Antibiotics, birth mode, and diet shape microbiome maturation … · MICROBIOME Antibiotics, birth...

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MICROB IOME

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Antibiotics, birth mode, and diet shape microbiomematuration during early lifeNicholas A. Bokulich,1 Jennifer Chung,1 Thomas Battaglia,1 Nora Henderson,1 Melanie Jay,1,2

Huilin Li,3 Arnon D. Lieber,1 Fen Wu,1,2 Guillermo I. Perez-Perez,1,4 Yu Chen,1,2 William Schweizer,5

Xuhui Zheng,4 Monica Contreras,1 Maria Gloria Dominguez-Bello,1 Martin J. Blaser1,4,6*

Early childhood is a critical stage for the foundation and development of both the microbiome and host. Early-lifeantibiotic exposures, cesarean section, and formula feeding could disrupt microbiome establishment and adverselyaffect health later in life. We profiledmicrobial development during the first 2 years of life in a cohort of 43 U.S. infantsand identified multiple disturbances associated with antibiotic exposures, cesarean section, and formula feeding.These exposures contributed to altered establishment of maternal bacteria, delayed microbiome development,and altered a-diversity. These findings illustrate the complexity of early-life microbiome development and its sensi-tivity to perturbation.

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INTRODUCTION

The establishment of stable microbial communities within the gastro-intestinal tract closely parallels host growth and immune developmentduring early life (1). The intestinal microbiome helps regulate host me-tabolism (2) and immune system development (3, 4) and thus couldplay an important role in directing host development. Delayed oraltered establishment of the intestinal microbiota in childhood, termedmicrobiota immaturity, has been associated with diarrhea and mal-nutrition in Bangladeshi children (5). The causes of these microbiotadisturbances and their consequences in other populations have not beenestablished, but they may be linked to host development.

Antibiotic use during childhood is prevalent in most parts of theworld, but the effects on maturation of microbiota and human healthare poorly characterized (6). The average U.S. child receives about threecourses of antibiotic by the age of 2 and 10 courses by the age of 10 (7).Antibiotics directly perturb the intestinal microbiota, leading to alteredcompositional states in children and adults (6, 8), but the consequencesof these changes on host physiology are not well understood. Antibioticexposure in children has been associated with increased risk of obesity(9), diabetes (10), inflammatory bowel disease (11), asthma (12), andallergies (13). We have shown previously that antibiotic exposure leadsto increased adiposity inmice (14), that early-life exposures lead to pro-longed effects on host metabolic characteristics (15, 16), and that thedisturbed intestinal microbiota mediates these host effects (16).

Other disturbances, including birth mode and infant diet (17), alsoaffect the intestinal microbiota during early life and are associated withlater-in-life adiposity and other clinical effects. Cesarean delivery hasbeen associated with asthma (18), allergies (19), type 1 diabetes (20),and obesity (21), possibly because of diminished exposure to maternalmicrobes during birth. Formula feeding similarly disrupts the intestinal

1Department of Medicine, New York University Langone Medical Center, New York, NY10016, USA. 2Department of Population Health, New York University Langone MedicalCenter, New York, NY 10016, USA. 3Division of Biostatistics, Department of PopulationHealth and Department of Environmental Medicine, New York University LangoneMedicalCenter, NewYork, NY10016, USA. 4Department ofMicrobiology, NewYorkUniversity LangoneMedical Center, NewYork, NY 10016, USA. 5Department of Obstetrics andGynecology,NewYork University LangoneMedical Center, NewYork, NY 10016, USA. 6New York Harbor Depart-ment of Veterans Affairs Medical Center, New York, NY 10010, USA.*Corresponding author. Email: [email protected]

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microbiota (17) andmay impair immune development (22) andnormalmetabolism (23).

Although the impacts of antibiotic exposures on intestinal dysbiosisin adults are well characterized, less attention has been given to theireffects on microbiota development during early childhood (6). We hy-pothesized that antibiotics and other early disturbances may alter mi-crobiome establishment during early life, potentially explainingassociations with emerging health issues. We examined the intestinalmicrobiota to model its development over 2 years of life in a cohortof 43 healthy urban U.S. infants. We then assessed the effects of birthmode, infant nutrition, and antibiotic exposures on intestinal micro-biota development in this population.

RESULTS

From the 53 mothers who initially enrolled in this study (table S1), atotal of 43 infants were enrolled for follow-up until 2 years of age(table S2). Stool samples were collected from these infants; stoolsamples, vaginal swabs, and rectal swabs were collected from theirmothers prepartum and postpartum. Birthmode, feeding, and systemicantibiotic exposures in the infants are shown in Table 1, and details ofprenatal, perinatal, and postnatal antibiotic usage are shown in tables S3to S5. The succession of bacterial taxa during the first 2 years of lifefollowed a predictable pattern (Fig. 1, A to E), consistent with previousstudies of the early-life microbiota (5, 24, 25). In the first month of life,stools were dominated by facultative aerobic Enterobacteriaceae beforeyielding to strict anaerobes—principally Bifidobacterium, Bacteroides,andClostridium (Fig. 1A). These taxa were gradually displaced betweenmonths 6 and 24 by a diverse mixture of Clostridiales, roughlycorresponding to the introduction and increased use of solid foods inthese infants (Fig. 1F). However, even among infants who received noantibiotics in the first 6months of life, those differing by birthmode andpredominant diet showed substantial early differences (Fig. 1, B and E,fig. S1, and table S6). During the first 2 years of life, themicrobiomewascharacterized by a period of gradual succession of different taxa. Althoughthe infantmicrobiotabegan toresembleanadult’smicrobiotaat about2yearsof age (see below), it had not yet achieved an adult-like state, character-ized by different alternative states that exist in quasi-equilibrium (26).

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Hence, we focused on the trajectory of microbiota development in chil-dren in the context of early disturbances.

