Delivery, Feeding and Gender Differently Contribute to Infant Gut Microbiota Development

Delivery, Feeding and Gender Differently Contribute to Infant Gut Microbiota Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Delivery, Feeding and Gender Differently Contribute to Infant Gut Microbiota Development Juan Ding, Xiao Ma, Hongyan Ren, Chenyang Zhi, Qi Xin, Zhen Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-828854/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To characterize gut microbiome of the infant during the first year of life and assess the different contributions of delivery mode, feeding mode and infant gender to gut microbial development. We collected 314 faecal samples from 80 infants at 5 time points of 0, 1st, 3rd, 6 th and 12 th months prospectively, and finally 213 samples completed Miseq sequencing and analysis. We characterized gut microbiome of the infant at the different phases and evaluated the different contributions of delivery mode, feeding mode and gender to gut microbial development. Delivery mode, gender and feeding mode were the strongest factors determining gut microbiome colonization at 0 months, from 1 month to 6 months and 12 months, respectively. Four genera including Bifidobacterium , Bacteroides , Parabacteroides and Phascolarctobacterium were increased, whereas 10 genera e.g. Salmonella and Enterobacter were reduced, in vaginal delivery versus cesarean section. Two genera including Peptostreptococcaceae incertae sedis and Anaerococcus were increased, whereas 3 genera e.g. Coriobacteriaceae uncultured were reduced, in exclusive breastfeeding versus combined feeding. This study indicated the contribution degrees of delivery mode, feeding mode and gender to gut microbial initiation and evolvement, and reported microbial differences induced by the different delivery mode, feeding mode and gender. Biomedical Engineering Obstetrics & Gynecology Infectious Diseases infant gut microbiota microbiota development delivery mode feeding mode infant gender. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The infant gut microbiota is critical to human health, playing significant roles in metabolism, immunity and development 1 , 2 . Colonization of the infant gut microbiota is a complex process dependent on multiple overlapping factors, such as delivery mode 3 , 4 , feeding mode 5 , 6 , gestational age 7 and environmental exposures 8 . There is growing evidence linking host health with the establishment of the infant gut microbiome. Cesarean delivery is associated with a number of human diseases, including obesity 9 , 10 , asthma 11 , celiac disease 12 , and type 1 diabetes 13 , 14 . Infant gut microbiota colonization patterns differ between infants experiencing vaginal delivery and those delivered by cesarean Sect. 15 . Cesarean-delivered infants have particularly low richness and diversity compared with vaginally delivered infants. Breastfeeding is related to fewer illnesses, such as obesity 9 , 10 , asthma 11 and metabolic syndrome 16 , than formula feeding. Feeding mode also influences the establishment of the infant gut microbiota. Breastfed infants are reportedly colonized with bacteria in breast milk and with greater numbers of Bifidobacterium than formula-fed infants 17 . Gender-associated differences are relevant to the prevalence, manifestations, and outcomes of malignancies and autoimmune and infectious diseases 18 . Gender-associated differences in the gut microbiota can influence susceptibility to disease 19 . There is an undeniable need to explore gut microbiota characteristics caused by gender differences. However, the contribution degrees of these factors to the development of the infant gut microbiota remain unknown. In this study, a total of 82 infants were prospectively enrolled, and fecal samples were separately collected at 0 months (meconium) and 1, 3, 6 and 12 months postpartum. Finally, 55 healthy infants were enrolled, and their samples were subjected to 16S rRNA gene sequencing. We characterized the gut microbiome at 5 time points during the first year of life, analyzed the bacterial community, and reported differences at crucial time points of infant development. Furthermore, we assessed the different contributions of delivery mode, feeding mode and infant gender to gut microbial development and compared microbial differences induced by the different delivery, feeding and gender. Material And Methods Study Participants The primary objective of this study