeutic effect.

[0139] Furthermore, methods for determining whether a subject has or is at risk of having enterotoxemia, Clostridium difficile infection, or food allergies, or whether administration of any of the pharmaceutical compositions described herein is required, are also within the scope of this disclosure.

[0140] Aspects of this disclosure relate to bacterial strains having a 16S rDNA sequence that has sequence identity to any one of the nucleic acid sequences of the bacterial strains or species described herein. As those skilled in the art will understand, a 16S rDNA sequence represents a DNA sequence corresponding to a 16S rRNA sequence. The terms “identical” or “identity” percentage in the context of two or more nucleic acid or amino acid sequences refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if, when compared and aligned over a specified region, they have a specified percentage of identical amino acid residues or nucleotides over a specified region of the nucleic acid or amino acid sequence, or over the entire sequence (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), measured using one of the subsequent sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity exists over a region of at least about 50 nucleotides in length, or more preferably over a region of 100 to 500 or 1000 nucleotides or longer. In some embodiments, identity exists over the length of the 16S rRNA or 16S rDNA sequence.

[0141] In some embodiments, a bacterial strain has at least 60%, at least 70%, 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 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or up to 100% sequence identity over a specified region or over an entire sequence compared to any of the strains or bacterial species described herein.

[0142] In some embodiments, the pharmaceutical composition comprises one or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), one or more of which contain a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8. In some embodiments, the pharmaceutical composition comprises two or more bacterial strains (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), two or more of which contain a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8. In some embodiments, the pharmaceutical composition comprises three or more bacterial strains (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein three or more bacterial strains contain a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8. In some embodiments, the pharmaceutical composition comprises four or more bacterial strains (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein four or more bacterial strains contain a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8. In some embodiments, the pharmaceutical composition comprises five or more bacterial strains (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each of which comprises a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8.In some embodiments, the pharmaceutical composition comprises six or more bacterial strains (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each containing a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8. In some embodiments, the pharmaceutical composition comprises seven or more bacterial strains (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each containing a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8. In some embodiments, the pharmaceutical composition comprises eight or more bacterial strains (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), the eight or more bacterial strains comprising a 16S rDNA sequence having at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs. 1, SEQ ID NOs. 2, SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8.

[0143] In some embodiments, the pharmaceutical composition comprises eight bacterial strains, each containing a 16S rDNA sequence having at least 97% sequence identity to the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8.

[0144] In some embodiments, the pharmaceutical composition comprises seven bacterial strains, each containing a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8. In some embodiments, the pharmaceutical composition comprises seven bacterial strains, each containing a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8. A composition containing a bacterial strain having a 16S rRNA sequence having 97% identity with the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8 may be referred to as "Composition VE416" or "VE416". Additional embodiments of a composition comprising a bacterial strain having a 16S rRNA sequence having 97% identity to the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8 are described in PCT Publication WO2019 / 094837.

[0145] A composition comprising a bacterial strain having a 16S rRNA sequence having 97% identity with the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8 may be referred to as "Composition VE303" or "VE303". Additional embodiments of the composition comprising a bacterial strain having a 16S rRNA sequence having 97% identity with the nucleic acid sequences indicated by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8 are described in PCT Publications WO2017 / 218680, WO2018 / 081550, and WO2018 / 112371.

[0146] In some embodiments, the pharmaceutical composition comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) bacterial strains, at least one of which comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises two or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) bacterial strains, at least one of which comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises three or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and at least one of the bacterial strains comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises four or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and at least one of the bacterial strains comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises five or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and at least one of the bacterial strains comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises six or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and at least one of the bacterial strains comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6.In some embodiments, the pharmaceutical composition comprises seven or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and at least one of the bacterial strains comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises eight or more bacterial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and at least one of the bacterial strains comprises a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6.

[0147] In some embodiments, the pharmaceutical composition comprises a bacterial strain containing a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6. In some embodiments, the pharmaceutical composition comprises a single bacterial strain containing a 16S rDNA sequence having at least 97% sequence identity with the nucleic acid sequence provided by SEQ ID NO: 6.

[0148] In addition, or separately, two or more sequences may be evaluated for alignment between them. In the context of two or more nucleic acid or amino acid sequences, the terms “alignment” or “alignment” percentage refer to two or more sequences or subsequences that are identical.

[0149] Two sequences are "substantially aligned" if, when compared and aligned over a specified region, they have a specified percentage of identical amino acid residues or nucleotides (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 or over the entire sequence, as measured by one of the subsequent sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the alignment exists over a region of at least about 50 nucleotides in length, or more preferably over a region of 100 to 500 or 1000 nucleotides or longer. In some embodiments, the identity exists over the length of the 16S rRNA or 16S rDNA sequence.

[0150] In sequence comparison, typically one sequence serves as a reference sequence, against which the test sequence is compared. Methods for aligning sequences for comparison are well known in the art. For example, see the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, the similarity search of Pearson and Lipman. Proc. Natl. Acad. Sci. USA 85:2444, 1988, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group. Madison. WI), or manual alignment and visual inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)). Two examples of suitable algorithms for determining sequence identity percentage and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990.

[0151] It should be understood that the terms “bacteria” and “best strain” as used herein are interchangeable. The compositions described herein, including multiple purified bacterial strains, may also be referred to as “live bacterial products.”

[0152] In some embodiments, the pharmaceutical composition described herein comprises one or more bacterial strains belonging to the Clostridium class. In some embodiments, the pharmaceutical composition described herein comprises one or more bacterial strains belonging to the Clostridium family. In some embodiments, the pharmaceutical composition described herein comprises one or more bacterial strains belonging to the Clostridium genus. In some embodiments, the pharmaceutical composition described herein comprises one or more bacterial strains belonging to Clostridium clusters IV, XIVa, and / or XVII. In some embodiments, the pharmaceutical composition comprises one or more bacterial strains belonging to Clostridium cluster XVII. In some embodiments, the composition described herein comprises one or more bacterial strains belonging to Clostridium clusters IV and / or XIVa.

[0153] In some embodiments, the compositions described herein contain 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%, The composition comprises bacterial strains belonging to Clostridium clusters XIVa, IV, and / or XVII in amounts of 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%. In some embodiments, the composition described herein comprises at least 50% bacterial strains belonging to Clostridium clusters IV and / or XIVa. In some embodiments, the compositions described herein contain at least 75% bacterial strains belonging to Clostridium cluster IV and / or XIVa.

[0154] In some embodiments, the pharmaceutical compositions described herein include one or more of the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavinofractor plautii. In some embodiments, the compositions include one or more of the following bacterial strains (e.g., 2, 3, 4, 5, 6, 7, or 8): Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavinofractor plautii.

[0155] In some embodiments, the pharmaceutical compositions described herein include one or more of the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum species. In some embodiments, the composition comprises one or more of the following bacterial strains (e.g., 2, 3, 4, 5, 6, 7, or 8): Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum species.

[0156] In some embodiments, the pharmaceutical composition comprises the following eight bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavinofractor plautii.

[0157] In some embodiments, the pharmaceutical composition comprises the following eight bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum species.

[0158] In some embodiments, the pharmaceutical composition is "VE303".

[0159] In some embodiments, the pharmaceutical compositions described herein include one or more of the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Clostridium innocuum, and Flavinofractor plautii. In some embodiments, the pharmaceutical compositions described herein include one or more of the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum species. In some embodiments, the composition comprises one or more of the following bacterial strains (e.g., 2, 3, 4, 5, 6, or 7): Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Clostridium innocuum, and Flavinofractor plautii.In some embodiments, the composition comprises one or more of the following bacterial strains (e.g., 2, 3, 4, 5, 6, or 7): Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum species.

[0160] In some embodiments, the compositions described herein include two or more of the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Clostridium innocuum, and Flavinofractor plautii. The compositions do not include Dorea longicatena. In some embodiments, the compositions described herein include two or more of the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum species. The compositions do not include Dorea longicatena.

[0161] In some embodiments, the composition comprises seven bacterial strains. In some embodiments, the composition comprises the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Clostridium innocuum, and Flavinofractor plautii. In some embodiments, the composition comprises the following bacterial strains: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena or Dracourtella massiliensis or Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Clostridium innocuum or Erysipelotrichaceae bacterium, and Flavinofractor plautii or Subdolinogranulum.

[0162] In some embodiments, the pharmaceutical composition is "VE416".

[0163] In some embodiments, the composition comprises one or more bacterial strains (e.g., 2, 3, 4, 5, 6, 7, 8, or more), at least one of which is Dorea longicatena. In some embodiments, the composition consists of Dorea longicatena. In some embodiments, the composition consists of a single bacterial strain that is Dorea longicatena.