Antibiotic exposures alter bacterial diversityTo assess the effects of disturbances on bacterial diversity, we measureda-diversity (that is, the species diversity in each individual sample), bac-terial richness [total number of unique operational taxonomic units(OTUs)], phylogenetic diversity (the relative amount of diverse phylo-genetic lineages detected), and species evenness in each sample (Fig. 2).Antibiotic use significantly diminished phylogenetic diversity immedi-ately after birth (P < 0.0001), but diversity subsequently recovered dur-ing the first year of life to resemble that of infants not exposed to

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antibiotics (Fig. 2, A to C, and table S6). However, a-diversity wasnot significantly suppressed in individual children immediately after an-tibiotic administration (P > 0.05) (fig. S2). Thus, antibiotic exposurealtered the trajectory of a-diversity changes during the first monthsof life, but transient effects were inconsistent.

b-Diversity, measuring similarities between samples as a function ofmicrobial composition, allowed us to assess potential impacts on thecomposition and recovery of an entire microbial ecosystem. Infantswho had not been exposed to antibiotics and those who had been ex-posed at any prior time differed significantly [weighted and unweightedUniFrac permutational multivariate analysis of variance (MANOVA),P < 0.001] (tables S7 and S8), although antibiotic exposure accounted

Table 1. Characteristics of the 43 children in the study including systemic antibiotic exposure, delivery mode, and diet.

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*The number of children (percentage of total) categorized by delivery mode, whether they were exposed to antibiotics at any time during the study, and their predominant diet during the first 3months of life.


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Fig. 1. Microbial and dietary succession viewed over the first 2 years oflife.Mean relative abundance (RA) of fecal bacteria at the genus level at each

32 subjects who were not antibiotic-exposed, organized by delivery mode(vaginal or cesarean) and predominant feeding mode (breast or formula).

month of life, for taxa with >1%mean RA across all samples. (A) All 43 infantsubjects during the first 2 years of life. (B to E) The first 6months of life for the

Vaginal-breast, n = 15; cesarean-breast, n = 7; vaginal-formula, n = 3; cesarean-formula, n = 7. (F) Dietary trends in all infants across the study period.

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for a small fraction of the overall variation among samples (R2 < 0.01).This observation provided evidence that antibiotics altered the intestinalmicrobiota in infants, but the effects were smaller than delivery mode(R2 = 0.02) and profoundly smaller than age (R2 = 0.14) (table S7). Eventhese factors accounted for a small fraction of community variation, andmost of the variation could not be explained by age, deliverymode, diet,or antibiotics (R2 > 0.60) (tables S7 and S8), stressing the need forbroader future studies that measure more factors that potentially con-tribute to microbiota development during early life.

Antibiotic exposure was associated with deficits in Clostridiales andRuminococcus from3 to 9months of life, butwith no consistent changesin other taxa (Fig. 3). Exposed subjects differed by age and by antibioticclass; thus, extensive variation in how the taxa were altered is notsurprising. Drug type, timing, route, duration, underlying conditions,number of exposures, and differences between individual subjects allmay be confounding factors.

Antibiotic exposures delay microbiota maturationDuring the first 2 to 3 years of life, intestinal microbiota undergoes agradual succession, and although a large degree of interindividual var-

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iation occurs, these age-dependent succession patterns share manyfeatures in different human populations (24). Microbiota maturation,then, is defined as the rate at which a child’s microbiota develops, asmeasured by these age-dependent successional stages; a “mature” mi-crobiota contains certain taxa that aremarkers for that child’s age group,whereas an “immature” or delayedmicrobiota resembles that of a youn-ger child. Delayed microbiota maturation, as defined in healthy chil-dren, mirrors physiological disturbances in the host (5) and occurs inmice exposed to antibiotics (15). Thus, we examined whether antibioticexposures and other disturbances similarly altered microbiota matura-tion in children, comparing relative maturation rates in a referencegroup (vaginally delivered, breast-fed, and unexposed to prenatal, peri-natal, or postnatal antibiotics), and antibiotic-exposed subjects using arandom forest (27) regression model to predict a child’s age as afunction of their microbial composition, as reported previously (5). Adefined microbiota maturation of the reference samples could be pre-dicted using 22 keyOTUs that explained the greatest degree of variation(pseudo-R2 = 82.1) in the model and, thus, are markers for normal de-velopment in this cohort (Fig. 4). Children exposed to antibioticsshowed delayedmicrobiota maturation compared to those not exposed


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Fig. 2. a-Diversity during the first 2 years of life in relation to antibi-otic treatment, delivery mode, and predominant diet. (A to I) Left:

shown for antibiotic use (A to C), delivery mode (D to F), and diet (G toI). Asterisks indicate significant linear longitudinal model (LLM) (P < 0.05)

Mean phylogenetic diversity ± SEM. Middle: Mean observed OTUs ±SEM. Right: Mean Shannon equitability (evenness) ± SEM. a-Diversity is

group differences at baseline or rate-of-change differences across ageranges (dotted lines demarcate time periods tested).




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to antibiotics (Fig. 4A and table S9). These effects were mostpronounced during months 6 to 12, and thereafter, no significant effectwas observed. A delayedmaturation pattern during early childhoodwasdue to depletion of specific OTUs, including constituents of Lachnos-piraceae and Erysipelotrichaceae (Fig. 4B).Within this cohort, intestinalbacterial communities followed a predictable pattern throughout thefirst 2 years (Fig. 1), but antimicrobials disturbed microbial successionat the OTU level, delaying microbial community development in theintestine relative to unexposed children (Fig. 4).

An important consequence of altered microbial patterns is changesto the functional gene repertoire presentwithin the gutmicrobiome.Wefound substantial differences in the maturation of gene functions in theimputed metagenome (28), with relation to perturbations includingantibiotics and predominant diet but not delivery mode (figs. S3 andS4 and table S10).

Delivery mode alters intestinal diversityThe first major microbial exposure for a vaginally born infant is in thebirth canal, a potentially important event for establishing a healthy mi-crobiome early in life. Cesarean section bypasses this exposure, alteringthe initial pool of microbes to which the neonate is exposed (29). Wesought to investigate the impact of delivery mode on microbiota matu-ration and diversity during the first 2 years of life.