was to use longitudinal sampling to monitor changes in the infant gut microbiome over time. Specific variables were examined, including delivery mode, feeding mode and infant gender. All study procedures were consistent with the ethical guidelines of the Helsinki Declaration. The study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University (2017-KY-12). Recruitment took place in China between July 2017 and August 2017. Infants were excluded if they had a health condition or received antibiotics. Written informed consent was obtained from all parents of the infants before collecting data and fecal samples. Basic demographics, delivery mode, feeding mode, and information concerning current or recent medications (including antibiotics) were obtained from hospital electronic medical records and questionnaires. Infants who had been breastfed and never given formula prior to fecal collection were assigned exclusively breastfed status. Infants who were fed both breast milk and formula prior to the time of stool collection were identified as having received combined feeding (both breast milk and formula milk). If an infant received formula milk at any collection time point, the infant was considered to have received combined feeding for the remainder of the study, as even short-term formula feeding causes profound and long-lasting shifts in the gut microbiome composition 44 . Sample Collection The study population was recruited before mothers gave birth. Meconium samples were collected in the hospital, and other fresh fecal samples were collected at their homes. Fecal samples were collected by the infant’s parents and stored in a household freezer (−20°C); samples were immediately shipped on dry ice to the laboratory, where they were placed at −80°C until DNA was extracted. DNA extraction DNA was extracted from the fecal samples using the E.Z.N.A. ® Stool DNA Kit (Omega Bio-tek, Inc., GA) according to the instructions provided by the manufacturer. DNA was quantified by a Qubit ® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States), and fragment sizes were determined using 1.5% agarose gel electrophoresis. PCR amplification The extracted DNA samples were amplified with a set of primers targeting the hypervariable V3-V4 region (341F/805R) of the 16S rRNA gene. The forward primer (341F) was 5’-CCTACGGGNGGCWGCAG-3’, and the reverse primer (805R) was 5’-GACTACHVGGGTATCTAATCC-3’. PCR was performed with an EasyCycler 96 PCR system (Analytik Jena Corp., AG) using the following program: 3 min of denaturation at 95 ℃; 21 cycles of 0.5 min at 94 ℃ (denaturation), 0.5 min for annealing at 58℃, and 0.5 min at 72 ℃ (elongation); and a final extension at 72 ℃ for 5 min. Paired-end sequencing was performed with the constructed sequencing libraries on the Illumina MiSeq platform by Shanghai Mobio Biomedical Technology Co., Ltd., China. The raw Illumina read data from all samples were stored in the European Bioinformatics Institute European Nucleotide Archive database (PRJNA665920). Operational taxonomic unit (OTU ) clustering and taxonomy annotation Random reads were chosen from all fecal samples with equal numbers, OTUs were binned by the UPARSE pipeline, and OTUs were binned by the UPARSE pipeline with the following steps: (i) abundant sequences and singletons were first removed, (ii) unique sequences were binned into OTUs with the command “usearch-cluster_ otus”, and (iii) randomly chosen sequences were aligned against OTU sequences with the command “usearch-usearch_global-id 0.97”. The identity threshold was set as 0.97, and then an OTU composition table was created. We annotated sequences by using RDP classifier version 2.6 45 . Bacterial diversity and taxonomic analysis Bacterial diversity was determined by sampling-based analysis of OTUs and represented by the Shannon index, Simpson index and Chao1 index, which were calculated using the R package “vegan” 46 . Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) based on OTUs were performed by the R package (http://www.r-project.org/) to analyze microbiome distances between samples. Unweighted UniFrac distances were calculated with the phyloseq package 47 . Taxonomic group comparisons, including comparisons of bacterial phyla, order, family and genera, were conducted using the Wilcoxon rank sum test. A heatmap that identified discriminatory variables was generated by a heatmap builder. The linear discriminant analysis (LDA) effect size (LEfSe) method was used to distinguish taxonomic types in microbial communities related to specific characteristics (http://huttenhower.sph.harvard.edu/lefse/) 48 . With a normalized relative abundance matrix, LEfSe used the Kruskal-Wallis rank sum test to detect the features related to markedly different abundances between assigned bacterial taxa and used LDA to assess the effect size of each feature 49 . Functional annotation of the 16S rRNA gene phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) version 1.0.0 pipeline 50 and human version 0.99 51 were used to establish KEGG orthology (KO) and KEGG pathway/module profiles and predict the functional profiles of the gut microbiota based on 16S rRNA gene sequences. Bioinformatics and statistical analysis Clean data were extracted from raw data using USEARCH 7.1 with the following criteria: (i) sequences shorter than 400 bp after the merge or containing a homopolymer longer than four bases were discarded; (ii) sequences from each sample were extracted using each index with fewer than two mismatches, and (iii) sequences with an overlap shorter than 16 bp were discarded. We evaluated the associations of delivery mode, feeding mode, and infant gender with microbiome community composition using generalized UniFrac analysis 52 . The phylogenetic tree required for UniFrac analysis was computed based on FastTree 53 and was midpoint rooted. Statistical comparisons of groups were conducted using ADONIS between groups with 10,000 permutations. We computed mean generalized UniFrac distances for both within and between delivery mode groups (vaginal delivery and cesarean section), feeding mode groups (exclusive breastfeeding and combined feeding) and infant gender. Statistical comparisons of mean UniFrac distances were performed using the Kruskal-Wallis rank sum test. The statistical significance threshold was set at P < 0.05. Results Study Population In this study, a total of 82 infants (314 fecal samples) were recruited, and infant fecal samples were collected at 0 months (meconium), 1 month (27 to 33 days), 3 months (87 to 93 days), 6 months (177 to 183 days) and 12 months (357 to 363 days) after birth. After confirmation, 2 infants without fecal samples, 3 premature infants, 5 infants with incomplete information, and 17 infants with drug therapy were excluded. After DNA extraction, 16S rRNA gene sequencing and data quality control, 38 fecal samples were further discarded. Finally, 55 healthy infants with 213 fecal samples at 0 months (n = 19), 1 month (n = 49), 3 months (n = 50), 6 months (n = 47) and 12 months (n = 48) were included in the final analysis. In total, 8,542,788 raw reads were obtained after sequencing. The mean read depth per sample was 40,107 ± 10,818 sequences per sample. After initial quality filtering, 6,904,876 sequences passed with a mean of 32,417 ± 7,111 sequences per sample. A flowchart describing the sample collection is shown in Fig. 1 . All infants included in the study were fed complementary food after fecal samples were collected at 6 months. The complementary food mainly included vegetables, porridge and pasta. Clinical information of the participants Comprehensive clinical information for each enrolled individual was recorded ( Table 1). We evaluated delivery and feeding characteristics in 55 infants ( Table 1) . Samples showed that 36 (65.5%) of enrolled infants had vaginal deliveries, and 19 (34.5%) infants had cesarean section deliveries. Thirty-six (65.5%) infants were exclusively breastfed, and 19 (34.5%) infants received combined feeding. Infant gut microbial diversity during the first year of life Bacterial diversity was determined by sampling-based analysis of OTUs as estimated by the Shannon index ( Fig. 2a ) and the Simpson index (Fig. 2b ). The fecal microbial diversity was significantly decreased at 0 months compared with the other 4 times ( p < 0.001 and p < 0.001, respectively, Supplementary Table S1 ). Microbial diversity was markedly increased at 12 months compared with the other 4 times ( p < 0.001 and p < 0.001, respectively, Supplementary Table S1 ). The microbial diversity presented no significant difference between 1, 3 and 6 months ( p = 0.4 and p = 0.87, respectively, Supplementary Table S2 ). These data suggested that gut microbial diversity was consistently increased from 0 months to 12 months with the development of the infant. To assess the overall diversity in bacterial composition, we performed PCoA and NMDS analysis based on unweighted UniFrac distances (Fig. 2c-d and Supplementary Fig. S1 , Supplementary Table S3 ) . The PCoA based on the unweighted UniFrac distances indicated that there was a notable separation of samples at 0, 1, 3, 6 and 12 months (Adonis p < 0.001, R 2 = 0.08, Supplementary Table S4 ). There was an obvious separation in gut microbiome composition between 0 months and 1 month (Adonis p = 0.001, R 2 = 0.04). Meanwhile, the microbial community at 12 months was clustered together and significantly separated from that at 6 months in the PCoA (Adonis p < 0.001, R 2 = 0.06). No obvious separation was observed in the microbiota between 1, 3 and 6 months (Adonis p = 0.22, R 2 = 0.02). These results suggested that infants’ gut microbiota was consistently altered from 0 months to 12 months with the development of the infant. In addition, we calculated the average UniFrac distances within each group to assess the microbial differences between the individual infants. We found the greatest average distance within each group between the infants at 0 months. The distance within the group was significantly decreased at 1, 3, and 6 months versus 0 months. Notably, the distance within the group remarkably increased from 6 months to 12 months (Fig. 2e, Supplementary Table S5 ) . These data demonstrated that individual microbial variation of the infants was decreased from 0 months to 1 month but then increased from 6 months to 12 months. Gut microbiota community structure during the first year of life A heatmap from hierarchical clustering analysis revealed a discriminatory gut microbiome during the first year of life. Comparison of fecal microbiomes demonstrated that 60 key OTUs were significantly different at 0, 1, 3, 6, and 12 months ( Supplementary Fig. S 2 ). We further analyzed the infant gut microbiota composition and alterations during the first year of life. The fecal bacterial composition in each sample at the phylum and genus levels is shown in Supplementary Fig. S 3 -4 , respectively ( Supplementary Table S6-S7 ). The average compositions of the microbial community at the phylum and genus levels during the first year of life are shown in F ig. 3a-b ( Supplementary Table S8 -S9 ). Moreover, bacterial compositions at the class and order levels during the first year of life are shown in Supplementary Fig. S 5a-b ( Supplementary Table S10-S11 ). The bacterial phyla Firmicutes, Proteobacteria , Bacteroidetes and Actinobacteria , together accounting for up to 99% of sequences on average, were the four dominant phyla at 0 months. Firmicutes, Proteobacteria and Actinobacteria , together accounting for greater than 85%, were the three dominant phyla at 1, 3, 6 and 12 months, respectively. The average abundance of the phylum Firmicutes increased from 16.34% at 0 months to 48.15% at 12 months ( p < 0.001, F ig. 3c, Supplementary Table S 12 ), while the abundance of the phylum Proteobacteria decreased gradually from 67.43% at 0 months to 15.82% at 12 months ( p < 0.001, F ig. 3d ). Moreover, the average relative abundance of the phylum Actinobacteria consistently increased from 4.59% at 0 months to 33.34% at 3 months ( p < 0.001) and then presented no significant alterations from 3 months to 12 months (26.25%) ( F ig. 3e ). Eighty-nine genera in the infant gut microbiota were detected during the first year of life. The infant gut microbiota was dominated by Bifidobacterium, Streptococcus, Escherichia−Shigella , Klebsiella , Bacteroides , Clostridium sensu stricto 1 , Veillonella , Lactobacillus and Lachnospiraceae incertae sedis , and their total abundance exceeded 70%. The average relative abundance of Bifidobacterium significantly elevated during the period from 0 months (2.66%) to 3 months (28.15) ( p < 0.001) and had no obvious increase from 3 months to 12 months (23.81%) ( Fig. 3f, Supplementary Table S 13 ) . The average amount of Streptococcus remarkably increased from 0 months (6.19%) to 1 month (14.11%) ( p < 0.001) and presented no significant changes from 1 month to 12 months (11.91%) (Fig. 3g) . Additionally, Veillonella consistently increased from 0 months (2%) and 1 month (3.22%) to 12 months (5.48%) ( p < 0.001, Fig. 3h) . To identify specific bacterial taxa associated with the time points, we compared the fecal microbiota using LEfSe. A cladogram representative of fecal microbial structures and their predominant bacteria displayed the greatest differences in taxa among different times (all p < 0.05, Supplementary Fig. S 6a-b, Supplementary Table S14 ). The