Gastroenterology 2014;146:1547–1553

Role of the Intestinal Microbiota in Resistance to Colonizationby Clostridium difficile

1 2,3 THEGU

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1Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan; 2Division ofInfectious Diseases, Department of Internal Medicine, 3Department of Microbiology and Immunology, University of Michigan,Ann Arbor, Michigan

Robert A. Britton Vincent B. Young

Antibiotic-associated infection with the bacterial pathogenClostridium difficile is a major cause of morbidity andincreased health care costs. C difficile infection followsdisruption of the indigenous gut microbiota by antibiotics.Antibiotics create an environment within the intestine thatpromotes C difficile spore germination, vegetative growth,and toxin production, leading to epithelial damage and co-litis. Studies of patients with C difficile infection and animalmodels have shown that the indigenous microbiota caninhibit expansion and persistence of C difficile. Although thespecific mechanisms of these processes are not known, theyare likely to interfere with key aspects of the pathogen’sphysiology, including spore germination and competitivegrowth. Increasing our understanding of how the intestinalmicrobiota manage C difficile could lead to better means ofcontrolling this important nosocomial pathogen.

Keywords: Colonization Resistance; C difficile; Microbiome;Microbial Ecology; Antibiotics.

he fulfillment of Koch’s postulates for Clostridium

Abbreviations used in this paper: CDI, Clostridium difficile infection; FMT,fecal microbiota transplantation.

© 2014 by the AGA Institute0016-5085/$36.00

http://dx.doi.org/10.1053/j.gastro.2014.01.059

Tdifficile in 1977 was one of the first formal recogni-tions of a microbiome-related disease.1 Although C difficileitselffit into themold of a typical bacterial pathogen, C difficileinfection (CDI) was unique in that previous antibiotic treat-ment of the patient was necessary for development of the fulldisease phenotype. We review C difficile as a pathogenicorganism and discuss the basic epidemiologic features of CDI,as well as its molecular pathogenesis. Research into thedeveloping story of how C difficile interacts with the indige-nous intestinal microbiota has provided important insightsinto the role of the microbiome in human health and disease.

C difficile and Antibiotic-AssociatedColitis

C difficile is a gram-positive, anaerobic, spore-formingbacterium that was first isolated from the feces of healthy

infants.2 Interestingly, the organism appears to be highlyprevalent in infants, who rarely show any clinical signs ofinfection with fully virulent strains.3,4 As the child andsubsequently the microbiome matures, C difficile is nolonger readily detected; in the healthy adult population,asymptomatic colonization with this organism is consideredto be a rare event.4,5 Rates of colonization are increasedprimarily among adults with frequent health care–associated contact and patients in chronic-care facilities.5

The pathogenesis of CDI involves production of 2members of the family of large clostridial toxins: TcdA andTcdB, which are products of genes located with a pathoge-nicity locus.6–8 In C difficile, these toxins are large proteinswith glycosyltransferase activities targeting the host gua-nosine triphosphatases Rho, Rac, and Cdc42, which regulateactin.9,10 Administration of purified toxin in model systems,including ligated ileal loops, is sufficient to replicate theintestinal damage that is characteristic of CDI.11 Further-more, there is evidence that these toxins have systemic ef-fects on the host.12 Although TcdA and TcdB are the mostwell-studied virulence factors of C difficile, other putativevirulence factors also have been described.13 A subset ofC difficile strains produce an additional toxin, often referredto as binary toxin, that is related to iota toxin from Clos-tridium perfringens.14,15 Recent interest in binary toxinlikely is related to the fact that the recent epidemic strainsof C difficile, exemplified by the NAP1/027/BI strain, oftenproduce this putative virulence factor.16

The epidemiology of CDI has been characterized by in-creases in incidence and severity that began around the year2000.17,18 It recently was estimated that CDI is the most

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common health care–associated infection, producing esti-mated annual hospital costs of more than $3 billion.19 Theincrease in virulence has been associated with theincreasing isolation of epidemic strains related to NAP1/027/BI.16,20 There has been debate as to whether epidemicstrains have enhanced virulence properties that result inmore severe disease or if these strains simply cause morecases of CDI without causing worse patient outcomes.21

However, the overall increase in incidence and severity ofCDI in the past decade has spurred research into C difficilepathogenesis. During the same period of time, the growinginterest in the role of the indigenous microbiota in humanhealth and disease, exemplified by projects such as theNational Institutes of Health’s Human Microbiome Proj-ect22,23 and the European Metagenomics of the Human In-testinal Tract consortium,24 has produced a scientificenvironment well suited for the study of CDI. There is nowmuch interest in the interactions between this pathogen inthe intestinal microbiome.

