DOI: 10.1542/peds.2012-1449; originally published online January 14, 2013; 2013;131;e550Pediatrics

Carolina de Weerth, Susana Fuentes, Philippe Puylaert and Willem M. de VosSignatures

Intestinal Microbiota of Infants With Colic: Development and Specific  

  http://pediatrics.aappublications.org/content/131/2/e550.full.html

located on the World Wide Web at: The online version of this article, along with updated information and services, is

 

of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.Boulevard, Elk Grove Village, Illinois, 60007. Copyright © 2013 by the American Academy published, and trademarked by the American Academy of Pediatrics, 141 Northwest Pointpublication, it has been published continuously since 1948. PEDIATRICS is owned, PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly

at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

Intestinal Microbiota of Infants With Colic: Developmentand Specific Signatures

WHAT’S KNOWN ON THIS SUBJECT: Colic affects many infants,with incidence rates of up to 25%. The pathogenesis is not wellunderstood. Initial studies based on traditional culturingapproaches and in infants .6 weeks of age point atabnormalities in intestinal microbiota.

WHAT THIS STUDY ADDS: Infants with colic showed lowermicrobiota diversity and stability than did control infants in thefirst weeks of life. Colic/control differences in the abundance ofcertain bacteria were also found at age 2 weeks. These microbialsignatures possibly explain the excessive crying.

abstractOBJECTIVES: To provide a comprehensive analysis of the fecal micro-biota in infants with colic, as compared with control infants, duringtheir first 100 days of life.

METHODS: Microbial DNA of .200 samples from 12 infants with colicand 12 age-matched control infants was extracted and hybridized toa phylogenetic microarray.

RESULTS: Microbiota diversity gradually increased after birth only inthe control group; moreover, in the first weeks, the diversity of the colicgroup was significantly lower than that of the control group. The sta-bility of the successive samples also appeared to be significantly lowerin the infants with colic for the first weeks. Further analyses revealedwhich bacterial groups were responsible for colic-related differencesin microbiota at age 1 or 2 weeks, the earliest ages with significantdifferences. Proteobacteria were significantly increased in infantswith colic compared with control infants, with a relative abundancethat was more than twofold. In contrast, bifidobacteria and lactobacilliwere significantly reduced in infants with colic. Moreover, thecolic phenotype correlated positively with specific groups ofproteobacteria, including bacteria related to Escherichia, Klebsiella,Serratia, Vibrio, Yersinia, and Pseudomonas, but negatively withbacteria belonging to the Bacteroidetes and Firmicutes phyla, thelatter of which includes some lactobacilli and canonical groupsknown to produce butyrate and lactate.

CONCLUSIONS: The results indicate the presence of microbial signa-tures in the first weeks of life in infants who later develop colic. Thesemicrobial signatures may be used to understand the excessive crying.The results offer opportunities for early diagnostics as well as fordeveloping specific therapies. Pediatrics 2013;131:e550–e558

AUTHORS: Carolina de Weerth, PhD,a Susana Fuentes,PhD,b Philippe Puylaert, MSc,b and Willem M. de Vos, PhDb,c

aBehavioural Science Institute, Radboud University, Nijmegen,Netherlands; bLaboratory of Microbiology, Wageningen University,Wageningen, Netherlands; and cDepartments of Bacteriology andImmunology and Veterinary Biosciences, University of Helsinki,Helsinki, Finland

KEY WORDScolic, intestinal microbiota, infants, excessive crying,development

ABBREVIATIONSHITChip—Human Intestinal Tract ChiprRNA—ribosomal RNA

Drs de Weerth and Fuentes contributed equally to this work.

Dr de Weerth conceptualized and designed the study, designedthe data collection instruments, coordinated and superviseddata collection, conducted basic statistical analyses, drafted theinitial manuscript, reviewed and revised the manuscript, andapproved the final manuscript as submitted; Dr Fuentesconducted the laboratory analyses, conducted and interpretedthe advanced statistical analyses, created the figures, reviewedand revised the manuscript, and approved the final manuscriptas submitted; Dr Puylaert conducted the laboratory analyses,critically reviewed the manuscript, and approved the finalmanuscript as submitted; and Dr de Vos coordinated andsupervised the laboratory analyses, interpreted the results,drafted, reviewed, and revised the manuscript, and approvedthe final manuscript as submitted.

www.pediatrics.org/cgi/doi/10.1542/peds.2012-1449

doi:10.1542/peds.2012-1449

Accepted for publication Sep 26, 2012

Address correspondence to Carolina de Weerth, PhD,Montessorilaan 3, PO Box 9104, 6500 HE, Nijmegen, Netherlands.E-mail: c.deweerth@psych.ru.nl

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).

Copyright © 2013 by the American Academy of Pediatrics

FINANCIAL DISCLOSURE: The authors have indicated they haveno financial relationships relevant to this article to disclose.

