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Official websites use. Share sensitive information only on official, secure websites. The rate of caesarean section delivery CSD is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered VD and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide LPS biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming. The effects of caesarean section delivery on mother-to-neonate transmission of microbiota are unclear. Here the authors show that caesarean section delivery can affect the transmission of specific microbial strains and the immunomodulatory potential of the microbiota. The past decades have witnessed steadily increasing rates in caesarean section deliveries CSD performed largely in the absence of medical necessity and reaching proportions of During vaginal birth, specific bacterial strains are transmitted from mothers to infants 3 — 6 and differences in microbial colonization in neonates born by CSD have been identified 7 — 10 as early as 3 days postpartum 7 , However, due to conflicting results, which principally imply a negligible impact of delivery mode on the colonizing neonatal microbiome in the gut 11 , it remains unclear whether disruption of mother-to-infant transmission of microbiota through CSD occurs and whether it affects human physiology early on, with potentially persistent effects in later life. To address these concerns, it is essential to determine if there are differences in the functional complement conferred by the earliest colonizing microbiota in relation to CSD, if any differences result from changes in the transmission of strains from mothers to neonates, and if these impact neonatal physiology. While the majority of studies so far indicate that delivery mode is the strongest factor determining early neonatal gut microbiome colonization 3 , 7 — 10 , 18 , these effects are either extenuated or largely absent in other studies 11 , In this context, it is important to consider that CSD may be performed as a result of underlying maternal or foetal medical conditions e. More specifically, CSD is most often accompanied by the administration of antibiotics to mothers due to local health regulations or hospital practices e. SGA neonates have an elevated propensity for developing metabolic disorders during childhood or adulthood, which has been associated with alterations to the gut microbiome 23 , and may be linked to the elevated rate of CSD in this population. Apart from confounding factors, the methods and study designs employed over the past years may in part explain some of the conflicting results regarding the effect of delivery mode on the early gut microbiome. Notably, taxonomic profiling based on 16S rRNA gene amplicon sequencing does not offer sufficient resolution to assess the direct effect of the delivery mode at the level of strain transmission, which is expected to be a determinant of succession. Although recent studies have focused on mother-to-neonate strain transfer and have shown that maternal strains do colonize the neonatal gut, non-vaginal delivery was not assessed comprehensively 4 — 6. In addition, although single nucleotide variants SNVs have been tracked over time, no such studies have so far covered the earliest time points after delivery days 0—5 in well-matched mother—neonate pairs in relation to a direct comparison of delivery modes. Consequently, there is a strong need for adequate high-resolution metagenomic analyses capable of resolving the vertical transmission of individual-specific strains and encoded functions from mothers to neonates on an individual basis, while also supplementing observed in silico findings with further in vitro validation experiments. Independent of whether prenatal colonization of the foetus takes place or not 24 , delivery marks the moment of extensive exposure to microbial communities of faecal, vaginal, skin and environmental origins and this event thereby has a profound impact on the colonization of the neonatal gut 9 , The initial low microbial biomass in the earliest neonatal stool samples 7 makes the sequencing data prone to the over-representation of putative artefactual reads 24 , 26 and may contribute to the generation of inconsistent results. Therefore, the removal of any artefactual sequences is essential to ensure unambiguous, high-resolution overviews of the earliest microbial colonization of the neonatal gut. Here, we performed a detailed analysis of the earliest microbial colonization of the neonatal gut using a combination of 16S rRNA gene amplicon sequencing and high-resolution metagenomics. Our results highlight differences in gut microbiome composition according to delivery mode as well as concurrent differences in the encoded functional potential, which in turn are linked to differences in the transfer of strains from mother to neonate. Based on the enrichment of the LPS biosynthesis pathway in VD neonates, we performed LPS extractions from neonatal stool samples, in vitro immune stimulation assays as well as extensive assessments of LPS purity. Taken together, we observe a microbiome-driven relationship between delivery mode and endotoxin-induced immune system priming with the potential for