INFECTION AND IMMUNITY, July 2011, p. 2856–2864 Vol. 79, No. 70019-9567/11/$12.00 doi:10.1128/IAI.01336-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Mouse Relapse Model of Clostridium difficile Infection�

Xingmin Sun,1§ Haiying Wang,1,2§ Yongrong Zhang,1,3 Kevin Chen,1Barbara Davis,1 and Hanping Feng1*

Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 015361; School of Bioscience and Biotechnology,South China University of Technology, Guangzhou, China2; and School of Bioengineering, East China University of

Science and Technology, Shanghai, China3

Received 19 December 2010/Returned for modification 8 February 2011/Accepted 30 April 2011

Clostridium difficile is the causative agent of primary and recurrent antibiotic-associated diarrhea and colitisin hospitalized patients. The disease is caused mainly by two exotoxins, TcdA and TcdB, produced by thebacteria. Recurrent C. difficile infection (CDI) constitutes one of the most significant clinical issues of thisdisease, occurs in more than 20% of patients after the first episode, and may be increasing in frequency.However, there is no well-established animal model of CDI relapse currently available for studying diseasepathogenesis, prevention, and therapy. Here we report the establishment of a conventional mouse model ofrecurrence/relapse CDI. We found that the primary episode of CDI induced little or no protective antibodyresponse against C. difficile toxins and mice continued shedding C. difficile spores. Antibiotic treatment ofsurviving mice induced a second episode of diarrhea, while a simultaneous reexposure of animals to C. difficilebacteria or spores elicited a full spectrum of CDI similar to that of the primary infection. Moreover, micetreated with immunosuppressive agents were prone to more severe and fulminant recurrent disease. Finally,utilizing this model, we demonstrated that vancomycin only delayed disease recurrence, whereas neutralizingpolysera against both TcdA and TcdB completely protected mice against CDI relapse. In conclusion, we haveestablished a mouse relapse CDI model that allows for future investigations of the role of the host immune responsein the disease’s pathogenesis and permits critical testing of new therapeutics targeting recurrent disease.

Clostridium difficile, a Gram-positive, anaerobic, and spore-forming bacterium, is an etiologic agent of pseudomembra-nous colitis and accounts for a quarter of all cases of antibiotic-associated diarrhea (10). With the recent emergence ofhypervirulent antibiotic-resistant strains, the incidence of C.difficile-associated diarrhea and intestinal inflammatory dis-ease (collectively designated CDI) has increased significantlyin both North America and Europe, causing lengthy hospital-izations and substantial morbidity and mortality (24, 26). CDIis now considered an important reemerging disease.

C. difficile produces metabolically dormant spores that areexcreted from infected patients. The infectious spores persistin the environment and are highly resistant to commonly useddisinfectants. Spores survive exposure to gastric acidity andgerminate in the gut. The use of antibiotics that spare C.difficile but suppress the intestinal microbiota allows C. difficileto proliferate and produce two exotoxins, TcdA and TcdB,which cause intestinal tissue damage and inflammation. There-fore, antibiotic exposure is the most significant risk factor forthe diseases (6). CDI ranges from mild diarrhea to life-threat-ening fulminant colitis (5, 8, 26). In addition to gastrointestinaldisease, systemic complications of infection like ascites (15),pleural effusion (7, 38), hepatic abscess (30), and renal failure(11) have also been reported. Standard treatment for CDI isuse of the antibiotic metronidazole or vancomycin, althoughneither of these antibiotics is fully effective (37), and an esti-

mated 20 to 35% of those who appear cured by the initialtreatment develop a second episode of the disease (4, 34). Therate of occurrence of further episodes of CDI in patients whohave already had one recurrence can be more than 50% (27),and a subset of patients will have multiple recurrences. Recur-rent CDI is not always due to infection with the same strain. Anew strain was found in 33 to 56% of recurrent episodes (3, 18,28, 33, 36). Important factors for the development of recurrentCDI include persistent disruption of the intestinal microflora,continuation of antimicrobial therapy, an inadequate antitoxinantibody response, and advanced age. Other factors were alsoreported to contribute to the recurrence of CDI such as longhospital stays and concomitant receipt of antacid medications(16). Recurrent CDI is a frustrating condition because it is notonly difficult to treat but may affect patients for months or evenyears (17).