FUNDING: Supported by the Netherlands Organization forScientific Research (NWO) by a personal Vidi grant to Dr deWeerth (grant 452-04-320) and an unrestricted Spinoza Award toDr de Vos.

e550 DE WEERTH et al at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

Infant colic, also referred to as excessivecrying, affects many infants. By usingthe widely used, modified Wessel’s cri-teria for defining colic1 (ie, crying foran average of .3 hours per day),St James-Roberts reported prevalencerates varying from 6.4% to #29%between 0 and 3 months of age.2 Coliccauses considerable concern and dis-tress to the parents, and professionalhelp is sought in 1 of 6 cases.3 Thepathogenesis of infant colic is not wellunderstood, and the underlying causesof colic remain unexplained. It has evenbeen proposed that, despite the name,colic may not have an intestinal originbut reflects the extreme end of thenormal distribution of infantile crying.4

A variety of physical, dietary, or drugtreatments are in use, but their efficacyis difficult to assess because the ex-cessive crying mostly stops by ∼4months of age.5 Moreover, a recentreview indicated that the clinical evi-dence for the effectiveness of thesetreatments is generally very low ormoderate.4

Aberrancies in the infant intestinalmicrobiota have been proposed to af-fect gut motor function and gas pro-duction, leading in turn to abdominalpain and colicky behavior.6 Some initialstudies indicated that the intestinalmicrobiota in infants with colic differedfrom that of healthy control infants. Forexample, infants with colic displayeda less diverse fecal microbiota, which isassociated with increased calprotectinlevels that are indicative of intestinalinflammation.7 Moreover, infants withcolic were found to have lower countsof lactobacilli and higher numbers ofGram-negative bacteria in their stools.7–9

However, these reports describeddifferences in infants already diag-nosed with colic and who were usually.6 weeks of age. Also, with 1 excep-tion,7 they were based on traditionalculturing approaches that do notrepresent the full complexity of the

intestinal microbiota.10 The recentdevelopment of high-throughput andmolecular approaches allows a view onthe intestinal microbiota and its func-tion in which bias is greatly reduced.11

The application of these molecularmethods has shown that the intestinaltract is colonized rapidly in early life bya developing microbial community thatdisplays specific succession patterns.12–14

Whereas at birth the intestinal tractis virtually sterile, already within a fewhours, bacteria start to appear in fecalsamples. These first settlers are oftenfacultative anaerobic bacteria that arerapidly replaced by anaerobes withdecreasing levels of oxygen tolerance.15

After several years of life, a complexmicrobial ecosystem is established, re-sembling that of adults.16 Although wedo not yet know the exact order andimpact of this microbial succession, itcan be envisaged that deviations in theearly intestinal colonization processcould have an impact in later life. Thesedeviations could also apply to the etiol-ogy of infant colic. If so, such deviationsshould appear early in life, precedingthe excessive crying that is known topeak at ∼6 weeks.17

The current study was initiated to pro-spectively follow the temporal de-velopment of the intestinal microbiota ina group of infants with and without colic.We collected samples in the first monthafter birth, which precedes the peak ofcolic at∼6weeks of age, and again at∼3to 4 months of age. By focusing on thefirst 100 days of life we looked at thebuildup of the colic phenotype as well asat a follow-up period, when colic hadmost probably resolved. In contrast toearlier studies that focused on specificand often cultured bacteria, we ad-dressed here the total intestinal micro-biota composition by extracting fecalDNA and analyzing it with a phylogeneticmicroarray that allows a global, andcomprehensive analysis of .1000 in-testinal phylotypes.18 We hypothesized

that infants with colic would displaya less diverse intestinal microbiota witha specific microbial signature and thatthese differences compared with controlinfants would be observable early in life.

METHODS

Participants and Study Design

This study is part of a prospectivelongitudinal project (termed the BiboStudy) that investigates the influencesof early-caregiving factors on the de-velopmentof children. Thedesignof thisstudy was described in detail else-where.19 All parents provided writteninformed consent, and the study wasapproved by the Ethical Committee ofthe Faculty of Social Sciences, RadboudUniversity Nijmegen (ECG/AvdK/07.563).For the current study, the parentsof a group of 160 healthy term infantscollected 9 stool samples from birthuntil ∼100 days of life. Inclusion crite-ria were as follows: an uncomplicatedand singleton pregnancy, no drug useduring pregnancy, no pre- and post-natal maternal physical health prob-lems (eg, diabetes and heart disease)or mental health problems (eg, majordepression), a term delivery ($37weeks) without major complications,and a normal 5-minute infant Apgarscore ($7). Four samples were col-lected in the first month of life at 2 (themeconium sample), 7, 14, and 28 daysand 5 samples were collected at 3 to 5months of age. The mean (SD) collec-tion days were as follows: 2.1 (1.0), 6.5(0.5), 13.8 (0.9), 27.6 (0.7), 83.9 (18.3),87.9 (18.2), 92.8 (18.3), 100.1 (18.6), and114.1 (18.2).