lasting effects in later life. To characterize the temporal patterns of earliest microbial colonization in relation to delivery mode, we recruited and sampled a total of 33 neonates Supplementary Data 1. The neonatal gut microbiome of some of these neonates had previously been characterized using a combination of 16S rRNA gene amplicon sequencing and quantitative real-time PCR 7. For a subset of neonates, well-matched neonatal and maternal samples were subjected to high-resolution metagenomic analyses. Extracted genomic DNA from all samples was subjected to 16S rRNA gene amplicon sequencing and extracted DNA from the samples of the subset of mother—neonate pairs were subjected to random shotgun sequencing. The 16S rRNA gene amplicon sequencing data were processed using NG-Tax 27 , while the resulting metagenomic data were processed using a reproducible, reference-independent bioinformatic pipeline Neonatal stool samples from days 1—5 postpartum contain limited amounts of microbial DNA 7 , and low-biomass samples are prone to over-representation of artefactual DNA that is introduced during the extraction procedure or preparation of sequencing libraries 24 , For the 16S rRNA gene amplicon sequencing data, any possible effect from putative artefactual reads was restricted by applying the methodology previously described in Wampach et al 7. To account for the presence of artefactual sequences in the metagenomic data, we devised an additional, combined in vitro and in silico strategy to identify and remove artefactual sequences from the metagenomic data Fig. For the in vitro part, DNA was extracted from a human gut epithelial cell line using the same procedure as for the neonatal stool samples and diluted to the levels of DNA extractable from the collected low-biomass samples Methods. The choice of human DNA as a negative control was based on the following criteria: i the inability to generate a sequencing library from blank water control samples due to the inherent very low amounts of DNA these are typically below the threshold for library construction ; ii the ability to clearly differentiate signal in the titration series: human sequences from artefacts non-human sequences ; microbial DNA was not chosen as the homology between contaminant and bona fide sequences may have confounded delineation; iii the removal of human sequences is common practice when performing metagenomic analyses on human samples and appropriate methods exist to distinguish between human and microbial sequences in silico; iv the blinding of the variability originating from the laboratory environment or sequencing facility due to the nature of the samples i. Our in silico workflow for the identification and removal of artefacts from metagenomic data Fig. It subsequently removes contigs from study samples that cluster with the artefactual contigs, i. After subsequent filtering steps and the successful removal of artefactual contigs from all study samples, we observed differences in the number of removed reads according to sample type Supplementary Fig. On the basis of this essential data curation step, sequences from Achromobacter xylosoxidans or Burkholderia spp. Using the curated metagenomic data, we obtained taxonomic profiles Supplementary Data 3 , functionally annotated gene sets Supplementary Data 4 , reconstructed genomes following binning 30 and strain-determining variant patterns 31 Supplementary Data 5. Curation of metagenomic data. The resulting data sets used for inter-sample comparisons are highlighted in grey. The 16S rRNA gene amplicon and the metagenomic sequencing data, which were generated for a subset of mother—neonate pairs, showed highly similar succession trends in terms of diversity, evenness and richness measures Supplementary Fig. The taxonomic profiles derived from the 16S rRNA gene amplicon and metagenomic sequencing were highly correlated Supplementary Fig. The differences in taxonomic profiles according to delivery mode reflected results from previous studies, notably the higher relative abundance in Bacteroides and Parabacteroides and lower levels in Staphylococcus in VD neonates at days 3 and 5 postpartum 7 , 10 Supplementary Data 6 to 8 ; Supplementary Note 3. In order to resolve the effect of delivery mode in relation to other potentially contributing factors such as maternal antibiotic intake prior to delivery, gestational age, feeding regime and sampling time point, differentially abundant taxa for both 16S rRNA gene amplicon and metagenomic sequencing data were determined separately using a multivariate additive general model approach MaAsLin Taking into account the effects of the above-mentioned factors, delivery mode was found to be the dominant driver of neonatal gut microbiome colonization, with other measured factors having considerably less of an effect Supplementary Note 4 ; Supplementary Data 9. To assess whether the apparent taxonomic differences between the gut microbiomes of VD and CSD neonates are reflected at the level of functional potential, we used the metagenomic sequencing data to calculate Jensen-Shannon divergences for all samples Supplementary Fig. Overall, comparison of the functional profiles of all neonates to the gut microbial