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SHORT COMMUNICATION

Efficacy of Fecal Microbiota Transplantation in 2 Children

With Recurrent Clostridium difficile Infection and its

Impact on Their Growth and Gut Microbiome

�1Ritu Walia, y1Shashank Garg, zYang Song, yMohit Girotra, §Carmen Cuffari,zWolfgang Florian Fricke, and jj�Sudhir K. Dutta

Received May 1, 2014; accepted July 7, 2014.From the �Division of Pediatric Gastroenterology, Department of

Pediatrics, West Virginia University, Charleston, the yDivision of Gas-troenterology, Department of Medicine, Sinai Hospital Program inInternal Medicine, the zInstitute for Genome Sciences, University ofMaryland School of Medicine, the §Division of Pediatric Gastroenter-ology, Department of Pediatrics, The Johns Hopkins University, thejjDivision of Gastroenterology, Department of Medicine, Sinai Hospitalof Baltimore, and the �Department of Medicine, University of MarylandSchool of Medicine, Baltimore, Maryland, USA.

Address correspondence and reprint requests to Sudhir K. Dutta, MD,FACP, MACG, Professor of Medicine, University of Maryland Schoolof Medicine, Division Director of Gastroenterology, Sinai Hospital ofBaltimore, 2411 W. Belvedere Ave, Suite 305, Baltimore, MD 21215,USA (e-mail: sdutta@lifebridgehealth.org, sudhirdutta@hotmail.com).

1 Drs Walia and Garg contributed equally to the article.The study received funding from Gastroenterology Research Fund, Division

of Gastroenterology, Department of Medicine, Sinai Hospital of Balti-more, Baltimore, Maryland, USA (Contributors: Weinberg Foundation,Eric Cowan Fund, Friedman & Friedman Law Firm Fund); and Institutefor Genome Sciences (IGS), University of Maryland, Baltimore, Mary-land, USA.

The contribution of each author is as follows: R.W.—acquisition and storageof data and clinical specimens; analysis, interpretation, and tabulation ofdata; enrolment of patients; and drafting and revisions of manuscript.S.G.—acquisition and storage of data and clinical specimens; analysis,interpretation, and tabulation of data; enrolment of patients; and draftingand revisions of manuscript. Y.S.—analysis and interpretation of micro-biota data; statistical analysis; and technical support. M.G.—acquisitionand storage of data and clinical specimens; analysis, interpretation, andtabulation of data; statistical analysis; enrolment of patients to the study;and drafting and revisions of manuscript. C.C.—clinical care of thepatients and drafting and revisions of manuscript. W.F.F.—handling ofthe clinical specimens; analysis and interpretation of microbiota data;critical revision of the manuscript for important intellectual content;statistical analysis; and technical support. S.K.D.—study concept anddesign; acquisition of data; analysis and interpretation of data; criticalrevision of the manuscript for important intellectual content; obtainedfunding; and study supervision.

The authors report no conflicts of interest.Copyright # 2014 by European Society for Pediatric Gastroenterology,

Hepatology, and Nutrition and North American Society for PediatricGastroenterology, Hepatology, and Nutrition

DOI: 10.1097/MPG.0000000000000495

JPGN � Volume 00, Number 00, Month 2014

ABSTRACT

Fecal microbiota transplantation (FMT) is recognized as an alternative

therapeutic modality for recurrent Clostridium difficile infection (RCDI);

however, data on its efficacy in children are lacking, including its effect on

their growth and fecal microbiota. We report 2 young children (<3 years old)

who failed available therapeutics for RCDI, however responded remarkably

well to FMT. Besides resolution of clinical features of C difficile infection

(CDI), FMT administration led to marked improvement in their growth,

along with increased microbiota diversity, especially proportion of Bacter-

oides. Our 2 cases illustrate the efficacy of FMT in children with RCDI and

its positive effect on their growth and gut microbiota.