Compared to vaginally born infants, cesarean-delivered infants ex-hibited significantly greater (P < 0.05) phylogenetic diversity, richness,and evenness at baseline. However, these declined significantly in cesarean-born infants during the first month after birth, and cesarean-born chil-dren subsequently displayed lower diversity and richness up to 2 years of

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age, especially after 8 months of age (Fig. 2, D to F, and table S6). Theseeffects were not due to differences in absolute bacterial abundance(fig. S5).

We also expected the comparative diversity between communities(b-diversity) to be altered in cesarean-delivered infants, reflecting bothdiffering microbial exposures during birth and the altered a-diversitythat was observed. For the babies’ first bowel movement (mean ±SD, 20.2 ± 18.3 hours of life), fecal b-diversity was not significantly dif-ferent, suggesting that the microbiota colonizing infants was of similarcomplexity. Thereafter, cesarean section significantly altered microbialb-diversity compared to vaginal delivery (unweighted UniFrac permu-tational MANOVA, P < 0.001) but accounted for a small fraction oftotal variation among samples (R = 0.02) (Fig. 5A and table S7). Forseveral common bacterial taxa, abundances were altered in cesarean-born infants compared to those vaginally born, underlying the differ-ences in a- and b-diversity (tables S6 and S7) (30). Most prominently,Bacteroides abundance was significantly lower in cesarean-delivered in-fants (Fig. 5, B and C, and fig. S6), regardless of predominant feedingmode. By 12 months, the balance of Bacteroides, Bifidobacterium, andEnterobacteriaceae that dominated the first year of life in all infants wasreplaced by a mixture of Firmicutes, primarily Clostridiales (Fig. 1). Al-though particular Clostridiales and Enterobacteriaceaewere significant-ly more abundant [linear discriminant analysis effect size (LEfSe), P <0.05] in cesarean-born infants during the first year, filling the void left byBacteroidales, few taxa were significantly different during the secondyear of life (Fig. 5C). This increasing similarity after 1 year in childrenborn vaginally or by cesarean section indicates that both communitiesundergo gradual maturation, eventually resembling the adult fecal


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Taxonomy 0 1.5 2 3 4 5 6 9 12 18 24

Bacteroidetes|Bacteroidia|Bacteroidales|Porphyromonadaceae|Parabacteroides

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Bacteroidetes|Bacteroidia|Bacteroidales|Porphyromonadaceae|Porphyromonas

Firmicutes|Clostridia|Clostridiales

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Firmicutes|Clostridia|Clostridiales|Lachnospiraceae

Firmicutes|Clostridia|Clostridiales|Lachnospiraceae|Ruminococcus

Firmicutes|Clostridia|Clostridiales|Lachnospiraceae|Dorea

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Proteobacteria|Deltaproteobacteria|Desulfovibrionales|Desulfovibrionaceae|Bilophila

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Fig. 3. Antibiotic exposure alters bacterial abundance. Antibiotic ex-posure significantly altered abundance of diverse bacterial taxa over the

were significantly (P < 0.05) more abundant in antibiotic-exposed in-fants at the given time points (columns); blue shading indicates more

first 2 years of life. On the basis of LEfSe analysis, red-shaded taxa (rows)
abundant taxa in unexposed infants.



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microbiota (Fig. 6). The taxa that dominated in the early months oflife—whether or not disturbed by cesarean delivery or antibiotics—declined as later-life taxa replaced them.

We hypothesized that the disruption of cesarean section could alsoalter microbiota maturation patterns in infants, similar to antibiotic ex-posure (Fig. 4). Using the microbiota maturation model described inthe Supplementary Materials, we found that cesarean- and vaginallydelivered infants demonstrated similar degrees of microbiota maturationduring the first 6 months of life. Subsequently, microbiota maturationstagnated in cesarean-delivered infants, with relative maturationdropping compared to vaginally born infants for the remainder of thestudy period (fig. S7).

Infant diet affects intestinal diversityTo assess the relationship between early diet and the microbiota, weroutinely surveyed infant nutrition, documenting the extent of breast-feeding or formula feeding and the timing of solid food introduction(Fig. 1F). We compared two major dietary groups that best describeddietary variation in this cohort: infants who were dominantly (>50% offeedings) breast-fed or dominantly formula-fed for the first 3months oflife. Phylogenetic diversity and bacterial richness growth rates were sig-nificantly decreased in formula-fed children during 12 to 24 months oflife (P < 0.05) (Fig. 2, G to I, and table S6). Formula feeding also alteredb-diversity (fig. S8A and table S7) and decreased microbiota maturationduring12 to 24monthsof life (fig. S8B).During this period,Lactobacillus,Staphylococcus, Megasphaera, and Actinobacteria were more abundantin breast-dominant children, and various genera of Clostridiales andPro-teobacteria weremore abundant in formula-dominant children (fig. S8C).

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Maternal bacteria populate the infant gut during early lifeThe earliest source of microbial colonizers in the infant gut is themother’s own microbiota, from passage through the birth canal tobreast-feeding and skin contact. However, the extent to which amother’s microbiota successfully establishes in her child, its dynamicsover time, and the relative contributions of bacteria from different bodysites have not been well described. We characterized the microbiota ofrectal swabs, vaginal swabs, and stool samples frommothers before andafter birth (fig. S9) to explore themicrobial relationships shared betweenmother-infant dyads, as well as unrelated individuals, in the context ofbirth mode.

