Subjects and sampling

A prospective cohort study was conducted to compare breastfed vaginally-delivered infants born at home to those born in the hospital. A total of 34 mother-baby dyads were enrolled in this study, including one set of twins (34 mothers, 35 infants). Fourteen babies delivered vaginally at home and 21 delivered vaginally in the hospital. Four of the babies born at home delivered directly into water. Seven of the babies born in the hospital were exposed via maternal treatment to either intrapartum antibiotic prophylaxis (IAP) for group B strep (n = 4) or peripartum antibiotics in the month before or after delivery (n = 7) for indications such as urinary tract and upper respiratory infections (see Supplementary Table S1). There was a high consistency of practice in home and in hospital births. This included deliveries attended by midwives, early skin-to-skin contact between mother and baby after birth, early initiation of breastfeeding, no vaginal or bowel cleansing prior to delivery, and limited use of sterile drapes, gowns and masks.

Maternal, infant, pregnancy and labor characteristics were well matched between cohorts including maternal ethnicity, age, education, pre-pregnant BMI, length of labor, duration of rupture of membranes, baby weight, baby gender, and timing of breastfeeding initiation. Women in the study were healthy and without significant complications during pregnancy including gestational diabetes. Lifestyle characteristics such as exercise and diet were also well matched, and there were no smokers in the study. Labor interventions varied little between home and hospital groups.

All mothers were enrolled during the last month of pregnancy. Recruitment continued from June, 2015 through June, 2016. Home births occurred in a 5-county area in the Hudson Valley Region of New York State and hospital births occurred in a community-level hospital in the same region. Study acceptance rate was 66%. Retention rate was 100%. Study participants met the following criteria: term gestation (>37 and <42 weeks), pregnancy without complications, infants without anomalies or treatment, women 21 years of age or older, women with the ability to understand and meaningfully consent to participation, and fluency in English. This study was approved by the NYU School of Medicine, Institutional Review Board (Study # i14-00377) and all methods were performed in accordance with the relevant guidelines and regulations. Participation was voluntary and included written informed consent.

A sterile, cotton-tipped swab was used for sampling babies’ feces and mothers’ feces and vaginal swabs. Mother and baby dyad samples were collected simultaneously. Sampling was performed at enrollment, the day of birth, followed by days 1, 2, 7, 14, 21, and 28. Infant fecal samples were obtained from feces in the current diaper. Infants delivered in the hospital were discharged between 48 and 60 hours post-partum. Day 0, 1, and 2 samples were collected in the hospital and the remaining samples were collected at home. The maternal vaginal microbiota samples at day 0 were collected during labor and up to two hours after delivery. Samples were kept less than one hour between sampling, and freezing.

Non-biological data consisted of a baseline questionnaire at enrollment that included demographic, medical, lifestyle and environmental information. A birth survey was completed at the time of delivery with labor, delivery, and infant characteristics. Follow-up questionnaires were completed at each of the subsequent sampling episodes to elicit information on current feeding method and changes in mother or baby health status. This information was either directly observed, elicited from the mother and/or confirmed in the medical record.

DNA isolation and analysis

We used approximately 5 grams of the feces collected from the diapers with sterile cotton tipped swabs and placed in sterile tubes. Samples were frozen within 1 hour of collection at <−18 °C until being transported to the lab for storage at −80 °C until processing. Total DNA was extracted from fecal samples using the Mobio Powersoil kit (MO BIO Laboratories, CA, USA), and the V4 region of the 16S rRNA gene was amplified with 515 F/806R V4 primers. The reverse primer contained the 3′ Illumina adapter, a unique 12-base error-correcting Golay barcode, a reverse primer pad, a two-base linker sequence (‘CC’) and the 806R primer for library construction. PCR amplification was carried out (in triplicate) using the Bio-Rad CFX 96 thermal cycler (Bio-Rad, CA, USA). Replicate amplicons were pooled, and DNA concentrations were determined using the Quant-iT PicoGreen dsDNA kit (Invitrogen, NY, USA). Amplicons were pooled in equimolar ratios and purified using QIA quick PCR purification kit (Qiagen Inc, CA, USA) and sequenced using Illumina MiSeq platform (Illumina, CA, USA) with paired-end technique. Sample processing, sequencing, and core amplicon data analysis were performed by the Earth Microbiome Project (www.earthmicrobiome.org), and all amplicon sequence data and metadata have been made public through the EMP data portal (qiita.microbio.me/emp; Study ID: 11575)27.

