Naturalization of the microbiota developmental trajectory of Cesarean-born neonates after vaginal seeding

Se Jin Song, Jincheng Wang, Cameron Martino, Lingjing Jiang, Wesley K Thompson, Liat Shenhav, Daniel McDonald, Clarisse Marotz, Paul R Harris, Caroll D Hernandez, Nora Henderson, Elizabeth Ackley, Deanna Nardella, Charles Gillihan, Valentina Montacuti, William Schweizer, Melanie Jay, Joan Combellick, Haipeng Sun, Izaskun Garcia-Mantrana, Fernando Gil Raga, Maria Carmen Collado, Juana I Rivera-Viñas, Maribel Campos-Rivera, Jean F Ruiz-Calderon, Rob Knight, Maria Gloria Dominguez-Bello, Se Jin Song, Jincheng Wang, Cameron Martino, Lingjing Jiang, Wesley K Thompson, Liat Shenhav, Daniel McDonald, Clarisse Marotz, Paul R Harris, Caroll D Hernandez, Nora Henderson, Elizabeth Ackley, Deanna Nardella, Charles Gillihan, Valentina Montacuti, William Schweizer, Melanie Jay, Joan Combellick, Haipeng Sun, Izaskun Garcia-Mantrana, Fernando Gil Raga, Maria Carmen Collado, Juana I Rivera-Viñas, Maribel Campos-Rivera, Jean F Ruiz-Calderon, Rob Knight, Maria Gloria Dominguez-Bello

Abstract

Background: Early microbiota perturbations are associated with disorders that involve immunological underpinnings. Cesarean section (CS)-born babies show altered microbiota development in relation to babies born vaginally. Here we present the first statistically powered longitudinal study to determine the effect of restoring exposure to maternal vaginal fluids after CS birth.

Methods: Using 16S rRNA gene sequencing, we followed the microbial trajectories of multiple body sites in 177 babies over the first year of life; 98 were born vaginally, and 79 were born by CS, of whom 30 were swabbed with a maternal vaginal gauze right after birth.

Findings: Compositional tensor factorization analysis confirmed that microbiota trajectories of exposed CS-born babies aligned more closely with that of vaginally born babies. Interestingly, the majority of amplicon sequence variants from maternal vaginal microbiomes on the day of birth were shared with other maternal sites, in contrast to non-pregnant women from the Human Microbiome Project (HMP) study.

Conclusions: The results of this observational study prompt urgent randomized clinical trials to test whether microbial restoration reduces the increased disease risk associated with CS birth and the underlying mechanisms. It also provides evidence of the pluripotential nature of maternal vaginal fluids to provide pioneer bacterial colonizers for the newborn body sites. This is the first study showing long-term naturalization of the microbiota of CS-born infants by restoring microbial exposure at birth.

Funding: C&D, Emch Fund, CIFAR, Chilean CONICYT and SOCHIPE, Norwegian Institute of Public Health, Emerald Foundation, NIH, National Institute of Justice, Janssen.

Keywords: Translation to humans.

Conflict of interest statement

Declaration of interests New York University has a U.S. patent (10357521) on behalf of M.G.D.-B., related to methods for restoring the microbiota of newborns.

Copyright © 2021. Published by Elsevier Inc.