Key Words: Clostridium difficile infection, fecal microbiota

transplantation, growth, recurrent

(JPGN 2014;00: 00–00)

he rate of Clostridium difficile infection (CDI)–related

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T pediatric hospitalizations in the United States has increasedfrom 7.24 per 10,000 in 1997 to 12.8 per 10,000 in 2006 (1). Despitetreatment with first-line antibiotics, namely metronidazole andvancomycin, up to 19.6% of children have a recurrence within 8weeks (2). Recent American Academy of Pediatrics guidelinessuggest that recurrent CDI (RCDI) can be treated with an additionalcourse of metronidazole, oral vancomycin, or pulsed and/or taperedvancomycin regimen (3); however, some children may not respondadequately to these antibiotics and experience antibiotic refractoryRCDI. Alternative therapies, viz., rifaximin, nitazoxanide, intrave-nous immunoglobulins, and probiotics, have not been studiedcarefully in children and hence not recommended by AmericanAcademy of Pediatrics (3).

To date, there are only 3 available reports of pediatric RCDItreated successfully with fecal microbiota transplantation (FMT)(4–6); however, these reports lacked any long-term follow-up orevaluation of the effect of FMT on growth and analysis of changesin microbiota in disease state and after FMT (Table 1). In our report,we describe 2 pediatric patients (<3 years of age) with RCDI treatedsuccessfully with FMT, with long-term follow-up and examinationof changes in bacterial microbiota. Sinai Hospital’s institutionalreview board (IRB) was consulted, and the board indicated thatbecause it is not a study and FMT is being performed for individualcases of RCDI, IRB approval was not required; however, aninformed consent was obtained from the legal guardian of eachpatient, in accordance with IRB suggestion (Table 2).

CASE 1A 20-month-old boy was referred from Johns Hopkins

Medical Center for consideration of FMT for antibiotic refractoryRCDI of 8-month duration. The patient’s medical history wassignificant for premature birth at 27 weeks of gestation, retinopathyof prematurity, and congenital gastroesophageal reflux diseasecomplicated by chronic lung disease presumably from silent aspira-tions. A Nissen fundoplication with gastrostomy tube (G-tube)placement was performed to prevent further aspiration, and thepatient was on tube feeds because of poor oral intake and failure to

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TABLE 1. Previously reported cases of fecal microbiota transplantation in young children

Age, mo Sex

Duration of

disease, mo

Inciting

antibiotic

No. of

recurrences

Treatment used

for RCDI Donor

Time to

resolution of

symptoms, days

Duration of

follow-up, mo

Follow-up

data

Microbiota

analysis

Russell

et al (4)

24 Girl Not provided Amoxicillin 8 Metronidazole,

vancomycin,

L rhamnosus GG,

rifaximin,

S boulardii,

nitazoxanide

Father 1.5 6 Not provided Not performed

Kahn

et al (5)

16 Boy 5 Azithromycin 6 Metronidazole,

vancomycin,

S boulardii

Mother 1 2 Not provided Not performed

Pierog

et al (6)

84 Boy Not provided Not provided >10 Metronidazole,

vancomycin,

IVIG

Mother Not provided Not provided Not provided Not provided

252 (adult

age group)

Girl Not provided Not provided 5 Metronidazole,

vancomycin,

rifaximin

Mother Not provided Not provided Not provided Not provided

21 Boy Not provided Not provided 5 Metronidazole,

vancomycin,

rifaximin

Father Not provided Not provided Not provided Not provided

144 Boy Not provided Not provided 4 Metronidazole,

vancomycin,

nitazoxanide

Father Not provided 9 wk Not provided Not provided

48 Girl Not provided Not provided 6 Metronidazole,

vancomycin,

rifaximin

Father Not provided Not provided Not provided Not provided

84 Boy Not provided Not provided 4 Metronidazole,

vancomycin,

fidaxomicin

Mother Not provided Not provided Not provided Not provided

Walia

et al, 2014AQ1120 Boy 8 Cefdinir 4 Metronidazole,

vancomycin,

IVIG, probiotics

Mother 4 27 Provided Performed

30 Boy 7 Amoxicillin–

clavulanate,

ciprofloxacin

5 Metronidazole,

vancomycin

including pulse-

tapered regimen,

probiotics

Grandmother <7 8 Provided Not performed

IVIG¼ intravenous immunoglobulins; RCDI¼ recurrent Clostridium difficile infection.