Microbial 16S rRNA sequencing and analyses

Fecal samples from the first month of life of 20 infants (not exposed to antibiotics or water birth; 10 born at home and 10 in the hospital), generated over 10 million sequences, with an average of 25,921 sequences per sample. Sequences were analyzed using the QIIME (v.1.9.1) pipeline28 based on an open-reference method that maps sequences with 97% identity to known sequences in the Greengenes database (v13_8)29 using UCLUST30 and PyNAST31 alignment algorithms. We used Usearch61 to identify chimeric sequences and performed permutational multivariate analysis of variance (PERMANOVA) to determine differences in beta diversity. To test for statistical significance, we used non-parametric t-tests with 10,000 Monte Carlo permutations in QIIME. The linear discriminant analysis (LDA) effect size (LEfSe) algorithm32 was used to identify significant differences OTU relative abundance. Spearman correlations were calculated using 10,000 permutations for bootstrapped p-value.

Infant sterile fecal water cell assay

The induction of inflammatory response of sterile fecal water was assayed using epithelial cells. This assay allows the determination of epithelial cell response to fecal metabolites33. Analysis of induction of epithelial gene expression, was performed on samples that had enough material remaining (>0.2 grams), which were those from 9 hospital and 10 home-born neonates. A doubled volume of pre-warmed 20% fetal bovine serum (FBS) in [Cat. No. 35-011-CV, CORNING, Manassas, VA 20109] PBS/4 mM EDTA was used to re-suspend stool (range 0.21–0.52 g) from swabs, suspensions were vortexed for 1–3 minutes to fully resuspend all stool material, incubated at 37 °C for 30 minutes, vortexed for 1–3 minutes, incubated at 4 °C overnight, centrifuged at 14,000 rcf (relative centrifugal force) for 10 minutes at room temperature, and the supernatant transferred to a sterile 2 mL tube. Samples were then centrifuged at 16,000 rcf for 30 minutes to remove solids before filtration of the supernatant through WhatmanTM 0.45 um PVDF Mini-UniPrep filters (Cat. No. US203NPEAQU, GE Healthcare Life Sciences) followed by filtration through 0.22 um filters (Cat. No. US203NPUAQU) to produce “sterile fecal water” (SFW) extracts for cell assays. Human colon epithelial HT-29 cells were seeded at a confluent density in 96-well plates in fresh McCoy’s 5A media (Cat. No. 16600-082; Thermo Fisher Scientific) supplemented with 10% heat-inactivated FCS (Cat. No. 9871-5244, USA Scientific), 100 U/ml penicillin-streptomycin (Cat. No. 10378016, Life Technologies, Carlsbad, CA) the day before treatment. After 24 hours, spent culture media was exchanged for fresh media containing SFW (final SFW concentration 25%) for 24 hours. Negative controls included media and SFW buffer. Total RNA was isolated with RNAqueous®-Micro Kit (Cat. No. AM1931, Thermo Fisher Scientific) and digested with DNase I. First-strand cDNA synthesis was performed using 300 ng RNA with High-Capacity RNA-to-cDNATM Kit (Cat. No. 4387406, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Real-time quantitative PCR

Real-time PCR was performed in triplicate with SYBR Green master mix (Cat. No. 4368577, Life Technologies) in the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster, CA). The PCR reaction conditions were as follows: 50 °C for 2 min, 95 °C for 10 min (1 cycle); 95 °C for 15 s and 60 °C for 1 min (40 cycles); and a final melting curve cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. The relative fold change of specific genes in expression is normalized to β-actin expression by the 2−∆∆CT (where C T is threshold cycle) method34. The forward and reverse primers used for PCR analysis of the investigated genes and β-actin are as follows.