Network analysis explores the relationships between samples andtheir constituent OTUs, revealing that the infant microbiota maturedfrom a neonatal state, associated withmaternal OTUs from both vaginaand rectum, to a post-infancy state resembling maternal stools (Fig. 6).In this analysis, sample nodes are connected by edges to all OTUs thatthey contain, indicating explicit connections between samples viashared OTUs. Edges are weighted on the basis of OTU abundance,causing samples with similar OTU composition to cluster togetheralong with their characteristic OTUs. These connections may be quan-titatively measured directly as shared OTU counts between samples(Fig. 7 and fig. S10) and indirectly as UniFrac distance (Fig. 6, B toD). Infant stool samples clustered together, associated stronglywith sev-eral key Bacteroidales, Clostridiales, Enterobacteriales, and Bifidobacter-iales OTUs (Fig. 6A). Infants cluster away from maternal stool samples,instead having more connections to maternal vaginal samples viarobust links with several Lactobacillales and Bifidobacteriales OTUs(Fig. 6A). As the children age beyond 12months, their fecalmicrobiota


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R.A. Z-score:

Enterococcaceae;g__Enterococcus;s__ 1111582Enterobacteriaceae;g__;s__ 1111294

Staphylococcaceae;g__Staphylococcus;s__ 1084865Veillonellaceae;g__Veillonella;s__dispar 342427

Erysipelotrichaceae;g__;s__ 262095Moraxellaceae;g__Acinetobacter;s__ 988314Veillonellaceae;g__Megasphaera;s__ 511378

Erysipelotrichaceae;g__[Eubacterium];s__dolichum 579851Ruminococcaceae;g__Oscillospira;s__ 581079

Lachnospiraceae;g__;s__ 564806Lachnospiraceae;g__Lachnospira;s__ 843553

Lachnospiraceae 338992Veillonellaceae;g__Dialister;s__ 403701

Ruminococcaceae;g__;s__ 357529Clostridiales;f__;g__;s__ 708680

Lachnospiraceae;g__;s__ 321902Lachnospiraceae;g__;s__ 529873

Ruminococcaceae;g__Faecalibacterium;s__prausnitzii 851865Lachnospiraceae;g__;s__ 363400

Ruminococcaceae;g__Faecalibacterium;s__prausnitzii 370287Ruminococcaceae;g__Faecalibacterium;s__prausnitzii 514940

Clostridiaceae;g__;s__ 628226

OTU IDTaxonomy

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Fig. 4. Antibiotic exposure delays microbiota maturation during earlylife. (A) Microbiota-by-age Z (MAZ) scores at each month of life between

drenwhohadnot beenpreviously exposed to systemic postnatal antibiotics,regardless of delivery mode and diet. This accounts for the observed devia-

antibiotic-exposed and unexposed infants (infants never exposed to system-ic pharmacologic antibiotic doses before the sampling time). MAZ scores in-dicate the number of SDs from the mean predicted age of age-matchedcontrol samples, as a function ofmicrobiotamaturation. Graymargins repre-sent 95%confidence interval (CI). Asterisks indicate significant (LLM, P<0.05)group differences at baseline or rate-of-change differences across ageranges (dotted lines demarcate timeperiods tested). The “unexposed”groupcontains both training set samples (from children who were never exposedto prenatal, perinatal, or postnatal systemic antibiotics; were vaginallydelivered; and were dominantly breast-fed) and all other samples from chil-

tion from a 0MAZ score in the unexposed group, as other factors influencedmaturation. (B) OTU abundance heatmaps illustrate the RA Z scores of 22maturation marker OTUs in the antibiotic-exposed and unexposed groupsthroughout the first 2 years of life. These OTUs were selected as those thatbest predict age of life in the control group and hence can be used as mar-kers of normal maturation. Substantial departures from the normal matura-tion profile alter predicted age of other samples. The color scale representsRA Z scores for each OTU (that is, the number of SDs from the mean RA ofthat OTU) across all samples at that age. Red text indicates OTUs that appearmost suppressed during months 6 to 12 after antibiotic exposure.





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middle left). See Fig. 7 for specific analyses. (B) Unweighted UniFrac dis-tance between maternal vaginal, rectal, and stool microbiota and child

nodes and OTU nodes is a function of shared microbial composition.Samples with a large degree of OTU overlap (weighted by the number ofobservations of that OTU) form clusters. Edges connect a sample to eachOTUdetected in that sample, revealing sharedOTUs between samples. Sam-ple nodes and edges are colored by sample type; the border of samplenodes is a function of the age of the child, including prepartum (negative)values for maternal samples (key at top left). OTU nodes are colored by taxo-nomic family affiliation; the size of each OTU node is a function of thatOTU’s overall abundance, registered as OTU count in all samples (key at

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stool microbiota as a function of child age. Shorter distance indicatesgreater similarity betweenmicrobial communities. (C) UnweightedUniFracdistance between stool microbiota from the same child (self) and otherchildren (nonself) as a function of the difference in age between sampling(D months). (D) Unweighted UniFrac distance between maternal vaginalmicrobiota and stool microbiota of vaginally born dyads, unrelated children,or cesarean-delivered dyads as a function of child age. For (B) to (D), linesindicate rolling average mean values, and gray shading is 95% CI. ANOVAP values are shown.




came to resemble maternal stool and rectal samples more closely(Fig. 6B), indicating that their microbiota matured to a maternal-like configuration associated with numerous Clostridiales and Bac-teroidales (Fig. 6A and fig. S10). Stool microbiotas from the samechild (“self”) were more similar to each other than those from un-related children (“nonself”) (unweighted UniFrac ANOVA, P <0.001), and children’s stool microbiotas were most similar to thoseof the same age, highlighting that the succession of the intestinal mi-crobiota occurs gradually (Fig. 6C). Conversely, at all ages, the chil-dren’s fecal microbiota was less similar to the mothers’ vaginal

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microbiota than to the mothers’ rectal and stool microbiotas (Fig.6B). The dissimilarity between a mother’s vaginal microbiotas andher child’s fecal microbiotas was significantly more pronounced incesarean-born children than in vaginally born mother-child pairs,whether dyads or unrelated (unweighted UniFrac ANOVA, P <0.001) (Fig. 6D). Considering two important genera, Bacteroides andBifidobacterium, there was a consistent diminution in the total OTUdiversity acquired in cesarean-born infants, as well as shared OTUs;dyads shared significantly more of these taxa than did unrelated pairs(fig. S10).