The Microbiota Affects C difficileInvasion

The concept that the indigenous gut microbiota mediatessome form of resistance against colonization by bacterialpathogens was proposed long before C difficile was associ-ated with antibiotic-associated pseudomembranous colitis.25

Figure 1. Cycle of CDI. Antibiotic administration alters the indipermits germination ofCdifficile spores andexpansion of thepathsymptoms. Antibiotics directed against C difficile can decreasemicrobiota to a state of colonization resistance cures CDI. Howezation by C difficile, then patients have recurring CDI. In certaineradicate the pathogen. In other cases, therapeutic restoration o

Productive infection of animals with enteric bacterial path-ogens often requires pre-exposure to antibiotics.26–28 How-ever, until recently, the specific mechanisms by which theindigenous microbiome could prevent colonization by path-ogenic organisms were not of widespread scientific interest.The requirement for antibiotic treatment before infectionwith a pathogen was regarded simply as a technical hurdle toovercome to investigate specific interactions between thepathogen in the host. However, increasing interest in the roleof the indigenous microbiome in maintaining intestinal ho-meostasis has focused attention on its interactions withpathogens. The development of tractable animal models forstudying CDI and the concurrent application of experimentalmicrobial ecology to host-associated bacterial communitieshas led to multiple reports of how they influence the devel-opment of CDI and its complications. Work in experimentalmodels of CDI and with clinical material from patients hasindicated that C difficile, the intestinal microbiota, and otherhost factors form a complex system; specific alterations tothis system can promote pathogenesis of CDI (Figure 1).

Studies in animals and human beings have shown thatantibiotics have profound and, in some cases, long-lastingeffects on the community structure of the gut micro-biota.29,30 Antibiotics affect not only the overall size of thegut bacterial community, but also the composition of thecommunity (they alter proportions of specific bacterialspecies). However, these effects do not necessarily occur

genous intestinal microbiota, producing an environment thatogen.Cdifficileproduces toxins that cause colitis and resultingthe load of the pathogen and toxin production. Returning thever, if the microbiota is unable to restore resistance to coloni-cases, repeat courses of anti–C difficile antibiotic therapy canf a diverse microbiota via FMT is required to overcome CDI.

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together.31 These effects on community structure are pre-sumed to alter functions of the community,32 and, for pur-poses of this discussion, might reduce resistance to CDI.

The hamster model of CDI initially was used to studyinteractions among C difficile, the microbiota, and host tis-sues,1 but mouse models are increasingly being used. In thehamster model of CDI, clindamycin is the standard antibioticused to generate a susceptible state.33 A recent study fol-lowed up the effect of clindamycin on the gut microbiota ofhamsters associated with experimental CDI.34 In the mousemodels of disease, a variety of antibiotics have been shownto lead to loss of resistance to C difficile colonization.35–38 Inaddition, specific effects of antibiotics on the mouse gutmicrobiome have been described, allowing researchers toinvestigate how specific members of the microbiomemediate resistance to colonization.39,40

Recent interest inmousemodels of CDI beganwith a 2008report in which Chen et al35 used a cocktail of 5 antibiotics,followed by a single dose of clindamycin, to render micesusceptible to colonization by C difficile and subsequentdevelopment of a severe colitis. A subsequent study by aseparate group, using this same model, associated suscepti-bility with a loss of the normal members of the cecal com-munity (mainly members of the family Lachnospiraceae) anda relative increase in the abundance of Enterobacteriaceae.37

Furthermore, the severity of disease was related to therelative dynamics of these bacterial families, with more se-vere outcomes associated with a failure of Lachnospiraceaeto return and a continued dominance with Enter-obacteriaceae. Formal tests of the relative roles of these or-ganisms in mediating colonization resistance wereaccomplished by monocolonization of germ-free mice withmurine representatives of each of these groups of bacteria.39

Colonization with a murine E coli isolate had no effect on theability of C difficile to colonize the intestine and induce colitis,whereas a Lachnospiraceae isolate significantly reduced thelevels of colonization by C difficile and the severity of colitis.

