Research Overview

Therapeutic Potential of theHuman Gastrointestinal MicrobiomeThomas J. Borody,1* Debra Peattie2 and Jordana Campbell1

1Centre for Digestive Diseases, Five Dock, NSW 2046, Australia2Pleiades Advisors, Lincoln, MA 01773, USA

Strategy, Management and Health Policy

EnablingTechnology,Genomics,Proteomics

PreclinicalResearch

Preclinical DevelopmentToxicology, FormulationDrug Delivery,Pharmacokinetics

Clinical DevelopmentPhases I-IIIRegulatory, Quality,Manufacturing

PostmarketingPhase IV

ABSTRACT Scientific breakthroughs in deciphering the human gut microbiome and the clinicalsuccess of fecal microbiota transplantation (FMT) to treat recurrent Clostridium difficile infection (R-CDI)are driving therapeutic advances based on human gut microbiota. Due to the powerful therapeuticcapacity of FMT and the keen interest for FMT-related products approved by regulatory agencies it istimely to review the growing field of therapeutics rooted in the human microbiome, emphasizing FMT butalso considering probiotics, vaccines, bacteriophages, and bioactive products. The diminishing effective-ness of antibiotics and the increasing rates of antibiotic resistance have renewed interest in findingalternative methods to combat bacterial infections. Despite pharmaceutical investment in developing newand more effective antibiotics, infectious disease experts warn of a compelling need to develop antibac-terial agents distinct from antibiotics. Probiotics have been recognized as beneficial to human health forover one hundred years. The most powerful probiotic of all, the gastrointestinal microbiota, housesapproximately 100 trillion species of bacteria, many of which produce a wealth of potent components,such as antimicrobial bacteriocins, metabolites, vitamins, and bacteriophages. The success of human gutmicrobiota in treating R-CDI and restoring gut homeostasis has highlighted the power of “nature’scomplete probiotic” and is propelling fecal microbiota along a therapeutic biologic regulatory path.Clinical use of FMT in R-CDI has also taught us that other conditions, e.g., ulcerative colitis, characterizedby superinfected and dysbiotic microbiomes may benefit from restoring gut homeostasis with normalmicrobiota, leading to active efforts to develop therapeutics from the human gut microbiome. Drug DevRes 74 : 385–392, 2013. © 2013 Wiley Periodicals, Inc.

Key words: microbiome; Clostridium difficile; microbiota; fecal microbiota transplantation; probiotics

INTRODUCTION

In considering the therapeutic potential of the gutmicrobiome, it is important to reflect on the composi-tion, size, and function of this human “tissue.” Thehuman gastrointestinal (GI) microbial population con-sists of 1013–1014 microorganisms, with a collectivegenome—the “microbiome”—containing at least 100times as many genes as the human genome [Gill et al.,2006]. Using molecular-based approaches to identifyand enumerate the numerous microbial species housedwithin the GI tract, we have begun to realize that thegut microbiome is much more than a waste by-product

of digestion. Indeed, human beings are an amalgam ofhuman genome attributes and microbial genome, i.e.,“second genome,” characteristics. The InternationalHuman Microbiome Consortium was created specifi-cally to characterize the species and genes of microbial

*Correspondence to: Thomas J. Borody, Centre for DigestiveDiseases, Level 1/229 Great North Road, Five Dock, NSW 2046,Australia.Email: thomas.borody@cdd.com.au

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ddr.21093

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communities that reside on and within the humanbody. A subset of this consortium, the MetaHIT(Metagenomics of the Human Intestinal Tract) project,is exploring and characterizing the microbial species ofour GI tract. MetaHIT has established a human gutmicrobial gene catalog and identified some 3.3 millionnonredundant microbial genes in 124 European fecalsamples [Qin et al., 2010]. Understanding the structureand function of the GI microbiome will likely revealdisease mechanisms and pave the way for creating noveltherapeutics. Numerous conditions previously thoughtto be “extra-intestinal” are being linked to alterations inthe GI microbiome. These include metabolic condi-tions such as diabetes [Qin et al., 2012] and obesity[Musso et al., 2011; Parks et al., 2013], neurological dis-orders such as Parkinson’s disease (PD) [Hawkes et al.,2009], multiple sclerosis (MS) [Berer et al., 2011], andautism [Finegold, 2011; Williams et al., 2012] as well asvarious autoimmune and allergic diseases [Borody andKhoruts, 2012].