CDI has been studied in a number of animal models, includ-ing hamsters, guinea pigs, rabbits, rats, germfree mice, conven-tional mice, and germfree piglets (1, 12, 13, 20, 29, 32). Thehamster model has been traditionally widely used, but recentlydeveloped mouse and piglet CDI models more closely resem-ble the disease symptoms in humans (9, 32). Hamsters areextremely sensitive to C. difficile, develop clinical signs of CDIrapidly, and die within 2 to 3 days of infection (25). Therefore,this model does not represent the usual course and spectrum ofCDI in human beings. The model was also explored to studyrelapses (2, 19), but because the animals are treated withantibiotics right after the bacterial inoculation and developonly one episode of clinic disease in these studies, they are nottrue relapses. In this study, we describe the establishment of aconventional mouse model of CDI relapse/recurrence, whichmimics the clinical symptoms of recurrent disease in humans.

* Corresponding author. Mailing address: Division of InfectiousDiseases, Tufts University Cummings School of Veterinary Medicine,200 Westboro Road, North Grafton, MA 01536. Phone: (508) 887-4252. Fax: (508) 839-7911. E-mail: hanping.feng@tufts.edu.

§ These authors have the same contribution to this work.� Published ahead of print on 16 May 2011.

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MATERIALS AND METHODS

Animals. C57BL/6 mice (5 to 6 weeks old) were purchased from JacksonLaboratory. All mice used in the experiments were housed in groups of 5 percage under the same conditions. Food, water, bedding, and cages were auto-claved. All procedures involving animals were conducted under protocols ap-proved by the Institutional Animal Care and Use Committee.

Preparation of inoculum. C. difficile UK1, an epidemic strain (kindly providedby Dale Gerding), was isolated during a 2006 outbreak at Stoke-MandevilleHospital in the United Kingdom. Sporulation of the C. difficile UK1 strain wasinduced on BHIS agar as described previously (31). Briefly, an overnight C.difficile culture in BHIS medium was diluted in fresh medium to an opticaldensity at 600 nm of 0.2. A 150-�l portion of this suspension was spread onto 5ml BHIS agar in each well of a six-well tissue culture dish. The culture wasincubated anaerobically for 4 to 7 days to induce sporulation. The spores werewashed off the plates with phosphate-buffered saline (PBS). The spore suspen-sion was then heated at 60°C for 20 min to kill vegetative cells. The sporesuspension was stored at 4°C, and the spore concentration was determined byserial dilution. In some experiments, vegetative bacteria of laboratory strain C.difficile VPI 10463 were also used for challenge as described previously (9).

Assessment of C. difficile challenge doses. The experimental design of differentchallenge doses of C. difficile UK1 spores is shown in Fig. 1A. To establish asuitable dose for C. difficile UK1 spores, mice were fed with an antibiotic mixturefollowed by intraperitoneal (i.p.) injection of clindamycin as described previously(9). Three different doses of spores (104, 105, and 106 CFU) were used forchallenge via gavage. Mice were observed daily for the duration of the experi-ment for the presence of diarrhea and other symptoms. Weights were measuredevery day. Animals judged to be in a moribund state were euthanized, and tissuesamples from the intestine were taken for histopathology analysis.

Establishment of the CDI relapse model in mice. The experimental scheme ofrelapse/recurrence CDI models is illustrated in Fig. 2A. Mice were initiallychallenged with 106 CFU of C. difficile UK1 spores after antibiotic cocktailtreatment (9). Thirty days later, mice that had recovered from primary CDI weredivided into 4 groups. Group 1 mice were i.p. injected with 3 consecutive dosesof clindamycin (10 mg/kg per dose per day) without a C. difficile spore challenge.Group 2 mice were rechallenged with 106 CFU of C. difficile UK1 spores after thesame antibiotic cocktail treatment as the primary infection. Group 3 was thesame as group 2, except that the mice also received dexamethasone in theirdrinking water (100 mg/liter) for 8 consecutive days and one subcutaneousinjection (1 �g/mouse) prior to reinfection. Group 4 mice were rechallenged with

106 CFU of C. difficile VPI10463 vegetative cells after receiving the same anti-biotic mixture treatment as for the primary infection. Also included was a controlgroup (group 5) which was treated with the antibiotic cocktail and dexametha-sone without C. difficile exposure. Animals were observed for CDI symptomssuch as weight loss, diarrhea, hunched posture, and death.