Then, Lawley et al40 showed that administration ofclindamycin permitted long-term, nonfatal colonization ofmice with C difficile.40 They identified 6 phylogeneticallydiverse species of bacteria that, when administeredtogether, could restore colonization resistance and preventchronic carriage of C difficile. It is interesting to note thatclindamycin has not been reported to disrupt resistanceagainst C difficile colonization in all genetically identicalmice. Two studies reported that clindamycin can overcomecolonization resistance in C57BL/6 mice,38,40 whereas athird group only achieved transient (2–3 days) colonizationof the same strain of mice with C difficile after an identicaldose of clindamycin.37 Baseline differences in microbiota ofmice from different colonies might cause these differencesin results. Comparative microbiome analysis of thesedifferent groups of mice could provide additional informa-tion about the roles of specific groups of bacteria in resis-tance to colonization with C difficile.

Experimental infection of human beings with C difficile isnot ethical, but studies of the microbiomes of patients withnaturally occurring C difficile infection have provided in-formation that correlates with findings from animal studies.

Antibiotics alter the structure of microbial communities,30

and fecal samples from patients who developed CDI afterantibiotic therapy have decreased diversity and otherchanges in the microbiota, compared with patients who didnot develop CDI.34 The intestinal microbiome of patientswith recurrent disease has lower diversity than patientswithout CDI or those whose CDI was treated successfullyand did not recur.41 Furthermore, patients with primary orrecurrent symptomatic CDI have fecal microbiomes thatdiffer in composition from patients with asymptomaticcolonization by C difficile.4 This observation makes it clearwhy fecal transplantation is so successful in the treatment ofrecalcitrant CDI (see fecal microbiota transplantation [FMT]review by Elaine Petrof and Alex Khoruts in this issue ofGastroenterology).42

Microbiome Structure andFunction in Resistance toC difficile Colonization

Although disruption of the healthy fecal microbiota cancreate an ecologic environment in which C difficile canflourish, little is understood about the changes in the envi-ronmental and microbial communities that occur in the in-testine. Host and microbial community factors are involveddirectly in C difficile invasion of a dysbiotic intestinal com-munity. C difficile selects specific environments for expansionand colonization that appear to occur via complementarymechanisms. The development of CDI may involve alteredbile acid metabolism and competition among microbes fornutrients, although other mechanisms by which the indige-nous microbiota resists colonization by pathogenic bacteriaexist.43

Bile Salts in C difficile SporeGermination and Vegetative Growth

Germination of C difficile spores can be stimulated by acomplex mixture of bile salts. The mechanisms by which in-dividual bile salts and amino acids affect spore germinationonly recently have been elucidated.44–47 Interestingly, theprimary bile acids cholate and chenodeoxycholate havedifferent effects on the spore germination process. The con-jugated and deconjugated forms of cholate, along with theamino acid glycine, function together to promote germinationof C difficile spores in vitro, whereas chenodeoxycholate is apotent inhibitor of germination.45,46 Importantly, physiolog-ically relevant levels of these bile acids are sufficient to affectgermination in vitro. Chenodeoxycholate competitively in-hibits germination at a concentration approximately 10-foldlower than the stimulatory concentration of cholate. So, un-der normal physiological conditions, chenodeoxycholatesuppresses C difficile invasion in vivo.

Cholate and chenodeoxycholate are metabolized into thesecondary bile acids deoxycholate and lithocholate, respec-tively. In line with the effects of primary bile acids on sporegermination, deoxycholate stimulates germination whereaslithocholate is a potent inhibitor. However, deoxycholate is

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toxic to vegetative C difficile; germination in the presence ofthis compound leads to quick death of bacteria. Therefore,an environment in which cholate levels are markedly higherthan these other primary and secondary bile acids wouldsupport C difficile germination and expansion.

Administration of antibiotics causes a large shift in thebile acid pool in the cecum of mice, which can disrupt thisbalance and lead to spore germination (Figure 2).48,49

Cholate is not detectable in the cecum of mice with anormal microbiota, but antibiotics increase its levels whilelevels of secondary bile acids remain low. Therefore,C difficile spores are not expected to germinate until theyare induced to do so by changes in concentrations of bileacids in the intestine.