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Fig. 7. Shared OTUs reveal microbial relatedness among mothers andchildren. (A) Shared OTU counts (median ± quartiles) between individual

related children at different times (D months). (D) Mothers’ rectal swabsand stool samples from their own children (dyad) or unrelated children.

stool samples (top), rectal swabs and stool samples (middle), and vaginalswabs and stool samples (bottom), represented in Fig. 6. Distributions repre-sent the total number of OTUs within a single sample (blue) or shared OTUsbetween samples from the same individual (self, yellow), another individual(nonself, white/black), or a mother-infant dyad (red). Lowercase letters indi-cate significantly different shared OTU count distributions [one-way ANOVA,P < 0.0001, followed by false discovery rate (FDR)–corrected Fisher’sprotected least significant difference (PLSD) test]. Key indicates coloringfor box plots in (A) or line plots in (B) to (I). (B to I) Shared OTU counts overtime between mothers and unrelated children, mother-infant dyads, andtotal OTUs in child stool samples. (C) Samples from the same child or un-

(E) Mothers’ vaginal swabs and stool samples from unrelated children ordyads of children delivered vaginally or by cesarean section. (F) Vaginaland rectal swabs from the samemother or other mothers. (G) Stool samplesfrom the same mother or other mothers. (H) Rectal swabs from the samemother or other mothers. (I) Vaginal swabs from the same mother or othermothers. (B), (D), and (E) compare mothers versus children, and x axes indi-cate the child’s age (months). For (C) and (F) to (I), x axes indicate the differ-ences in child age (Dmonths) between the times when these samples wereobtained. Lines indicate rolling average mean values, and gray shading isequal to 95% CI. *P < 0.0001, ANOVA, followed by FDR-corrected Fisher’sPLSD test.





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Because microbial composition differs widely between adults andchildren, and between samples from different body sites, quantitationof shared OTUs is a metric to assess microbial transmission betweenthese loci (Fig. 7). Samples withmore sharedOTUs are expected to havecloser relationships, whether OTUs are transmitted directly betweensamples or both have a common external source. Maternal microbiotatransmission implies that more maternal OTUs are expected in herchild than in unrelated children, except when OTUs are highly dis-persed among mothers. We examined the total number of OTUs ineach sample, and the number shared between samples belonging tothe same individual (self) or other individuals (nonself), or betweenmother-infant dyads (Fig. 7). Results support expectations that OTUsare more shared between self samples, and a mother’s rectal microbiotasharesmoreOTUswith her own child’s stool than that of other children(Fig. 7A). Although vaginal swabs follow this trend, effects are not sig-nificant when examining samples from all time points and birthmodes.

Child stool OTU counts gradually increased with time (Fig. 7B),approaching OTU counts in mothers after 2 years of age (compareFig. 7, A and B). Unrelated mothers and children shared far fewer stoolOTUs throughout this time, but, surprisingly, still shared more OTUson average than mother-infant dyads (Fig. 7B). The shared OTU countincreased as children grew older—for both dyads and unrelatedmother-infant pairs—further evidence that children’smicrobiotas grad-ually mature into an adult-like state. After 2 years of age, a-diversity(total OTU counts) approached adult levels (compare Fig. 7, A andB), and b-diversity became lower between children and adults (Fig. 6B),but OTUs shared with adults remained low, indicating that differentstrains colonized children and adults even when similar bacterial taxawere present.

A child’s stool shared more OTUs with their other stools that werecollected <14 months apart, compared to nonself stool samples; as timebetween sampling increased, shared OTUs decreased until self and non-self stools had similar shared OTU counts (occurring for samples >14months apart) (Fig. 7C), indicating the dynamism of themicrobiota dur-ing the first 2 years of life. As expected, the number of OTUs shared be-tween self-stool samples gradually decreased as the time betweensampling increased, indicating gradual succession of OTUs. As childrenaged, they also sharedmoreOTUswithmaternal rectal swabswhether ornot theywere related (Fig. 7D).Dyads sharedmoreOTUs across all times(Fig. 7A) but were not significant at individual time points (Fig. 7D).Mothers shared significantly more vaginal OTUs with their children ifthey had been vaginally delivered, compared to both cesarean-deliveredandunrelated infants (Fig. 7E), an effect only significant after 1 year of life,peaking between18 and24months of life.VaginalOTUs, relevant duringearly infancy, were detected less frequently in fecal specimens in laterchildhood, leading to the decline in shared OTUs. As expected, peripar-tummaternal stool, vaginal, and rectal samples also showed fewer sharedOTUs with samples from the same mother as the interval betweensampling increased (Fig. 7, F to I). As sampling interval grew, maternalsamples shared as many OTUs with samples from other mothers as theydid with self. Stool samples were the exception, with more shared self-OTUs across sampling than from other mothers (Fig. 7G).

DISCUSSION

The purpose of this study was to characterize early-life microbial devel-opment in the context of antibiotic use, cesarean section, and formula

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feeding. Each of these has been associated with conditions emerginglater in life, including obesity (9), diabetes (10, 20), and allergies(13, 19). The cause of these relationships is unclear, but altered patternsof microbiota assembly during early life are plausible. We profiledmicrobiota development during the first 2 years of life and documen-ted disturbances related to antibiotic treatment, cesarean section,and diet.

Intestinal bacterial communities undergo a gradual succession dur-ing early life (Fig. 1A), after a predictable, age-dependent pattern that isconserved between disparate human populations and stabilizes after3 years of age (5, 24, 25). These events may reflect a coevolutionary re-lationship in which normal maturation of the gut microbiome during acritical window contributes to host development; disturbances mayinterrupt this delicate choreography, with consequences for long-termhost health (16).

We observed three distinct phases in the childhood microbiomeduring the first years of life. During the first month of life, Entero-bacteriaceae dominated the microbiota (Fig. 1), suggesting that thesefacultative anaerobes can exploit the naïve conditions of the neonatalintestine. The intermediate stages of development, from about 1 to24months of life, weremore dynamic and appeared to be sensitive to dis-turbances from birth mode, predominant nutrition, and antibiotic use.We speculated that even transient effects during this sensitive, develop-mental window could lead to long-lasting effects—as we have shownwith short-term antibiotic exposures in mice (15, 16) and as has beenobserved in human children, associating antibiotic exposures to illnessoutcomes (9–13, 31). Finally, as children reached 2 years of age, the mi-crobiota gradually stabilized toward an adult-like community (Fig. 6),characterized by both higher diversity and greater resilience to change.This period paralleled dietary transition from liquid to solid foods (Fig.1F), an important catalyst for microbiota maturation in childhood (17)and a source of introduced microbes. Alternative hypotheses for thehigher diversity include the diminution of the constraint imposed bycesarean section onmicrobial transmission between themother and ne-onate (fig. S9) and between breast-feeding and its strong selective effects(32). Higher immunological tolerance during early life (33) may beending, adding new constraints to acquiring nonfounding microbes.These conserved transitions may be important and suggest that dis-turbances during the first 2 years of life are likely to have strongeffects on development of the microbiota and potentially for hosthealth.