Despite (or perhaps because of) the new antibiot-ics developed to curtail the problem, antibiotic-resistantbacteria are increasingly significant global threats.Global antibiotic use over the past 50 years has enabledsome bacteria to evolve escape mechanisms that allowthem to resist new classes of antibiotics, even those towhich they have not been previously exposed [Davies,1994]. In addition, novel and highly utilized antibioticsdamage the GI microbiota, thereby permitting oppor-tunistic pathogens to establish and thrive. We may facereturn to a pre-antibiotic era, with fewer effective treat-ments available to treat potentially deadly epidemicssuch as Clostridium difficile infection (CDI). As acounterpoint to antibiotics, other measures are beingexplored to maintain a lead in this bacterial “arms race.”FMT is a promising therapeutic approach with stunningclinical efficacy capable of eradicating infections thatthe current antibiotic repertoire cannot, and it teachesus that the gut microbiota offer therapeutic riches.Here, we review some therapeutic agents derivedfrom the gut microbiota and explore potential futuredevelopments.

GUT MICROBIOTA AS A TRANSPLANTABLEBIOLOGIC AGENT

Fecal Microbiota Transplantation (FMT)

FMT is the infusion of a fecal suspension derivedfrom a healthy donor into the intestine of a recipientsuffering from an intestinal-related disease or disorder.Such “gut dysbioses” include CDI, Crohn’s disease, andulcerative colitis. As critical components of the GImucosal defense barrier, intestinal microbiota are wellsuited to play a therapeutic role. Studies have demon-

strated that animals bred in germ-free environmentsare more susceptible to pathogenic infections thananimals that acquire and maintain a complex intestinalmicrobiota population [Inagaki et al., 1996; Smith et al.,2007]. A rich GI microbial population provides ameans, termed “colonization resistance,” to counteractinfections, whereby commensal bacteria compete withpathogens for nutrients and attachment sites. Further,the microbiota produce compounds that inhibit growthof pathogens and other transient incoming organisms,thereby limiting opportunistic invasion. The potentiallyfatal CDI is an excellent example of pathogenic conse-quences arising from a breach in the colonization resis-tance provided by normal gut microbiota. Followingintestinal microbiota disruption, often induced by acourse of antibiotics, C. difficile can colonize the GItract, where it produces toxins and can cause increasedintestinal permeability, diarrhea, inflammation, pseudo-membranous colitis, renal failure, and life-threateninghypotension.

CDI has been growing steadily in incidence, mor-bidity, and mortality across North America and Europeover the past decade. In the US alone, an estimated3 million new acute C. difficile infections (A-CDI) arecurrently diagnosed annually [Sailhamer et al., 2009].Patients with CDI have deficiencies in fecal flora com-position, particularly of Bacteroides and Firmicutes[Tvede and Rask-Madsen, 1989; Khoruts et al., 2010].Of those infected, a subgroup will progress to developfulminant CDI (F-CDI), which results in approximately300 deaths/day or almost 110 000 deaths/year in UShealth care facilities [Jarvis et al., 2009]. A larger sub-group will experience recurrent CDI (R-CDI), whichoccurs when CDI persists despite repeated rounds ofantibiotic therapy. Reconstituting the gut microbialecology with FMT, thereby re-establishing the widediversity of intestinal microbiota, has shown promise insevere CDI patients [Trubiano et al., 2012; Weingardenet al., 2013] and provides rapid clinical cure in R-CDIpatients [Borody and Khoruts, 2012]. Studies for up to24 weeks following FMT have shown that donor floracan offer durable implantation and correct intestinalfloral deficiencies [Floch, 2010; Grehan et al., 2010].