[0164] In one embodiment, the 16S rDNA sequence of a purified bacterial strain was compared with the 16S rDNA sequences of known bacterial species / strains in a bacterial genome database to identify the known and related bacterial species most closely related to the bacterial strain disclosed herein. It should be understood that multiple bacterial strains of the compositions disclosed herein may have the same most closely related bacterial species.

[0165] In one embodiment, as shown herein (for example in the Examples), the compositions and methods described herein include the following bacteria: Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum, and Flavinofractor plautii. Exemplary bacterial strains of the compositions disclosed herein may also be identified by their 16S rRNA sequences (SEQ ID NOs: 1-8). Identifying bacteria by these sequences allows for the further identification of additional bacterial strains identical or very similar to the exemplary bacteria. For example, the 16S rRNA sequences of bacterial strains were used to identify the closest related species (based on identity percentage) through whole-genome sequencing and by comparison of these sequences with 16S databases (Table 1). In addition, based on whole-genome sequencing and comparison of whole genomes with whole-genome databases, bacterial strains with 16S rRNA sequences provided by SEQ ID NOs: 1-8 are most closely related to the following bacterial species: Clostridium bolteae 90A9, Anaerotruncus colihominis DSM 17241, Dracourtella massiliensis GD1, Clostridium symbiosum WAL-14163, Clostridium bacterium UC5.1-1D4, Dorea longicatena CAG:42, Erysipelotrichaceae bacterium 21_3, and Clostridium orbiscindens 1_3_50AFAA (see, for example, Table 1). Therefore, in one embodiment, it should be understood that the bacterial strains in each row of Table 1 are very similar and / or identical. In some embodiments, in the context of this disclosure, the names of the bacterial strains in the rows of Table 1 may be used interchangeably.

[0166] Therefore, for example, in some embodiments, the present disclosure provides methods and compositions comprising the following bacteria, which may be referred to as “Composition VE303”: Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Erysipelotrichaceae bacterium, and Subdolinogranulum species. See also PCT Publication WO2017 / 218680.

[0167] In some embodiments, the present disclosure provides methods and compositions comprising the following bacteria: Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Erysipelotrichaceae bacterium, and Subdolinogranulum species.

[0168] The homology based on whole-genome analysis is shown in Table 1. Table 1: Bacterial strains [Table 1]

[0169] In some embodiments, this disclosure provides methods and compositions comprising the following bacterial strains, which may be referred to as “Composition VE202”: Clostridium saccharogumia (Clostridium ramosum JCM 1298), Flavonifractor plautii (Pseudoflavonifractor capillosus ATCC 29799), Clostridium hathewayi (Clostridium saccharolyticum WM1), Blautia coccoides (Lachnospiraceae bacterium 6_1_63FAA), Clostridium species (Clostridium bolteae ATCC BAA-613), cf. Clostridium species MLG055 (Erysipelotrichaceae bacterium 2_2_44A), Clostridium indolis (Anaerostipes caccae DSM 14662), Anaerotruncus colihominis (Anaerotruncus colihominis DSM 17241), Ruminococcus species ID8 (Lachnospiraceae bacterium 2_1_46FAA), Clostridium lavalense (Clostridium asparagiforme DSM 15981), Clostridium symbiosum (Clostridium symbiosum WAL-14163), Clostridium ramosum, Eubacterium contortum (Clostridium species D5), Clostridium scindens (Lachnospiraceae bacterium 5_1_57FAA), Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1), Clostridium species 316002 / 08 (Clostriales bacterium 1_7_47FAA), and Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1).Such bacterial strains are described, for example, in PCT Publication WO2013 / 080561, which is incorporated herein by reference in its entirety. See, for example, Table 4, which also provides OTU and 16S sRNA sequences. Such bacterial compositions are also described, for example, in Atarashi et al. Science (2011) 331(6015):337-341 and Atarashi et al. Nature (2013) 500(7361):232-236. The sequence of the bacterial strain VE202 is also published in PCT Publication WO2013 / 080561. It should be understood that alternative names for the bacterial strains may be used.

[0170] In some embodiments, the present disclosure relates to Clostridium saccharogumia (Clostridium ramosum JCM 1298), Flavonifractor plautii (Pseudoflavonifractor capillosus ATCC 29799), Clostridium hathewayi (Clostridium saccharolyticum WM1), Blautia coccoides (Lachnospiraceae bacterium 6_1_63FAA), Clostridium sp. (Clostridium bolteae ATCC BAA-613), cf. colihominis DSM 17241), Ruminococcus species ID8 (Lachnospiraceae bacterium 2_1_46FAA), Clostridium lavalense (Clostridium asparagiforme DSM 15981), Clostridium symbiosum (Clostridium symbiosum WAL-14163), Clostridium ramosum, Eubacterium contortum (Clostridium species D5), Clostridium scindens (Lachnospiraceae bacterium 5_1_57FAA), Lachnospiraceae bacterium A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1), Clostridium species 316002 / 08 (Clostriales bacterium 1_7_47FAA), and Lachnospiraceae bacterium The present invention provides a composition comprising a purified bacterial mixture containing A4 (Lachnospiraceae bacterium 3_1_57FAA_CT1).

[0171] In one embodiment, this disclosure provides compositions and methods for the treatment of infectious pathogens. In some embodiments, this disclosure provides compositions and methods for the treatment of Clostridium difficile infection. Clostridium difficile has been renamed Clostridioides difficile (e.g., Lawson et al., Anaerobe. 2016 Aug;40:95-9. doi:10.1016 / j.anaerobe.2016.06.008. Epub 2016 Jun 28). Clostridium difficile and Clostridioides difficile are used interchangeably herein. In some embodiments, this disclosure provides compositions and methods for the treatment of primary Clostridium difficile infection. In some embodiments, this disclosure provides compositions and methods for the treatment of recurrent Clostridium difficile infection. Any of the pharmaceutical compositions described herein may contain one or more Clostridium difficile inhibitory strains. As used herein, “Clostridium difficile inhibitory strain” or “a strain capable of inhibiting C. difficile” refers to a bacterial strain that inhibits (directly or indirectly) the presence and / or amount of C. difficile in the target microbiome. In some embodiments, the Clostridium difficile inhibitory strain directly inhibits the presence and / or amount of C. difficile in the target microbiome. In some embodiments, the Clostridium difficile inhibitory strain indirectly inhibits the presence and / or amount of C. difficile in the target microbiome. The C. difficile inhibitory strain may inhibit the presence and / or amount of C. difficile in the target microbiome by any of a variety of mechanisms. For example, the C. difficile inhibitory strain may kill C. difficile, inhibit the growth / replication of C. difficile, and / or prevent colonization by C. difficile.Examples of Clostridium difficile inhibitory strains, and compositions containing such strains, are provided, for example, in PCT Publication WO2017 / 218680.

[0172] In some embodiments, the presence and / or amount of C. difficile in the target microbiome is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the presence of one or more C. difficile inhibitors compared to the presence and / or amount of C. difficile in the target microbiome in the absence of one or more C. difficile inhibitors.

[0173] In one embodiment, the present disclosure provides compositions and methods for the treatment or suppression of food allergies.

[0174] In some embodiments, one or more bacterial strains are of human origin, meaning that one or more bacterial strains were obtained from or identified from a human or human-derived sample (e.g., a human donor). In some embodiments, all bacterial strains are of human origin. In some embodiments, the bacterial strains are derived from two or more human donors.

[0175] The bacterial strains used in the pharmaceutical compositions provided herein are generally isolated from the microbiome of healthy individuals. In some embodiments, the pharmaceutical compositions include strains originating from a single individual. In some embodiments, the pharmaceutical compositions include strains originating from multiple individuals. In some embodiments, the pharmaceutical compositions are obtained from multiple individuals, isolated, and grown individually. The individually grown bacterial compositions may then be combined to provide the pharmaceutical compositions of this disclosure. It should be understood that the origin of the bacterial strains of the pharmaceutical compositions provided herein is not limited to the human microbiome from healthy individuals. In some embodiments, the bacterial strains originate from humans with an enterotoxemia microbiome. In some embodiments, the bacterial strains originate from non-human animals or the environment (e.g., soil or surface water). In some embodiments, the combination of bacterial strains provided herein originates from multiple sources (e.g., humans and non-human animals).

[0176] In some embodiments, the pharmaceutical composition contains one or more anaerobic bacteria. In some embodiments, the pharmaceutical composition contains only anaerobic bacteria. In some embodiments, the pharmaceutical composition contains one or more facultative anaerobic bacteria. In some embodiments, the pharmaceutical composition contains only facultative anaerobic bacteria. In some embodiments, the pharmaceutical composition contains one or more obligate anaerobic bacteria. In some embodiments, the pharmaceutical composition contains only obligate anaerobic bacteria.