The particular microbial species and the specific gene pathwaysthat best define “microbial age” can be used to track how an infant’sgut microbiome matures. In that sense, they can be markers, parallel-ing how weight-for-height tracks a child’s development, and are simi-larly vulnerable to disruptions affecting health (5, 15). Although theprecise role that these bacteria play in development is unclear, identi-fyingmaturationmarkers actually linked to host processes at key junc-tures during an infant’s development has great potential. Bysuppressing early-life bacterial markers including Lachnospiraceae,Enterobacteriaceae, Erysipelotrichaceae, and numerous predictedgene pathways, antibiotic exposures effectively stall the developmentof the intestinal microbiome, causing the intestinal communities inthese infants to appear less developed (younger) than unexposedcounterparts (Fig. 4).

Lachnospiraceae sp. and other Clostridiales appeared particularlysensitive to antibiotic exposures, with significant depletion observedin antibiotic-treated infants often throughout early life (Fig. 3). These





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findings confirm previous studies of adult human andmousemicrobio-tas (8, 34). Lachnospiraceae live almost exclusively in the mammaliangastrointestinal tract, often producing butyrate and other short-chainfatty acids (35) that regulate host immunity via epithelial cell signaling,colonic T regulatory cells (36), and macrophages (37). Butyrate synthe-sis could explain how Lachnospiraceae induce T regulatory cells, sup-pressing colitis and allergic diarrhea inmice (38), and is consistent withprotective associations against type 1 diabetes development in infants(39). Butyrate, serving as an energy source for host epithelial cells, alsoregulates the cell growth and differentiation-related AP-1 (activatorprotein 1) signaling pathway (40), potentially explaining links betweenbutyrate producers (including Lachnospiraceae sp.) and body weight(41). Lachnospiraceae OTUs were also implicated as markers of intes-tinal microbiota maturation in Bangladeshi infants (5), suggesting thattheir blooms may be an important event in the developing infant gutacross continents.

a-Diversity, an important ecosystem characteristic, was disturbed bydelivery mode, antibiotic use, and diet (Fig. 2). During early life, overalldiversity rapidly increases in the developing infant gut (Fig. 2) (24, 25) aschildren acquire bacterial strains encompassing greater phylogenetic di-versity from diet, human contact, and environment. Because this pro-cess is occurring during a sensitive period when acquired intestinalbacteria train the nascent immune system (33), increasing diversitymay be relevant to normal development (1). Disturbance from antibi-otic use, formula feeding, or cesarean section, as well as the potential forcumulative damage from multiple disturbances, might affect intestinalhomeostasis and long-termhealth. Decreased intestinala-diversity dur-ing infancy precedes type 1 diabetes onset (39) and allergic manifesta-tions (42) and has been reported in obese adults (41).

Whether intestinal a-diversity influences disease development oris only a marker, functional diversity differences could contribute tointestinal homeostasis (26). The substantial functional redundancy inthe healthy human microbiome (26, 43) may provide insurance thatthe gut ecosystem can recover from temporary disturbances, main-taining productivity by minimizing risk of functional loss. In thismodel, a healthy microbiota with sufficiently high diversity can with-stand normal environmental fluctuations, for example, due to dietchange or illness, and maintain key functions. However, disturbancesduring development may reduce diversity below thresholds sufficientto maintain an essential functional repertoire. Such lack of resiliencemay delay ecosystem recovery or lead to a new stable state (26); eitherevent may promote later disease development. One challenge to thetranslation of this theory will be identifying microbial ecosystemfunctions important for critical developmental steps; as targets for fu-ture investigation, we identify several gene pathways affected by earlydisturbances (see the Supplementary Materials).

In another study in this issue, Yassour et al. (44) have shown thatantibiotic exposures also affect intestinal microbiota stability and diver-sity at the level of individual strains, highlighting the importance ofstrain-level dynamics to microbiome establishment during early life.If specific strains have unique functional niches in the developing intes-tine, their displacement could affect host-microbial interactions.

Another notable effect of cesarean section was a marked, persistentdecrease in Bacteroides populations. Although several studies have not-ed decreased phylum Bacteroidetes in cesarean-delivered infants(45, 46), we now show that this effect persists throughout the first yearof life and is associated with an altered metagenomic landscape in thegut, consistent with the findings of Yassour et al. (44). Because some

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Bacteroides sp. help regulate intestinal immunity (47), this long-lasting,large-scale disturbance could partially explain cesarean section–associatedhealth consequences, including asthma, obesity, and allergies (18, 21). Thedeficiency of Bacteroides after cesarean section, with commensurate ex-pansion of other taxa, may disrupt tolerogenic feedback loops (26), po-tentially contributing to development of inflammation and obesity.Disturbances to microbial metabolism and cell motility pathwaysin cesarean-born children suggest mechanisms by whichmicrobial per-turbation could influence the host; targets for future investigation in-clude the altered production of short-chain fatty acids or otherimmunomodulatory metabolites (36).

The strong evidence for maternal transmission of early-life taxa ingeneral, and specific dominant taxa in particular, provides support forthe importance of the composition of the maternal microbiota forhealthy development. Widespread practices that affect this reservoir in-clude antibiotic therapies and prophylaxes but also cleansings (for ex-ample,maternal vaginal lavage, skin cleansing of neonate, and umbilicalcord cleansing) that were aimed for neonates in developing countriesbut have recently spread to more developed populations (48).