potential of their respective mothers highlighted that the neonatal gut microbiota were more divergent from the maternal vaginal microbiota than the corresponding gut microbiota Supplementary Fig. Maternal and neonatal gut microbiome functional profiles. Colour key indicates row-wise z-scores. These presented the same directionality of change using the R package DESeq2 33 with a linear model considering the different collection time points containing at least 1, KOs days 3 and 5 as covariates Fig. Among the differentially abundant genes, there was an enrichment in genes involved in LPS biosynthesis Fig. Results from the multivariate analyses demonstrated that delivery mode was the strongest determining factor in both data sets i. Whilst not statistically significant, the trends for the predicted microbial pathways obtained with PanFP were largely concordant with the enriched pathways in VD neonates based on the differential analysis of the metagenomic data. Nevertheless, predictions of functional potentials based on 16S rRNA gene amplicon sequencing data are likely unreliable as a significant fraction of the gut microbiome i. In contrast, the metagenomic data, through resolving the actual functional gene complement, allows a detailed comparison of the functional potential of the earliest gut microbiomes, as well as the tracking of individual-specific single-nucleotide variants SNVs. To determine if the observed differences in microbial functions were encoded by specific strains that were vertically transferred from the mother to the neonate, we mined the metagenomic sequencing data to identify microbial taxa and strains that both members of any of the 16 maternal—neonatal pairs, for which we had generated metagenomic data, had in common. We devised an ensemble approach to link reconstructed genomes on the basis of taxonomic annotations 30 , , similarity of phylogenetic marker genes and the presence of SNVs 31 Methods. This enabled the tracking of specific strains from mothers to neonates Supplementary Data 5. Given the high degree of specificity, the presence of transferred strains is highly relevant on a pair-by-pair, individual basis to assess mother-to-neonate transfer. This is all the more important given the extensive inter-individual variability of the neonatal gut microbiome Supplementary Fig. While the reconstructed genomes of 25 taxa belonging to the phyla Proteobacteria and Firmicutes were identified in maternal—neonatal pairs for all birth modes mostly the skin-derived and upper-gastrointestinal tract-inhabiting genera Streptococcus and Staphylococcus spp. Notably, in the case of vaginal delivery, multiple strains of Gram-positive bacteria e. Transmission of functions by distinct microbial strains. The level of evidence of transmission is indicated by the shading colour, with darker shading for stronger evidence. A taxon without link describes a taxon that was found in the maternal samples, but not shared between mother and neonate. Neonates C and C are twins. Phylum-level colour key is given in a. Encircled symbols highlight strains that are shared with the respective mother. Circles and triangles represent maternal and neonatal faecal samples, respectively. Our results reflected a shift in population structures during the transfer from mothers to neonates. The differences in relative abundance of taxa corresponded to the inferred routes of transmission linked to birth mode. While the same strain of B. Taken together, these results are consistent with the transmission of strains from the maternal gut microbiome during vaginal delivery, resulting in relatively stable colonization of the neonatal gut during the earliest days, in contrast to CSD neonates. To assess whether the transferred strains conferred specific functional traits to the neonate or not, we assessed the genomic complements of the earliest microbiota. Analysis of reconstructed genomes that were linked to maternal metagenomes showed that vertically transmitted strains were more likely to be enriched in functions that were depleted in CSD neonates odds ratio OR 5. Among these strains, Bacteroidetes B. Most strikingly, a B. By contrast, strains of Staphylococcus aureus , an uncharacterized Actinobacterium and S. The reconstructed genome of S. These results indicate that vaginal delivery not only favours the vertical transfer of enteric strains from mother to neonate, but also results in the transfer of specific functional traits to the neonate, which are involved in important microbial pathways such as LPS biosynthesis and may be relevant in stimulating the developing immune system during the first days of life. As LPS forms part of the outer membrane of Gram-negative bacteria, the attributed Gram staining information of microorganisms directly corresponds to their propensity to synthetize LPS. Consequently, an apparent higher microbial synthesis of LPS likely results in an increased immunostimulatory potential of the developing gut microbiome. To test whether the VD-associated colonizing gut microbiota, which encode a specific functional complement including an enrichment in genes involved in LPS biosynthesis , drives early physiological differences in VD neonates, we focussed