[0177] In some embodiments, at least one of the bacterial strains in the pharmaceutical composition (e.g., one, two, three, four, five, six, seven, eight, or more) is a spore-forming bacterium. In some embodiments, at least one of the bacterial strains in the pharmaceutical composition (e.g., one, two, three, four, five, six, seven, eight, or more) is spore-forming. In some embodiments, at least one of the bacterial strains in the pharmaceutical composition (e.g., one, two, three, four, five, six, seven, eight, or more) is non-spore-forming. In some embodiments, at least one of the bacterial strains in the pharmaceutical composition (e.g., one, two, three, four, five, six, seven, eight, or more) is vegetative. As discussed above, spore-forming bacteria can also be vegetative. In some embodiments, at least one of the bacterial strains in the pharmaceutical composition (e.g., one, two, three, four, five, six, seven, eight, or more) is spore-forming, and at least one of the bacterial strains in the pharmaceutical composition (e.g., one, two, three, four, five, six, seven, eight, or more) is vegetative. In some embodiments, at least one bacterial strain is considered capable of spore formation (i.e., a spore-forming bacterium) but is present in the composition in vegetative form. In some embodiments, at least one bacterial strain considered capable of spore formation is present in the pharmaceutical composition in both spore-forming and vegetative forms. In some embodiments, each of the bacterial strains is vegetative.

[0178] The bacterial strains of the pharmaceutical compositions provided herein are assumed to be alive and to be alive when they reach the target area (e.g., the intestines). In this regard, bacterial spores are considered alive. In some embodiments, bacteria administered as spores may grow in the target area (e.g., the intestines). It should be further understood that not all bacteria are alive, and the compositions may contain a non-living percentage (e.g., by weight). In addition, in some embodiments, the compositions contain bacterial strains that are not alive when administered or when the compositions reach the target area (e.g., the intestines). Non-living bacteria are assumed to still be useful by providing some nutrients and metabolites to other bacterial strains in the composition.

[0179] In some embodiments of any of the live bacterial products provided herein, the bacterial strains are purified. In some embodiments of any of the live bacterial products provided herein, the bacterial strains are isolated. Any of the bacterial strains described herein may be isolated and / or purified from a source such as a culture or microbiome sample (e.g., fecal matter). The bacterial strains used in the compositions provided herein are generally isolated from the microbiome of healthy individuals. However, bacterial strains may also be isolated from individuals considered unhealthy. In some embodiments, the compositions include strains originating from multiple individuals. As used herein, the term “isolated” in relation to bacteria refers to bacteria isolated from one or more undesirable components, such as another bacterium or bacterial strain, one or more components of a growth medium, and / or one or more components of a sample such as a fecal sample. In some embodiments, bacteria are substantially isolated from the source such that other components of the source are not detected (e.g., below detection level). Similarly, as used herein, the term “purified” refers to a bacterial strain or composition containing such a product isolated from one or more components, such as contaminants. In some embodiments, the bacterial strains are substantially free of contaminants. In some embodiments, one or more bacterial strains of the composition may be purified independently from one or more other bacteria produced and / or present in the culture or sample containing the bacterial strain. In some embodiments, the bacterial strain is isolated or purified from the sample and then cultured under conditions suitable for bacterial replication, for example, under anaerobic conditions. Bacteria grown under conditions suitable for bacterial replication can then be isolated / purified from the culture grown therein.

[0180] In some embodiments, specific combinations of one or more bacterial strains of the compositions described herein result in synergistic effects that promote the reduction of enterotoxemia, restoration of the microbiome, recovery of a healthy microbiome after an enterotoxemia-induced event, protection of the microbiome, and / or colonization of the microbiome of interest. In some embodiments, the synergistic effect is brought about by the ability of this combination to metabolize specific nutrients. In some embodiments, the synergistic effect is brought about by the ability of this combination to provide specific metabolites to the environment. Such specific metabolites may inhibit the growth of pathogens and / or stimulate the growth of non-pathogens. In some embodiments, the synergistic effect is brought about by the ability of this combination to provide short-chain fatty acids to the environment. In some embodiments, the synergistic effect is brought about by the ability of this combination to provide specific short-chain fatty acids to the environment. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce butyrate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce acetate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce lactate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce propionate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce succinate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce multiple metabolites. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce multiple short-chain fatty acids. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce both butyrate and acetate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce both butyrate and lactate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce both butyrate and propionate. In some embodiments, the synergistic effect is brought about by the ability of this combination to produce both butyrate and succinate.In some embodiments, the synergistic effect is brought about by the ability of this combination to produce butyrate, acetate, and additional short-chain fatty acids.

[0181] In some embodiments, specific combinations of one or more bacterial strains of the compositions provided herein are superior to other strains (e.g., pathogens) in nutrient utilization and transplantation, thereby protecting and / or restoring the microbiome, for example, by inhibiting the growth of pathogens. In some embodiments, specific combinations of one or more bacterial strains of the compositions provided herein induce an immune response in a subject that promotes reduction of enterotoxosis, restoration of the microbiome, recovery of a healthy microbiome after an enterotoxosis-induced event, protection of the microbiome, and / or colonization of one or more of the bacterial strains of the pharmaceutical composition into the subject's microbiome.

[0182] Compositions, such as pharmaceutical compositions, for administration to a target are also within the scope of this disclosure. In some embodiments, the composition comprises one of the bacterial strains described herein.

[0183] In one embodiment, the present disclosure provides a pharmaceutical composition comprising one of the bacterial strains described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for rectal administration. In some embodiments, the pharmaceutical composition is formulated for delivery to the intestines. In some embodiments, the pharmaceutical composition is formulated for delivery to the colon.

[0184] In some embodiments, the pharmaceutical composition described herein comprises one or more bacterial strains. In some embodiments, the pharmaceutical composition may be lyophilized. In some embodiments, the pharmaceutical composition is in the form of a capsule. In some embodiments, the pharmaceutical composition further comprises a pH-sensitive composition comprising one or more enteric-coated polymers.

[0185] In some embodiments, one or more bacterial strains of the pharmaceutical composition are spray-dried. The spray-drying process refers to creating a dry powder from a liquid containing the bacterial composition. (See, for example, Ledet et al., Spray-Drying of Pharmaceuticals in "Lyophilized Biologics and Vaccines," pp. 273-194, Springer). Generally, this process involves rapidly drying the bacterial composition with a high-temperature gas.

[0186] Any of the compositions described herein, including pharmaceutical compositions and food products, wherein the bacterial strain is in any form, e.g., an aqueous form such as a solution or suspension, embedded in a semi-solid form, in powder form, or in lyophilized form. In some embodiments, the composition or bacterial strain is lyophilized. In some embodiments, a subset of the bacterial strain is lyophilized. Methods for lyophilizing compositions, particularly compositions containing bacteria, are well known in the art. See, for example, U.S. Patent Nos. 3,261,761, 4,205,132, PCT Publications WO2014 / 029578 and WO2012 / 098358, which are incorporated herein by whole reference. The bacteria may be lyophilized as a combination, and / or the bacteria may be lyophilized separately and combined before administration. A bacterial strain may be combined with a pharmaceutical excipient before being combined with another bacterial strain, or multiple lyophilized bacteria may be combined while they are in their lyophilized form, and this bacterial mixture may then be combined with a pharmaceutical excipient at the time of combination. In some embodiments, the bacterial strain is a lyophilized cake. In some embodiments, the composition containing one or more bacterial strains is a lyophilized cake.

[0187] In some embodiments, one or more bacterial strains of the composition, including pharmaceutical compositions and food products, are spray-dried. In some embodiments, a subset of bacterial strains is spray-dried. The spray-drying process refers to creating a dry powder from a liquid containing a bacterial composition (see, for example, Ledet, et al., Spray Draying of Pharmaceuticals in "Lyophilized Biologics and Vaccines," pp. 273-294, Springer). Generally, this process involves rapidly drying the bacterial composition with a hot gas. Bacterial strains may be combined with pharmaceutical excipients before being combined with other bacterial strains, or multiple spray-dried bacteria may be combined while in the spray-dried form, and this mixture of bacteria may then be combined with pharmaceutical excipients at the time of combination.

[0188] Bacterial strains can be produced using fermentation techniques well known in the art. In some embodiments, bacteria are grown or produced using anaerobic fermenters, which can support the rapid growth of anaerobic bacterial species. The anaerobic fermenter may be, for example, a stirred-tank reactor or a disposable wave bioreactor. Culture media such as BL medium and EG medium, or similar media without animal components, can be used to support the growth of bacterial species. Bacterial products can be purified and concentrated from the fermentation broth by conventional techniques such as centrifugation and filtration, and optionally dried and freeze-dried by techniques well known in the art.

[0189] In some embodiments, the live bacterial product may be formulated for administration as a pharmaceutical composition. As used herein, the term “pharmaceutical composition” means a product resulting from a mixture or combination of at least one active ingredient, such as one of the bacterial strains described herein, and one or more inactive ingredients, which may include one or more pharmaceutically acceptable excipients.