Until this past year, only in vitro studies had shown thatbile acids were able to impact the germination of C difficilespores. Recently, researchers have shown that bile acidderivatives that inhibit spore germination in vitro reducethe ability of C difficile to colonize the intestines of mice.53

Furthermore, in an elegant genetic screen, Francis et al54

identified a putative protease (CspC) that is required forspore germination in vitro and could be a long-sought-afterspore germination receptor. cspC mutants of C difficilecannot germinate in the presence of cholate and glycine, andare unable to colonize and cause disease in hamsters.Therefore, the ability to sense bile is critical for C difficilecolonization of the intestine. These findings support a rolefor bile metabolism in C difficile colonization of the intestine.

Ecologic Niche Expansion:Availability of Food Sources

The intestinal microbiota also can impact colonization bypathogens such as C difficile by providing competition for

Figure 2. Bile acids affect the ability of C difficile to colonize theand chenodeoxycholate (CDCA) are high in the small intestine, ais less well absorbed than chenodeoxycholate in the terminchenodeoxycholate, resulting in increased spore germination.50

exposed to the toxic effects of deoxycholate (DCA), further prevemicrobiota is disrupted with antibiotics, bacterial transformatiosuppressed. In this case, CA levels remain high while bacterialincreased CA-mediated spore germination and a loss of inhibitois that expansion of this pathogen will occur readily in the colo

resources. Antibiotic disruption of the intestinal microbiotaalters sources of nutrients in at least 2 ways. First, byreducing the diversity of the intestinal microbiota, antibi-otics decrease competition for limited resources and free uppreviously unavailable ecologic niches. Second, bacterial celllysis releases carbon sources that the remaining communitycan consume. Despite the large number of C difficile ge-nomes that have been sequenced, there have been relativelyfew studies of the metabolic networks used by C difficile tocolonize the intestine. In vitro studies have shown thatC difficile can grow on simple sugars, amino acids, peptides,and complex carbohydrates.55–58

Wilson and Perini59 used continuous-flow cultures toinvestigate whether a complex microbiota suppressesC difficile infection by competing for available resources. Ingrowth medium formulated from feces of germ-free mice,cultivation of the cecal microbiota from hamsters gave riseto a community that not only resisted C difficile invasion butalso could displace an already established C difficile infec-tion. When hamster cecal communities were grown on arich synthetic medium, they lost their ability to resistC difficile, although they were still diverse. To investigatewhat components of the germ-free mouse fecal mediumpromoted expansion of intestinal bacteria that suppressesC difficile, the investigators added back individual carbonsources that were major components of this medium.Interestingly, they found that components of mucin (sialicacids, N-acetylglucosamine, and N-acetylneuraminic acid,which are abundant in the fecal medium) fully restored theability to suppress C difficile to communities cultured in richmedium.

In support of these findings, C difficile recently was re-ported to use host-derived sialic acids to proliferate in theintestines of mice.60 C difficile was found to be able to break

intestinal tract. In the healthy microbiota, levels of cholate (CA)nd chenodeoxycholate inhibits germination. Because cholateal ileum, the net effect is an increased ratio of cholate:

–52 As vegetative C difficile cells move into the colon, they arenting C difficile from gaining a foothold in the colon. When then of primary bile acids (such as CA to DCA) can be greatlytransformation of CA to DCA does not occur. This results inry effects of deoxycholate on C difficile growth. The net effectn.

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down sialic acids and use them as a carbon source; mutantC difficile that lacked this activity had a 4-fold reduction incolony-forming units per gram of feces. Although these re-sults show that C difficile can metabolize sialic acids to reachhigh densities in the intestine, additional factors are likely tobe required for expansion of C difficile in the intestine.

Freter et al61 proposed a chemostat model for pathogencolonization, based on observations made while studyingthe persistence of individual microbes in a complex micro-biota. This model might explain how C difficile persists in theintestine after antibiotic treatment and how competingmicrobes could displace C difficile during bacteriotherapytreatment.62 For example, if 2 organisms are in directcompetition for a limited nutrient, the organism that can usethis nutrient most efficiently will outcompete the other. Thismodel also predicts that when one organism already fullyoccupies an intestinal niche, it is difficult for other organ-isms to invade.

