1. Precision Microbiome Reconstitution Restores Bile Acid Mediated Resistance to Clostridium difficile
By Janna Seto

2. GI Tract: Microbial Communities
Roles of Microbes in GI Tract:
Mutualistic & Commensal relationship
Break down sugar polymers
Fermenting dietary fiber
Synthesizing vitamins
Gastrointestinal tract is home to primarily anerobic bacteria.
Mutualistic = both humans and bacteria benefit
Commensal =only beneficial to bacteria & doesn’t harm the host
Bacteria in the digestive system assist in nutrient metabolism, vitamin production, and waste processing.
C. Scindens = anaerobic bacteria found in the human colon & can be found in the soil as well.
Convert bile acids to secondary bile acids (inhibit the growth of C. diff)
Convert glucocorticoids to androgens

3. Antibiotics: Microbial Communities
Patients treated with antibiotics:
Destroy intestinal & disease causing microbes
Increase susceptibility to intestinal pathogens
Overuse can lead to antibiotic resistance
Most common intestinal pathogen from antibiotics?
Clostridium difficile

4. Clostridium difficile (C. difficile)
Commonly affects:
Long term patients in health care facilities
Frequent antibiotic medications
Common symptoms:
Diarrhea
Fever
Loss of appetite
Inflammation of the colon (Life- threatening)
Severe dehydration, abdominal pain, loss of appetite, kidney failure
C. Diff are found in soil, air, water, & feces, food products (processed meats)
Some people carry C. diff in their large intestine with no effects
Treatment includes: Antibiotics, fecal transplant, probiotics

5. Microbiota-Based Therapies
Infection resistance is currently undefined:
microbiome-encoded genes
Biosynthetic gene clusters
This study identifies microbiome- mediated C. difficile resistance and targets therapeutics for C. diff infections.
Although treatments such as a fecal transplant is available, the microbiome-encoded genes and biosynthetic
gene clusters critical for infection resistance remain largely undefined.

6. Mice Models Mice Models: C57BL/6J female mice
10 mice administered clindamycin
10 mice administered ampicillin
10 mice administered enrofloxacin
Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile.
Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans.

7. C. difficile Infection Susceptibility
Clindamycin
Ampicillin
Enrofloxacin
long-lasting susceptibility, transient susceptibility, no increase susceptibility to C. difficile infection.
C. difficile toxin expression correlated significantly with C. difficile abundance in the intestine.
recovery of secondary bile acids and the abundance of the
gene family responsible for secondary bile acid biosynthesis (as predicted
using PICRUSt15) correlatedwith C. difficile resistance

8. Impact of Antibiotics on Intestinal Microbiota
Clindamycin
Ampicillin
Enrofloxacin
16S ribosomal RNA (rRNA) gene amplicon sequencing
Illumina MiSeq platform
16S ribosomal RNA (rRNA) gene amplicon sequencing revealed that the three antibiotics had distinct impacts on intestinal microbiota composition
Infection susceptibility correlated with decreased microbiota alpha diversity (diversity within individuals)
Amplicons of the V4-V5 16S rRNA region were amplified and sequenced using an Illumina MiSeq
platform for samples in the in vivo and ex vivo adoptive transfer experiments.

9. OTUs & Susceptibility to C. difficile Infection
Correlated resistance with individual bacterial species abundances:
operational taxonomic units (OTUs, $97% ≥ 16S sequence similarity)
11 bacterial OTUs that correlated strongly with infection resistance
The OTU with the strongest resistance correlation C. scindens

10. Human Clinical Studies
Human Models:
Allogeneic hematopoietic stem-cell transplantation (allo-HSCT):
12 diagnosed with C. difficile infection
12 C. difficile carriers without clinical infection
patients were diagnosed with a haematological malignancy and received chemotherapy and/or total body irradiation as well as antibiotics
during transplantation
To facilitate comparisons across data sets, we clustered murine and human microbiota together to define OTUs that together
accounted for a majority of both the human and mouse microbiota structure
identified bacteria displaying strong inhibition against C. difficile.

11. C. difficile Interactions: Human & Murine
the human model identified two C. difficile-inhibitingOTUs that were conserved in the murine model, the strongest of
whichwas C. scindens

12. Transferred Resistance Associated Bacteria
Evaluate causality between intestinal bacteria and infection resistance
Representative consortium of four intestinal bacterial isolates:
4 Species-level 16S similarity to OTUs
After antibiotics & before infection:
Phosphate-buffered saline (PBS)
4 Bacteria
C. scindens
The abundance of C. scindens correlated significantly with infection resistance.
suggesting that improving bacterial engraftment efficiency may enhance the protective effects of the adoptive transfer.

13. Secondary Bile Acid Biosynthesis
Bile acid dependent C. scindens mediated inhibition of C. difficile quantified ex vivo
introduction of C. scindens significantly inhibited C. difficile in the intestinal content from antibiotic-exposed animals.
C. Scindensmediated inhibition of C. difficile is dependent upon accessing and modifying endogenous bile salts and recapitulates a natural mechanism
of microbiota-mediated infection resistance.

14. Conclusion
A fraction of the intestinal microbiota confers infection resistance by synthesizing C. difficile-inhibiting metabolites from host-derived bile salts.
C. scindens-mediated inhibition of C. difficile is dependent upon accessing and modifying endogenous bile salts.
The replenishment of secondary bile acids and/or biosynthesis- competent bacteria) may contribute to the therapeutic efficacy of fecal microbiota transplant.