The vancomycin treatment-induced relapse experimental scheme is illustratedin Fig. 3A. In group 1, mice (n � 10) were orally administered vancomycin for 9consecutive days (50 mg/kg/day via gavage) starting on day 2 after C. difficilechallenge. Group 2 was the same as group 1, except that mice were rechallengedwith 106 CFU of C. difficile UK1 spores on day 11 (after withdraw of vancomycintreatment). Mice were monitored for symptoms of disease.

Treatment of relapse CDI. For the experimental design of treating CDI re-lapse/recurrence to examine the therapeutic effects of vancomycin or antibodieson relapse disease, see Fig. 7A. One month after primary infection, survivorswere rechallenged with 106 CFU of C. difficile UK1 spores. For treatment, micewere i.p. injected once with 100 �l of anti-TcdA and anti-TcdB polysera fromalpaca (a domesticated species of South American camelid) or the same volumeof presera (sera collected prior to immunization of alpaca with glucosyltrans-ferase-deficient holotoxin aTcdA or aTcdB) as a control at 4 h postinfection.Vancomycin treatment (50 mg/kg/day via gavage) was started on the day of therechallenge and was continued for 5 days. Animals were monitored for CDIsymptoms.

Fecal cytotoxicity. After the primary and secondary challenges with C. difficilespores, feces were collected and dissolved in an equal volume (g/ml) of sterilePBS containing protease inhibitor cocktail and the supernatants were collectedafter centrifugation and stored at �80°C. To measure toxin-mediated cytotoxic-ity in fecal samples, the supernatants were filtered and serially diluted beforeaddition to CT26 monolayers, and cell rounding was observed under a phase-contrast microscope. Toxin titers were defined as the highest dilution to cause100% cell rounding after 24 h of incubation. Goat anti-TcdA and -TcdB polysera(Techlab Inc.) were used to determine the specific activity caused by C. difficiletoxins.

Bacterial culture. Fecal samples were dissolved in an equal volume of sterilePBS. Samples were serially diluted and plated on TCCFA (taurocholate-cefoxi-tin-cycloserine-fructose agar) plates and cultured anaerobically for 24 to 48 h at37°C. The Remel PRO Disc test (Remel, Lenexa, KS) was used to confirm thepresence of C. difficile by following the manufacturer’s instructions. For straintyping, DNA was isolated and digested with HindIII. The fragments were sep-arated on 0.7% agarose, and DNA fragment patterns were compared with thoseof the C. difficile strains used for infection.

Antibody titers and neutralizing activity. IgG titers in sera and IgA titers infeces were measured by standard enzyme-linked immunosorbent assay againstpurified recombinant holotoxins. Positive control sera were obtained from miceimmunized with glucosyltransferase-deficient holotoxins aTcdA and aTcdB (gen-erated in our laboratory). To assess in vitro neutralizing activities of the serumsamples, we used a mouse intestinal epithelial cell line, CT26, which is sensitiveto both TcdA and TcdB. The serum neutralizing titer is defined as the maximumdilution that still blocks cell rounding induced by toxin at a given concentration.This given concentration is 4 times the minimum dose of the toxin that causes allCT26 cells to round after a 24-h exposure to the toxin. Wild-type TcdA at 1.25ng/ml or TcdB at 0.0625 ng/ml causes rounding of 100% of CT26 cells after 24 hof toxin exposure. Therefore, TcdA at 5 ng/ml or TcdB at 0.25 ng/ml was mixedwith serially diluted serum samples and then applied to CT26 cells and cellrounding was observed under a phase-contrast microscope after 24 h of incuba-tion.

Histopathological analysis. Histopathological analysis was performed to eval-uate mucosal damage and inflammation induced by the toxins. Resected colon orcecum tissues were fixed in 4% formaldehyde buffered with PBS and thenembedded in paraffin. Deparaffinized 6-�m-thick sections were stained withhematoxylin and eosin for histological analysis.