[0190] An “acceptable” excipient is an excipient that must be compatible with the active ingredient and must not be harmful to the recipient. In some embodiments, pharmaceutically acceptable excipients are selected based on the intended route of administration of the composition; for example, a composition for oral or nasal administration may contain different pharmaceutically acceptable excipients than a composition for rectal administration. Examples of excipients include sterile water, saline, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, fragrances, aromatics, excipients, vehicles, preservatives, binders, diluents, isotonic modifiers, mitigating agents, bulking agents, disintegrants, buffers, coatings, lubricants, colorants, sweeteners, thickeners, and solubilizers.

[0191] The pharmaceutical compositions of the present invention can be prepared according to methods that are well known and routinely practiced in the art (see, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed. 2000). The pharmaceutical compositions described herein may further contain any carrier or stabilizer 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 agents, and / or surfactants. The pharmaceutical compositions are preferably manufactured under GMP conditions. This pharmaceutical composition can be used orally, nasally, or parenterally in the form of, for example, capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated formulations, pellets, lozenges, sublingual formulations, chewables, oral formulations, pastes, syrups, suspensions, elixirs, emulsions, topical preparations, ointments, plasters, poultices, transdermal absorption systems, lotions, inhalants, aerosols, injections, suppositories, etc. In some embodiments, this pharmaceutical composition can be used by injection, such as intravenous, intramuscular, subcutaneous, or intradermal administration.

[0192] In some embodiments, the composition containing the bacterial strain is formulated for oral delivery. In some embodiments, the composition containing the bacterial strain is formulated for delivery to the intestines (e.g., the small intestine and / or colon). In some embodiments, the composition containing the bacterial strain may be formulated using an enteric coating to increase the survival of bacteria in the harsh environment of the stomach. The enteric coating is a coating that resists the action of gastric juice in the stomach, allowing the bacteria of the composition within it to pass through the stomach and enter the intestines. The enteric coating may readily dissolve upon contact with intestinal fluid, so that the bacteria encapsulated in the coating are released into the intestinal tract. The enteric coating may consist of commercially available polymers and copolymers well known in the art, such as EUDRAGIT (Evonik Industries). (See, for example, Zhang, AAPS PharmSciTech, 2016, 17(1), 56-67).

[0193] The composition containing the bacterial strain may also be formulated for rectal delivery to the intestines (e.g., the colon). Therefore, in some embodiments, the composition containing the bacterial strain may be formulated for delivery by suppositories, colonoscopy, endoscopy, sigmoidoscopy, or enema. Pharmaceutical preparations or formulations, and in particular pharmaceutical preparations for oral administration, may contain additional components that enable efficient delivery of the composition of this disclosure to the intestines (e.g., the colon). A variety of pharmaceutical preparations can be used to allow delivery of the composition to the intestines (e.g., the colon). Examples include pH-sensitive compositions, more specifically, buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after passing through the stomach. When a pH-sensitive composition is used in the formulation of a pharmaceutical preparation, the pH-sensitive composition is preferably a polymer whose pH threshold for decomposition of the composition is about 6.8 to about 7.5. Such a numerical range is the range in which the pH shifts to the alkaline side in the distal part of the stomach, and is therefore suitable for use in delivery to the colon. Furthermore, it should be understood that different parts of the intestine (e.g., the duodenum, jejunum, ileum, cecum, colon, and rectum) have different biochemical and chemical environments. For example, parts of the intestine have different pH levels, which allows for targeted delivery by compositions having specific pH sensitivity. Therefore, 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 appropriate pH sensitivity. (See, for example, Villena et al., Int J Pharm 2015, 487(1-2):314-9).

[0194] Pharmaceutical compositions for administration by additional or alternative routes are also within the scope of this disclosure. In some embodiments, the pharmaceutical compositions are formulated for sublingual administration. In some embodiments, the pharmaceutical compositions are formulated for administration by injection.

[0195] In some embodiments, the pharmaceutical composition may include additional components that enable efficient delivery of the composition of the disclosure to a desired site, such as the gastrointestinal tract (e.g., the colon).

[0196] In some embodiments, the pharmaceutical composition includes an adjuvant associated with providing benefits in the treatment of allergies. In some embodiments, the pharmaceutical composition includes one or more components of an oral immunotherapy agent, a transdermal immunotherapy agent, or a sublingual immunotherapy agent.

[0197] Another embodiment of a pharmaceutical preparation useful for the delivery of a composition to the intestines (e.g., the colon) ensures delivery to the colon by delaying the release of the contents (e.g., bacterial strain) by approximately 3 to 5 hours, corresponding 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 hydrates and swells upon contact with gastrointestinal fluid, resulting in the effective release of the contents (primarily in the colon). Delayed-release dosage forms include drug-containing compositions having a material that coats or selectively coats the drug or active ingredient to be administered. Examples of such selective coating materials include in vivo degradable polymers, gradual hydrolyzable polymers, gradual water-soluble polymers, and / or enzymatically degradable polymers. A wide variety of coating materials are available for efficiently delaying release, and these include, for example, cellulosic polymers such as hydroxypropylcellulose, acrylic polymers and copolymers such as methacrylic polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

[0198] Additional examples of pharmaceutical compositions that allow delivery to the intestines (e.g., the colon) include bioadhesive compositions that adhere specifically to the colonic mucosa (e.g., polymers described in U.S. Patent No. 6,368,586), and compositions that incorporate protease inhibitors to protect biopharmaceutical preparations in particular within the gastrointestinal tract from degradation due to protease activity.

[0199] Another example of a system that enables delivery to the intestines (e.g., the colon) is a system that delivers a composition to the colon by a pressure change that releases the contents by utilizing the pressure change caused by gas production during bacterial fermentation in the distal part of the stomach. Such systems are not particularly limited, and a more specific example is a capsule in which the contents are dispersed in a suppository base and coated with a hydrophobic polymer (e.g., ethylcellulose).

[0200] Further examples of systems that enable the delivery of compositions to the intestines (e.g., the colon) include compositions comprising a coating that can be removed by enzymes present in the gastrointestinal tract (e.g., the colon), such as carbohydrate hydrolases or carbohydrate reductases. Such systems are not particularly limited, and more specific examples include systems using food ingredients such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

[0201] The compositions provided herein can also be delivered to specific target areas, such as the intestines, by delivery via an opening (e.g., a nasal tube) or by surgery. In addition, compositions provided herein, formulated for delivery to specific areas (e.g., the cecum or colon), may be administered by tube (e.g., directly into the small intestine). Combining mechanical delivery methods, such as tubes, with chemical delivery methods, such as pH-specific coatings, makes it possible to deliver the compositions provided herein to desired target areas (e.g., the cecum or colon).

[0202] A composition containing bacteria is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosing regimen is adjusted to provide the optimal desired response (e.g., prophylactic or therapeutic effect). In some embodiments, the dosage form of the composition is a tablet, pill, capsule, powder, granule, solution, or suppository. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition contains a bacterial strain and is formulated so that the bacteria or a portion of them remain viable after passing through the stomach of the subject. In some embodiments, the pharmaceutical composition is formulated for rectal administration, for example, as a suppository. In some embodiments, the pharmaceutical composition is formulated for delivery to the intestine or a specific region of the intestine (e.g., the colon) by providing a suitable coating (e.g., a pH-specific coating, a coating that can be degraded by a target-region-specific enzyme, or a coating that can bind to receptors present in the target region).

[0203] The dosage of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective in achieving a desired pharmaceutical response for a particular subject, composition, and mode of administration without being toxic or having adverse effects on the subject. The selected dosage level is based on a variety of factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and prior medical history of the subject being treated, and similar factors.

[0204] A physician, veterinarian, or other trained practitioner may begin administering the pharmaceutical composition at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect (e.g., treatment of Clostridium difficile infection, treatment of allergies, or modulation of one or more immune responses associated with allergies) is achieved. In general, the effective dose of the composition of the present invention for the prophylactic treatment of groups of people such as those described herein varies depending on many different factors, including the route of administration, the physiological state of the subject, whether the subject is human or animal, other drugs administered, and the desired therapeutic effect. To optimize safety and efficacy, the dose may need to be titrated. In some embodiments, the administration regimen requires oral administration of any dose of one of the compositions described herein. In some embodiments, the administration regimen requires oral administration of multiple doses of one of the compositions described herein. In some embodiments, any of the compositions described herein is administered to a subject once, twice, three times, four times, five times, five times, six times, seven times, eight times, nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, or more. In some embodiments, any of the compositions described herein is administered to a subject in multiple doses at regular intervals, such as daily, every two days, every three days, every four days, every five days, every six days, weekly, every two weeks, monthly, every two months, every three months, every four months, every five months, every six 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., on consecutive days). In some embodiments, one dose of any of the compositions described herein is administered, and each of the additional doses of the composition is administered on consecutive days (e.g., the first dose on day one, the second dose on day two, the third dose on day three, etc.).