Determination of Colic

The parents reported crying by meansof a well-validated 4-day diary at 6weeks of age (416 5 days).20 The meandaily minutes of crying (ie, sum ofcrying, fussing, and unsoothable cry-ing) were calculated. Criteria for colicwere fulfilled in 25.4% of the group (ie,

ARTICLE

PEDIATRICS Volume 131, Number 2, February 2013 e551 at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

modified Wessel’s criteria for colic1:crying for an average of.180 minutesper day over the 4 diary days). From thegroup of infants for whom a completeseries of fecal samples was available(N = 106, 24.7% fulfilling the criteria forcolic), the 12 infants with the highestlevels of daily crying and the 12 with thelowest levels of daily crying were se-lected. The 2 groups showed large dif-ferences in the daily time spent crying(Table 1). Other than the excessive cry-ing in 1 of the groups, the infants werehealthy and the cohort representative ofnormally delivered infants in a Westerncountry with low antibiotic use. Themost salient health issues during thestudy period were as follows: antibioticuse (1 control infant), chickenpox (1infant with colic), and 1-week hospitali-zation for fracture (1 control infant).

Fecal DNA Extraction andPhylogenetic Microarray Analysis

Fecal samples were collected by theparents at home and stored at220°C.The samples were transported in cool-

ers with freezing cartridges or dry icefor further processing. Total DNA wasextracted from fecal material by therepeated bead beating procedure byusing a modified protocol of the QiaAmpDNA Mini Stool Kit (Qiagen, Hilden,Germany) essentially as described pre-viously.21 The subsequent analysis of 16Sribosomal RNA (rRNA) bacterial ampli-cons by using the Human Intestinal TractChip (HITChip) was performed in dupli-cate as described earlier.18 The HITChipis a comprehensive and highly re-producible phylogenetic microarraythat enables the parallel profiling andsemiquantitative analysis of 1140 phylo-types representing all major intestinalphyla grouped in 131 genus-like taxa de-scribed for the human intestinal micro-biota.18 This high-throughput HITChiphas been benchmarked with ultra-deeppyrosequencing of 16S rRNA22 and next-generation parallel sequencing of in-testinal metagenomes.23 Its taxonomicread-outs are linked to a recent overviewof the human intestinal microbiota24 andhave been described in detail.18

The HITChip microarray hybridizationwas considered satisfactory if 2 in-dependent hybridizations for eachsample correlated .95% (Pearson’scorrelations). The HITChip microarraysshowed a dynamic range of .10 000-fold, and .200 independent micro-array readouts were used in this study.Ward’s minimum variance method wasused for the generation of hierarchicalclustering of the total microbiota probeprofiles, whereas the distance matrixbetween the samples was based oncomplete observation correlations.

Quantification of total bacteria by real-time quantitative polymerase chainreaction was performed as describedpreviously.21

Data Analysis

The stability of the total microbiota com-position was assessed by calculatingPearson’s moving window correlation

between the log-transformed hybrid-ization signals for 3699 unique HITChipprobes18 obtained for pairs of 2 con-secutive samples in time.

The diversity of the total microbiota orits subgroups was assessed by cal-culating the Simpson’s reciprocal in-dex of diversity (1/D).25 The diversitywas calculated at the HITChip probelevel as detailed previously.18 Toevaluate the significance of thedifferences between the colic andcontrol microbiota, 2-tailed Student’st tests were calculated.

Wilcoxon signed-rank test corrected forfalse-discovery rate by the Benjaminiand Hochberg method was applied todetermine significant differences ofindividual genus-level groups betweenthe study groups.

For comprehensive multivariate statisti-cal analyses, Canoco software for Win-dows 4.5 (Wageningen, The Netherlands)was used.26 Redundancy analysis wasused to assess correlations betweenthe microbial groups detected by theHITChip analysis and the sample char-acteristics. The log-transformed hy-bridization signals of 131 genus-levelphylogenetic groups targeted by theHITChip were used as biological vari-ables. As environmental confoundervariables we included breastfeeding(weeks), gender, birth weight, homeversus hospital delivery, sampling agein days, and crying (infants with colicor control infants). The Monte CarloPermutation Procedure was used toassess significance of the variation inlarge data sets.

RESULTS

Colic and Control InfantCharacteristics

The 12 infants with colic and 12 controlinfants showed highly similar genderdistribution, birth weight, place andmode of delivery, and breastfeedingduration (Table 1). From all infants, 9fecal samples were obtained.

TABLE 1 Characteristics of the InfantCohort Described in This Study

Colic Group(n = 12)

Control Group(n = 12)

Gender of infantMale 7 8Female 5 4

Place of deliveryHome 5 4Hospital 7 8

Type of deliveryVaginal,

unassisted10 11

Vacuum pump 1 1Cesarean

delivery1 0

Birth wt, g 3686.3 (417.7) 3603.0 (383.9)Breastfeeding, moa 4.3 (3.8) 6.3 (4.5)Center-based childcareYes 8 5No 4 7

Crying, min/db 221.3 (30.5) 70.9 (19.0)*

Data are presented as means (SD) or n.a Four infants from the colic group and 2 from the controlgroup received no breastfeeding at all.b Sum of crying, fussing, and unsoothable crying.* Significant difference between the groups (T = 214.5,P , .001).

e552 DE WEERTH et al at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

Phylogenetic Profiling, TemporalClustering, and CompositionAnalysis of the Global FecalMicrobiota

Six of the 216 total fecal samples did notcontain enough fecal material foranalysis. Total DNA was readily re-covered from the remaining 210 sam-ples, but from the meconium the totalbacterial count as based on the quan-titative polymerase chain reactionanalysis was generally 100 to 1000-foldlower than from the other samples.