Statistical analysis. Data were subjected to Kaplan-Meier survival analysis,analysis of variance, and t-test analysis using the StatView statistical softwareprogram (Abacus Concepts, Berkeley, CA). Results are expressed as means �standard errors unless otherwise indicated.

RESULTS

The severity of CDI depends on the C. difficile challengedose. CDI most likely initiates from and is transmitted throughspores. In recent years, the incidence and severity of CDI havesharply increased, in part due to outbreaks caused by the hy-

FIG. 1. Development of primary CDI after C. difficile spore chal-lenge. (A) Experimental design of different challenge doses of C.difficile UK1 spores. After antibiotic cocktail treatment, groups of micewere orally challenged with different doses of C. difficile UK1 spores bygavage: 104 (solid line), 105 (dashed line), and 106 (dotted line) CFU(n � 10). Mice were monitored for signs of disease and euthanizedwhen they became moribund. (B) Kaplan-Meier survival plot of miceinfected with different doses of C. difficile spores. (C) Mean relativeweights of surviving mice. Relative weight is based on the weight onday 0. Error bars show means � standard errors.

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pervirulent strains of type NAP1/BI/027. We therefore choseto challenge mice with C. difficile UK1 (an epidemic NAP1/BI/027 strain isolated from a patient) spores to mimic the diseaseoccurrence in humans. The experimental scheme is shown inFig. 1A. Mice were challenged with different doses of spores.Challenge with 106 CFU of spores led to 50% of the micebecoming moribund by day 4 (Fig. 1B), and all of the micedeveloped diarrhea within 2 to 3 days postchallenge. The mor-

tality and diarrhea rates were 30% (Fig. 1B) and 70%, respec-tively, for those challenged with 105 CFU of spores. All micesurvived a challenge with 104 CFU of spores (Fig. 1B), while30% of the mice developed diarrhea. Figure 1C shows themean relative weight of all surviving mice and indicates a sharpdecline within 2 to 3 days postinfection. After day 3 or 4, thesurviving mice showed clinical recovery from diarrhea andother signs of CDI, began to gain weight, and by day 6 had

FIG. 2. Induction of recurrence/relapse CDI in mice. (A) Experimental scheme of relapse/recurrence CDI models. Thirty days after the initialinfection, surviving mice were divided into 4 groups. Group 1 (n � 9), clindamycin only; group 2 (n � 10), antibiotic cocktail plus spore challenge;group 3 (n � 10), antibiotic cocktail plus dexamethasone (Dex) plus spore challenge; group 4 (n � 10), strain VPI10463 challenge. A control group(group 5, n � 10), which was treated with antibiotic cocktail and dexamethasone without C. difficile exposure, was also included. Data on thesurvival (B), weight loss (C), and diarrhea (D) of mice treated with antibiotics only (n � 10, solid line) or treated with antibiotics and then givenan initial spore challenge (n � 85, dashed line) are shown. Also shown are data on deaths (E), weight loss (F), and diarrhea (G) in mice withrecurrent CDI (group 1, solid line; group 2, dashed line; group 3, dotted line; all of the mice in group 5 appeared normal, without weight loss ordeath, and their data are not shown here for clarity). Data on the survival (H) and weight loss (I) of mice challenged with strain VPI10463 areshown at the bottom. Relative weight is based on the weight on day 0. The data shown are means � standard errors, and asterisks show significantdifferences between the two groups.

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returned to a normal weight. Based on these data, a C. difficiledose of 106 CFU was chosen as a challenge that would causesevere CDI in a substantial proportion of mice but not univer-sal lethality.

Development of recurrence/relapse CDI in mice. The exper-imental scheme is illustrated in Fig. 2A. In the primary infec-tion, 85 mice were challenged with 106 CFU of C. difficile UK1spores after antibiotic mixture and clindamycin administration.A control group of mice were treated with antibiotic cocktailonly without C. difficile infection. Animals were monitored fordeath (Fig. 2B), weight loss (Fig. 2C), and diarrhea (Fig. 2D).In the C. difficile-infected group, 81 mice (95%) developeddiarrhea and lost weight and 42 (50%) of these mice weremoribund (Fig. 2B). The remaining 39 surviving mice eventu-ally recovered from diarrhea, weight loss, and other signs ofCDI. Four mice did not develop any signs of CDI and wereexcluded from subsequent experiments. All mice treated withthe antibiotic cocktail alone did not develop any signs of dis-ease, although the treatment reduced mouse weight slightly(Fig. 2C).