on the early immunostimulatory potential of LPS from the neonatal gut. Based on our data, the microbial composition differed most strongly in VD and CSD neonates on day 3 postpartum and thereby may critically affect the developing immune system at this time We subsequently used several approaches to assess the purity of the isolated LPS fractions Supplementary Note 5; Methods. Using agarose and polyacrylamide gel electrophoresis, we successfully visualized the isolated LPS and did not find traces of protein contamination but observed minor traces of fragmented DNA. Some microbial products detected by hTLR2 e. Consequently, the composition of the different isolated LPS fractions played a role in the subsequently triggered immune response. To assess the stimulatory effect at the interface between the neonatal innate and adaptive immune systems 36 , we stimulated monocyte-derived dendritic cells MoDCs from 12 adult donors with neonatal LPS extracts Methods. In parallel, a panel of additional cytokines was measured using an approach for quantifying and normalizing the employed LPS fractions Methods. Based on the outcome of all applied methods, we observed that the nature and composition of the different LPS subtypes contributed to the level of immune activation triggered by the LPS fractions from the different samples. Taken together, these results demonstrate higher immunostimulatory potentials of the earliest gut microbiome in neonates born by VD compared to CSD. Cytokines of monocyte-derived dendritic cells after stimulation with LPS from neonatal stool and in neonatal plasma. MoDCs were stimulated with the exact same LPS volume that was extractable from the same initial amount of faecal material from each neonate sample Methods. Exact numbers of donors used per sample are given in the plot. Positive control: LPS isolated from E. Circles correspond to neonates with metagenomic data, crosses represent neonates without metagenomic data. To test whether potential effects of differential immunostimulation are apparent early on in vivo, we assessed cytokine levels in plasma samples from a total of 31 neonates Supplementary Data 1. Plasma samples were collected on the same day as the neonatal faecal samples from which LPS was isolated, i. Here we employed high-resolution, artefact-curated metagenomic analyses of paired, high-quality samples from mothers and neonates to resolve the neonatal gut microbiome over the first few days of life. While previous studies have used analogous analytical approaches 16S rRNA gene amplicon sequencing and metagenomics to resolve the early neonatal gut microbiome, these studies did not involve the systematic collection and appropriate preservation of paired mother—neonate samples 11 , they did not specifically track vertical strain transfer 11 , 18 , they did not include provisions for the removal of artefactual sequences 3 , 5 , 6 , 11 , 18 , they did not focus on the earliest time points after delivery 3 , 18 , nor did they resolve differences in functional potential according to delivery mode 3 , 5 , 6 , 11 , However, consideration of these factors is essential to assess the effect of delivery mode on the earliest transfer of community structure and function, subsequent microbiome colonization patterns and the resulting effects on neonatal physiology. Our cohort included paired sample sets from mothers i. All microbiome samples yielded high-resolution, artefact-curated sequencing data, which was analysed at the strain level. As earliest neonatal gut microbiome samples are naturally of low biomass, the accurate identification and curation of potential artefactual sequences is essential. In the absence of appropriate controls, sequences derived from contaminant taxa in reagents may be relatively prominent, thereby masking actual signals and confounding results regarding in particular the transfer of taxa and functions from mothers to neonates. In our study, adequate controls were included and putative artefactual reads removed based on a combined in vitro and in silico workflow. In order to reach the required specificity to unambiguously address the question of vertical transmission of microbial community structure and function from mother to neonate, the use of curated, high-resolution metagenomic sequencing data rather than solely performing 16S rRNA gene amplicon sequencing is imperative. More specifically, the applied methodological approach allows the highly specific tracking of individual microbial functions and strains from mother to neonates on a case-by-case basis. Our results based on both 16S rRNA gene amplicon and metagenomic sequencing, and supported by multivariate analyses, demonstrate that early differences exist in the gut microbiomes of neonates and that these differences are predominantly driven by the mode of delivery. Our data agrees with previously reported differences in microbial composition related to birth mode, notably the increased relative abundance of Bacteroides and Parabacteroides in VD neonates as well as OTUs assigned to Staphylococcus being enriched in CSD neonates 3 , 4 , 7 , In line with the broad taxonomic differences between VD and CSD, our findings demonstrate that CSD significantly