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TABLE 2. Sinai Hospital protocol for donor screening and fecal filtrate reparation for fecal microbiota transplantation

Laboratory testing of the donor:

Blood-borne pathogens:

1. Human immunodeficiency virus (HIV)

2. Syphilis enzyme immunoassay

3. Hepatitis A immunoglobulin M

4. Hepatitis B surface antigen and core IgM antibody

5. Hepatitis C antibody

6. Helicobacter pylori antibody

7. HTLV 1 and 2AQ12Fecal-borne pathogens:

1. Pathogenic bacteria such as Salmonella, Shigella, Campylobacter, Yersinia by culture and sensitivity

2. Ova and parasites

3. Clostridium difficile toxin

4. Noro virus

Fecal filtrate preparation and administration:� A total of 25 to 30 g of donor fecal sample (3 scoops of tongue depressor) is mixed with 250 cm3 of sterile saline in a dedicated blender, and feces

is broken down at a low speed into a smooth slurry� The slurry is filtered through surgical gauze (4 in� 4 in and pore size 200–360 mm), to remove large particles. Saline is added to wash the filter� The filtrate from the gauze is filtered through a coffee filter (with pore size ranging 8–20 mm) into a funnel. Whenever the filter gets clogged,

it is replaced with a new filter. Fecal slurry is passed through this filter twice, and volume of the filtrate was made up to approximately 450 cm3 by

adding saline� The filtrate is divided into 5 aliquots of 90 mL each. These aliquots were labeled and sent to GI diagnostic center for the FMT procedure� During the colonoscopy, 4 aliquots (360 cm3) was instilled into the colon—180 cm3 into right colon and 90 cm3 in transverse and upper descending

colon using spray catheter

FMT¼ fecal microbiota transplantation; GI¼ gastrointestinal; HTLV¼ human T-cell lymphotropic virus; IgM¼ immunoglobulin MAQ13 .

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thrive. The patient received cefdinir antibiotic at 10 months of agefor an ear infection, after which the patient developed bloodydiarrhea and the patient’s fecal tested positive for C difficile toxinby enzyme immunoassay. The patient received a 10-day course ofmetronidazole, but diarrhea returned after its discontinuation,necessitating a second course of metronidazole and then a 2-weekoral vancomycin course, but diarrhea persisted, along with poorappetite and positive fecal C difficile toxin. Additional diagnostictests to exclude alternative causes of diarrhea were unrewarding,which included serologies for celiac and thyroid diseases, plasmaimmunoglobulin levels for immunodeficiency and food allergy,fecal culture, ova and parasites, and esophagogastroduodenoscopyand colonoscopy for inflammatory bowel disease. AlternativeRCDI treatments including intravenous immunoglobulin and pro-biotic (Saccharomyces boulardii) were also administered withoutany improvement.

Physical examination before FMT revealed a toddler withweight less than fifth percentile and length less than third percentilefor age (Fig. 1), which was markedly less compared with pre-RCDIvalues (weight >25th percentile, length 10–25th percentile). Thechild’s mother agreed for FMT as an alternative therapy for RCDI,and informed consent was obtained from the patient. A pre-FMTfecal sample from the child was obtained for microbiota analysis.The patient’s mother was screened for potential infectious diseases,as per our institutional protocol for adults (7), and 200 mL ofprocessed filtrate prepared from the patient’s fecal was instilledinto child’s right colon via colonoscopy (7). The toddler hadcomplete resolution of clinical symptoms along with disappearanceof fecal C difficile toxin. Three months after FMT, the childdemonstrated a remarkable recovery in physical growth (weightincreased to 50th percentile and length reached 3rd percentile forage; Fig. 1). One-year post-FMT, fecal sample was obtained fromthe child for microbiota analysis. The patient has not had a CDIrecurrence during 2 years of clinical follow-up.

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CASE 2A 30-month-old child with RCDI was referred from

Lancaster, Pennsylvania, for FMT. The patient’s medical historyincluded premature birth at 26 weeks of gestation, surgicalcorrection of patent ductus arteriosus at 3 weeks of age, andNissen fundoplication with G-tube for gastroesophageal refluxdisease/failure to thrive at 3 months of age. Because of poorresponse to tube feeds, the patient received long-term totalparenteral nutrition. At 23 months of age, the patient developedan upper respiratory infection requiring amoxicillin–clavulanateand ciprofloxacin and subsequently developed diarrhea withpositive fecal C difficile toxin. After failing a 10-day course ofmetronidazole, and 3 successive courses of oral vancomycin, thepatient was treated with a 5-month pulse-tapered vancomycin withprobiotic (S boulardii); however, the patient’s diarrhea returnedafter discontinuation of antibiotics. Other causes of chronic diarrheawere ruled out as in case 1.