In support of this idea, colonization of hamsters with anontoxigenic strain of C difficile strain before challenge witha toxigenic strain can completely prevent C difficile–associated colitis.63,64 These findings indicate that the non-toxigenic strain occupies a niche that cannot be taken overby a new strain. In a prospective study, patients who werefollowed up until they developed CDI were found to havereduced proportions of the family Clostridiales IncertaeSedis XI just before infection, compared with individualswho did not develop CDI.65 C difficile is a member of thefamily Clostridiales Incertae Sensu XI—loss of the Clos-tridiales Incertae Sedis XI could create a niche that C difficileis able to occupy. However, because of other alterations inthe intestinal microbiota of these patients, we cannotconclude that loss of these microbes is directly responsiblefor the subsequent C difficile colonization.

Therapeutic Interventions WithDefined Microbial Communities

Administration of defined microbes, usually single-strainprobiotics, has had only limited success in ameliorating thesymptoms of CDI. This is not surprising given recent animaland human studies associating large-scale alterations inmicrobial communities with antibiotic use and CDI. FMT isbecoming more accepted for the treatment of recurrent CDI(reviewed by Petroff and Khoruts in this issue of Gastro-enterology).42 Despite its success, the undefined and com-plex nature of stool makes it difficult to envision widespreadFMT therapy for individuals other than those with severerecurrent CDI.

Defined communities of microbes have been shown to beeffective against recurrent CDI in patients and animalmodels. Interestingly, the 3 defined communities that areeffective against recurrent CDI are remarkably simple,compared with the diversity of the intestinal microbiota. Inaddition, each community shares few organisms at thegenus level, indicating there will be many combinations thatcan suppress CDI. Tvede and Rask-Madsen66 reported that acocktail of 10 facultative aerobes and anaerobes was

effective against recurrent CDI in 5 patients. They noted thatall of the patients were deficient in Bacteroides before thecocktail was administered, and then afterward the genuswas detected in fecal samples from the patients. Petrofet al67 reported using a 33-strain combination of microbescultured from human fecal samples to treat 2 patients withrecurrent CDI. The strains were selected based primarily ontheir antibiotic susceptibility and safety, while the re-searchers attempted to generate as much taxonomic di-versity as possible. Both patients recovered from CDI,although it is not clear this was a direct result of populationof the intestine with the defined microbes or an indirectresult of stimulation of the indigenous microbiota.Sequencing analysis of microbial communities from fecalsamples of the patients indicated that the 33 strainsadministered to the patients were gradually lost.

Although Petrof et al67 only included 2 patients in theirtrial, the concept that the indigenous microbiota can bealtered through addition of microbes was supported by theeffects of a 6-species community of microbes administeredto a mouse model of CDI.40 Interestingly, none of the 6strains appeared to be able to colonize the mice over longtime periods, but a single administration of these microbesincreased the microbial diversity in the intestines of mice. Ittherefore appears that the defined communities might actby stimulating a suppressed indigenous microbiota.

ConclusionsThe ability of FMT to cure recurrent CDI in up to 95% of

patients provides support for strategies aimed at restoringthe diversity of the intestinal microbiota. The efficacy of fecalcommunities from a variety of donors with diverse micro-biotas indicates that many microbial community structureswill be able to prevent or treat CDI. For most cases, the fecalmaterial for transplantation spends a significant amount oftime being prepared under aerobic conditions, thus it is likelythat a substantial proportion of the microbes being admin-istered in FMTs do not remain viable. Studies are underwayto understand how feces, as well as defined microbial com-munities, are able to interfere with CDI. It is also critical tounderstand not only how these communities suppress Cdifficile invasion, but how the implantation of new commu-nities may affect other aspects of human health. Studies havelinked the intestinal microbiota with diseases such as dia-betes, colon cancer, obesity, and atherosclerosis. If we use themicrobiota to treat disease, we inadvertently may increasethe risk of others. Continued research into the roles of theintestinal microbiome in health and disease might lead toways to manipulate this community for benefit while mini-mizing unintended consequences.

References

1. Bartlett JG, Onderdonk AB, Cisneros RL, et al. Clinda-

mycin-associated colitis due to a toxin-producing spe-cies of Clostridium in hamsters. J Infect Dis 1977;136:701–705.

1552 Britton and Young Gastroenterology Vol. 146, No. 6

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2. Hall IC, O’Toole E. Intestinal flora in new-born infantswith the description of a new pathogenic anaerobe,Bacillus difficilis. Am J Dis Child 1935;49:390–402.