Figures

Figure 1.. Fecal microbiota development during the…
Figure 1.. Fecal microbiota development during the first year of life in babies discordant to birth mode/exposure.
(a) Compositional Tensor Factorization (CTF) first principal component (Y-axis) of infant samples over age in days (X-axis). (b) Convex hull volume (Y-axis) on the first three Principal Coordinates (unweighted UniFrac distances) in mothers (purple) and infants by birth mode or exposure (X-axis). Infants show highest volumes in Cesarean born and lowest in Vaginally born, with Cesarean-seeded babies showing intermediate volumes; all pairwise comparisons are significant using Mann-Whitney test with Bonferroni corrections at 0.05 level (Table S3). (c-e) Songbird differentials shown for day 2, 30, and 180 after birth; ternary plots of the inverse additive log-ratio transform (inverse-ALR) of Songbird differentials give the estimated probability of a microbe being observed in Cesarean (left-axes; red), Vaginal (bottom-axes; blue), or Cesarean-seeded (right-axes; green). The color of the dots depicts the seeding effectiveness, with yellow color indicating effectively seeded/suppressed and black indicating not effectively seeded. Below each triangle, bar plots of top and bottom 20% Songbird differentials summarized at genus-level taxa between Cesarean-seeded and Cesarean born babies; a positive value indicates higher association with the Cesarean-seeded group, a negative value indicates higher with Cesarean. Bars are colored by the ASVs’ seeding effectiveness. The majority of taxa discordant overrepresented in the Cesarean-seeded group over the Cesarean group are yellow-orange, indicating ASVs effectively seeded in the Cesarean seeded group, and these are observed at all ages. See alsoFigure S4, Supplementary Methods S2–S7.
Figure 2.. Oral microbiota development during the…
Figure 2.. Oral microbiota development during the first year of life in babies discordant to birth mode/exposure.
(a) Compositional Tensor Factorization (CTF) first principal component (Y-axis) of infant samples over age in days (X-axis). (b) Convex hull volume (Y-axis) on the first three Principal Coordinates (unweighted UniFrac distances) in mothers (purple) and infants by birth mode or exposure (X-axis). Infants show highest volumes in Cesarean born and lowest in Vaginally born, with Cesarean-seeded babies showing intermediate volumes; all pairwise comparisons are significant using Mann-Whitney test with Bonferroni corrections at 0.05 level (Table S3). (c-e) Songbird differentials shown for day 2, 30, and 180 after birth; ternary plots of the inverse additive log-ratio transform (inverse-ALR) of Songbird differentials give the estimated probability of a microbe being observed in Cesarean (left-axes; red), Vaginal (bottom-axes; blue), or Cesarean-seeded (right-axes; green). The color of the dots depicts the seeding effectiveness, with yellow color indicating effectively seeded/suppressed and black indicating not effectively seeded. Below each triangle, bar plots of top and bottom 20% Songbird differentials summarized at genus-level taxa between Cesarean-seeded and Cesarean born babies; a positive value indicates higher association with the Cesarean-seeded group, a negative value indicates higher with Cesarean. Bars are colored by the ASVs’ seeding effectiveness. The majority of taxa discordant overrepresented in the Cesarean-seeded group over the Cesarean group are yellow–orange, indicating ASVs effectively seeded in the Cesarean seeded group, and these are observed at all ages. See alsoFigure S4, Supplementary Methods S2–S7.
Figure 3.. Skin microbiota development during the…
Figure 3.. Skin microbiota development during the first year of life in babies discordant to birth mode/exposure.
(a) Compositional Tensor Factorization (CTF) first principal component (Y-axis) of infant samples over age in days (X-axis). (b) Convex hull volume (Y-axis) on the first three Principal Coordinates (unweighted UniFrac distances) in mothers (purple) and infants by birth mode or exposure (X-axis). Infants show highest volumes in Cesarean born and lowest in Vaginally born, with Cesarean-seeded babies showing intermediate volumes; all but one pairwise comparison are significant using Mann-Whitney test with Bonferroni corrections at 0.05 level (Table S3). (c-e) Songbird differentials shown for day 2, 30, and 180 after birth; ternary plots of the inverse additive log-ratio transform (inverse-ALR) of Songbird differentials give the estimated probability of a microbe being observed in Cesarean (left-axes; red), Vaginal (bottom-axes; blue), or Cesarean-seeded (right-axes; green). The color of the dots depicts the seeding effectiveness, with yellow color indicating effectively seeded/suppressed and black indicating not effectively seeded. Below each triangle, bar plots of top and bottom 20% Songbird differentials summarized at genus-level taxa between Cesarean-seeded and Cesarean born babies; a positive value indicates higher association with the Cesarean-seeded group, a negative value indicates higher with Cesarean. Bars are colored by the ASVs’ seeding effectiveness. The majority of taxa discordant overrepresented in the Cesarean-seeded group over the Cesarean group are yellow–orange, indicating ASVs effectively seeded in the Cesarean seeded group, and these are observed at all ages. See alsoFigure S4, Supplementary Methods S2–S7.
Figure 4.. Microbial source tracking of the…
Figure 4.. Microbial source tracking of the neonate microbiome (first month) through fast expectation-maximization microbial source tracking (FEAST).
Contributions (y-axes) of various maternal sources (rows) to the infant microbial community (columns) are estimated across age in days (x-axes) for the first month of life, in 15 mother-baby pairs. Error bars show 95% confidence interval of the mean calculated by bootstrapping; Dunn test based on Kruskal-Wallis were performed on each time points by each maternal source for each baby sink, significant differences are marked by different letters in each panel. The vaginal source -prominent in day “0” for oral and skin in babies exposed to vaginal fluids (vaginal and CS-seeded; panel e, f)- as not prominent later in any baby site. Baby site specific communities resemble the corresponding maternal site (panels a, h, o), consistent with specific site selection of bacteria. The maternal right areola appears as a source for baby oral bacteria (panel k), which likely means that baby oral bacteria is transmitted to the mother’s areola during lactation.
Figure 5.. Proportions of bacterial vaginal ASVs…
Figure 5.. Proportions of bacterial vaginal ASVs shared with other body sites in the mothers of the current study, at the day of delivery (a) and in non-pregnant women (b).
(a) V4 sequences from vaginal swabs and gauzes obtained from 97 parturient mothers form this study at the day of birth. Current study data were sequenced by Illumina HiSeq and processed by QIIME2 using the same pipeline as for the HMP data. (b) HMP V4 data from vaginal swabs obtained from 105 non-pregnant women; ASVs included in the analyses were present in at least 10% of the samples in the respective body site. Roche 454 V3V5 sequences were trimmed to obtain the V4 region. See alsoFigure S2 b–c, Supplementary Methods S13–S14.

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