[0205] In one embodiment, the Disclosure provides a method comprising administering multiple doses of the pharmaceutical composition. In some embodiments, the Disclosure provides a method comprising administering an antibiotic (e.g., vancomycin) followed by multiple doses of the pharmaceutical composition. In some embodiments, multiple doses of the pharmaceutical composition described herein enhance the colonization (engraftment) of one or more bacterial strains of the pharmaceutical composition compared to a single dose of the pharmaceutical composition. In some embodiments, multiple doses of the pharmaceutical composition described herein enhance the recovery of one or more bacterial strains of the pharmaceutical composition compared to a single dose of the pharmaceutical composition. In some embodiments, multiple doses of the pharmaceutical composition increase the abundance of one or more bacterial strains of the pharmaceutical composition compared to a single dose of the pharmaceutical composition. In some embodiments, multiple doses of the pharmaceutical composition described herein increase the number of subjects to which all of the bacterial strains of the pharmaceutical composition colonize compared to a single dose of the pharmaceutical composition. In some embodiments, administration of multiple doses of the pharmaceutical composition described herein results in the establishment of one or more bacterial strains of the pharmaceutical composition (e.g., up to 6 months) compared to administration of a single dose of the pharmaceutical composition. In some embodiments, administration of multiple doses of the pharmaceutical composition described herein results in the establishment of all bacterial strains of the pharmaceutical composition (e.g., 6 months) compared to administration of a single dose of the pharmaceutical composition. Furthermore, it should be understood that combinations of the results described may occur with multiple doses. For example, in some embodiments, administration of multiple doses of the pharmaceutical composition described herein results in increased establishment (engraftment) and increased recovery rate of one or more bacterial strains of the pharmaceutical composition compared to administration of a single dose of the pharmaceutical composition.

[0206] In some embodiments, administration of multiple doses of the pharmaceutical composition described herein enhances the colonization (engraftment) of one or more bacterial strains of the pharmaceutical composition compared to administration of a single dose of the pharmaceutical composition. As shown in Figures 6 and 7, administration of multiple doses of the pharmaceutical composition enhances the colonization (engraftment) of each bacterial strain of the pharmaceutical composition and increases their abundance. In some embodiments, administration of a single dose of the pharmaceutical composition results in the same or similar level of engraftment (e.g., total bacteria) as administration of multiple doses of the pharmaceutical composition, but the engraftment may be occupied by only one bacterial strain or a subset of bacterial strains of the pharmaceutical composition.

[0207] Any of the methods described herein may further include administering an antibiotic to the subject before administering the pharmaceutical composition described herein. In some embodiments, the antibiotic is vancomycin, fidaxomicin, or ridinirazole. Non-limiting examples of antibiotics that may be used in any of the methods provided herein include the cephalosporin antibiotics cefaloxine, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cephamandol, cefoxitin, cefprodil, ceftoviprole, clindamycin, ceftriaxone, cefotaxime, cefazolin, cefoperazone, cefuroxime, cefmetazole, fluoroquinolone, ciprofloxacin, Levaquin, phloxine, tequin, avelox, norflox, tetracy This includes cinnamide, minocycline, oxytetracycline, doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, benzylpenicillin, carbenicillin, vancomycin, and methicillin), ertapenem, doripenem, imipenem / cilastatin, meropenem, clavulanate, tazobactam, piperacillin, ceftriaxone, cefotaxime, cefazolin, fluoroquinolone, imipenem, meropenem, metronidazole, fidaxomyxin, or ridinirazole. In some embodiments, any of the methods described herein may further include administering vancomycin to the subject before administering the pharmaceutical composition described herein. In some embodiments, the method does not include administering vancomycin to the subject before administering the pharmaceutical composition described herein. Vancomycin administration is known to alter the composition of the human gut microbiota. See, for example, Reijnders et al. Cell Metabolism (2016) 24(1):63-72. While we do not wish to be bound by any particular theory, it is thought that vancomycin administration may help the colonization of the bacterial strain(s) of the pharmaceutical composition described herein by eliminating other microorganisms present in the gastrointestinal tract, for example.

[0208] In some embodiments, vancomycin is administered to the subject as a single dose. In some embodiments, vancomycin is administered to the subject in multiple doses. In some embodiments, vancomycin is administered to the subject in at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more doses. The multiple doses of vancomycin may be administered to the subject at regular intervals before administering any of the pharmaceutical compositions described herein. In some embodiments, each of the multiple doses of vancomycin is administered on consecutive days (e.g., the first dose on day one, the second dose on day two, the third dose on day three, etc.). In some embodiments, vancomycin is administered to the subject for two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more consecutive days. In some embodiments, vancomycin is administered to the subject daily for three consecutive days. In some embodiments, vancomycin is administered to the subject daily for five consecutive days. In some embodiments, vancomycin is administered to the subject daily for one day. In some embodiments, vancomycin is administered to the subject daily for seven consecutive days. In any of the embodiments described herein, the subject may be administered one or more doses of the first antibiotic, followed by one or more doses of the second antibiotic.

[0209] In some embodiments, the first dose in a single-dose or multi-dose treatment regimen is administered on the same day as the final dose of vancomycin. In some embodiments, the first dose in a single-dose or multi-dose treatment regimen is administered the day following the final dose of vancomycin. In some embodiments, the first dose in a single-dose or multi-dose treatment regimen is administered two days after the final dose of vancomycin. In some embodiments, the method provided herein anticipates a drug-free day between the final dose of vancomycin and the first dose of the pharmaceutical composition. In some embodiments, the first dose in a single-dose or multi-dose treatment regimen is administered three, four, five, six, ten, or more days after the final dose of vancomycin. In some embodiments, the method provided herein anticipates multiple drug-free days between the final dose of vancomycin and the first dose of the pharmaceutical composition.

[0210] Each dose of vancomycin may be the same amount of vancomycin or different amounts of vancomycin. In some embodiments, vancomycin is administered in an amount sufficient to allow colonization of one or more bacterial strains of the pharmaceutical composition described herein. In some embodiments, the subject is administered about 50 mg to 1 g, 100 mg to 750 mg, 100 mg to 500 mg, 200 mg to 750 mg, 200 mg to 500 mg, 300 mg to 750 mg, 300 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, 100 mg to 200 mg, 200 mg to 400 mg, 200 mg to 300 mg, or 450 mg to 550 mg of vancomycin per day. As will be understood by those skilled in the art, the total amount of vancomycin administered to the subject per day may be administered as a single dose or as multiple doses totaling the total amount of vancomycin per day.

[0211] In some embodiments, the subject is administered approximately 500 mg of vancomycin per day prior to administration of any of the pharmaceutical compositions described herein. In some embodiments, 500 mg of vancomycin per day is administered as a single dose (e.g., 500 mg). In some embodiments, 500 mg of vancomycin per day is administered as multiple doses (e.g., two, three, four, five, or more times) totaling 500 mg of vancomycin per day. In some embodiments, 500 mg of vancomycin is administered as four doses of 125 mg of vancomycin per day. In some embodiments, 500 mg of vancomycin is administered to the subject for one day. In some embodiments, 500 mg of vancomycin is administered to the subject daily for two days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for three days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for four days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for 5 days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for 6 days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for 7 days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for 8 days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for 9 days. In some embodiments, 500 mg of vancomycin is administered to the subject daily for 10 days.

[0212] In some embodiments, the subject is administered approximately 250 mg of vancomycin per day prior to administration of any of the pharmaceutical compositions described herein. In some embodiments, 250 mg of vancomycin per day is administered as a single dose (e.g., 250 mg). In some embodiments, 250 mg of vancomycin per day is administered as multiple doses (e.g., two, three, four, five, or more times) totaling 250 mg of vancomycin per day. In some embodiments, 250 mg of vancomycin is administered as two doses of 125 mg of vancomycin per day. In some embodiments, 250 mg of vancomycin is administered to the subject for one day. In some embodiments, 250 mg of vancomycin is administered to the subject daily for two days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for three days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for four days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for 5 days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for 6 days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for 7 days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for 8 days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for 9 days. In some embodiments, 250 mg of vancomycin is administered to the subject daily for 10 days.

[0213] In some embodiments, the subject is administered approximately 125 mg of vancomycin per day prior to administration of any of the pharmaceutical compositions described herein. In some embodiments, this 125 mg of vancomycin per day is administered as a single dose (e.g., 125 mg). In some embodiments, this 125 mg of vancomycin per day is administered as multiple doses (e.g., two, three, four, five, or more times) totaling 125 mg of vancomycin per day. In some embodiments, 125 mg of vancomycin is administered to the subject for one day. In some embodiments, 125 mg of vancomycin is administered to the subject daily for two days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for three days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for four days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for five days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for six days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for 7 days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for 8 days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for 9 days. In some embodiments, 125 mg of vancomycin is administered to the subject daily for 10 days.