gut microbial community metabolic function profiles and the predominant microbial functions at 0, 1, 3, 6 and 12 months were analyzed by a cladogram and LDA ( Supplementary Fig. S 7a -b, Supplementary Table S15 ). Gut microbial differences of the infants at the crucial development time points Compared with that at 1 month, as estimated by the Shannon index, Simpson index and Chao1 index, fecal microbial diversity at 0 months was significantly decreased ( p < 0.001, p < 0.001 and p < 0.05, respectively, Supplementary Fig. S 8a-c, Supplementary Table S16 ). A heatmap revealed a discriminatory gut microbiome and demonstrated that 15 key OTUs were significantly different between 0 months and 1 month ( Supplementary Fig. S 9 ) . We analyzed differences in bacterial taxonomic compositions at the phylum and genus levels between 0 months and 1 month.(all p < 0.05, Supplementary Fig. S 10a - b, Supplementary Table S17-S18 ). The predominant fecal microbiome at 0 months and 1 month was determined by LEfSe and LDA ( all p 3, Supplementary Fig. S 11a-b, Supplementary Table S 19 ). The predominant fecal microbial functions at 0 and 1 month were visualized by a cladogram and LDA (all p 2, Supplementary Fig. S 12a-b, Supplementary Table S20 ). Compared with that at 12 months, fecal microbial diversity at 6 months was significantly decreased (all p < 0.001, Supplementary Fig. S 13 a-c, Supplementary Table S21). A heatmap revealed a discriminatory gut microbiome and demonstrated that 35 key OTUs were significantly different between 6 months and 12 months ( Supplementary Fig. S 14 ). We analyzed differences in bacterial taxonomic compositions at the phylum and genus levels (all p < 0.05, Supplementary Fig. S 15a-b , Supplementary Table S22-S23 ). The predominant fecal microbiome at 6 months and 12 months was determined by LEfSe and LDA (all p 2, Supplementary Fig. S 16a-b, Supplementary Table S 24 ) . The fecal predominant microbial functions at 6 months and 12 months were visualized by a cladogram and LDA (all p 2, Supplementary Fig. S 17a-b, Supplementary Table S25 ). These data revealed significant differences in the gut microbiota between these two groups. Delivery, feeding and gender contribute differently to microbiota development Each infant has unique gut microbial characteristics. We analyzed gut microbial similarity within each group, compared the within-group average UniFrac distances between infants within specific delivery mode, feeding mode and gender groups, and revealed individual microbial differences of the infant. Greater average UniFrac distances were observed between infants who were born by vaginal delivery versus cesarean delivery (Fig. 4a, Supplementary Table S 26 ) . A greater average distance was observed between infants who received combined feeding versus those exclusively breastfed (Fig. 4b, Supplementary Table S 26 ). Within-group distances revealed no significant difference between boys and girls (Fig. 4c, Supplementary Table S 26 ) . These data suggested that vaginal delivery and combined feeding played an important role in the development of the infant gut microbiota. We further calculated between-group average UniFrac distances to assess the microbial compositional similarities. After birth, the UniFrac distance between vaginal delivery and cesarean section was the largest, followed by the distances between feeding modes and gender differences (Fig. 5a, Supplementary Table S 27 ) . At 1, 3 and 6 months, the average distance was greater between boys and girls than between vaginal delivery and cesarean section, as well as exclusively breastfed and combined feeding (Fig. 5b-d, Supplementary Table S 27 ). By 12 months, the contributions to the development of gut microbiota had changed. The greatest distance was observed between exclusively breastfed and combined feeding; then between vaginal delivery and cesarean section, as well asbetweenboys and girls (Fig. 5e, Supplementary Table S 27 ). These data demonstrated that for the development of the infant gut microbiota, the delivery mode was the main contribution at birth, gender accounted for the dominant contribution at 1, 3 and 6 months, and feeding mode played the main role after 12 months. The effect of delivery, feeding and gender on the gut microbiota We analyzed fecal bacterial composition and differences between vaginal delivery and cesarean section. A heatmap revealed a discriminatory gut microbiome between vaginal delivery and cesarean section, and