Full-Spectrum Microbiota

What therapeutic products can raw fecal materialoffer? Although we are well acquainted with tissue-derived therapies—such as human blood products orporcine insulin—fecal microbiota have yet to beregarded or exploited as a tissue in standard clinicalpractice. That being said, however, freshly collectedstool blended with saline and roughly filtered to removelarge particulate matter has emerged as a therapeutic

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form of fecal microbiota [Aroniadis and Brandt, 2013]and is starting to yield more refined products. The Uni-versity of Minnesota group led by Khoruts andSadowsky has produced a standardized filtrate com-posed almost entirely of viable fecal bacteria preparedfrom a carefully selected, highly screened “universaldonor” [Hamilton et al., 2012]. This preparation, whichlacks the strong odor of raw feces, retains all repre-sented microbial groups of healthy human GI flora,thereby warranting the name “Full-Spectrum Micro-biota” (trademark pending, CIPAC Limited, Malta). Itis as effective as crude homogenized flora at restoringdeficient fecal components in R-CDI patients undergo-ing FMT via colonoscopy [Hamilton et al., 2013]. Pre-served at −80°C prior to use, full-spectrum microbiotahas shown excellent long-term viability [Khoruts andSadowsky, personal communication] and clinical utility[Hamilton et al., 2012] to date. CIPAC Limited, astart-up company working with the University of Min-nesota, is developing this first-generation FMT productfor R-CDI. Rebiotix Inc. (Roseville, MN) has report-edly initiated a nonrandomized, open-label US trial forCDI using a similar suspension for enema delivery fromdonors, albeit with no published information or dataabout screening standards, method of preparation, orproduct efficacy. Monarch Labs (Irvine, CA) plans tocommercialize two FMT-based products: a cGMP pro-cessing and banking service for autologous FMT trans-plantation and Medical Microbiota™ for allografttransplantation. CIPAC, Rebiotix, and Monarch Labsare pursuing FMT products based on the full spectrumof healthy GI microbiota; other commercial entities arepursuing cultured, low-diversity, or nonbacterial prod-ucts (see Table 1).

Using the full spectrum of gut microbiota as atherapeutic entity offers advantages over approachesbased on use of selected cultured bacteria. The advan-tages derive from three facts: (i) many gut microbescannot be cultured; (ii) culturing leads to loss of adhe-sive ability (i.e., implantability) over time; and (iii)only the full-microbial spectrum can deliver the com-plete microbial genomic repertoire, including thefull GI virome [Reyes et al., 2010]. First, based onDNA sequencing and 16S ribosomal RNA analysis ofintact microbial communities, we know that currentlyculturable bacteria represent only a small fraction of themicrobiota [Flint et al., 2010; Stewart, 2012]. Second,we know that repeated passaging and culturingdecreases bacterial genome size and compromises criti-cal aspects of microbial function [Nilsson et al., 2005].Jorup-Rönström et al. [2012] demonstrated this point inthirty-two CDI patients treated with “stable,” humanfecal extract that was cultured and re-cultured for up toten years under anaerobic conditions. In contrast to the

93% cure rate achieved with one dose of purified, frozenfull-spectrum microbiota delivered by colonoscopy[Hamilton et al., 2013], only 68% (15/22) of enema-treated patients and 80% (4/5) of colonoscopy-treatedpatients were cured with one dose of continuallyre-cultured organisms. More generally, we know thatoral probiotics, which are often mass-produced fromstock cultures, implant only transiently for approxi-mately 7–17 days after ceasing oral administration[Saxelin et al., 2010]. Finally, by studying the structureand assembly of complex microbial communities, Burkeet al. [2011] found that bacterial gene function corre-lates with communities of organisms rather than withspecies. This implies that a complete, full-spectrummicrobial community can provide the (as yet unidenti-fied) gene products necessary to eradicate CDI in largepatient populations, while a selected, restricted commu-nity will be less efficacious due to its restricted generepertoire. In addition, the varied microbial deficienciespresent in different gut dysbioses and individuals [Tvedeand Rask-Madsen, 1989; Chang et al., 2008; Khorutset al., 2010; Petrof et al., 2013] means that tailoringcultured replacements could prove challenging.