Subsequently, total bacterial 16S rRNAgenes were amplified from the extrac-ted DNA and labeled and hybridized tothephylogeneticmicroarraycontaining3699 distinct HITChip oligonucleotideprobes.18 All samples were analyzedtwice in a dye swap experiment, and inall cases a high reproducibility (.95%Pearson’s coefficient) was obtained,which substantiated the accuracy ofthe used high-throughput approach.Moreover, hierarchical clustering re-vealed high similarities between thefecal samples from the same infantexcept for the meconium samples,which often clustered together (datanot shown). Typically, such unsuper-vised clustering exposed a propertemporal ordering of the samples withthe meconium sample being the mostdifferent (Fig 1A). The immediate andpermanent presence of actinobacteria(mainly bifidobacteria; see below) wasevident. Whereas bacilli were found inearly samples, their abundance wasreduced after 75 days when the pro-teobacteria and Bacteroides increasedin numbers. This shift was furtherquantified in the overall compositionbased on the cumulative signals for thelevel 1 groups, representing genus- orgroup-level taxa (Fig 1B). A total of 210phylogenetic microarray analyses wereperformed, and the overall bacterialcomposition of the samples revealeda highly variable temporal composition(Supplemental Figs 5 and 6). Careful

inspection indicated that, in many cases,most of the different members ofFirmicutes, including many strict anaer-obes, were already present at a lowlevel even in the first week of life (seealso Fig 1). As early as in the first weeksof life, significant differences could befound in the microbiota of controlinfants and infants with colic (see Sup-plemental Table 3). Bacteroidetes ap-peared to be lower in the infants withcolic for the entire study period, withvarious levels of significance in the first2 months. In contrast, proteobacteria(including bacterial groups related toEscherichia coli, Enterobacter aero-genes, Pseudomonas, or Yersinia) weresignificantly increased in infants withcolic compared with control infantsin the first 2 months, with a morethan doubled relative abundance after2 weeks. Remarkably, the intestinalbifidobacteria and lactobacilli (includ-ing bacteria related to Lactobacillusgasseri and Lactobacillus plantarum)were found to be similarly increased incontrol infants compared with infantswith colic after 1 and 2 weeks, re-spectively (see Supplemental Table 4).

Diversity and Stability AnalysesRevealed Early Differences BetweenColic and Control Samples

To further address the differences be-tween infants with colic and controlinfants, the microbiota diversity wasdetermined on the basis of all hybrid-ization signals. In the control infants,microbiota diversity increased slightlywith time. However, the diversity of themicrobiota in the infants with colicappeared to have a different temporaldevelopment and stayed rather lowduring thefirst 100 days of life (Fig 2). Onpostnatal days 14 and 28, the diversity ofthe microbiota was significantly lowerin infants with colic than in controlinfants (P , .02 and P , .01, re-spectively). This difference could bemainly attributed to the decreasedbacterial evenness in the colic sam-

ples (ie, how similar the amounts ofthe different bacterial groups in thesamples are), whereas the richness(ie, number of different species foundin the samples) was similar to that inthe control infants (data not shown).

A further analysis on the stability of themicrobiota was performed by com-paring the similarity between succes-sive samples. The lowest similarity in allinvestigated samples is that betweenthemeconiumsampleand thefirst fecalsample. The results indicated that thecontrol infants showed a higher sta-bility than did the infantswith colic (seeFig 3). Moreover, the similarity betweenthe samples taken at 1 and 2 weeks ofage was significantly lower in theinfants with colic compared with thecontrol infants (P , .04, Fig 3).

Multivariate Statistical Analysis ofFecal Microbiota in Infants WithColic and Control Infants

To detail the differences in the micro-biota between infants with colic andcontrol infants, we performed a multi-variate cluster analysis on the micro-biota composition and other data sets.Comparisons were performed withdata from all time points. These ana-lyses revealed significant differencesoccurring at 2 weeks of age (Fig 4); atthat age, the microbiota of both groupscould be separated with the environ-mental variable “crying,” which signif-icantly influenced the sampledistribution (P = .03, Monte Carlo Per-mutation Procedure). The variablecrying was also found to be associated(although not significantly) with theinfants with colic and specific bacterialgroups (notably the proteobacteria) inthe earlier life samples. The separationof the colic and control groups and thebacterial associations were not asmarked in later life samples as at age 2weeks (data not shown).

Noneof theothervariables(gender,birthweight, breastfeeding, home/hospital

ARTICLE

PEDIATRICS Volume 131, Number 2, February 2013 e553 at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

delivery, and sampling age) significantlyinfluenced the sample separation at thistime point (data not shown). The ob-served separation (Fig 4) was found toexplain $18% of the variation in theabundance of 34 bacterial groups.These bacterial groups are included inthe plot (gray arrows) and listed se-parately (Table 2). The 8 bacterialgroups that were positively associatedwith crying included potentially patho-genic Gram-negative bacteria such as

bacteria related to Escherichia, Klebsi-ella, Serratia, Vibrio, Yersinia, and Pseu-domonas. In contrast, the groups thatwere negatively associated with cryingcontained relatively abundant bacteria.These included bacteria belonging to thebutyrate-producing species Butyrivibriocrossotus, Eubacterium rectale, andEubacterium hallii, which were foundto be consistently more (∼1.5-fold) abun-dant in healthy infants than in the infantswith colic (data not shown).

