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4 Using gut microbes as therapy for C.difficile infection and beyond Emma Allen-Vercoe University of Guelph PHO Rounds Microbiology October 8 th 2015

5 Presenter disclosure Faculty: Emma Allen-Vercoe Name of the commercial interest: Nubiyota LLC Nature of the relationship: Co-founder and scientific advisor NuBiyota Better ecosystem, better health

6 Only very few microbes are pathogens In fact, human health depends on microbiota health We are the vessels for our microbial passengers

7 There are more bacteria living in your gut than there are people on the planet Gut: bacterial species Also Archaea as well as yeasts & other microscopic eukaryotes

8 Everyone is different Gut microbial ecosystems are highly variable in composition and abundance profiles between people

9 How do we acquire our microbes? Toh & Allen-Vercoe, MEHD Feb 2015

10 How do we acquire our microbes? Toh & Allen-Vercoe, MEHD Feb 2015

11 What do our gut microbes do for us? Metabolic Fermentation of non-digestible substrates Production of vitamins, SCFA Removal of toxins, carcinogens Differentiation of IECs Protective Colonization resistance Innate and adaptive immunity Inflammatory cytokine oversight Antimicrobial secretion Competition for sites and nutrients Immune system and barrier function Energy Structural Intestinal villi and crypts Tight junctions siga production Mucus secretion After Cryan et al.

12 We exist with our microbes in a delicate state of equilibrium

13 Maintaining the equilibrium High diversity of species: Healthy ecosystem Balance Functional redundancy Resistance to disease Low diversity of species: Sick ecosystem Imbalance Functional disability Susceptibility to disease

14 Extinction events may impact health Hygiene hypothesis We are preventing proper colonization by being too clean Missing microbiota hypothesis Blaser & Falkow Nature Reviews Microbiology 2009 We are disturbing proper colonization across generations through e.g. antibiotic use, poor diet, over-sanitary lifestyle Antimicrobial use (especially in early childhood) may be particularly problematic Highly refined Western-style diet exacerbates the problem further

15 Several studies have shown: Gut microbiota changes significantly with antibiotic use Takes a long time afterwards to return to baseline Sometimes does not return to baseline at all Repeated hits cause vast changes from which the ecosystem does not recover Looft et al., PNAS 2012; Robinson & Young Gut Microbes. 2010; Jakobsson et al. PLoS One. 2010; Antonopoulos et al., Infect Immun. 2009; Dethlefsen et al. PLoS Biol

16 Lack of microbial diversity Loss of keystone species Overgrowth of opportunistic pathogens Poor diet/lifestyle Drug interactions Dysbiosis How do we look inside the black box? How do we study it? DISEASE

17 Examples of diseases associated with reduced gut microbiota diversity (published research) Inflammatory bowel diseases Infant colic Eczema Autism Colorectal cancer Allergic asthma Celiac disease Obesity Neonatal necrotizing enterocolitis Irritable Bowel Syndrome Clostridium difficile infection

18 What s inside the black box? The Good Lactic Acid Bacteria (LAB) E.g. Bifidobacterium and Lactobacillus spp. Butyrate-producing bacteria E.g. Faecalibacterium prausnitzii, Roseburia spp. The Bad Opportunistic pathogens E.g. E.coli, Pseudomonas aeruginosa, Clostridium difficile, Bacteroides fragilis Sulfate-reducing bacteria E.g. Desulfovibrio spp. The Ugly: it really is not that clear-cut

19 Some microbes are like bad teenagers in a subway station In a crowded environment they tend to behave well When the crowds are gone, they tend to start behaving in antisocial ways Wikimedia commons images

20 C.difficile: a bad teenager Normal colon Non-spore-forming anaerobes predominate; C.difficile absent or numbers low Return to normal Cessation of therapy Antibiotics -clindamycin -cephalosporins -ampicillin Reduction in major genera of anaerobes: C.difficile grows to high numbers Production of exotoxins A and B Diarrhea Symptoms abate Ulceration of colon Vancomycin or metronidazole Wikimedia commons Death C. Carlucci, A-V lab, 2012

21 So, C.difficile is a pathogen that needs to be destroyed! We need to throw our best chemical weapons at it to rid ourselves of the scourge! NO, this is NOT the best strategy

22 We simply need to repopulate the subway but how do we do that?

23 The human gut microbiota is a complex microbial ecosystem Its function and behaviour is best studied as a whole

24 Microbes in a microbiology lab Almost always exist on their own as part of a pure culture Usually have to adapt to survive this way Are often grown logarithmically Are usually given access to rich nutrient sources Microbes in nature Almost always exist as part of microbial communities Benefit from their microbial friends (& host) Rarely grow logarithmically Rarely have access to rich nutrient sources

25 How do we study the black box? The human colon is a type of chemostat thus, chemostats can be used to emulate the human colonic environment

26 Our chemostats ( Robogut ) Seeded with fresh feces or defined communities and set to model the distal gut ecosystem Host-free system Can be used to culture the unculturable Can support whole gut microbial ecosystems for several weeks at a time

27 Microbes talk each other using small molecules Many of these small molecules are also absorbed by the host Wikimedia commons images

28 Liquid gold Allows us to eavesdrop on microbial conversations

29 Kathleen Schroeter and Julie McDonald Different people s microbiota responds to antibiotic disturbance in different ways Patterns of perturbation differ between donors

30 Defined microbial ecosystems As well as studying fecal ecosystems, we can culture diversity from fecal samples Reconstitute into defined experimental ecosystems Strain composition is known Genomes, transcriptomes, metabolomes etc. known or measureable Strain composition can be easily (and cleanly) altered We can look at deliberate changes in composition and how they affect ecosystem function

