Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns Maria G. Dominguez-Belloa,1,2, Elizabeth K. Costellob,1,3, Monica Contrerasc, Magda Magrisd, Glida Hidalgod, Noah Fierere,f, and Rob Knightb,g aDepartment of Biology, University of Puerto Rico, San Juan, Puerto Rico 00931; bDepartment of Chemistry and Biochemistry, eDepartment of Ecology and Evolutionary Biology, and fCooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO 80305; cCenter of Biophysics and Biochemistry, Venezuelan Institute for Scientific Research, Caracas 1020A, Venezuela; dAmazonic Center for Research and Control of Tropical Diseases, Puerto Ayacucho 7101, Amazonas, Venezuela; and gThe Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80305 Edited by Jeffrey I. Gordon, Washington University School of Medicine, St. Louis, MO, and approved May 24, 2010 (received for review March 2, 2010) Upon delivery, the neonate is exposed for the first time to a wide (12–15), metabolism of xenobiotics (16, 17), pathogen coloni- array of microbes from a variety of sources, including maternal zation resistance (18, 19), and the maturation of the intestine bacteria. Although prior studies have suggested that delivery and the immune system (20, 21), and similarly important roles mode shapes the microbiota’s establishment and, subsequently, are likely played by the microbiotas of nongut body habitats, its role in child health, most researchers have focused on specific although the influence of delivery mode on the bacterial com- bacterial taxa or on a single body habitat, the gut. Thus, the initi- munities found in these habitats is unknown. Delivery mode may ation stage of human microbiome development remains obscure. lead to differences in the microbiota’s development, which The goal of the present study was to obtain a community-wide may then contribute to variations in normal physiology or to perspective on the influence of delivery mode and body habitat on disease predisposition. the neonate’s first microbiota. We used multiplexed 16S rRNA The age-related successional mechanisms involved in the dif- gene pyrosequencing to characterize bacterial communities from ferentiation of the human microbiota across body habitats are mothers and their newborn babies, four born vaginally and six only beginning to be understood (6), and defining the pioneer MICROBIOLOGY born via Cesarean section. Mothers’ skin, oral mucosa, and vagina colonizers is a first step toward elucidating the initial stages of were sampled 1 h before delivery, and neonates’ skin, oral mu- microbiota development. It is thought that the initial microbial cosa, and nasopharyngeal aspirate were sampled <5 min, and me- exposure is important in defining the successional trajectories conium <24 h, after delivery. We found that in direct contrast to leading to more complex and stable adult ecosystems (10, 22), the highly differentiated communities of their mothers, neonates and additionally, initial communities may serve as a direct source harbored bacterial communities that were undifferentiated across of protective or pathogenic bacteria very early in life. Here, we multiple body habitats, regardless of delivery mode. Our results use cultivation-independent, molecular-phylogenetic techniques also show that vaginally delivered infants acquired bacterial com- to characterize the first bacterial assemblages associated with munities resembling their own mother’s vaginal microbiota, dom- full-term babies born vaginally or by C-section, and the inated by Lactobacillus, Prevotella, or Sneathia spp., and C-section assemblages associated with their mothers, across multiple body infants harbored bacterial communities similar to those found on habitats near the time of delivery. the skin surface, dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp. These findings establish an important Results and Discussion baseline for studies tracking the human microbiome’s successional Sampling for this study was performed over 4 d at the obstetrics development in different body habitats following different deliv- unit of the Puerto Ayacucho hospital, Amazonas State, Ven- ery modes, and their associated effects on infant health. ezuela. A total of nine women, aged 21 to 33 y, and their 10 newborns participated in the study. Four women (two Mestizo host–microbe interactions | human microbiome | neonatal bacterial and two Amerindians) delivered vaginally, giving birth to three assemblages | pioneer community males and one female. Five women (four Mestizo and one Amerindian) delivered via C-section, giving birth to three he healthy human fetus is thought to develop within a bacteria- females and three males, including male dizygotic twins. Mothers