To induce relapse/recurrent CDI, 39 mice that had recov-ered from primary CDI were divided into 4 groups (Fig. 2A).A control group (group 5) was included which was treated withthe antibiotic cocktail and dexamethasone without C. difficileexposure. In group 1, three doses of clindamycin injectioninduced all 9 mice to develop diarrhea, which quickly resolvedin 1 or 2 days and they survived (Fig. 2E). Mice lost weight but

not significantly (Fig. 2F). In group 2, all 10 mice surviving theprimary infection developed CDI symptoms, including diar-rhea (Fig. 2G) and weight loss (Fig. 2F), after a spore rechal-lenge. Forty percent of the mice were moribund (Fig. 2E), andtheir weight change showed a pattern similar to that seen afterthe primary C. difficile challenge. Recurrent CDI occurs moreoften in immunocompromised individuals (16, 17). Therefore,in group 3, we used dexamethasone to suppress the mouseimmune system, mimicking one of the immunocompromisedsituations. All dexamethasone-treated mice (n � 10) devel-oped severe diarrhea (data not shown) and became moribund(Fig. 2E) after a rechallenge with C. difficile. All of the mice ingroup 5 appeared normal without diarrhea or death. Theseresults indicate that immunosuppressed mice were more sus-ceptible to severe disease and death. Recurrent CDI is notalways due to reinfection with the same bacterial strain as inthe primary infection (16). Therefore, we investigated whethera different strain of C. difficile induced relapse CDI after micerecovered from primary C. difficile UK1 infection. In group 4,10 mice were challenged with vegetative cells of C. difficilestrain VPI10463 after treatment with the antibiotic cocktailand all of them developed diarrhea and lost weight (Fig. 2I) ina pattern similar to that seen after the primary infection (Fig.2C), with 20% mortality (Fig. 2H).

To more closely mimic what occurs in humans treated forCDI, 2 groups of mice were orally administered vancomycinfor 9 consecutive days after they experienced clinical symptoms

FIG. 3. Induction of relapse by vancomycin treatment. (A) Experimental scheme. Group 1 mice (n � 10) were orally administered vancomycinfor 9 consecutive days starting on day 2 after C. difficile challenge; group 2 received the same treatment as group 1 plus a C. difficile rechallengeon day 11 (after withdraw of vancomycin treatment). Data on the diarrhea (B), survival (C), and weight loss (D) of the two groups of mice areshown.

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of CDI (2 days postinfection), and one of the two groups wasrechallenged with C. difficile UK1 spores after withdraw ofvancomycin (Fig. 3A). Vancomycin treatment did not reducethe diarrhea rate or other clinical symptoms associated withthe initial C. difficile infection (data not shown), possibly be-cause mice usually start to recover at around day 3 postinfec-tion even without any treatment intervention. Three days afterwithdraw of vancomycin, 30% of the mice developed milddiarrhea (Fig. 3B) but recovered 1 day later and all of the micesurvived (Fig. 3C). Mice lost weight but not as severely as inthe initial infection (Fig. 3D). Rechallenge with C. difficile aftervancomycin treatment increased the rate of diarrhea to 80%(Fig. 3B) but did not induce any deaths (Fig. 3C) or significantweight loss compared with the group treated with vancomycinalone (Fig. 3D). These results suggest that vancomycin treat-

ment is not enough to fully disrupt intestinal microflora, al-lowing C. difficile to induce severe disease in mice.