Some limitations are inherent to our study. Our sample size lackssufficient power to account for complex interactions between manypotentialmicrobial disturbances during early life. Larger and longer stu-dies may assess whether early-life microbiota disruptions are indeedcumulative, counteractive, or independent. Longer studies can deter-mine how community assembly during childhood transitions to climax(for example, adult) communities, characterized by diverse alternativestable states (49). Dissimilar trajectories of microbiota maturation dur-ing childhood may lead to different stable states in adulthood, withoutnecessarily affecting the host. Microbial disruptions primarily delayedmicrobiota maturation during the first year, indicating transient effectsrather than permanent alterations. Transient antibiotic disturbancesduring early life durably alter host development in mice (14–16), butour results do not necessarily imply that this effect extends to humans.An alternative possibility is that disruptions to microbiota compositionand maturation are nullified if they are replaced by other functionallysimilar taxa. The disparate microbiota states observed in adults havefunctional overlap despite compositional similarity, suggesting thatcommunity function may be more relevant for predicting host interac-tions (26, 49). Our results suggest that community function and func-tional maturation are altered by antibiotics, cesarean section, andformula feeding [using PICRUSt (Phylogenetic Investigation of Com-munities by Reconstruction of Unobserved States)–predicted metagen-omes], but metagenomic and transcriptomic studies will be needed toassess whether early-life disruptions influence community function andbehavior.

Our results identify multiple disturbances to microbial develop-ment during early life. These alterations were not linked to health out-comes, which we did not survey, but are potential targets for ongoinginquiry. The microbial populations and dynamics deserve furtherscrutiny for their role in host-microbial communication, host devel-opment, and health. Mounting evidence associates antibiotic use, cesar-ean section, and formula feeding with metabolic and immunologicaldisorders later in life, possibly as a consequence of disturbed micro-biome development. Although these interventions often strongly bene-fit recipients, the hidden costs imply the need for careful considerationin children with less severe illnesses. Our findings warrant studies toestablish whether and how early-life microbial disturbances associatedwith antibiotic use, cesarean section, and formula feeding influence

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health outcomes. Recent studies of Canadian and Finnish children pro-vide evidence that such early-life disturbances are important for asthmarisk (12, 50, 51).


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MATERIALS AND METHODS

Study summaryAn Institutional Review Board–approved study was conducted inhealthy, pregnant mothers in New York City from 2011 to 2014.Maternal vaginal, rectal, and fecal specimens and fecal specimensfrom their infants were obtained from birth to the age of 3 years,chiefly in the first 2 years of life, and analyzed by microbiome sequen-cing. Information about home environment, delivery mode, infantdiet, and antibiotic exposures was obtained for each mother-babydyad. Complete details are provided in the Supplementary Materials.

Study designThe Early ChildhoodAntibiotics and theMicrobiome (ECAM) study isa longitudinal study aiming to examine the development of the intesti-nal microbiota in early childhood. Enrollment of mothers, which wasspecific for this study, began inDecember 2011 and ended inDecemberof 2014 at New York University (NYU) School of Medicine. Inclusioncriteria included healthy pregnantmothers 18 to 45 years old. For bettergeneralizability of the results and to reduce the likelihood of loss tofollow-up, we excluded mothers who were at high risk for prematuredelivery (that is, incompetent cervix, premature labor, bed rest), hadfetal anomalies detected in utero, and/or were unable to meet study ob-ligations. We excluded infants who required treatment more than 7 daysin a neonatal intensive care unit, premature infants of less than 38weeksgestation, and those with genetic diseases and/or birth-related injuriesrequiring intensivemonitoring.We sought to enroll at least 60mothers,so that, taking dropouts into account, we would have a cohort of at least40 babies who could be followed for at least 1 year. We assumed a ce-sarean section rate of ~50%, which would provide 20 babies in eachgroup to examine the chief outcome variables. In total, 53mothers wereenrolled (table S1); after early dropouts, follow-up of their infantsoccurred in 43 cases. The mothers who continued with the study didnot significantly differ from the initial enrollees (table S1). Their babieswere followed up for up to the first 3 years of life (table S2). Study surveyswere obtained at enrollment; 6 weeks postpartum; 4, 6, and 9 monthsduring year 1; and every 6 months thereafter (Table 1). Maternal fecaland vaginal swab samples were collected at two separate times prepartumand once postpartum. Stool samples were collected from infants 12 to24 hours after delivery, every month for the first year, and every othermonth for the second and third years of the study (see Sample collection).With 43 infants, we would have 80% power to detect a 0.82 standard-ized effect (standardized to sample SD of the outcome, such as diversityindices) in relation to a dichotomized factor, such as antibiotic use or de-livery methods, with a prevalence of 0.50. This is a conservative estimatebased on t test, because analyses based on longitudinal data have greaterpower. This study was approved by the Institutional Review Board atthe NYU School of Medicine.

Sample collectionMaternal fecal and vaginal swab samples were collected at twoseparate times prepartum (4 to 110 days before parturition) andonce postpartum (18 to 188 days postpartum, typically ~6 weeks;

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average, 51.4 days). Samples were collected by the obstetrician and/orthrough self-collection by the mothers using sterile cotton-tippedswabs; these were collected during routine prenatal visits and werekept chilled at 2° to 8°C immediately after collection and deliveredwithin 2 hours to the laboratory, where they were immediately fro-zen at −80°C until processing. Stool samples were collected oncefrom each mother, 1 to 63 days postpartum, typically within 1 to3 weeks of parturition, with only one sample collected 16 daysbefore parturition and an additional sample from one mother at232 days postpartum. Stool samples were collected from infants12 to 24 hours after delivery, every month for the first year, andevery other month for the second and third years of study. Mothers,when possible, also collected stool samples from their childrenbefore, during, and after each course of antibiotic use. Mothers wereprovided with sterile supplies and instructions for sanitary collectionand storage of feces from their own children. Mothers were alsoprovided with ice packs and insulated cooler bags; samples were keptchilled at 2° to 8°C immediately after collection and delivered within24 to 48 hours to the laboratory, where they were stored at −80°Cuntil processing.