3. Thompson CM Jr, Gilligan PH, Fisher MC, et al. Clos-tridium difficile cytotoxin in a pediatric population. Am JDis Child 1983;137:271–274.

4. Rousseau C, Poilane I, De Pontual L, et al. Clostridiumdifficile carriage in healthy infants in the community: apotential reservoir for pathogenic strains. Clin Infect Dis2012;55:1209–1215.

5. Rea MC, O’Sullivan O, Shanahan F, et al. Clostridiumdifficile carriage in elderly subjects and associatedchanges in the intestinal microbiota. J Clin Microbiol2012;50:867–875.

6. Voth DE, Ballard JD. Clostridium difficile toxins: mecha-nism of action and role in disease. Clin Microbiol Rev2005;18:247–263.

7. Pruitt RN, Lacy DB. Toward a structural understanding ofClostridium difficile toxins A and B. Front Cell InfectMicrobiol 2012;2:28.

8. Shen A. Clostridium difficile toxins: mediators of inflam-mation. J Innate Immun 2012;4:149–158.

9. Hecht G, Pothoulakis C, LaMont JT, et al. Clostridiumdifficile toxin A perturbs cytoskeletal structure and tightjunction permeability of cultured human intestinalepithelial monolayers. J Clin Invest 1988;82:1516–1524.

10. Just I, Fritz G, Aktories K, et al. Clostridium difficile toxinB acts on the GTP-binding protein Rho. J Biol Chem1994;269:10706–10712.

11. Kelly CP, Becker S, Linevsky JK, et al. Neutrophilrecruitment in Clostridium difficile toxin A enteritis in therabbit. J Clin Invest 1994;93:1257–1265.

12. Hamm EE, Voth DE, Ballard JD. Identification of Clos-tridium difficile toxin B cardiotoxicity using a zebrafishembryo model of intoxication. Proc Natl Acad Sci U S A2006;103:14176–14181.

13. Vedantam G, Clark A, Chu M, et al. Clostridium difficileinfection: toxins and non-toxin virulence factors, andtheir contributions to disease establishment and hostresponse. Gut Microbes 2012;3:121–134.

14. Rupnik M, Grabnar M, Geric B. Binary toxin producingClostridium difficile strains. Anaerobe 2003;9:289–294.

15. Stiles BG, Wigelsworth DJ, Popoff MR, et al. Clostridialbinary toxins: iota and C2 family portraits. Front CellInfect Microbiol 2011;1:11.

16. McDonald LC, Killgore GE, Thompson A, et al. Anepidemic, toxin gene-variant strain of Clostridium diffi-cile. N Engl J Med 2005;353:2433–2441.

17. Pepin J, Valiquette L, Alary ME, et al. Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to2003: a changing pattern of disease severity. CMAJ2004;171:466–472.

18. Freeman J, Bauer MP, Baines SD, et al. The changingepidemiology of Clostridium difficile infections. ClinMicrobiol Rev 2010;23:529–549.

19. Dubberke E. Clostridium difficile infection: the scope ofthe problem. J Hosp Med 2012;7(Suppl 3):S1–S4.

20. Goorhuis A, Bakker D, Corver J, et al. Emergence ofClostridium difficile infection due to a new hypervirulent

strain, polymerase chain reaction ribotype 078. ClinInfect Dis 2008;47:1162–1170.

21. Walk ST, Micic D, Jain R, et al. Clostridium difficileribotype does not predict severe infection. Clin Infect Dis2012;55:1661–1668.

22. Proctor LM. The Human Microbiome Project in 2011 andbeyond. Cell Host Microbe 2011;10:287–291.

23. Human Microbiome Project C. A framework for humanmicrobiome research. Nature 2012;486:215–221.

24. Arumugam M, Raes J, Pelletier E, et al. Enterotypes ofthe human gut microbiome. Nature 2011;473:174–180.

25. Freter R. The fatal enteric cholera infection in the guineapig, achieved by inhibition of normal enteric flora. J InfectDis 1955;97:57–65.

26. BohnhoffM,DrakeBL,Miller CP. Effect of streptomycin onsusceptibility of intestinal tract to experimental Salmonellainfection. Proc Soc Exp Biol Med 1954;86:132–137.

27. Wadolkowski EA, Laux DC, Cohen PS. Colonization ofthe streptomycin-treated mouse large intestine by a hu-man fecal Escherichia coli strain: role of growth in mucus.Infect Immun 1988;56:1030–1035.