[0214] In some embodiments, vancomycin is administered according to a tapering pulse regime. See, for example, Sirbu et al., Clinical Infectious Diseases (2017) 65:1396-1399.

[0215] In some embodiments, vancomycin is administered to the subject one, two, three, four, five, six, seven, or more days before administration of any of the pharmaceutical compositions described herein. In some embodiments, the administration of vancomycin is completed at least one day (e.g., one, two, three, four, five, or more) before administration of any of the pharmaceutical compositions described herein.

[0216] In some embodiments, additional antibiotics are administered in combination with the vancomycin regime provided herein.

[0217] It should be understood that in some embodiments, either the vancomycin dose or administration regimen may be combined with any of the pharmaceutical composition doses or administration regimens provided herein.

[0218] In some embodiments, the Disclosure provides a method comprising administering one or more antibiotics to a subject, followed by administering one, two, three, four, five, six, seven, eight, nine, or at least ten times to the subject. In some embodiments, the Disclosure provides a method comprising administering one or more antibiotics to a subject, followed by administering one of the bacterial compositions described herein in multiple doses at regular intervals, such as every two weeks, monthly, every two months, every three months, every four months, every five months, every six months, or more. In some embodiments, a single dose of one of the compositions described herein is administered, followed by a second dose of the composition on the following day (e.g., on consecutive days). In some embodiments, one dose of one of the compositions described herein is administered, followed by each of the additional doses of the composition on consecutive days (e.g., the first dose on day 1, the second dose on day 2, the third dose on day 3, etc.).

[0219] In one embodiment, the Disclosure provides a method comprising administering one or more antibiotics as a target, followed by administering one of the bacterial compositions as a daily dose of the Pharmaceutical Composition. In some embodiments, the Pharmaceutical Composition is administered daily for 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 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 longer.

[0220] In one embodiment, the Disclosure provides a method comprising administering an antibiotic (e.g., vancomycin) followed by the administration of a pharmaceutical composition provided herein, wherein a single or multiple dose of the pharmaceutical composition is administered after the administration of the antibiotic (e.g., vancomycin). In some embodiments, administering an antibiotic (e.g., vancomycin) followed by a single or multiple dose of the pharmaceutical composition increases the abundance (engraftment) of the bacterial strain of the pharmaceutical composition in the target microbiota compared to administering the pharmaceutical composition without administering the antibiotic. In some embodiments, administering an antibiotic (e.g., vancomycin) followed by a single or multiple dose of the pharmaceutical composition increases the duration of establishment of the bacterial strain of the pharmaceutical composition in the target microbiota compared to administering the pharmaceutical composition without administering the antibiotic (e.g., up to 6 months).

[0221] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by a single or multiple dose of the pharmaceutical composition increases the initial colonization rate of the bacterial strains of the pharmaceutical composition in the target microbiome by 10 to 100 times (e.g., within the first 48 hours) compared to administering the pharmaceutical composition without antibiotic administration.

[0222] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by a single or multiple dose of the pharmaceutical composition results in a greater number (quantity) of all bacterial strains of the pharmaceutical composition present in the microbiome compared to administering the pharmaceutical composition without antibiotics.

[0223] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by multiple doses of the pharmaceutical composition increases the abundance (engraftment) of the bacterial strain of the pharmaceutical composition in the target microbiota compared to administering a single dose of the pharmaceutical composition. In some embodiments, the disclosure provides a method comprising administering the pharmaceutical composition provided herein, wherein administering multiple doses of the pharmaceutical composition increases the abundance (engraftment) of the bacterial strain of the pharmaceutical composition in the target microbiota compared to administering a single dose of the pharmaceutical composition.

[0224] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by multiple doses of the pharmaceutical composition increases the initial engraftment rate of the bacterial strain of the pharmaceutical composition in the target microbiota compared to administering a single dose of the pharmaceutical composition. In some embodiments, the present disclosure provides a method comprising administering the pharmaceutical composition provided herein, wherein administering multiple doses of the pharmaceutical composition increases the initial engraftment rate of the bacterial strain of the pharmaceutical composition in the target microbiota compared to administering a single dose of the pharmaceutical composition.

[0225] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by multiple doses of the pharmaceutical composition results in a greater abundance of the bacterial strain of the pharmaceutical composition in the target microbiome compared to administering a single dose of the pharmaceutical composition. In some embodiments, the disclosure provides a method comprising administering the pharmaceutical composition provided herein, wherein administering multiple doses of the pharmaceutical composition results in a greater abundance of the bacterial strain of the pharmaceutical composition in the target microbiome compared to administering a single dose of the pharmaceutical composition.

[0226] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by multiple doses of the pharmaceutical composition results in a greater number (amount) of subjects in the microbiome containing all of the bacterial strains of the pharmaceutical composition compared to administering a single dose of the pharmaceutical composition. In some embodiments, the disclosure provides a method comprising administering the pharmaceutical composition provided herein, where administering multiple doses of the pharmaceutical composition results in a greater number (amount) of subjects in the microbiome containing all of the bacterial strains of the pharmaceutical composition compared to administering a single dose of the pharmaceutical composition.

[0227] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by multiple doses of the pharmaceutical composition accelerates the recovery of the microbiome compared to administering a single dose of the pharmaceutical composition (e.g., an increase in Bacteroidetes and / or Firmicutes bacterial species and / or a decrease in Proteobacteria). In some embodiments, the disclosure provides a method comprising administering the pharmaceutical composition provided herein, wherein administering multiple doses of the pharmaceutical composition accelerates the recovery of the microbiome compared to administering a single dose of the pharmaceutical composition (e.g., an increase in Bacteroidetes and / or Firmicutes bacterial species and / or a decrease in Proteobacteria).

[0228] In some embodiments, administering an antibiotic (e.g., vancomycin) followed by a single or multiple dose of the pharmaceutical composition accelerates the recovery of the microbiome (e.g., an increase in Bacteroidetes and / or Firmicutes bacterial species and / or a decrease in Proteobacteria) compared to administering the antibiotic without the pharmaceutical composition.

[0229] Compositions comprising the pharmaceutical compositions disclosed herein include compositions comprising a selected bacterial strain. The amount of bacteria, including the amount of each bacterium of the bacterial strain in the composition comprising the pharmaceutical composition, can be expressed in terms of weight, number of bacteria, and / or CFU (colony forming units). In some embodiments, the composition comprising the pharmaceutical composition comprises, per dosage, about 10, about 10 2 about 10 3 about 10 4 about 10 5 about 10 6 about 10 7 about 10 8 about 10 9 about 10 10 about 10 11 about 10 12 about 10 13 or more of each of the bacterial strains. In some embodiments, the composition comprising the pharmaceutical composition comprises, per dosage, about 10, about 10 2 about 10 3 about 10 4 about 10 5 about 10 6 about 10 7 about 10 8 about 10 9 about 10 10 about 10 11 about 10 12 about 10 13 or more total bacteria. It should be further understood that each bacterium of the bacterial strain may be present in a different amount. Thus, for example, by way of non-limiting example, the composition may comprise 10 3 of bacterium A, 10 4 of bacterium B, and 10 6 of bacterium C. In some embodiments, the composition comprising the pharmaceutical composition comprises, per dosage, about 10, about 10 2 about 10 3 about 10 4 about 10 5 about 10 6 about 10 7 about 10<​​​​​​​​​​and each of the bacterial strains of one or more CFUs. In some embodiments, the composition comprising the pharmaceutical composition comprises, per dosage, for all of the combined bacterial strains, in total, about 10 1 about 10 2 about 10 3 about 10 4 about 10 5 about 10 6 about 10 7 about 10 8 about 10 9 about 10 10 about 10 11 about 10 12 about 10 13 or more CFUs. As discussed above, each bacterium of the bacterial strain may be present in a different amount. In some embodiments, the composition comprising the pharmaceutical composition comprises each bacterium of the bacterial strain in the composition, per dosage, about 10 -7 about 10 -6 about 10 -5 about 10 -4 about 10 -3 about 10 -2 about 10 -1 or more grams. In some embodiments, the composition comprising the pharmaceutical composition comprises, per dosage, for all of the combined bacterial strains, in total, 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.

[0230] In some embodiments, the dosage is one means of administration (e.g., tablet, pill, or capsule). In some embodiments, the dosage is the amount administered at one time, which may be in the form of two or more means of administration (e.g., multiple tablets, pills, or capsules). In some embodiments, the dosage is the amount administered over a particular period (e.g., one day or one week).

[0231] As described herein, any of the pharmaceutical compositions described herein may be administered as a single dose. In some embodiments, the pharmaceutical 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 the administration of multiple capsules. In some embodiments, each dose is administered in the form of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more capsules.