affects the functional gene complement of the earliest neonatal gut microbiome by impeding the vertical transfer of specific bacterial strains from the maternal gut microbiome to the neonate. Consequently, the gut of CSD neonates is most likely colonized by strains derived from other sources, such as breast milk, skin or saliva, as suggested in previous studies 40 — Notably, a selection of enteric strains from the mother was found to be exclusively transferred to the VD neonate e. When measuring population differentiation F ST , we found evidence that strains acquired from the mother are capable of quickly adapting to the new environment, as observed previously 5. In contrast, vaginal strains harboured a low potential to stably colonize the neonatal gut thereby further adding to recently published data demonstrating that vaginal taxa do not play a prominent role in the initial colonization of the neonatal gut 5. This may in part be explained by the distinct niches of the anaerobic gastrointestinal environment and the microaerophilic vaginal environment. With respect to ongoing clinical interventions aimed at restoring the earliest neonatal gut microbiome in the case of caesarean section 43 , our findings raise questions over the expected efficiency of microbiota engraftment from a purely vaginal source and suggest that gut-derived strains may be more efficacious. Independent of the precise mechanism of strain transfer, we observed that several functional pathways were significantly under-represented in CSD neonates, while these were in turn enriched in VD neonates and linked to vertically transmitted strains, in particular the LPS biosynthesis pathway Fig. LPS, an outer surface membrane component of Gram-negative bacteria, promotes the secretion of pro-inflammatory cytokines and thereby sits at the interface of the earliest gut microbiome colonization and neonatal immune system priming. Following the apparent enrichments in LPS biosynthesis in VD neonates due to higher amounts of Gram-negative bacteria, the subsequent extraction and quantification of LPS from neonatal stool and stimulation of primary human immune cells therewith demonstrated a reduced immunostimulatory potential of the earliest gut microbiome in CSD neonates. The differences in earliest immune system priming may result in persistent effects on human physiology in later life, which has also been recently suggested based on work in a murine model On the basis of the observed immunogenicity of the purified LPS fractions, it has not escaped our attention that other factors, including the actual LPS composition, may additionally contribute to the difference in the immunostimulatory potential of the colonizing gut microbiome Fig. Furthermore, other bacterial products, triggering for example TLR2, may contribute towards the observed higher immunostimulatory potential of faecal LPS from VD neonates Supplementary Fig. Considering the potential repercussions on neonatal physiology, the detailed elucidation of these additional factors will be the subject of future work. Our study highlights differences in immunostimulatory potential of the earliest gut microbiome according to delivery mode. This occurs during a critical window of immune system priming. Notably, alterations to early immune system stimulation may be linked to the higher propensity of CSD infants to develop chronic diseases in later life 2. In this context, LPS is likely closely involved in the priming of the neonatal immune system and the subsequent tolerance towards the colonizing gut microbiome during a most critical window in early neonatal life 12 — Using a mouse model, it has been shown that strongly immunostimulatory LPS can contribute to the protection from immune-mediated diseases such as diabetes 47 and that disruption of host-commensal interactions in early-life can lead to persistent defects in the development of specific immune subsets Investigations of the longer-term consequences of these differences between CSD and VD neonates will be necessary to assess their possible impact on the development of chronic diseases in later life. Apart from LPS biosynthesis, other pathways that were significantly enriched in the gut microbiome of VD neonates included genes involved in membrane transport, i. On the one hand this may reflect the adaptation of the colonizing microbiome of VD neonates to the gut environment through enhanced nutrient intake. On the other hand, associated ABC transporter proteins for both Gram-positive and Gram-negative bacteria have previously been shown to be immunogenic 48 , which may suggest that they play a role in the activation of the neonatal immune system. Additionally, enrichments in pathways relating to bacterial motility were observed. These included the two-component system pathway, which is an important mediator of signal transduction, flagellar assembly and bacterial chemotaxis. These pathways are essential for bacterial motility in response to external stimuli and consequently also for competition with other members of the gut microbiome Additionally, flagellin, the main structural component of the flagellum, is an effective stimulator of innate immunity 50 and promotes mucosal immunity through