28. Wadolkowski EA, Burris JA, O’Brien AD. Mouse model forcolonization and disease caused by enterohemorrhagicEscherichia coli O157:H7. Infect Immun 1990;58:2438–2445.

29. Antonopoulos DA, Huse SM, Morrison HG, et al.Reproducible community dynamics of the gastrointes-tinal microbiota following antibiotic perturbation. InfectImmun 2009;77:2367–2375.

30. Dethlefsen L, Huse S, Sogin ML, et al. The pervasiveeffects of an antibiotic on the human gut microbiota, asrevealed by deep 16S rRNA sequencing. PLoS Biol 2008;6:e280.

31. Robinson CJ, Young VB. Antibiotic administration altersthe community structure of the gastrointestinal mico-biota. Gut Microbes 2010;1:279–284.

32. Robinson CJ, Bohannan BJ, Young VB. From structureto function: the ecology of host-associated microbialcommunities. Microbiol Mol Biol Rev 2010;74:453–476.

33. Lusk RH, Fekety R, Silva J, et al. Clindamycin-inducedenterocolitis in hamsters. J Infect Dis 1978;137:464–475.

34. Peterfreund GL, Vandivier LE, Sinha R, et al. Successionin the gut microbiome following antibiotic and antibodytherapies for Clostridium difficile. PLoS One 2012;7:e46966.

35. Chen X, Katchar K, Goldsmith JD, et al. A mouse modelof Clostridium difficile-associated disease. Gastroenter-ology 2008;135:1984–1992.

36. Theriot CM, Koumpouras CC, Carlson PE, et al. Cefo-perazone-treated mice as an experimental platform toassess differential virulence of Clostridium difficilestrains. Gut Microbes 2011;2:326–334.

37. Reeves AE, Theriot CM, Bergin IL, et al. The interplaybetween microbiome dynamics and pathogen dynamicsin a murine model of Clostridium difficile infection. GutMicrobes 2011;2:145–158.

38. Buffie CG, Jarchum I, Equinda M, et al. Profound alter-ations of intestinal microbiota following a single dose ofclindamycin results in sustained susceptibility to

May 2014 Intestinal Microbiota and C difficile 1553

THEGU

TMICRO

BIOM

EAN

DDISE

ASE

Clostridium difficile-induced colitis. Infect Immun 2012;80:62–73.

39. Reeves AE, Koenigsknecht MJ, Bergin IL, et al. Sup-pression of Clostridium difficile in the gastrointestinaltracts of germfree mice inoculated with a murine isolatefrom the family Lachnospiraceae. Infect Immun 2012;80:3786–3794.

40. Lawley TD, Clare S, Walker AW, et al. Targeted restora-tion of the intestinal microbiota with a simple, definedbacteriotherapy resolves relapsing Clostridium difficiledisease in mice. PLoS Pathog 2012;8:e1002995.

41. Chang JY, Antonopoulos DA, Kalra A, et al. Decreaseddiversity of the fecal microbiome in recurrent Clostridiumdifficile-associated diarrhea. J Infect Dis 2008;197:435–438.

42. Petrof A, Khoruts E. From stool transplants to next-generation microbiota therapeutics. Gastroenterology2014;146:1573–1582.

43. Britton RA, Young VB. Interaction between the intestinalmicrobiota and host in Clostridium difficile colonizationresistance. Trends Microbiol 2012;20:313–319.

44. Howerton A, Ramirez N, Abel-Santos E. Mapping in-teractions between germinants and Clostridium difficilespores. J Bacteriol 2011;193:274–282.

45. Sorg JA, Sonenshein AL. Bile salts and glycine as co-germinants for Clostridium difficile spores. J Bacteriol2008;190:2505–2512.

46. Sorg JA, Sonenshein AL. Chenodeoxycholate is an in-hibitor of Clostridium difficile spore germination.J Bacteriol 2009;191:1115–1117.

47. Wilson KH. Efficiency of various bile salt preparations forstimulation of Clostridium difficile spore germination.J Clin Microbiol 1983;18:1017–1019.

48. Antunes LC, Finlay BB. A comparative analysis of theeffect of antibiotic treatment and enteric infection on in-testinal homeostasis. Gut Microbes 2011;2:105–108.