The primary and relapse CDIs exhibit similar intestinaldiseases. We investigated whether recurrent CDI in mice dis-plays an intestinal histopathology similar to that seen in theprimary disease. Histology shows that intestinal sections frommice with relapse CDI exhibited mild-to-severe necrotizingtyphylitis and colitis. A remarkable result was the consistencyand extent of submucosal to transmural (all layers) edema(Fig. 4). Cecal tissues from mice treated with an antibioticcocktail alone appeared normal (Fig. 4A and B), while tissuesfrom mice with recurrent CDI showed extensive ulceration,hemorrhage, neutrophilic infiltration, and edema (Fig. 4C andD). Histological examination of ceca obtained from mice withprimary CDI showed similar findings (Fig. 4E and F). The

FIG. 4. Histology of mouse intestinal tissues. Magnifications: A, �100; B, �200. Cecum tissue from a mouse treated with antibiotic cocktailonly shows intact mucosa. Slight separation of the submucosa is a sectioning artifact. Magnifications: C, �100; D, �400. Cecum from a mouse withrelapse CDI that became moribund displays extensive ulceration, hemorrhage, neutrophilic infiltration, and edema. (E, F) Cecal tissue from amouse with severe primary CDI shows transmural inflammation, loss of superficial mucosal surface (erosions), an expanded submucosal layer(edema), and extravasated red blood cells (hemorrhage) with neutrophilic infiltrates. Magnifications: E, �40; F, �200. (G) Colonic tissue froma mouse treated with antibiotic cocktail alone shows a normal mucosal structure (magnification, �40). (H) Colonic tissue from a mouse withrelapse CDI that became moribund exhibits marked disruption of the mucosa and significant inflammation, congestion, and hemorrhage(magnification, �100).

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colons of infected mice showed marked disruption of the mu-cosa and significant inflammation, congestion, and hemor-rhage (Fig. 4H) in comparison with those of noninfected mice(Fig. 4G). Mouse small intestines were not significantly af-fected in either primary or recurrent CDI (data not shown).

Presence of C. difficile bacteria and toxins in feces. Fecalsamples from mice that experienced relapse CDI were exam-ined for the presence of bacteria and toxins. In C. difficilerechallenge-induced relapse, the presence of the toxins wasdetected in mouse feces from day 1 to day 9 postinfection (Fig.5A), with peak cytotoxicity seen on days 2 and 3. Toxins werealso detected in the feces of clindamycin-treated mice (Fig. 5B)but at a level significantly lower than that in mice rechallengedwith C. difficile (Fig. 5A). We further examined C. difficilebacterial colonization and found that three consecutive dosesof clindamycin induced a high level of C. difficile spore shed-ding at around day 3 (Fig. 5C) and mice continued to shedspores over a 30-day experimental period, which is in agree-ment with the recent finding (23).

Immune response to C. difficile infection in mice. AntitoxinIgG titers in sera and IgA titers in feces were measured on days15 and 30 after primary infection to assess the host immuneresponse to the toxins. All surviving mice had no or low levelsof IgG against TcdA or TcdB on both days 15 and 30 (data for

day 15 are not shown), whereas mice immunized with bothglucosyltransferase-deficient holotoxins (aTcdA and aTcdB)developed a significant antitoxin IgG response (Fig. 6A). FecalIgA against TcdA and TcdB was also determined and showeda pattern similar to that of IgG levels in sera (Fig. 6B). Theserum titers of neutralizing antibodies against TcdA and TcdBwere barely detectable in C. difficile-infected mice comparedwith those in sera from aTcdA- and aTcdB-immunized mice(Fig. 6C).

Neutralizing antibodies to TcdA and TcdB, but not vanco-mycin, prevent relapse CDI. We examined the therapeuticeffects of vancomycin and antitoxin polysera on recurrent/relapse CDI in mice. The experimental design is illustrated inFig. 7A. Twenty-eight mice that recovered from primary CDIwere challenged with C. difficile spores and then divided into 3groups. All mice in the preserum-treated group developeddiarrhea (Fig. 7B), and 40% of them were moribund within 4days postinfection (Fig. 7C), whereas antibody-treated miceshowed no signs of disease during the entire course of theexperiment. In the vancomycin-treated group, one mouse diedon day 2 postinfection and the remaining 7 mice did not de-velop CDI symptoms during therapy. However, 2 days aftervancomycin treatment was discontinued, all 7 mice developedsevere diarrhea (Fig. 7B) and 2 of them died. In total, about

FIG. 5. C. difficile spore and toxin shedding in the feces of mice with recurrent CDI. The experimental scheme is illustrated in Fig. 2A. Shownare toxin levels in feces from mice experiencing a relapse induced by antibiotic treatment and a C. difficile rechallenge (A) or by clindamycintreatment alone (B) and C. difficile spore shedding in mice treated with clindamycin (C).