[0232] In some embodiments, each capsule contains 10 to 10 capsules. 13 , 10 2 ~10 13 , 10 3 ~10 13 , 10 4 ~10 13 , 10 5 ~10 13 , 10 6 ~10 13 , 10 7 ~10 13 , 10 8 ~10 13 , 10 9 ~10 13 , 10 10 ~10 13 , 10 11 ~10 13 , 10 12 ~10 13 , 10~10 12 , 10 2 ~10 12 , 10 3 ~10 12 , 10 4 ~10 12 , 10 5 ~10 12 , 10 6 ~10 12 , 10 7 ~10 12 , 10 8 ~10 12 , 10 9 ~10 12 , 10 10 ~10 12 , 1011 ~10 12 、10~10 11 、10 2 ~10 11 、10 3 ~10 13 、10 4 ~10 13 、10 5 ~10 13 、10 6 ~10 13 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10~10 9 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9、 10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9、 10~10 8 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8、 10 5 ~10 8 、106 ~10 8 , 10 7 ~10 8 , 10~10 7、 10 2 ~10 7 , 10 3 ~10 7 , 10 4 ~10 7 , 10 5 ~10 7 , 10 6 ~10 7 , 10~10 6 , 10 2 ~10 6 , 10 3 ~10 6 , 10 4 ~10 6 , 10 5 ~10 6 , 10~10 5 , 10 2 ~10 5 , 10 3 ~10 5 , 10 4 ~10 5 , 10~10 4 , 10 2 ~10 4 , 10 3 ~10 4 , 10~10 3 , 10 2 ~10 3 , or 10-10 2 It includes each of the bacterial strains.

[0233] In some embodiments, each capsule contains 10 to 10 capsules. 13 , 10 2 ~10 13 , 10 3 ~10 13 , 10 4 ~10 13 , 10 5 ~10 13 , 10 6 ~10 13 , 10 7 ~10 13 , 10 8 ~10 13 , 10 9 ~1013 、10 10 ~10 13 、10 11 ~10 13 、10 12 ~10 13 、10~10 12 、10 2 ~10 12 、10 3 ~10 12 、10 4 ~10 12 、10 5 ~10 12 、10 6 ~10 12 、10 7 ~10 12 、10 8 ~10 12 、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10~10 11 、10 2 ~10 11 、10 3 ~10 13 、10 4 ~10 13 、10 5 ~10 13 、10 6 ~10 13 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、109 ~10 10 、10~10 9 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9 、10~10 8 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8、 10 5 ~10 8 、10 6 ~10 8 、10 7 ~10 8 、10~10 7 、10 2 ~10 7 、10 3 ~10 7 、10 4 ~10 7 、10 5 ~10 7 、10 6 ~10 7 、10~10 6 、10 2 ~10 6 、10 3 ~10 6 、10 4 ~10 6 、10 5 ~10 6 、10~10 5 、10 2 ~10 5 、10 3 ~10 5 、10 4 ~10 5 、10~10 4 、10 2 ~10 4、 10 3 ~10 4, 10~10 3 , 10 2 ~10 3 , or 10-10 2 Contains total bacteria. In some embodiments, each capsule contains 10 7 ~10 9 , 10 7 ~10 8 , or 10 8 ~10 9 Contains total bacteria. In some embodiments, each capsule contains approximately 1.0 × 10 7 , 2.0×10 7 , 3.0×10 7 , 4.0×10 7 , 5.0×10 7 , 6.0×10 7 , 7.0×10 7 , 8.0×10 7 , 9.0×10 7 , 1.0 × 10 8 , 2.0×10 8 , 3.0×10 8 , 4.0×10 8 , 5.0×10 8 , 6.0×10 8 , 7.010 8 , 8.0×10 8 , 9.0×10 8 , 1.0 × 10 9 , 1.1 × 10 9 , 1.2 × 10 9 , 1.3 × 10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9×10 9 , 2.0×10 9 , 2.1 × 10 9 , 2.2 × 10 9 , 2.3 × 10 9 , 2.4×10 9 , 2.5×10 9 , 2.6×10 9 , 2.7×10 9 , 2.8×10 9 , 2.9×10 9 , 3.0×109 , 3.1×10 9 , 3.2×10 9 , 3.3×10 9 , 3.4×10 9 , 3.5×10 9 , 3.6×10 9 , 3.7×10 9 , 3.8×10 9 , 3.9×10 9 , 4.0×10 9 , 4.1×10 9 , 4.2×10 9 , 4.3×10 9 , 4.4×10 9 , 4.5×10 9 , 4.6×10 9 , 4.7×10 9 , 4.8×10 9 , 4.9×10 9 , 5.0×10 9 It contains the total number of bacteria. In some embodiments, each capsule contains approximately 8.0 × 10⁶ 8 It contains the total number of bacteria. In some embodiments, each capsule contains approximately 1.6 × 10 9 It contains the total number of bacteria. In some embodiments, each capsule contains approximately 8.0 × 10⁶ 8 Contains CFU of 1.6 × 10⁶. In some embodiments, each capsule contains approximately 1.6 × 10⁶. 9 This includes CFU.

[0234] In some embodiments, each capsule contains 10 to 10 capsules. 13 , 10 2 ~10 13 , 10 3 ~10 13 , 10 4 ~10 13 , 10 5 ~10 13 , 10 6 ~10 13 , 10 7 ~10 13 , 10 8 ~10 13 , 10 9 ~10 13 , 10 10 ~10 13 , 10 11 ~1013 、10 12 ~10 13 、10~10 12 、10 2 ~10 12 、10 3 ~10 12 、10 4 ~10 12 、10 5 ~10 12 、10 6 ~10 12 、10 7 ~10 12 、10 8 ~10 12 、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10~10 11 、10 2 ~10 11 、10 3 ~10 13 、10 4 ~10 13 、10 5 ~10 13 、10 6 ~10 13 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10~10 9 、10 2~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9 、10~10 8 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8 、10 5 ~10 8 、10 6 ~10 8 、10 7 ~10 8 、10~10 7、 10 2 ~10 7 、10 3 ~10 7 、10 4 ~10 7 、10 5 ~10 7 、10 6 ~10 7 、10~10 6 、10 2 ~10 6 、10 3 ~10 6 、10 4 ~10 6 、10 5 ~10 6 、10~10 5 、10 2 ~10 5 、10 3 ~10 5 、10 4 ~10 5 、10~10 4 、10 2 ~10 4 、10 3 ~10 4 、10~10 3 、10 2 ~10 3, or 10-10 2 Includes each of the bacterial strains.