the activation of TLR5 Another functional pathway that is potentially interacting with the human immune system early on is the resistance to cationic antimicrobial peptides CAMP. While this resistance has been observed in all major commensal phyla and across all members of the phylum Bacteroidetes, this pathway is essential to evade detection by the human immune system through the modification of the microbial LPS structure In the context of our study, an enrichment in CAMP resistance may therefore prevent the dominant colonizers i. Future studies are needed to assess whether the gut microbiome of VD neonates harbours more modified LPS moieties linked to CAMP resistance and which potential effects the altered LPS structures may have on the neonatal immune system. In accordance with the observation of an apparent enrichment in flagellar biosynthesis, bacterial chemotaxis and CAMP resistance, other microbiota-derived molecular factors, apart from LPS, may be involved in early immune system priming. Our results imply that a more comprehensive understanding of the effect of the earliest microbial exposure on innate and adaptive immune responses and the different molecular factors involved in neonatal immune system priming is necessary. Future long-term follow-up studies based on larger cohorts, high-resolution multi-omic analyses, detailed immunological screening and tracking of health status will be essential to unravel the interdependencies between mode of delivery, other potential confounding factors, mother-to-neonate transmission, microbiome colonization, exposure to microbial factors, immune system priming and long-term health status. Furthermore, additional sources of maternal strains of importance in relation to microbiome-conferred molecular factors, besides the maternal vagina and gut, have to be considered to assess their relative importance in relation to their impact on neonate physiology. For this, additional samples may be obtained from maternal milk, skin, the oral cavity, and the hospital environment 5 , 11 , 40 — An additional focus should be placed on uncovering the source and mode of transfer of gut strains from mothers to neonates. Such mechanistic understanding will be important for devising future clinical interventions principally aimed at restoring a VD-like pioneering microbiota in the case of CSD. An alternative approach may consist of ensuring appropriate early priming of the neonatal immune system by the controlled provision of microbial antigens. Both avenues may provide the basis for the development of preventative strategies for adverse health effects in CSD neonates in the future. Written informed consent was obtained before specimen collection from all enrolled mothers after a detailed consultation. Exclusion criteria for mother—neonate pairs included the administration of antibiotics to neonates immediately postpartum, birth prior to 34 weeks of gestation, and maternal gestational diabetes. Clinical metadata for all the analysed time points days 1, 3 and 5 postpartum are listed in Supplementary Data 1. Recorded metadata include information on the delivery mode, classification of caesarean section as elective or emergent, birth weight, gestational age, identification of the neonate as small for gestational age SGA status where relevant, gender, body length, weight and feeding regime. If a neonate received formula milk at any collection time point, the neonate was considered having received combined feeding for the remainder of the study, as even short-term formula feeding has been shown to cause profound and long-lasting shifts in the gastrointestinal microbiome composition Samples and data were collected at the CHL until day 3 after birth; subsequent samples were collected at home by trained study nurses. From the 33 neonates that were recruited into the study, the gut microbiome of 15 Supplementary Data 1 had previously been characterized using a combination of 16S rRNA gene amplicon sequencing and quantitative real-time PCR 7. For a subset of neonates, the mother was sampled additionally. This step was repeated until all lysate had passed through the filter. This latter step was omitted for the low input neonatal samples. Selected DNA samples of maternal vaginal and faecal extracts , neonatal faecal extracts at days 1, 3 and 5 and cell-culture origins were subjected to random shotgun sequencing Supplementary Data 1. Illumina TruSeq3-PE-2 adapter sequences were trimmed from the reads in the pre-processing step including the removal of human reads , and the de novo assembly step used the MEGAHIT 54 metagenome assembler. The IMP parameters were customized for different sample types: default parameters were retained for maternal faecal samples; for low-biomass samples maternal vaginal swabs, neonatal faecal samples from days 1, 3 and 5 and cell culture sample , the integrated VizBin 29 sequence cut-off length was set to 1. To identify and exclude artefactual sequences in the low biomass samples, contigs were assembled from the sequencing reads obtained from the DNA extracts of the Caco-2 cells after the removal of human reads. Then, metagenomic reads from each study sample were mapped against these contigs using Bowtie 2 