DISCUSSION

In this study we followed the temporaldevelopment of the intestinal micro-biota from birth onward in infants withand without colic by using the HITChip,a phylogenetic microarray that allowsa highly reproducible and comprehen-sive analysis of the known intestinalmicrobiota.18 Colic was determined at6 weeks of age with a cry diary. Theinfants with colic were characterizedby a microbiota that developed slower

FIGURE 1A, Hierarchical clustering with a heatmap of the HITChip profiles of a representative control infant (infant A) taken at days 1, 7, 14, 28, 75, 79, 84, 91, and 106. Thedarkness of the lines represents thebacterial abundance in the sample. Thehighest phylogenetic levels representedareshownon the right side of thefigure. B,Temporal dynamics of the relative abundance (%) of the most abundant phyla/classes in fecal samples from a representative control infant taken at the 9different time points.

e554 DE WEERTH et al at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

than that of the control infants andthat also showed a reduced temporalstability. Infants with colic also showeda significantly reduced microbiotadiversity at 14 and 28 days of life. Inaddition, already in the first 2 weeksof life, specific significant differencesbetween both groups were found;

proteobacteria were increased ininfants with colic, with a more thandoubled relative abundance, whereasbifidobacteria and lactobacilli were in-creased in control infants. Moreover,samples from infants with colic werefound to contain fewer bacteria re-lated to butyrate-producing species.

The observed differences confirm andextend earlier studies at older ages (ie,at ∼6 weeks) with traditional culturingapproaches and a focus on specificbacteria and revealed an increasedlevel of coliform bacteria (notably gas-producing Escherichia and Klebsiellaspp.) in infants with colic.7–9 Theresults are also in line with those of thefirst study showing that colic is linkedto reduced lactobacilli,27 and those ofa recent study that found that colic islinked to inflammation and reducedmicrobial diversity.7 In addition, a recentstudy that assessed continuous levels ofcrying in healthy infants reported thatthe abundance of Lactobacillus spp. at 3weeks of age was inversely associatedwith total infant distress at 7 weeks.28

However, the type of lactobacilli was notspecified in that study.

Our results indicate the importance ofthe first weeks of life for microbiotadevelopment. The reduced diversity andspecific microbiota signature observedin infants with colic already in the firstweeks of age could suggest a role ofmicrobiotadevelopment in theetiologyofcolic, as bothwell precede theusual colicpeak (ie, ∼6 weeks after birth). More-over, the findings may aid the futuredevelopment of tests designed to predictthe development of colic as well asspecific therapies for prevention of colic.The results could also help explain whythe administration of probiotics can re-sult in a decrease in colic symptoms29,30;

FIGURE 2Temporal development of the inverse Simpson’s diversity index (highest observed value set to 100 for each individual) in the first 100 days of life of controlinfants and infants with colic, calculated with all of the HITChip probe signals. Diversity was reduced in the colic group and was significantly different (*)between groups at days 14 and 28 (P = .03 and P = .02, respectively).

FIGURE 3Movingwindow analysis of similarity indexes of themicrobial composition of control infants and infantswith colic (indexes calculated for pairs of successive sampling days). The colic group revealed a lessstable pattern in the first weeks, and the similarity indexeswere significantly different (*) in the controlgroup between 7 and 14 days of age (P = .04).

ARTICLE

PEDIATRICS Volume 131, Number 2, February 2013 e555 at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

the probiotics might change the micro-biota thereby displacing the colic-associated bacteria.

With respect to the mechanisms un-derpinning this possible relation, thetwofold relative abundance of proteo-bacteria may be of relevance. These in-cluded bacteria known as potentialpathogens that might cause inflamma-tion. Remarkably, we have previouslydescribed that the abundance of similarpathobionts at the other extreme end oflife,namely incentenarians, isassociatedwith inflammation and a lower abun-dance of butyrate-producing bacteria.31

The butyrate-producing bacteria identi-fied here as correlating with the ab-sence of crying (Butyrivibrio crossotus

et rel., Coprococcus eutactus et rel.)appear to be different species than inthose described in the study in cen-tenarians. However, the mechanism bywhich they actmay be similar because ithas been shown that butyrate reducesthe pain sensation in adults.32 The sub-stantially reduced level of lactobacilliin infants with colic compared withhealthy infants is of specific interest,notably because this reduction wasfound to be limited to bacteria related toL. gasseri and L. plantarum. Both ofthese are considered to be mucosallactobacilli, suggesting that they maysignal to the host. We recently estab-lished that exponentially growing L.plantarum cells induce expression of

antiinflammatory genes in the upperintestinal tract of adults.33 Moreover,strains of L. gasseri are known toinduce an antiinflammatory response.Hence, we propose that the excessivecrying may be caused by increasedinflammation by an increased level ofpathogens and by a reduction in anti-inflammatory lactobacilli.