Tfree environment. Upon delivery, the neonate is exposed to who delivered vaginally were not given antibiotics and had not a wide variety of microbes, many of which are provided by the consumed antibiotics during pregnancy, except for one Mestizo mother during and after the passage through the birth canal, an women, who declared having taken antibiotics in the seventh ecosystem heavily colonized by a relatively limited set of bacterial month of pregnancy. Women who delivered via C-section were taxa (1, 2). Babies are born with immunological tolerance that is instructed by the mother by preferential induction of regulatory T lymphocytes (3), which might allow the baby to become colonized Author contributions: M.G.D.-B. designed research; M.G.D.-B., E.K.C., M.C., M.M., G.H., fi N.F., and R.K. performed research; M.G.D.-B., E.K.C., N.F., and R.K. analyzed data; and by this rst inoculum. However, only a subset (if any) of the M.G.D.-B., E.K.C., and R.K. wrote the paper. microbes to which the newborn is initially exposed will perma- The authors declare no conflict of interest. nently colonize available niches and contribute to the distinctive This article is a PNAS Direct Submission. microbiotas harbored by the body habitats of adults (4–7). Database deposition: The sequence reported in this paper has been deposited in the Many modern human babies are not exposed to vaginal Sequence Read Archive database (ID 020060). microbes at birth. In the United States, for example, more than 1M.G.D.-B. and E.K.C. contributed equally to this work. 30% of all live births in 2007 were Cesarean section (C-section) 2To whom correspondence should be addressed. E-mail: [email protected]. deliveries (http://www.cdc.gov/nchs/births.htm), and differences 3Present address: Department of Microbiology and Immunology, Stanford University in delivery mode have been linked with differences in the in- School of Medicine, Stanford, CA 94305. – testinal microbiota of babies (8 11). Mutualistic relationships This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with intestinal bacteria are known to influence energy balance 1073/pnas.1002601107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1002601107 PNAS Early Edition | 1of5 Downloaded by guest on September 28, 2021 administered cephalosporin several hours before the C-section. We found evidence that infant delivery mode also affected the The mothers’ skin (ventral forearms), oral mucosa, and vagina direct transmission of initial bacteria from mother to newborn. were sampled 1 h before delivery, and their babies’ skin (before Using the weighted UniFrac distance metric, which takes into removing the vernix caseosa), oral mucosa, and nasopharyngeal account both membership and the relative abundance of lineages aspirate were sampled <5 min, and meconium <24 h, after de- (26), we calculated the overall difference between bacterial com- livery. For each sample, variable region 2 (V2) of the bacterial munities from mothers and (i) their own babies, (ii) other babies 16S rRNA gene was PCR-amplified using a primer-set with with the same delivery mode, and (iii) other babies with a different a unique error-correcting barcode (23, 24). Multiplexed pyrose- delivery mode. For vaginally delivering mothers, comparisons were quencing of 34 maternal and 46 infant samples yielded 157,915 made to their vaginal microbiota, and for C-section mothers, com- partial (∼250 bp) 16S rRNA gene sequences, with ∼2,000 parisons were made to their skin microbiota. In three of four sequences per sample. We did not compare bacterial loads in the vaginal deliveries, the mother’s vaginal bacterial community was collected samples, as we could not effectively control for dif- significantly more similar to her own baby’s microbiota than to ferences in the sampled area or volume; instead, we focus here the microbiota of other vaginally delivered babies (Student’s t test, on shifts in bacterial community structure and diversity. P < 0.01) (Fig. 2A), suggesting that the vaginal community [which is In the mothers, bacterial communities were structured pri- unique to each mother (Fig. 1C and Table S2)] is vertically trans- marily by body habitat, with distinct oral, skin, and vaginal mitted to the baby. In contrast, skin bacterial communities of assemblages (Fig. 1A and Table S1), as has been reported in C-section mothers were no more similar to their own babies than to previous studies (4, 5). Accordingly, the mothers’ aggregate the other babies born via C-section (Fig. 2B), even though previous bacterial communities were dominated by taxa typical of these work has demonstrated that adult skin communities are highly habitats; for example, Streptococcus