55 version 2. Matching sequences were excluded prior to taxonomic profiling of metagenomic reads by phylogenetic markers As the artefactual sequences identified in the control samples did not represent full genomes, we further used a binning-based approach to identify additional potential artefactual sequences of the same organism among the de novo assembled contigs of the study samples. Bins were identified based on VizBin embeddings 56 using density-based spatial clustering of applications with noise DBSCAN , without correction for the depth of coverage and completeness. Genes were predicted from contigs assembled with IMP and, after removing artefactual contigs, these genes were functionally annotated with hidden Markov models HMMs 56 trained for all KO 57 groups. The best hit KO if multiple KOs could be assigned to a gene, the KO with the highest bit score was chosen for every gene was assigned if the bit score was higher than the binary logarithm of the number of target genes. The reconstructed genomes of all samples belonging to a mother—neonate pair were merged into a union set. For each sample set, predicted amino acid sequences were searched against and annotated using a defined set of essential marker genes 61 using HMMER 3. Protein sequences assigned to 35 specific marker genes that form the cross-section of previously suggested sets of phylogenetic marker genes 61 , 62 were selected. The frequencies of genes from different genome reconstructions co-occurring in the same clusters were determined. A simple graph network representation was constructed with the reconstructed genomes as nodes and counts of co-occurrences between two reconstructed genomes as weighted, undirected edges. The resulting reconstructed genomes from a given sub-network were manually inspected, and the taxonomy of reconstructed genomes was assigned using PhyloPhlAn 30 Supplementary Data 5. Strains that occurred in multiple samples were determined with StrainPhlAn 31 , using the pre-processed sequencing read data and reconstructed genomes. For each sample, taxonomic profiles were generated from pre-processed reads with MetaPhlAn2 65 using default settings. Strain reconstructions were extracted with the sample2markers. StrainPhlAn was used to extract the clades detected in all samples and to construct reference databases for each clade. The sample-based strain reconstructions and reference databases of each clade and all reconstructed genomes were analysed with StrainPhlAn to build multiple sequence alignments and phylogenetic trees. The neonatal samples were considered to share strains with maternal samples if the cophenetic distance between the neonatal microbiome read-based or reconstructed genome-based markers and the maternal markers was less than the distance to the markers of any other individual. To reduce bias stemming from variation in coverage, alignments were down-sampled to a median coverage of 20X using Picard tools version 1. SNV calling was performed with FreeBayes 68 version 1. When reads of two or more samples mapped with sufficient coverage to the same genome, the fixation index F ST was calculated, reflecting the population differentiation between a pair of samples. F ST is defined as one minus the average intra-population diversity of the samples divided by the nucleotide diversity between the samples inter-population diversity. POGENOM was tuned to include only the loci recovered in all samples mapped to the same reference genome, assuring a valid comparison of the intra-species variation. To exclude sequencing artefacts, only dominant phylotypes were examined by removing OTUs that were represented by fewer than 10 reads in the study samples. Final Gram staining was assessed by main staining trends per genus and manually curated at the family and order levels. The R statistical software package version 3. Sum normalization and calculations of taxon richness number of metagenomic OTUs mOTUs for metagenomic data or OTUs for amplicon sequencing data , diversity Shannon , evenness Pielou indices and Spearman correlation coefficients were performed using the vegan R package. To discover differences in the data sets between the birth modes at the different collection time points postpartum, Wilcoxon rank-sum tests were applied, with FDR multiple-testing adjustment if applicable. Differential taxonomic abundances according to delivery mode were also calculated using ANCOM 71 with Benjamini-Hochberg multiple testing correction at 0. Principal coordinate analysis PCoA graphs were generated using Jensen-Shannon distances as implemented in the R package phyloseq Differentially abundant pathways were detected through pathway enrichment analysis using a custom R script LPS was isolated from 16 selected neonatal faecal samples on the basis of availability of sufficient material. For the E. LPS was isolated from cell pellets by the same protocol as above. From the 16 neonatal faecal samples, 11 produced measurable amounts of LPS, whereas 5 were under the detection limit Supplementary Data An extraction blank was generated using the same LPS isolation protocol. Primer sequences, annealing temperatures and cycle details are specified in Supplementary Data Reaction