But why do certain infants show thesedelays and aberrancies in microbiotacolonization patterns? Possible early-life candidate factors that could bedistinguishing future infantswith colicfrom those without colic are genetics,epigenetics (eg, from the prena-tal environment), postnatal environ-ment (eg, household and caregiving

FIGURE 4Redundancyanalysis of samples takenat age14days fromcontrol infants (bluecircles)and infantswithcolic (redsquares). Capital letters indicate thedifferentinfants. Gray arrows indicate the bacterial groups associated with the different samples (see Table 2). The first and second ordination axes are plotted andexplained 17.5% of the variability in the data set. Crying was the only environmental variable significantly related to the sample distribution (P = .03, MonteCarlo Permutation Procedure with forward selection).

e556 DE WEERTH et al at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

factors), and even fortuitous encoun-ters with specific bacteria in the neo-natal period. Future prospectiveresearch concentrating on the early-life microbiota and host functionsshould shed light on this question.

Remarkably, the differences betweencolic and control microbiota were allseen in the first month of life, before thecolic peak takes place. At∼3 to 4months

of age, when the colic phenotype hasusually disappeared, there were nolonger detectable differences betweenour 2 study groups. This could indicatethat the colic phenotype is associatedwith a delayed and somewhat aberrantmicrobiota development, but that it isonly temporary and not indicative of apermanently altered intestinal micro-biota. However, longitudinal studies

with samples taken at later ages arerequired to clarify this issue.

ACKNOWLEDGMENTSWe are grateful for the continuousefforts of the familieswhokindly partic-ipate in the Bibo Study. We also thank allthe research assistants, master stu-dents, and PhD students, for their as-sistance with data collection.

REFERENCES

1. St James-Roberts I, Conroy S. Do preg-nancy and childbirth adversities predictinfant crying and colic? Findings and rec-ommendations. Neurosci Biobehav Rev.2005;29(2):313–320

2. St James-Roberts I. The Origins, Preventionand Treatment of Infant Crying and Sleep-ing Problems: An Evidence-Based Guide for

Healthcare Professionals and the FamiliesThey Support. New York, NY: Routledge/Taylor & Francis Group; 2012

3. Morris S, James-Roberts IS, Sleep J,Gillham P. Economic evaluation of strate-gies for managing crying and sleep-ing problems. Arch Dis Child. 2001;84(1):15–19

4. Lucassen P. Colic in infants. Clin Evid. 2010;02:309

5. Lehtonen L. From colic to toddlerhood. In:Barr RG, St James-Roberts I, Keefe MR, eds.New Evidence on Unexplained Early InfantCrying: Its Origins, Nature and Management.New Brunswick, NJ: Johnson and JohnsonPediatric Institute; 2001:259–272

TABLE 2 Bacterial Groups (and Related Species) That Are Negatively (Left) or Positively (Right) Associated With the Environmental Variable Crying

Negatively Associated Positively Associated

Phylum and Group No. Phylum (Class)/Genus-like Phylum and Group No. Phylum (Class)/Genus-like

Actinobacteria Actinobacteria Proteobacteria Proteobacteria1 Eggerthella lenta et rel. 26 Anaerobiospirillum spp.

Bacteroidetes Bacteroidetes 27 Enterobacter aerogenes et rel.2 Bacteroides intestinalis et rel. 28 Escherichia coli et rel.3 Bacteroides plebeius et rel. 29 Haemophilus4 Bacteroides splachnicus et rel. 30 Klebsiella pneumoniae et rel.5 Bacteroides vulgatus et rel. 31 Pseudomonas spp.6 Prevotella oralis et rel. 32 Serratia spp.7 Prevotella tannerae et rel. 33 Vibrio spp.

Firmicutes Bacilli 34 Yersinia et rel.8 Gemella spp.9 Streptococcus bovis et rel.10 Streptococcus intermedius et rel.11 Streptococcus mitis et rel.12 Weisella spp.

Clostridium cluster I13 Clostridia

Clostridium cluster IV14 Clostridium orbiscindens et rel.

Clostridium cluster IX15 Peptococcus niger et rel.16 Veillonella

Clostridium cluster XIVa17 Bryantella formatexigens et rel.18 Butyrivibrio crossotus et rel.19 Clostridium symbiosum et rel.20 Coprococcus eutactus et rel.21 Eubacterium ventriosum et rel.22 Lachnospira pectinoschiza et rel.23 Ruminococcus gnavus et rel.

Clostridium cluster XVI24 Eubacterium cylindroides et rel.

Proteobacteria Proteobacteria25 Oxalobacter formigenes et rel.

Numbers correspond to species arrows in Fig 4.