FIG. 6. Mouse IgG and IgA responses after C. difficile infection. Mouse serum and fecal samples (n � 10, M1 to M10) were collected 30 daysafter an initial spore challenge. Serum IgG titers (A), fecal IgA titers (B), and serum neutralizing titers (C) were measured. Gray and open barsshow anti-TcdA and anti-TcdB levels, respectively. Immunization of glucosyltransferase-deficient holotoxins A (aTcdA) and B (aTcdB) inducedhigher levels of IgG. “Immunized” represents sera from mice immunized with aTcdA and aTcdB. “Infected” represents sera from mice infectedwith C. difficile.

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40% of the mice in the vancomycin-treated group died(Fig. 7C).

Figure 7D shows the mean relative weight of all survivingmice (up to when they became moribund). The preserum-treated mice showed significant weight loss from day 1 andreached their lowest weight on day 2. Surviving mice showedrecovery from diarrhea and other signs of CDI and began togain weight by day 3 to 4. By day 7, these mice had returned toa normal weight. The vancomycin-treated group had slightweight loss during treatment but developed signs of CDI afterdiscontinuation of the therapy. By day 7 (2 days after vanco-mycin treatment), significant weight loss was evident, and bodyweight reached the lowest level on day 8, but thereafter thesurviving mice gradually recovered (Fig. 7D). This result issimilar to that reported in the hamster model, except that thedisease occurred faster in mice after withdraw of vancomycintreatment (19). In the antibody-treated group, mice did notlose weight (Fig. 7D) or develop diarrhea (Fig. 7B) during orafter therapy. Therefore, antibodies against the two toxins, butnot vancomycin, prevented relapse CDI, which is also similarto what occurred in hamsters (19).

On necropsy, the intestines of the antibody-treated miceappeared normal (data not shown). Histology of the ceca fromantibody-treated mice showed intact mucosa, without apparent

edema or inflammation (Fig. 7E and F). However, mice thatwere moribund after discontinuation of vancomycin had severececal mucosal damage and inflammation (data not shown).

Since the dexamethasone-treated mice are more susceptibleto severe relapse CDI and all of them died (Fig. 2E), weinvestigated whether passive antibody treatment can preventfulminant and fatal CDI in these mice. Surviving mice from thefirst episode of C. difficile infection were administered dexa-methasone, rechallenged with C. difficile spores, and then di-vided into 2 groups with or without antitoxin polyserum ther-apy, respectively (Fig. 8A). As expected, all mice withoutantibody treatment developed severe CDI symptoms and weremoribund (Fig. 8B and C). In contrast, all mice treated with asingle dose of antitoxin antibodies survived (Fig. 8B) eventhough they lost weight (Fig. 8C). Only 20% of these micedeveloped mild diarrhea (Fig. 8D) but recovered 1 day later.Thus, neutralizing antibodies against the two toxins preventmice from developing fulminant relapse CDI.

DISCUSSION

Recurrence/relapse is a significant issue faced by cliniciansin the management of CDI patients. In this study, we reportedthe establishment of a conventional mouse model of CDI re-

FIG. 7. Antitoxin serum but not vancomycin treatment prevents recurrent CDI. (A) Scheme of experimental design for treating relapse/recurrence CDI. Groups: I (n � 10), antitoxin polyserum treatment (solid lines); II (n � 10), presera as a control (dotted lines); III (n � 8),vancomycin treatment (dashed lines). Mouse diarrhea (B), deaths (C), and weight loss (D) were monitored. Asterisks show the significantdifferences between groups I and II and groups I and III. (E, F) Representative histology of ceca from antitoxin serum-treated mice showed intactmucosa, no edema or inflammation (E; magnification, �100), or occasionally mild inflammation without edema with only mild changes in themucosa (F; magnification, �100).

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lapse/recurrence. We believe that this model provides a much-needed tool to investigate the pathogenesis and host immuneresponse to recurrent CDI and to evaluate new strategies ofinterventions against the disease.