31 Defined communities from ulcerative colitis patients UC1 Rifaximin treatment effects vary according to community UC3 Kaitlyn Oliphant

32 Marc Aucoin, U Waterloo Sandi Yen 1 H NMR profiling of liquid gold Liquid gold derived from different donors produces metabolic profiles unique to the respective hosts Yen et al., Journal of Proteome Research, 2015

33 Back to CDI FMT: one way to put the people back on the subway platform Fresh homogenate instilled into patient within a few hrs of preparation Rectal enema Colonoscopy Nasoduodenal tube Poop pills Results in cure of the patient in >90% of cases Rapid resolution of CDI Only rare recurrence of disease

34 But not without risk Known Potential to transmit infection Risks of procedure Colonoscopy and associated sedation Unknown Changes in microbiome that increase risk of other diseases (e.g., IBD, cancer, obesity, etc) Risk of acquiring new pathogens or infection Development of autoimmune disorders Long-term risks: UNKNOWN

35 Case report: sudden and unexpected weight gain after FMT Alang and Kelly, 2014 Sciencedaily.com

36 What if we could create synthetic stool to treat CDI?

37 Good idea why hasn t anyone done this before? They have! Early studies used small number of gut microbial isolates to treat CDI Tvede & Rask Madsen, Lancet 1989 Fell out of favour with increased antibiotic use Difficult to do most gut anaerobes (erroneously) thought to be unculturable Prior lack of understanding of the importance of the gut microbiota to health

38 Steps to MET 1. Find a healthy donor 2. Use chemostat to isolate microbial diversity 3. Characterize, curate and preserve isolates 4. Test for, and remove, undesirable components Antibiotic resistance, presence of virulence genes, toxicity, etc. 5. Assemble, test and optimize ecosystem Determine optimal growth conditions/scalability

39 RePOOPulate Acidaminococcus intestinalis Bacteroides ovatus Bifidobacterium adolescentis (x2) Bifidobacterium longum (x2) Collinsella aerofasciens Dorea longicatena (x2) Escherichia coli Eubacterium eligens Eubacterium limosum Eubacterium rectale (x4) Eubacterium ventriosum Faecalibacterium prausnitzii Lactobacillus casei Lactobacillus paracasei Parabacteroides distasonis Raoultella sp. Roseburia faecalis Roseburia intestinalis Ruminococcus torques (x2) Streptococcus mitis Likely novel species (x5) Likely novel genus & species (x1) (Closest species by full-length 16S alignment)

40 Acidaminococcus intestinalis Bacteroides ovatus Bifidobacterium adolescentis (x2) Bifidobacterium longum (x2) Collinsella aerofasciens Dorea longicatena (x2) Escherichia coli Eubacterium eligens Eubacterium limosum Eubacterium rectale (x4) Eubacterium ventriosum RePOOPulate Faecalibacterium prausnitzii Lactobacillus casei Lactobacillus paracasei Parabacteroides distasonis Raoultella sp. Roseburia faecalis Roseburia intestinalis Ruminococcus torques (x2) Streptococcus mitis Likely novel species (x5) Likely novel genus & species (x1) Lachnospiraceae and Ruminococcaceae family species (Generally lacking in CDI, IBD)

41 RePOOPulate proof-of-principle trial 2 elderly ladies with severe, recurrent C.diff infections were treated (April and June 2011) RePOOPulate made fresh at Guelph, driven to KGH, and administered via colonoscopy 1 dose, 100mLs Both patients recovered within 2 days and have remained C.diff-free ever since (despite numerous subsequent antibiotic exposures)

42 2013: 1 (1): 6

43 Petrof et al. Microbiome, 2013 In both patients, RePOOPulate signatures could be seen 6 months following administration: colonization had taken place

44 Why only n=2? Trial temporarily stopped by Health Canada Is RePOOPulate/MET1 a probiotic or a biologic drug? Health Canada (& FDA): should be considered as a biologic Potentially easier to regulate than fecal bacteriotherapy But so far, no guidelines for us! From Health Canada Guidelines for FMT

45 Where we currently are with MET Developing MET1+ as well as other METs from a range of healthy donors Creating a QC pipeline Based on metagenomics, metabolomics/metabonomics and other proprietary measures Scale up and stabilization of product For encapsulation and simple, oral delivery Sounds easy, but it isn t! Gearing up for new clinical trial

46 The challenges What s the definition of a healthy gut microbiota? Defining species content is likely not enough What determines success or failure of engraftment? And how can we modulate this? What are the long-term risks? (Much easier to measure with a defined MET) Can the MET approach be applied for other disease indications?

47 Do we need to match patient to ecosystem? Consider different people s bodies as different models of car Consider the gut microbiota as an engine Wikimedia commons images

48 Maybe we should just replace the faulty part of the engine Birchall et al. in prep Ushering in the era of personalized medicine

49 I foresee a time when People will regularly have a microbiome check-up Antibiotic use will be minimal and personalized to reduce risk of microbiota damage Microbial Ecosystem Therapeutics will enter mainstream medicine Symbiontology will become a new medical specialty A merger of Gastroenterology, microbial ecology, nutrition science, and many other disciplines

50 Acknowledgements EA-V lab members Christian Ambrose Erin Bolte Christian Carlucci Kyla Cochrane Michelle Daigneault Kaitlyn Oliphant Rafael Peixoto Kathleen Schroeter Mariia Taguer Mike Toh Sandi Yen And alumni Eric Brown Ian Brown Julie McDonald Queen s U Elaine Petrof Adriana Breen Curtis Noordhof KGH clinical staff Western U Greg Gloor Jean Macklaim U Waterloo Marc Aucoin NuBiyota Better ecosystem, better health

51 I found the problem, Mr. Smith. Instead of probiotics, you have been taking amateur biotics.