ARTICLE

PEDIATRICS Volume 131, Number 2, February 2013 e557 at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

6. Gupta SK. Is colic a gastrointestinal disor-der? Curr Opin Pediatr. 2002;14(5):588–592

7. Rhoads JM, Fatheree NY, Norori J, et al.Altered fecal microflora and increased fe-cal calprotectin in infants with colic. JPediatr. 2009;155(6):823–828, e1

8. Lehtonen L, Korvenranta H, Eerola E. Intes-tinal microflora in colicky and noncolickyinfants: bacterial cultures and gas-liquidchromatography. J Pediatr GastroenterolNutr. 1994;19(3):310–314

9. Savino F, Cordisco L, Tarasco V, CalabreseR, Palumeri E, Matteuzzi D. Molecularidentification of coliform bacteria fromcolicky breastfed infants. Acta Paediatr.2009;98(10):1582–1588

10. Rajili�c-Stojanovi�c M, Heilig HG, Molenaar D,et al. Development and application of thehuman intestinal tract chip, a phylogeneticmicroarray: analysis of universally con-served phylotypes in the abundant micro-biota of young and elderly adults. EnvironMicrobiol. 2009;11(7):1736–1751

11. Zoetendal EG, Rajilic-Stojanovic M, de VosWM. High-throughput diversity and func-tionality analysis of the gastrointestinal tractmicrobiota. Gut. 2008;57(11):1605–1615

12. Favier CF, Vaughan EE, De Vos WM, AkkermansAD. Molecular monitoring of succession ofbacterial communities in human neonates.Appl Environ Microbiol. 2002;68(1):219–226

13. Palmer C, Bik EM, DiGiulio DB, Relman DA,Brown PO. Development of the human infantintestinal microbiota. PLoS Biol. 2007;5(7):e177

14. Koenig JE, Spor A, Scalfone N, et al. Suc-cession of microbial consortia in the de-veloping infant gut microbiome. Proc NatlAcad Sci USA. 2011;108(suppl 1):4578–4585

15. Scholtens PA, Oozeer R, Martin R, Amor KB,Knol J. The early settlers: intestinal micro-biology in early life. Annu Rev Food SciTechnol. 2012;3:425–447

16. Yatsunenko T, Rey FE, Manary MJ, et al.Human gut microbiome viewed across age

and geography. Nature. 2012;486(7402):222–227

17. Barr RG, Trent RB, Cross J. Age-related in-cidence curve of hospitalized Shaken BabySyndrome cases: convergent evidence forcrying as a trigger to shaking. Child AbuseNegl. 2006;30(1):7–16

18. Rajili�c-Stojanovi�c M, Heilig HG, Molenaar D,et al. Development and application of thehuman intestinal tract chip, a phylogeneticmicroarray: analysis of universally con-served phylotypes in the abundant micro-biota of young and elderly adults. EnvironMicrobiol. 2009;11(7):1736–1751

19. Beijers R, Jansen J, Riksen-Walraven M, deWeerth C. Attachment and infant nightwaking: A longitudinal study from birththrough the first year of life. J Dev BehavPediatr. 2011;32(9):635–643

20. Barr RG, Kramer MS, Boisjoly C, McVey-White L, Pless IB. Parental diary of infantcry and fuss behaviour. Arch Dis Child.1988;63(4):380–387

21. Salonen A, Nikkilä J, Jalanka-Tuovinen J,et al. Comparative analysis of fecal DNAextraction methods with phylogenetic mic-roarray: effective recovery of bacterialand archaeal DNA using mechanical celllysis. J Microbiol Methods. 2010;81(2):127–134

22. Claesson MJ, O’Sullivan O, Wang Q, et al.Comparative analysis of pyrosequencingand a phylogenetic microarray for explor-ing microbial community structures in thehuman distal intestine. PLoS ONE. 2009;4(8):e6669

23. Arumugam M, Raes J, Pelletier E, et al;MetaHIT Consortium. Enterotypes of thehuman gut microbiome. Nature. 2011;473(7346):174–180

24. Rajili�c-Stojanovi�c M, Smidt H, de Vos WM.Diversity of the human gastrointestinaltract microbiota revisited. Environ Micro-biol. 2007;9(9):2125–2136

25. Simpson EH. Measurements of diversity.Nature. 1949;163(4148):688

26. Lep�s J, �Smilauer P. Multivariate Analysis ofEcological DataUsing CANOCO. 1st ed.Cambridge, UK: Cambridge UniversityPress; 2003

27. Savino F, Cresi F, Pautasso S, et al. Intestinalmicroflora in breastfed colicky and non-colicky infants. Acta Paediatr. 2004;93(6):825–829

28. Pärtty A, Kalliomäki M, Endo A, SalminenS, Isolauri E. Compositional developmentof Bifidobacterium and Lactobacillusmicrobiota is linked with crying andfussing in early infancy. PLoS ONE. 2012;7(3):e32495

29. Savino F, Cordisco L, Tarasco V, et al. Lac-tobacillus reuteri DSM 17938 in infantilecolic: a randomized, double-blind, placebo-controlled trial. Pediatrics. 2010;126(3).Available at: www.pediatrics.org/cgi/con-tent/full/126/3/e526

30. Savino F, Pelle E, Palumeri E, Oggero R,Miniero R. Lactobacillus reuteri (AmericanType Culture Collection Strain 55730) ver-sus simethicone in the treatment of in-fantile colic: a prospective randomizedstudy. Pediatrics. 2007;119(1). Available at:www.pediatrics.org/cgi/content/full/119/1/e124

31. Biagi E, Nylund L, Candela M, et al. Throughageing, and beyond: gut microbiota andinflammatory status in seniors and cen-tenarians. PLoS ONE. 2010;5(5):e10667