TABLE 1. Companies Developing Microbiome-Related Therapeutics[Derived from Olle, 2013]

Company Founded Focus

CIPAC LimitedMalta

2012 Full-spectrum microbiota for CDIand other indications

Rebiotix Inc.(formerly MicroBeX)Roseville, MN

2011 Microbiota suspension for“microbiota restoration therapy”for CDI

Monarch LabsIrvine, CA

2005 Service for autologous cGMPFMT product with follow-onmedical microbiota for allograftuse

Osel Inc.Mountain View, CA

1998 Single strains of native andgenetically engineered bacteriafor urogenital and GI diseaseindications

EnterologicsSt Paul, MN

2009 Bacterial strain previously used indietary supplements

AvidBioticsSan Francisco, CA

2005 Targeted bactericidalnon-antibody proteins

GT BiologicsAberdeen, Scotland

Unknown IBD therapies frommicrobiome-based molecules

Seres HealthCambridge, MA

2012 Selected, cultured GI bacteria forrestoring healthy gutmicrobiome in CDI and otherindications

Second GenomeSan Bruno, CA

2010 Exploring role of gut microbes inulcerative colitis

Vedanta BioscienceBoston, MA

2010 Immunomodulating therapiesbased on human microbiome

CDI, Clostridium difficile infection; FMT, fecal microbiota transplan-tation; GI, gastrointestinal; IBD, inflammatory bowel disease.

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Cultured Microbiota Products

Given the demonstrated effectiveness of FMT forrecurrent C. difficile infection (R-CDI), researchers areworking to develop standardized cultured products totreat CDI. This approach offers two key advantagesover the “full-spectrum” approach: (i) it eliminates theneed to find and screen healthy donors on an ongoingbasis, and (ii) it allows all bacterial species and strainsto be defined within a product. Canadian researchershave reported an “artificial stool” containing thirty-three selected bacterial strains obtained, purified,and cultured from a single donor. This approach curedCDI in the two patients treated, although only 25–36%of the implanted donor strains remained after sixmonths [Petrof et al., 2013]. As noted in Table 1, SeresHealth (Cambridge, MA) is working to develop

selected, cultured GI bacteria to restore healthy gutmicrobiota to treat CDI. Whether cultured bacteria willlose their ability to adhere and implant within the gutover time and whether their limited genomic repertoirecan eradicate CDI in a large patient population is yet tobe determined.

Full-Spectrum versus Selected,Cultured Microbiota

Unsurprisingly, the two main categories ofFMT-related microbial therapies—the full-spectrumapproach and the selected, cultured approach—offerdistinct profiles of advantages and disadvantages(Table 2).

TABLE 2. Full-Spectrum Microbiota versus Selected, Cultured Microbiota

Therapeutic approach Advantage Disadvantage

Full-spectrummicrobiota

Safety and efficacy extensively studied and confirmedin Clostridium difficile infection in hundreds ofpatients

Requires identifying healthy donors and screeningon ongoing basis

Emerging clinical evidence of safety and efficacy fortreating a wide range of diseases, including ulcerativecolitis, Crohn’s disease, diabetes, obesity, Parkinson’sdisease, and multiple sclerosis

Less scalable than cultured approach

Minimal odor relative to crude stool Higher cost of goods than cultured approachStable at −80°C storage Long-term benefits unknownProvides complete gene repertoire of gut microbial

population, thereby addressing microbial variationinindividuals and clinical indications

Allows for synergistic population effects driven bycomplete microbe collection, which includesthe normal human virome (including naturalbacteriophages that may play a role in efficacy)

Both microbial “mother liquor” and microbes can beused to provide cellular products such as vitamins,metabolites, and antibiotics

Semi-defined cell populationCan be assayed for pathogens

Selected, culturedmicrobiota

Clinical evidence of safety and efficacy for treatingClostridium difficile infection in two patients

Passaging effect decreases colonization capacityand efficacy over time due to adherence andimplantability issues and to gene deletion andrearrangement

Minimal odor relative to crude stool Provides limited gene repertoire of gut microbialpopulation, thereby limiting ability to addressmicrobial variation in individuals and clinicalindications

Stable at −80°C storage Contamination issues can be significantEasily scalable Potential long-term effects unknownDefined cell population Absence of normal virome and natural

bacteriophagesLower cost of goods than full-spectrum approachCan be assayed for pathogens

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The FMT-based therapeutic approaches dis-cussed above focus on using the normal gut microbiotato restore homeostasis and missing componentsand homeostasis to dysbiotic guts. The therapeuticapproaches described below focus on identifying spe-cific abnormal gut microbes and devising ways to targetand destroy them.