Physical examination revealed that toddler was <3rd per-centile in length and at 50th percentile in weight-for-age. Thepatient’s mother provided the informed consent for FMT; however,the patient tested positive for fecal C difficile, and therefore thechild’s grandmother donated the fecal sample for FMT. Processedfecal filtrate (200 mL) was instilled into the right colon duringcolonoscopy, and the toddler had complete resolution of diarrheaalong with disappearance of fecal C difficile toxin. Four monthspost-FMT, the patient demonstrated favorable changes in thegrowth (increase in weight to 84th percentile, although lengthremained <3rd percentile).

Microbiota Analysis

Microbiota analysis of the fecal samples of case 1 pre-FMTand 1-year post-FMT was performed using 16S ribosomal RNAamplicon using Illumina MiSeq platform (San Diego, CA) (8,9).

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FIGURE 1. Growth chart for case 1 showing catch-up growth in length (upper panel) and weight (lower panel) after FMT (arrow).

Walia et al JPGN � Volume 00, Number 00, Month 2014

This analysis confirmed some of the findings reported in our earlierstudy in adult patients (9). Microbiota diversity was reduced in thechild’s pre-FMT fecal sample (Fig. 2A) with an increased relativeabundance of Proteobacteria (Escherichia species) and Actinobac-teria (Eggerthella species) as compared with the mother’s fecalsample (Fig. 2B and C). Moreover, a dominance of Blautia speciesfrom the Lachnospiraceae family (Firmicutes phylum) was found inthe child’s pre-FMT fecal sample. One year post-FMT, the micro-biota diversity increased in the child’s fecal sample, with areduction in Proteobacteria and Actinobacteria and an increase inBacteroides species (Bacteroidetes phylum, 22.4%) as comparedwith the patient’s pre-FMT fecal sample (1.7%).

DISCUSSIONOurs is the first documented report of 2 pediatric patients

with antibiotic refractory RCDI treated successfully with FMT,which illustrates a >6-month clinical follow-up and patient–donorfecal microbiota analysis pre- and post-FMT. Furthermore, thesechildren are arguably the youngest ones to receive FMT, if their ageis corrected for prematurity. Our patients are unique because ofprematurity and its related comorbidities as compared with thepreviously reported 7 cases in the literature (Table 1).

Both of our patients had similar clinical course since birth,which likely affected their development of gastrointestinal micro-biota. Both of the children were born premature and delivered bycesarean section. Penders et al reported that infants born throughcesarean section had lower numbers of Bifidobacteria and Bacter-oides and higher C difficile colonization as compared with vaginallyborn infants (10). Furthermore, prematurity and prolonged perinatalhospitalization was associated with greater C difficile colonization(10). Moreover, both of the children in our series were not breastfed.There is emerging evidence that breast-feeding may be crucial forestablishment of normal gastrointestinal microbiota in children. Jostet al demonstrated that mothers of infants who were being breastfed

4

had similar microbiota in their breast milk and fecal samples.Moreover, the microbiota in the infant fecal samples resembledtheir respective mother’s fecal and breast milk microbiota (11).Breastfed infants have been shown to have lower C difficilecolonization rates compared with formula-fed infants (14% vs30%) (10,12). Interestingly, both of the children were not able totransition to solid food because of G-tube feeding. Palmer et alsuggested that proportion of Bacteroides increases in intestines ofinfants/children after introduction of solid food (13). This transitionis associated with maturation of pediatric microbiota into adult-typemicrobiota by 2 to 3 years of age (13). Moreover, tube feeds aresterile in preparation and may not affect the gut microbiota asregular food does. Lastly, both of the children were exposed tomultiple antibiotic courses for recurrent infections. Research hasclearly demonstrated a reduction in fecal microbiota diversity ininfants after antibiotic administration (14). Therefore, the effect ofrepeated antibiotic exposure may have caused a long-lasting micro-biota change or may have interfered with the establishment ofnormal microbiota in our patients.