Recurrent/relapse CDI exhibits a disease profile similar tothat of the first episode of CDI. Primary infection with 106 C.difficile UK1 spores routinely caused more than 95% of theanimals to develop diarrhea, with a mortality rate of around50%. After antibiotic exposure and rechallenge of survivorswith the same dose of spores 1 month later, mice developedrelapse CDI with morbidity and mortality rates comparable tothose of primary CDI. Similar histopathology was also evidentin the intestines of mice that experienced the first and secondepisodes of CDI, both of which resemble the CDI pathology inhumans. The most consistent and significant inflammatory le-sions were observed in the cecum, with occasional inflamma-tion in the colon but seldom in the small intestine. Mice de-veloped mild-to-severe typhylitis and colitis. In addition tointestinal inflammation and injury, a portion of the mice de-veloped acute and systemic fatal disease and died within 2 to 3days after rechallenge. The toxins may be associated with thedevelopment of the systemic disease since the presence oftoxin was detected in sera from these mice using an ultrasen-sitive immunocytotoxicity assay we have described previously(14; data not shown).

Although the mechanisms whereby recurrent CDI occurshave not been completely understood, the intimate associationof CDI with prior or concurrent antibiotic use indicates thatdisruption of normal intestinal microflora and a decrease incolonization resistance are critical factors in the developmentof recurrent CDI (17). Factors other than endogenous micro-flora may also contribute to recurrent CDI. Immune responsesto C. difficile and/or its toxins are likely to play a role. Investi-gators previously have reported an association between inad-

equate immune responses to TcdA and recurrent CDI (21, 22,35). Patients with recurrent CDI were shown to have signifi-cantly lower serum levels of IgG antibody against TcdA (21).Recurrent CDI in humans can be induced by the same ordifferent C. difficile strains (17). Our model mimics these fea-tures of relapse CDI in humans in the following ways. First, wetreated mice with an antibiotic cocktail before the primary andsecondary infections to disrupt the normal microflora; second,the toxin-neutralizing antibodies were low or absent prior torecurrent CDI; and third, the relapse/recurrent CDI can beinduced by challenging with either the same strain of C. difficilethat caused the initial infection or a different strain. Despitethese similarities, there is a significant difference in the induc-tion of relapse between mice and humans. In patients, thestandard treatment with vancomycin or metronidazole is oftenassociated with a high rate of relapse CDI and the diseaseseverity ranges from mild diarrhea to fulminant colitis anddeath. Vancomycin treatment of mice resulted in only a milddisease, whereas the antibiotic cocktail induced CDI with a fullspectrum of severity seen in CDI patients. The mouse gutmicroflora may be very different from that of humans, andvancomycin alone may not be enough to precipitate severeCDI in mice.

Using this model, we demonstrated that antitoxin antibodiesagainst both toxins, but not vancomycin, prevent recurrentCDI. After a single dose of antibody treatment, mice have notdeveloped any signs of recurrent CDI for months until thecompletion of the experiments. On necropsy, the treated miceshowed no gross signs of intestinal inflammation (data notshown). Histology of the ceca from antibody-treated miceshowed intact mucosa, no inflammation, or occasionally slightchanges in the mucosa but without edema. Vancomycin treat-ment, on the other hand, only delayed the onset of recurrentCDI. All mice developed severe recurrent disease after discon-

FIG. 8. Antitoxin antibodies prevent mice from fulminant relapse CDI. (A) Scheme of the experimental design. (B) Kaplan-Meier survival plotof mice with or without antitoxin antibody treatment. Also shown are data on weight loss (C) and diarrhea (D) in the two groups of mice (n �10). Dex, dexamethasone.

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tinuation of the antibiotic treatment. The mouse relapse CDImodel thus allows us to evaluate the new treatment strategiesagainst recurrent CDI.

We found that dexamethasone-treated mice were more sus-ceptible to severe and fulminant disease, indicating an impor-tant role of the immune response in preventing recurrent CDI.Consistent with this finding, immunocompromised individualsare more likely to develop severe C. difficile-associated diseaseand relapse (16, 17). It is likely that specific elements of theimmune system are compromised in such individuals, makingthem susceptible to more severe disease and relapse, althoughfurther studies are necessary to address this hypothesis.

ACKNOWLEDGMENTS

The project described here was supported by NIH grantsR01AI088748, R01DK084509, and K01DK076549.

We thank Joseph Sorg for providing C. difficile UK1 spores.

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