MICROBIOTA AS IMMUNIZING AGENTS

Enterotoxigenic Escherichia coli (ETEC)diarrhea vaccine

An oral vaccine is being developed to treat ETECdiarrhea, a leading cause of illness among internationaltravelers to developing countries [Black, 1990]. ETECis also the primary cause of diarrhea in children livingin low- and middle-income countries, resulting in 400million diarrheal episodes and approximately 300 000child deaths per year [Fleckenstein et al., 2010]. Thenovel vaccine consists of four inactivated E. coli bacte-rial strains of intestinal origin and the subunit B of thecholera toxin, which has shown a good response in aPhase I study [Svennerholm and Lundgren, 2012].

E. coli Vaccine against Recurrent UrinaryInfections—URO-VAXOM

URO-VAXOM, a purified extract from lysates ofeighteen heat-killed uropathogenic E. coli strains origi-nating from the GI microbiome, is the only non-antimicrobial urinary tract infection (UTI) prophylacticregimen recommended by the European Association ofUrology [Grabe et al., 2013]. The extract has beenshown to stimulate macrophages [Bessler et al., 2010],B-lymphocytes [Van Pham et al., 1990], and immuno-competent cells in Peyer’s patches, and to increase IgAlevels against E. coli in intestinal secretions [Baier et al.,1997]. A meta-analysis of five double-blind, placebo-controlled studies showed that URO-VAXOM signifi-cantly reduced recurrent UTIs without significant sideeffects [Bauer et al., 2002; 2005].

Clostridium bolteae and AutisticSpectrum Disorders

Chronic GI disorders such as diarrhea and consti-pation correlate strongly with autistic spectrum disor-ders (ASDs) [Finegold et al., 2010], with documentedoccurrence rates greater than 90% in one landmarkstudy [Parracho et al., 2005]. Several studies (e.g.,Finegold et al., [2002]) have reported an abundance ofpathogenic intestinal flora in autistic children compared

with healthy controls. Because clostridial speciesproduce metabolic end-products that can alter motilityof the GI tract, variations in Clostridium spp. levels canaffect GI function. In other studies, Finegold et al.[2010], Finegold [2011], and Sandler et al. [2000] foundstatistically significant differences of C. bolteae andClostridium clusters between autistic and control chil-dren and documented improved behavioral, cognitive,and GI aspects of autism following oral vancomycin,which is not absorbed from the GI tract.

One approach being considered by someresearchers [S. Finegold and J. Adams, personal com-munication] to address ASD symptoms is to provideaffected individuals with the full spectrum of healthygut microbiota to restore the gut microbiome anderadicate “rouge” Clostridium spp. For children, oraladministration—perhaps by adding freeze-driedmicrobiota to food or drink—is more suitable thannasogastric or rectal administration. Whether a single“oral delivery” of FMT” could induce rapid and positivelong-term effects on ASD-related GI symptoms, as tra-ditional FMT does with over 90% efficacy for recurrentCDI, remains to be seen. The success of an FMT-restored microbiome to improve behavioral and/or cog-nitive aspects of ASD (theoretically possible due toaltering the profile of toxins, neurotransmitters, andother molecules secreted by gut microbes) remains tobe determined. Another approach to address ASDwould be to identify, target, and eliminate “rogue”gut microbes associated with ASD symptoms suchas Clostridium bolteae, recently reported as an over-abundant bacterium in the guts of autistic children suf-fering with GI ailments [Pequegnat et al., 2013]. Theseresearchers plan to pursue development of a capsularpolysaccharide-based vaccine to prevent C. bolteaeinfection, in the hope of addressing GI and potentiallyother symptoms of ASDs.

Vaccine Candidates to Prevent RecurrentC. difficile Infection

Vaccines targeting C. difficile are in various stagesof development. Viropharma Inc. is developing an oralvaccine candidate (VP 202621) containing nontoxigenicC. difficile spores to prevent CDI recurrence after aninitial acute infection. A Phase 1 safety study of VP202621 showed good overall tolerance and GI tractcolonization, particularly in patients with antibiotic-disrupted microbiota [Villano et al., 2012]. A Phase 2study for safety and efficacy in preventing recurrentCDI is currently underway. In partnership with Massa-chusetts Biological Laboratories Inc. and Medarex,Merck is developing MK-3415A, a combination oftherapeutic antibodies targeting C. difficile toxins A and

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B. Currently in Phase 3 trials, actoxumab/bezlotoxumabis being evaluated for preventing R-CDI.