It is likely that some or all of these factors contributed to thedevelopment of altered gut microbiota resulting in predisposition toC difficile colonization in both of our patients. We suspect thataltered microbiota in these children reduced colonization resistanceto CDI. Moreover, the altered microbiota may also have contributedto multiple CDI recurrences following discontinuation of antibiotictherapy. Pre-FMT microbiota diversity was decreased in the child’sfecal sample, with a decreased proportion of Bacteroides and adominance of Lachnospiraceae (Blautia), Coriobacteriaceae(Eggerthella), and Enterobacteriaceae (Escherichia). Post-FMT,the microbiota diversity and the proportion of Bacteroides increasedin the child’s fecal sample with a concomitant decrease in pro-portion of Lachnospiraceae, Coriobacteriaceae, and Enterobacter-iaceae. The changes in the composition of fecal microbiota afterFMT, including increased proportion of Bacteroides after FMT,may have led to the resolution of RCDI. Enterobacteriaceae has

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Infant CDI

Infant,1 year post-FMT

Adult donor,processed stool

Adult donor

A B

C D

1.00

0.75

0.50

Verrucomicrobia

ProteobacteriaFirmicutesBacteroidetes

Actinobacteria

0.25

0.00

PC2 (29%)

Relative abundance

BlautiaCoprococcusFaecelibacterium[Ruminococcus]Bacteroides

Streptococcus[Coprobacillaceae, unknown]KlebsiellaEggerthellaPep strep to coccaxeae, unknown

Colin sellaRuminococcus

Ruminococcaceae, unknown

Ruminococcaceae, unknownCoriobacteriaceae, unknownEscherichia

Lachnospiraceae, unkhown

[Ruminococcus]strptococcus

Lachnospiraceae, unkhownPhascolarctobacteriumDorea

PC3 (11%)

Infan

t, CDI

Adult d

onor

,

proc

esse

d sto

ol

Adult d

onor

Infa

nt 1

year

post-

FMT

Bacteroides

FaecalibateriumLachnospiraceae,unknown

CoprococcusRuminococcus

Blautia

Eggerthella

Collinsella

PC1 (60%)

Infant, 1 yearpost-FMT

Adult donor

Adult donor,processed stool

Infant, CDI

0 10000

050

100

Num

ber

of O

TU

s

Rel

ativ

e ab

unda

nce

150

200

20000

Sampled sequences

30000

Infa

nt, C

DI

Adult d

onor

Adult d

onor

,

proc

esse

d sto

ol

Infa

nt, 1

year

post-

FMT

FIGURE 2. Microbiota analysis. Fecal microbial communities of the child before and 1 year after FMT and of the adult donor sample before and

after processing for use in FMT were compared using (A) microbiota diversity estimations based on the Shannon diversity index; (B) histograms

showing relative abundances of microbiota members at the taxonomic phylum level; (C) heat maps of the 25 most abundant microbiota membersat the genus level and sample relation based on hierarchical clustering (low confidence genus assignments in parentheses); and (D) phylogenetic

distance calculations between all of the samples taking the evolutionary relation of all of the microbiota members in all of the samples into account,

based on the unweighted UniFrac metricAQ10 . FMT¼ fecal microbiota transplantation; CDI¼Clostridium difficile infection.

JPGN � Volume 00, Number 00, Month 2014 Long-Term Impact of FMT on Growth of Children With RCDI

previously been reported as often increased in adult patients withRCDI (9). Furthermore, proportion of Lachnospiraceae in case 1pre-FMT was high in contrast to that in adult patients with RCDI,who have been shown to have decreased proportion of Lachnospir-aceae (9). This indicates that the RCDI pathogenesis may bedifferent in children when compared with that in adults. It mayalso point toward variable effects of different species within thefamily Lachnospiraceae on C difficile colonization or infection.

Safety concerns regarding use of FMT in children have beenraised, and Vandenplas et al recently described a case of early-onsetnon-CDI inflammatory colitis in a 1-year-old child who developed atransient systemic inflammatory response after FMT (15); however,our patients did not have any underlying inflammatory colitis andFMT may be safer in children without underlying inflammatorycolitis as compared with that in children with it. Randomizedcontrolled studies are needed to examine the efficacy and long-term safety of use of FMT in children for any indication.

