Methods of decreasing dysbiosis and restoring a microbiome

1. A method for treating or preventing Clostridium difficile infection in a subject, comprising

administering vancomycin to the subject, and

administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a purified bacterial mixture, the purified bacterial mixture comprising:

(i) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1;

(ii) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 2;

(iii) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3;

(iv) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4;

(v) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 5;

(vi) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6;

(vii) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7; and

(viii) a bacterial strain comprising a 16S rDNA sequence with at least 99% sequence identity to the nucleotide sequence of SEO ID NO: 8,

wherein the vancomycin is administered in multiple doses prior to administering the pharmaceutical composition; wherein the pharmaceutical composition is administered in multiple doses, and wherein a total of at least 4.0×10 10 CFUs (colony forming units) or 1.1×10 11 CFUs of bacteria are administered to the subject.

2. The method of claim 1 , wherein the multiple doses comprise administering the pharmaceutical composition to the subject at least 4 times and/or wherein the multiple doses are administered at regular intervals.

3. The method of claim 1 , wherein the multiple doses of the pharmaceutical composition comprise a total of at least 4.0×10 10 CFUs of bacteria and are administered in five doses.

4. The method of claim 1 , wherein the multiple doses of the pharmaceutical composition comprise a total of at least 1.1×10 11 CFUs of bacteria and are administered in fourteen doses.

5. The method of claim 1 , wherein each of the multiple doses of the pharmaceutical composition is administered on consecutive days.

6. The method of claim 1 , wherein each of the multiple doses is administered in 10 capsules comprising the pharmaceutical composition.

7. The method of claim 1 , wherein the method is for preventing recurrence of C. difficile infection.

8. The method of claim 1 , wherein the purified bacterial mixture consists of the bacterial strains of (i)-(viii).

9. The method of claim 1 , wherein the vancomycin is administered at 500 mg per day or 250 mg per day.

10. The method of claim 9 , wherein the vancomycin is administered in 4 doses of 125 mg per day or 2 doses of 125 mg per day.

11. The method of claim 1 , wherein the vancomycin is administered on five consecutive days up to two days prior to the day of administration of the pharmaceutical composition, and wherein the method includes a washout day prior to administration of the pharmaceutical composition.

12. The method of claim 1 , wherein one or more of the bacterial strains are lyophilized or spray-dried.

13. The method of claim 1 , wherein one or more of the bacterial strains are in spore form and/or one or more of the bacterial strains are in vegetative form.

14. The method of claim 1 , wherein each of the one or more bacterial strains are in spore form or each of the bacterial strains are in vegetative form.

15. The method of claim 1 , wherein the pharmaceutical composition further comprises one or more enteric polymers.

16. The method of claim 1 , wherein the administration is oral administration.

17. The method of claim 1 , wherein the pharmaceutical composition is formulated for oral delivery or rectal delivery; and/or the pharmaceutical composition is formulated for delivery to the intestine, optionally for delivery to the colon.

18. The method of claim 1 , wherein one or more of the bacterial strains are detected in the microbiome at least four weeks after initial administration of the pharmaceutical composition.

19. The method of claim 1 , further comprising assessing colonization of one or more bacterial strains of the pharmaceutical composition in a microbiome of the subject, optionally wherein the assessing comprises:

(i) isolating nucleic acid from a sample of the microbiome of the subject;

sequencing the isolated nucleic acid to obtain a plurality of nucleotide sequences of the isolated nucleic acid; and

determining the presence of at least one bacterial strain of the bacterial composition by comparing the plurality of nucleotide sequences to a plurality of genomic markers for each bacterial strain of the bacterial composition;

wherein if a genomic marker for a bacterial strain is present in the plurality of nucleotide sequences, the microbiome is colonized with the bacterial strain; or

(ii) isolating nucleic acid from a sample of the microbiome of the subject; and

determining the presence of at least one bacterial strain of the bacterial composition by amplifying a nucleotide sequence of a genomic marker for the at least one the bacterial strains in the isolated nucleic acid;

wherein if a genomic marker for a bacterial strain is present in the amplified nucleotide sequences, the microbiome is colonized with the bacterial strain.

20. The method of claim 19 , wherein if the genomic marker for the bacterial strain is absent in the plurality of nucleotide sequences or absent in the amplified nucleotide sequences, the method further comprises administering one or more additional doses of the pharmaceutical composition to the subject.