BIOACTIVE AGENTS ORIGINATING INTHE GUT MICROBIOTA

Bacteriocins

Numerous bacteria produce antimicrobial sub-stances called bacteriocins that confer competitiveadvantage by killing or inhibiting the growth of otherbacteria, usually species closely related to thebacteriocin-producer. Originally thought to be pro-duced predominantly by Lactobacillus, bacteriocinswere subsequently found to be produced by most bac-teria [Bakkal et al., 2012; Cotter et al., 2013]. Thesesmall molecules have not yet reached clinical applica-tion, although the global food industry has used nisin,the most well-research bacteriocin, for over 50 years toextend the shelf life of food products. Nisin wasapproved in 1988 as a food additive in canned goods toprevent the growth of Clostridium botulinum [Joneset al., 2005].

Probiotic Bacteria

Probiotics are living bacteria or other microorgan-isms that, when consumed, can benefit the health ofhumans or animals. Beneficial bacteria commonly usedas probiotics include Lactobacillus and Bifidobacteriumspp. and other lactic acid bacteria. Two examples ofprobiotic bacteria sourced from the human gutmicrobiome include Lactobacillus GG (LGG orCulturelle) and E. coli Nissle 1917 (Mutaflor).Lactobacillus rhamnosus GG (ATCC 53103) is a strainof L. rhamnosus isolated from the intestinal tract ofa healthy human being [Silva et al., 1987] and laterfound useful for treating diarrhea, UTI, eczema, andother conditions. Nissle isolated E. coli in 1917 from thefeces of a soldier who, unlike his many comrades, didnot develop enterocolitis during an outbreak of theinfection [Nissle, 1918]. Numerous double-blind,placebo-controlled trials have demonstrated the thera-peutic success of probiotic E. coli Nissle 1917 in man-aging GI infections and conditions [Konturek et al.,2009; Kruis et al., 2012]. Several head-to-head trialscomparing E. coli Nissle 1917 to mesalazine in inflam-matory bowel disease have found it to be equivalentto mesalazine in inducing and maintaining remissionand in preventing disease relapse [Schultz, 2008;Katz, 2010].

Bacteriophages

Bacteriophages (or phages) viruses that infect andreplicate within bacteria are capable of selectively

eradicating pathogenic bacterial strains. First isolatedfrom human stool in 1915 by Twort and then in 1917 byd’Herelle [Lederberg, 1996] who described “an invis-ible, antagonistic microbe to dysentery bacilli,” theyhave been proposed to combat bacterial infections inhumans [Ackermann, 2011]. The immense viral com-ponent of the human microbiome, the human virome,has just begun to be elucidated and appreciated [Reyeset al., 2010; Minot et al., 2011]. While it is clear thatinterpersonal variation of the gut virome between indi-viduals is high and that the intrapersonal diversitywithin an individual is low [Reyes et al., 2010], theeffect that bacteriophage chromosomes have on thefunctional genomic repertoire of the human gutmicrobiome is unknown.

CONCLUSIONS

We have only started to recognize and develop thetherapeutic potential of the human GI microbiome. Inthe near term we anticipate refined, regulated thera-peutics for fecal microbiota transplants to addressrecurrent CDI and to evaluate for treating other dis-eases and disorders. In the longer term we envisiontherapeutic products emerging in a variety of forms,including lyophilized encapsulated human microbiomebiologic agents, specialized anaerobic human probioticconsortia, and enteric vaccines. Now that we havebegun to appreciate the function and power of ourindividual and collective microbiomes, we can begin toleverage what they can teach and offer us. The humanGI microbiome offers therapeutic riches waiting to berealized.

FINANCIAL AND COMPETINGINTERESTS DISCLOSURE

T. J. Borody has a financial interest in the Centrefor Digestive Diseases, where fecal microbiota trans-plantation is a treatment option; in addition, he has filedpatents in this field. D. Peattie has a financial interest inCIPAC Limited, a privately held company developing atherapeutic product for fecal microbiota transplanta-tion. J. Campbell has no financial interest or affiliationwith any institution, organization, or company relatingto the manuscript.

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