Summary of statistical methodsMicrobiome sequencing data, bacterial composition, and analyses ofa-diversity (observed OTUs, phylogenetic diversity, and species even-ness) and b-diversity [UniFrac (30) phylogenetic distance betweensamples] were calculated in QIIME (Quantitative Insights into Micro-bial Ecology) (52). Random forest (27) regression models, using micro-biota composition as a predictor of subject age, were used to examinerelative rates of microbial maturation, as described previously (5).Significant differences in b-diversity were tested by permutationalMANOVA (53). Significant longitudinal changes in a-diversity, mi-crobiota maturation, and PICRUSt-predicted (28) metagenome mat-uration were tested by piecewise LLM (54). Significant differences inRA of microbial taxa were tested by LEfSe (55).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/343/343ra82/DC1Materials and MethodsFig. S1. LEfSe analysis of differentially abundant taxa between vaginally born, predominantlybreast-fed (n = 15) and cesarean-delivered, predominantly formula-fed children (n = 7) from1 to 6 months of life.Fig. S2. Antibiotic exposures did not alter short-term a-diversity in individual subjects.Fig. S3. Functional maturation of the microbiome is delayed by antibiotic exposure.Fig. S4. Functional maturation of the microbiome is altered by formula feeding.Fig. S5. Enumeration of 16S ribosomal RNA genes in fecal specimens in children at 1 and 12 monthsof age, by quantitative polymerase chain reaction.Fig. S6. Average RA of Bacteroides in children in relation to feeding and delivery mode in thefirst 2 years of life.Fig. S7. Cesarean section exerts a modest impact on microbiota maturation.Fig. S8. Infant diet alters microbiota composition and maturation over the first 2 years of life.Fig. S9. RA heatmap of major taxa in 296 maternal samples.Fig. S10. Development of diversity of Bifidobacterium and Bacteroides OTUs in children in earlylife.Table S1. Baseline characteristics of the 53 mothers in the study.Table S2. Baseline characteristics of the 43 infants in the study.Table S3. Prenatal antimicrobial use by class and purpose.Table S4. Perinatal antibiotic use by class and indication.Table S5. Postnatal antimicrobial use by class and age of child.Table S6. LLM estimates of antibiotic treatment, diet, and delivery effects on a-diversity.Table S7. Permutational MANOVA scores of antibiotic, diet, and delivery effects on unweightedUniFrac distance b-diversity.


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Table S8. Permutational MANOVA scores of antibiotic, diet, and delivery effects on weightedUniFrac distance b-diversity.Table S9. LLM estimates of antibiotic, diet, and delivery effects on microbiome maturationMAZ scores.Table S10. LLM estimates of antibiotic, diet, and delivery effects on PICRUSt-predicted meta-genome maturation MAZ scores.References (56–65)


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Acknowledgments: We thank S. Bedi and A. Radin for the clinical assistance. Funding: This studywas supported by grant R01 DK090989 from NIH, Public Health Service Institutional ResearchTraining Award T32 AI007180, the Diane Belfer Program for Human Microbial Ecology, the Danieland Leslie Ziff Fund, and theC&D fund.Author contributions:M.J.B.,M.J., Y.C., G.I.P.-P., andM.G.D.-B.planned and designed the longitudinal study design and the analytical experiments. N.H. and W.S.recruited study subjects. J.C. performed microbiome sequencing. X.Z. and M.C. performed quanti-tativepolymerase chain reaction experiments. N.H., J.C., and T.B.managed the study data.N.A.B., T.B.,A.D.L., F.W., J.C., and H.L. performed all analyses. N.A.B. and M.J.B. wrote the manuscript. All authorscontributed to and reviewed the final manuscript. Competing interests: N.A.B. is a co-founder ofMicroTrek Inc., a company focused on microbial analysis for food, beverage, health, and other in-dustries. MicroTrek was not involved in funding, design, or interpretation of this study. M.G.D.-B.,together with NYU Langone Medical Center (LMC), has intellectual property related to the res-toration of the microbiome of newborns, which has been licensed to Commense Inc. M.G.D.-B.and M.J.B. are co-founders and equity holders in Commense Inc., which is focused on the res-toration of depletedmicrobiota in early life. Commense was not involved in the funding, design,or interpretation of this study. M.J.B. serves as a consultant to Johnson & Johnson, which fundsunrelated studies in his laboratory at NYU LMC. The other authors declare that they have nocompeting interests.Data andmaterials availability:All sequencing data are publicly availablein QIITA database (https://qiita.microbio.me/) under study ID 10249.

Submitted 31 July 2015Accepted 27 May 2016Published 15 June 201610.1126/scitranslmed.aad7121

Citation: N. A. Bokulich, J. Chung, T. Battaglia, N. Henderson, M. Jay, H. Li, A. D. Lieber, F. Wu,G. I. Perez-Perez, Y. Chen, W. Schweizer, X. Zheng, M. Contreras, M. G. Dominguez-Bello,M. J. Blaser, Antibiotics, birth mode, and diet shape microbiome maturation during earlylife. Sci. Transl. Med. 8, 343ra82 (2016).

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Antibiotics, birth mode, and diet shape microbiome maturation during early life

Dominguez-Bello and Martin J. BlaserWu, Guillermo I. Perez-Perez, Yu Chen, William Schweizer, Xuhui Zheng, Monica Contreras, Maria Gloria Nicholas A. Bokulich, Jennifer Chung, Thomas Battaglia, Nora Henderson, Melanie Jay, Huilin Li, Arnon D. Lieber, Fen

DOI: 10.1126/scitranslmed.aad7121, 343ra82343ra82.8Sci Transl Med

whether these disturbances influence the health of these babies.those babies who were born vaginally, breast-fed, or unexposed to antibiotics. Future studies will determineis altered in children who are delivered by cesarean section, fed formula, or treated with antibiotics, compared to

now show that this pattern of developmentet al.undergoes many changes during the first 2 years of life. Bokulich The intestinal ''microbiota,'' that is, the community of microbes inhabiting the human intestinal tract,

Snapshots of the developing infant gut microbiota

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