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In summary, our 2 cases illustrate the efficacy of FMT inchildren with RCDI and its positive effect on their growth. Uniquefeatures in the child’s microbiota pre- and post-FMT suggestpotential for more specific and targeted FMT-based therapies.

Acknowledgments: The authors thank the following for theirtime and contribution toward our study: Ms Kenda Koerner and MsBarbara Bodner in the Department of Pathology, Sinai Hospital ofBaltimore, for their assistance in preparing fecal samples intofiltrate for administration during the FMT procedure; Ms DaynaSherba in the Division of Gastroenterology, Sinai Hospital ofBaltimore, for helping the study team with procedure schedulingand managing follow-up visits of our patients with FMT; Dr DeepaDutta, Director of Department of Microbiology and Pathology,Sinai Hospital of Baltimore, for the patient’s personnel andresource management in preparation of fecal filtrates for

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administration during the present study; and nursing staff at theGastrointestinal Diagnostic Center at Sinai Hospital for theirexcellent patient care and collection of fecal specimens.

REFERENCES1. Zilberberg MD, Tillotson GS, McDonald C. Clostridium difficile in-

fections among hospitalized children, United States, 1997–2006. EmergInfect Dis 2010;16:604–9.

2. Khanna S, Baddour LM, Huskins WC, et al. The epidemiology ofClostridium difficile infection in children: a population-based study.Clin Infect Dis 2013;56:1401–6.

3. Schutze GE, Willoughby RE. Clostridium difficile infection in infantsand children. Pediatrics 2013;131:196–200.

4. Russell G, Kaplan J, Ferraro M, et al. Fecal bacteriotherapy for relapsingClostridium difficile infection in a child: a proposed treatment protocol.Pediatrics 2010;126:e239–42.

5. Kahn SA, Young S, Rubin DT. Colonoscopic fecal microbiota transplantfor recurrent Clostridium difficile infection in a child. Am J Gastro-enterol 2012;107:1930–1.

6. Pierog A, Mencin A, Reilly NR. Fecal microbiota transplantation inchildren with recurrent Clostridium difficile infection. Pediatr Infect DisJ 2014 [Epub ahead of print].

7. Sadahiro S, Ohmura T, Yamada Y, et al. A case of cecocolic intussus-ception with complete invagination and intussusception of the appendixwith villous adenoma. Dis Colon Rectum 1991;34:85–8.

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8. Fadrosh DW, Ma B, Gajer P, et al. An improved dual-indexing approachfor multiplexed 16S rRNA gene sequencing on the Illumina MiSeqplatform. Microbiome 2014;2:6.

9. Song Y, Garg S, Girotra M, et al. Microbiota dynamics in patientstreated with fecal microbiota transplantation for recurrent Clostridiumdifficile infection. PLoS One 2013;8:e81330.

10. Wolff M, Ahmed N. Epithelial neoplasms of the vermiform appendix(exclusive of carcinoid). II. Cystadenomas, papillary adenomas, andadenomatous polyps of the appendix. Cancer 1976;37:2511–22.

11. Jost T, Lacroix C, Braegger CP, et al. Vertical mother–neonate transferof maternal gut bacteria via breastfeeding. Environ Microbiol 2013[Epub ahead of print].

12. Benno Y, Sawada K, Mitsuoka T. The intestinal microflora of infants:composition of fecal flora in breast-fed and bottle-fed infants. MicrobiolImmunol 1984;28:975–86.

13. Palmer C, Bik EM, DiGiulio DB, et al. Development of the humaninfant intestinal microbiota. PLoS Biol 2007;5:e177.

14. Koenig JE, Spor A, Scalfone N, et al. Succession of microbial consortiain the developing infant gut microbiome. Proc Natl Acad Sci U S A2011;108 (suppl 1):4578–85.

15. Vandenplas Y, Veereman G, van der Werff Ten Bosch J, et al. Fecalmicrobial transplantation in a one-year-old girl with early onset coli-tis—caution advised. J Pediatr Gastroenterol Nutr 2014 [Epub ahead ofprint].

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