32. Vanhoutvin SA, Troost FJ, Kilkens TO, et al.The effects of butyrate enemas on visceralperception in healthy volunteers. Neuro-gastroenterol Motil. 2009;21(9):952–e76

33. van Baarlen P, Troost FJ, van Hemert S,et al. Differential NF-kappaB pathways in-duction by Lactobacillus plantarum in theduodenum of healthy humans correlatingwith immune tolerance. Proc Natl Acad SciUSA. 2009;106(7):2371–2376

e558 DE WEERTH et al at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

Wang et al. Cotransplantation of Allogeneic Mesenchymal and HematopoieticStem Cells in Children With Aplastic Anemia. Pediatrics. 2012;129(6):e1612–e1615

An error occurred in this article by Wang et al, titled “Cotransplantation of Al-logeneic Mesenchymal and Hematopoietic Stem Cells in Children With AplasticAnemia” published in the June 2012 issue of Pediatrics (2012;129[6]:e1612–e1615;originally published online May 7, 2012; doi:10.1542/peds.2011-2091). On pagee1613, under the heading of Table 1 Patient Characteristics, lines 8–10, this reads:“in column Nucleated Cells, subcolumn BM, the listed numbers are 9.79, 6.09, and22.” This should have read: “in column Nucleated Cells, the numbers 9.79, 6.09,and 22 should be sequentially listed in subcolumn PBSC.”

doi:10.1542/peds.2012-2593

Maguire et al. Estimating the Probability of Abusive Head Trauma: A PooledAnalysis. Pediatrics. 2011;128(3):e550–e564

An error occurred in this article by Maguire et al, titled “Estimating the Probabilityof Abusive Head Trauma: A Pooled Analysis” published in the September 2011issue of Pediatrics (2011; 128:e550–e564; doi:10.1542/peds.2010-2949). On pagenumber e553, in Table 2, on line 1, this reads: “Bechtel et al12; Ettaro et al13; Hettlerand Greenes14; Hobbs et all9; Kemp et al15; Vinchon et al16”. This should have read:“Bechtel et al12; Ettaro et al13; Vinchon et al16; Hettler and Greenes14; Hobbs et all9;Kemp et al15.”

doi:10.1542/peds.2012-3300

Rochow et al. Misclassification of Newborns Due to Systematic Error inPlotting Birth Weight Percentile Values. Pediatrics. 2012;130(2):e347–e351

Two errors occurred in this article by Rochow et al, titled “Misclassification ofNewborns Due to Systematic Error in Plotting Birth Weight Percentile Values”published in the August 2012 issue of Pediatrics (2012;130[2]:e347–e351; origi-nally published online July 23, 2012; doi:10.1542/peds.2011-3884). On page e347,under the Abstract Results section, line 1, the copy reads: “Fourteen of the 16identified publications contained the systematic error in plotting.” This shouldhave read: “Twelve of the 16 identified publications contained the systematicerror in plotting.”

On page e349, under the Results/Literature Search section, lines 5–6, the copyreads: “The plotting error was identified in 14 of these 16 publications.2–10”This should have read: “The plotting error was identified in 12 of these 16publications.2–4,6–9,11–15”

doi:10.1542/peds.2012-3472

ERRATA

PEDIATRICS Volume 131, Number 2, February 2013 361

ERRATA

at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from

DOI: 10.1542/peds.2012-1449; originally published online January 14, 2013; 2013;131;e550Pediatrics

Carolina de Weerth, Susana Fuentes, Philippe Puylaert and Willem M. de VosSignatures

Intestinal Microbiota of Infants With Colic: Development and Specific  

ServicesUpdated Information &

tmlhttp://pediatrics.aappublications.org/content/131/2/e550.full.hincluding high resolution figures, can be found at:

Supplementary Material

9/peds.2012-1449.DCSupplemental.htmlhttp://pediatrics.aappublications.org/content/suppl/2013/01/0Supplementary material can be found at:

References

tml#ref-list-1http://pediatrics.aappublications.org/content/131/2/e550.full.hat:This article cites 29 articles, 8 of which can be accessed free

Rs)3Peer Reviews (PPost-Publication

  http://pediatrics.aappublications.org/cgi/eletters/131/2/e550

R has been posted to this article: 3One P

Subspecialty Collections

ntal:behaviorhttp://pediatrics.aappublications.org/cgi/collection/developmeDevelopmental/Behaviorthe following collection(s):This article, along with others on similar topics, appears in

Errata

htmlhttp://pediatrics.aappublications.org/content/131/2/361.2.full.see:An erratum has been published regarding this article. Please

Permissions & Licensing

tmlhttp://pediatrics.aappublications.org/site/misc/Permissions.xhtables) or in its entirety can be found online at: Information about reproducing this article in parts (figures,

Reprints http://pediatrics.aappublications.org/site/misc/reprints.xhtml

Information about ordering reprints can be found online:

rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.Grove Village, Illinois, 60007. Copyright © 2013 by the American Academy of Pediatrics. All and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elkpublication, it has been published continuously since 1948. PEDIATRICS is owned, published, PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly

at UNIV OF CHICAGO on May 2, 2013pediatrics.aappublications.orgDownloaded from