mixture, measurements and calculations of bacterial load nanograms bacterial DNA per milligram stool and nanograms E. The proportion of E. Primary human monocytes were isolated from blood samples obtained from the Luxembourg Red Cross originating from twelve healthy adult donors. Human neonatal dendritic cells DCs were previously shown to be competent in MHC class I antigen processing and presentation to the same extent than adult DCs As we started from the same amount of material for all the neonatal stool samples and used the exact same extraction protocol to isolate all LPS fractions for all samples, we assumed that if we treated MoDCs from the same donor with the exact same volume of yielded LPS independent of the concentration of LPS present , we would realistically emulate the microbial LPS load which immune cells would be exposed to in vivo and thus be representative of the immunostimulatory potential of a given sample at 3 days postpartum. To stimulate MoDCs, 7. For the negative control, MoDCs were incubated with 7. Treatments were performed in duplicates and tested on at least three different donors. For the second method, we verified our results using the bacterial load for normalizing LPS concentration values. In order to assess if the differences that we observed before with equal volumes of LPS see above were due to the fact that some samples have a much lower bacterial load or if also the bacterial composition and proportion of Gram-negative bacteria plays a role in the immunostimulation, we normalized the amount of LPS used to stimulate MoDCs with the bacterial load. Therefore, this load was divided by the load in each other sample to yield a normalization factor. For other samples, the LPS load was calculated by multiplying 2. Statistical significance between the different cohorts was determined using the Wilcoxon rank-sum test. On the basis of availability of sufficient extracted LPS material, 0. As positive controls, 0. A precast gel was loaded with the LPS samples and stained with Coomassie Imperial protein stain, ThermoFisher, Belgium to check for protein contaminations. To check if LPS extracts were contaminated with immunostimulatory nucleic acids, 0. In addition, 0. As controls, bands of E. In addition, a purification blank was generated. While the hTLR4 receptor only recognizes LPS, hTLR2 recognizes peptidoglycan, lipoteichoic acid and lipoprotein from gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from the yeast cell wall, the receptor NOD1 binds to bacterial molecules containing the D-glutamyl-meso-diaminopimelic acid iE-DAP moiety and NOD2 recognizes bacterial molecules peptidoglycans and stimulates an immune reaction. In silico analyses presented in this paper were carried out using the HPC facilities of the University of Luxembourg We are grateful to all the parents and neonates who participated in the study. We thank the dedicated clinical staff and neonatologists of the paediatric clinic and gynaecologists at the CHL for participant recruitment and sample collection, especially Alain Noirhomme and all involved study nurses of the Clinical and Epidemiological Investigation Center CIEC who performed sample and data collection at the CHL and at home as well as the scientific staff of the IBBL for sample storage. Sample collection, processing and storage were co-funded by the Integrated BioBank of Luxembourg under the Personalised Medicine Consortium Diabetes programme. Both L. All authors read and approved the final manuscript. The pre-processed, non-human metagenomic sequencing data and the amplicon sequencing data generated during the current study are available from NCBI under bioproject accession number PRJNA A reporting summary for this Article is available as a Supplementary Information file. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Nat Commun. Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential Linda Wampach Linda Wampach 1 Luxembourg Centre for Systems Biomedicine, University of Luxembourg, avenue des Hauts-Fourneaux 7, Esch-sur-Alzette, Luxembourg. Find articles by Linda Wampach. Find articles by Anna Heintz-Buschart. Find articles by Javier Ramiro-Garcia. Find articles by Janine Habier. Find articles by Malte Herold. Find articles by Shaman Narayanasamy. Find articles by Anne Kaysen. Find articles by Angela H Hogan. Find articles by Lutz Bindl. Find articles by Jean Bottu. Find articles by Rashi Halder. Find articles by Patrick May. Find articles by Anders F Andersson. Find articles by Carine de Beaufort. Find articles by Paul Wilmes. Contributed equally. Received Aug 3; Accepted Nov 13; Collection date Open in a new tab. Supplementary Information 3. Peer Review File Supplementary Data 1 Supplementary Data 2 Supplementary Data 3 Supplementary Data 4 5MB, xlsx. Supplementary Data 5 Supplementary Data 6 Supplementary Data 7 28KB, xlsx. Supplementary Data 8 Supplementary Data 9 Supplementary Data 10 Supplementary Data 11 4MB, xlsx. Supplementary Data 12 Supplementary Data 13 Supplementary Data 14 Supplementary Data 15 Supplementary Data 16 52KB, xlsx. Supplementary Data 17 Reporting Summary Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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