49. Giel JL, Sorg JA, Sonenshein AL, et al. Metabolism of bilesalts in mice influences spore germination in Clostridiumdifficile. PLoS One 2010;5:e8740.

50. Krag E, Phillips SF. Active and passive bile acid ab-sorption in man. Perfusion studies of the ileum andjejunum. J Clin Invest 1974;53:1686–1694.

51. Mekhjian HS, Phillips SF, Hofmann AF. Colonic absorp-tion of unconjugated bile acids: perfusion studies in man.Dig Dis Sci 1979;24:545–550.

52. Schiff ER, Small NC, Dietschy JM. Characterization ofthe kinetics of the passive and active transport mecha-nisms for bile acid absorption in the small intestine andcolon of the rat. J Clin Invest 1972;51:1351–1362.

53. Howerton A, Patra M, Abel-Santos E. A new strategy forthe prevention of Clostridium difficile infection. J InfectDis 2013;207:1498–1504.

54. Francis MB, Allen CA, Shrestha R, et al. Bile acidrecognition by the Clostridium difficile germinant recep-tor, CspC, is important for establishing infection. PLoSPathog 2013;9:e1003356.

55. Bouillaut L, Self WT, Sonenshein AL. Proline-dependentregulation of Clostridium difficile Stickland metabolism.J Bacteriol 2013;195:844–854.

56. Jackson S, Calos M, Myers A, et al. Analysis of prolinereduction in the nosocomial pathogen Clostridium diffi-cile. J Bacteriol 2006;188:8487–8495.

57. Karasawa T, Ikoma S, Yamakawa K, et al. A definedgrowth medium for Clostridium difficile. Microbiology1995;141:371–375.

58. Nakamura S, Nakashio S, Yamakawa K, et al. Carbohy-drate fermentation by Clostridium difficile. MicrobiolImmunol 1982;26:107–111.

59. Wilson KH, Perini F. Role of competition for nutrients insuppression of Clostridium difficile by the colonicmicroflora. Infect Immun 1988;56:2610–2614.

60. Ng KM, Ferreyra JA, Higginbottom SK, et al. Microbiota-liberated host sugars facilitate post-antibiotic expansionof enteric pathogens. Nature 2013;502:96–99.

61. Freter R, Brickner H, Botney M, et al. Mechanisms thatcontrol bacterial populations in continuous-flow culturemodels of mouse large intestinal flora. Infect Immun1983;39:676–685.

62. Wilson KH. The microecology of Clostridium difficile. ClinInfect Dis 1993;16(Suppl 4):S214–S218.

63. Merrigan M, Sambol S, Johnson S, et al. Susceptibility ofhamsters to human pathogenic Clostridium difficile strainB1 following clindamycin, ampicillin or ceftriaxoneadministration. Anaerobe 2003;9:91–95.

64. Sambol SP, Merrigan MM, Tang JK, et al. Colonizationfor the prevention of Clostridium difficile disease inhamsters. J Infect Dis 2002;186:1781–1789.

65. Vincent C, Stephens DA, Loo VG, et al. Reductions inintestinal Clostridiales precede the development ofnosocomial Clostridium difficile infection. Microbiome2013;1:18.

66. Tvede M, Rask-Madsen J. Bacteriotherapy for chronicrelapsing Clostridium difficile diarrhoea in six patients.Lancet 1989;1:1156–1160.

67. Petrof EO, Gloor GB, Vanner SJ, et al. Stool substitutetransplant therapy for the eradication of Clostridiumdifficile infection: ‘RePOOPulating’ the gut. Microbiome2013;1:3.

Received October 14, 2013. Accepted January 30, 2014.

Reprint requestsAddress requests for reprints to: Vincent B. Young, MD, PhD, Division ofInfectious Diseases, Department of Internal Medicine, University of Michigan,Ann Arbor, Michigan. e-mail: youngvi@umich.edu.

Conflicts of interestThis author discloses the following: Vincent Young is a recipient of anAdvancing Science Through Pfizer-Investigator Research Exchange (ASPIRE)research award. The remaining author discloses no conflicts.

FundingResearch in the laboratories of the authors was supported by National Instituteof Allergy and Infectious Diseases/Division of MIcrobiology and InfectiousDiseases of the National Institutes of Health under award numbersU19AI090871 (V.B.Y) and U19090872 (R.A.B.).