[0235] In some embodiments, the pharmaceutical composition contains 10 to 10 per dose. 13 , 10 2 ~10 13 , 10 3 ~10 13 , 10 4 ~10 13 , 10 5 ~10 13、 10 6 ~10 13 , 10 7 ~10 13 , 10 8 ~10 13 , 10 9 ~10 13 , 10 10 ~10 13 , 10 11 ~10 13 , 10 12 ~10 13 , 10~10 12、 10 2 ~10 12 , 10 3 ~10 12 , 10 4 ~10 12 , 10 5 ~10 12、 10 6 ~10 12 , 10 7 ~10 12 , 10 8 ~10 12 , 10 9 ~10 12 , 10 10 ~10 12 , 10 11 ~10 12 , 10~10 11 , 10 2 ~10 11、 10 3 ~10 13 , 10 4 ~10 13 , 10 5 ~10 13 , 10 6 ~10 13、 10 7 ~1011 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10~10 9 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9、 10 8 ~10 9 、10~10 8 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8 、10 5 ~10 8 、10 6 ~10 8 、10 7 ~10 8 、10~10 7 、10 2 ~10 7 、10 3 ~10 7 、10 4 ~10 7 、10 5 ~10 7 、10 6 ~107 , 10~10 6 , 10 2 ~10 6 , 10 3 ~10 6 , 10 4 ~10 6 , 10 5 ~10 6 , 10~10 5、 10 2 ~10 5 , 10 3 ~10 5 , 10 4 ~10 5 , 10~10 4 , 10 2 ~10 4 , 10 3 ~10 4 , 10~10 3 , 10 2 ~10 3 , or 10-10 2 Each of the CFU bacterial strains is included. In some embodiments, the pharmaceutical composition contains 10 to 10 per dose. 13 , 10 2 ~10 13 , 10 3 ~10 13 , 10 4 ~10 13 , 10 5 ~10 13 , 10 6 ~10 13 , 10 7 ~10 13 , 10 8 ~10 13 , 10 9 ~10 13 , 10 10 ~10 13 , 10 11 ~10 13 , 10 12 ~10 13 , 10~10 12 , 10 2 ~10 12 , 10 3 ~10 12 , 10 4 ~10 12 , 10 5 ~10 12 , 10 6~10 12 、10 7 ~10 12 、10 8 ~10 12 、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10~10 11 、10 2 ~10 11 、10 3 ~10 13 、10 4 ~10 13 、10 5 ~10 13 、10 6 ~10 13 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10~10 10 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10~10 9 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9、 10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9, 10 to 10 8 , 10 2 to 10 8 , 10 3 to 10 8 , 10 4 to 10 8 , 10 5 to 10 8 , 10 6 to 10 8 , 10 7 to 10 8 , 10 to 10 7 , 10 2 to 10 7 , 10 3 to 10 7 , 10 4 to 10 7 , 10 5 to 10 7 , 10 6 to 10 7 , 10 to 10 6 , 10 2 to 10 6 , 10 3 to 10 6、 10 4 to 10 6 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​、1.6×10 8 、1.7×10 8 、1.8×10 8 、1.9×10 8 、2.0×10 8 、2.1×10 8 、2.2×10 8 、2.3×10 8 、2.4×10 8 、2.5×10 8 、2.6×10 8 、2.7×10 8 、2.8×10 8 、2.9×10 8 、3.0×10 8 、3.1×10 8 、3.2×10 8 、3.3×10 8 、3.4×10 8 、3.5×10 8 、3.6×10 8 、3.7×10 8 、3.8×10 8 、3.9×10 8 、4.0×10 8 、4.1×10 8 、4.2×10 8 、4.3×10 8 、4.4×10 8 、4.5×10 8 、4.6×10 8 、4.7×10 8 、4.8×10 8 、4.9×10 8 、5.0×10 8 、5.1×10 8 、5.2×10 8 、5.3×10 8 、5.4×10 8 、5.5×10 8 、5.6×10 8 、5.7×10 8 、5.8×10 8 、5.9×10 8 、6.0×10 8 、6.1×10 8 、6.2×10 8 、6.3×10 8 、6.4×10 8 、6.5×10 8、6.6×10 8 、6.7×10 8 、6.8×10 8 、6.9×10 8 、7.0×10 8 、7.1×10 8 、7.2×10 8 、7.3×10 8 、7.4×10 8 、7.5×10 8 、7.6×10 8 、7.7×10 8 、7.8×10 8 、7.9×10 8 、8.0×10 8 、8.1×10 8 、8.2×10 8 、8.3×10 8 、8.4×10 8 、8.5×10 8 、8.6×10 8 、8.7×10 8 、8.8×10 8 、8.9×10 8 、9.0×10 8 、9.1×10 8 、9.2×10 8 、9.3×10 8 、9.4×10 8 、9.5×10 8 、9.6×10 8 、9.7×10 8 、9.8×10 8 ...

Claims

1. A pharmaceutical composition for use in a method for reducing the level of primary bile acids and / or increasing the level of secondary bile acids in a subject, and for preventing Clostridium difficile infection in a subject, the method being: The procedure includes administering vancomycin to the subject, followed by administering a therapeutically effective dose of the pharmaceutical composition to the subject. Here, the pharmaceutical composition is as follows: (i) A purified bacterial strain belonging to the species Clostridium bolteae; (ii) A purified bacterial strain belonging to the species Anaerotruncus colihominis: (iii) Purified bacterial strains belonging to the species Dracoutella massiliensis or Sellimonas intestinalis: (iv) A purified bacterial strain belonging to the species Clostridium symbiosum: (V) Purified bacterial strains belonging to the species Blautia producta: (Vi) Purified bacterial strains belonging to the species Dorea longicatena: (Vii) Purified bacterial strains belonging to the species Clostridium innocuum: and (Viii) A purified bacterial strain belonging to the species Flavonifractor plautii: Includes, Here, vancomycin is administered in multiple doses before administering the pharmaceutical composition. Here, the pharmaceutical composition is administered in multiple doses over multiple days, where at least 4.0 × 10 １０ CFU (colony-forming unit) or at least 1.1 × 10 １１ The total bacterial count for CFU is targeted and administered. The aforementioned pharmaceutical composition.

2. A pharmaceutical composition for use in a method for reducing the level of primary bile acids and / or increasing the level of secondary bile acids in a subject, and for preventing Clostridium difficile infection in a subject, the method being: The procedure includes administering vancomycin to the subject, followed by administering a therapeutically effective dose of the pharmaceutical composition to the subject. Here, the pharmaceutical composition is as follows: (i) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with respect to the nucleotide sequence of Sequence ID No. 1; (ii) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of Sequence ID No. 2: (iii) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of Sequence ID No. 3: (iv) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of Sequence ID No. 4: (V) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of Sequence ID No. 5: (Vi) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: (Vii) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of Sequence ID No. 7: and (Viii) A purified bacterial strain containing a 16S rDNA sequence having at least 99% sequence identity with the nucleotide sequence of Sequence ID No. 8: Includes, Here, vancomycin is administered in multiple doses before administering the pharmaceutical composition. Here, the pharmaceutical composition is administered in multiple doses over multiple days, where at least 4.0 × 10 １０ CFU (colony-forming unit) or at least 1.1 × 10 １１ The total bacterial count for CFU is targeted and administered. The aforementioned pharmaceutical composition.

3. The pharmaceutical composition according to claim 1 or 2, wherein the C. difficulte infection is a recurrent C. difficulte infection.

4. The pharmaceutical composition contains at least 4.0 × 10 １０ A pharmaceutical composition according to any one of claims 1 to 3, comprising total bacteria of CFU, wherein the pharmaceutical composition is administered in five doses.

5. The pharmaceutical composition contains at least 1.1 × 10 １１ A pharmaceutical composition according to any one of claims 1 to 3, comprising total bacteria of CFU, wherein the pharmaceutical composition is administered in 14 doses.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein a total bacterial strain of at least 4.0 × 10¹⁰ CFUs is administered in at least five doses over at least five days.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein a total bacterial strain of at least 1.1 × 10¹¹ CFUs is administered in at least 14 doses over at least 14 days.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein each of the multiple doses of the pharmaceutical composition contains at least 8.0 × 10⁹ CFU total bacterial strains.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein each of the multiple doses of the pharmaceutical composition is administered on consecutive days.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein each dose of the pharmaceutical composition contains 10 capsules.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein each of the multiple doses of vancomycin is administered on consecutive days.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein vancomycin is administered at a dose of 500 mg per day.

13. The pharmaceutical composition according to claim 12, wherein vancomycin is administered in four doses of 125 mg per day.

14. The pharmaceutical composition according to any one of claims 1 to 11, wherein vancomycin is administered at a dose of 250 mg per day.

15. The pharmaceutical composition according to claim 14, wherein vancomycin is administered in two doses of 125 mg per day.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein vancomycin is administered for five consecutive days up to two days before the administration date of the pharmaceutical composition, and the method includes a drug-free day before the administration of the pharmaceutical composition.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the bacterial strain is freeze-dried or spray-dried.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein one or more of the bacterial strains are of the spore type.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein one or more of the bacterial strains are in the vegetative form.

20. A pharmaceutical composition according to any one of claims 1 to 19, wherein the pharmaceutical composition is formulated for delivery to the intestines.

21. A pharmaceutical composition according to any one of claims 1 to 20, wherein the pharmaceutical composition is formulated for delivery to the colon.

22. A pharmaceutical composition according to any one of claims 1 to 21, wherein the pharmaceutical composition is administered orally.

23. The pharmaceutical composition according to claim 22, further comprising one or more enteric polymers.

24. A pharmaceutical composition according to any one of claims 1 to 23, wherein the pharmaceutical composition is administered rectally.

25. A pharmaceutical composition according to any one of claims 1 to 24, wherein the method further comprises evaluating the colonization of one or more bacterial strains of the pharmaceutical composition in a target microbiome, and the evaluation is as follows: (i) Isolating nucleic acids from a sample of the target microbiome; Sequence isolated nucleic acids to obtain multiple nucleotide sequences of the isolated nucleic acids; and Determining the presence of at least one bacterial strain in a pharmaceutical composition by comparing multiple nucleotide sequences with multiple genomic markers for each bacterial strain in the pharmaceutical composition; If genomic markers for a bacterial strain are present in multiple nucleotide sequences, then the bacterial strain is established in the microbiome; or (ii) Isolating nucleic acids from a sample of the target microbiome; and Determining the presence of at least one bacterial strain in a pharmaceutical composition by amplifying the nucleotide sequence of a genomic marker for at least one bacterial strain in isolated nucleic acids; If a genomic marker for a bacterial strain is present in the amplified nucleotide sequence, then the bacterial strain is established in the microbiome. The pharmaceutical composition comprising the above.

26. The pharmaceutical composition according to claim 25, wherein if the genomic marker for the bacterial strain is not present in multiple nucleotide sequences or amplified nucleotide sequences, the method further comprises administering one or more additional doses of the pharmaceutical composition to the target.

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