demonstrated that 17 key OTUs were significantly different between the two groups ( Supplementary Fig. S 18 ). The average composition of the microbial community at the phylum and genus levels between the two groups is shown in Fig. 6a and Supplementary Fig. S 19a ( Supplementary Table S 28-S29 ). At phylum level, 2 phyla including Actinobacteria and Bacteroidetes were increased, whereas 2 phyla including Firmicutes and Proteobacteria reduced, in vaginal delivery versus cesarean section (all p < 0.05, Fig. 6b, Supplementary Table S 30 ). At genus level, 4 genera including Bifidobacterium , Bacteroides , Parabacteroides and Phascolarctobacterium were increased, whereas 10 genera mainly including Clostridium sensu stricto 1, Salmonella and Enterobacter reduced, in vaginal delivery versus cesarean section (all p < 0.05, Fig. 6c , Supplementary Table S 31 ). The predominant fecal microbiome for vaginal delivery and cesarean section were determined by LEfSe ( Supplementary Fig. S 20a ). Based on LDA, 14 bacteria mainly including Bacteroidetes , Bacteroidales , Bacteroidia and Actinobacteria were enriched, while 14 bacteria mainly including Firmicutes , Proteobacteria and Clostridiales , were reduced for vaginal delivery compared with cesarean section (all p 2.5, Supplementary Fig. S 20b, Supplementary Table S32 ). The predominant fecal microbial functions for vaginal delivery and cesarean section were analyzed by a cladogram ( Supplementary Fig. S 21a ). Based on LDA, 81 functions mainly including Genetic Information Processing, Replication and Repair, Metabolism and Translation were enriched, while 40 functions mainly including Environmental Information Processing, Membrane Transport and Transporters, were reduced for vaginal delivery compared with cesarean section (all p 2, Supplementary Fig. S 21b, Supplementary Table S33 ). We further analyzed bacterial taxonomic compositions and alterations at the phylum level between vaginal delivery and cesarean section infants under fixed exclusive breastfeeding ( p < 0.05, Supplementary Fig. S 22a-b, Supplementary Table S 34-S35 ) .The predominant fecal microbiome associated with delivery mode under fixed exclusive breastfeeding was determined by LEfSe and LDA (all p 2, Supplementary Fig. S 23a-b, Supplementary Table S36 ). We also analyzed gut microbial composition and alterations between exclusive breastfeeding and combined feeding. A heatmap revealed a discriminatory gut microbiome between exclusive breastfeeding and combined feeding, and demonstrated that 17 key OTUs were significantly different between the two groups ( Supplementary Fig. S 24 ). The average composition of the microbial community at the phylum and genus levels between the two groups is shown in Supplementary Fig. S 19b and Fig. 6d ( Supplementary Table S 37-S38 ). We found no significant between two groups at phylum level (all p < 0.05, Supplementary Table S 39 ). At genus level, 2 genera including Peptostreptococcaceae incertae sedis and Anaerococcus were increased, whereas 3 genera including Lachnospiraceae incertae sedis , Coriobacteriaceae uncultured and Erysipelotrichaceae norank reduced, in exclusive breastfeeding versus combined feeding (all p < 0.05, Fig. 6e , Supplementary Table S 40 ). The predominant fecal microbiome for vaginal delivery and cesarean section were determined by LEfSe ( Supplementary Fig. S 25a ). Based on LDA, 3 bacteria including Anaerococcus , Serratia and Peptostreptococcaceae were enriched, while 2 bacteria including Lachnospiraceae and Incertae Sedis , were reduced for exclusive breastfeeding compared with combined feeding (all p 2, Supplementary Fig. S 25b, Supplementary Table S41 ). The predominant fecal microbial functions for exclusive breastfeeding compared with combined feeding were analyzed by a cladogram ( Supplementary Fig. S 26a ). Based on LDA, 2 functions including Fatty acid metabolism and Glycine serine and threonine metabolism were enriched, while 2 functions including Sporulation and Methane metabolism, were reduced for exclusive breastfeeding compared with combined feeding (all p 2, Supplementary Fig. S 26b, Supplementary Table S42 ). We further analyzed bacterial taxonomic compositions and alterations at the phylum level between exclusive breastfeeding and combined feeding infants under fixed vaginal delivery ( p < 0.05, Supplementary Fig. S 27, Supplementary Table S 43-S44 ) .The predominant fecal microbiome associated with feeding mode under fixed vaginal delivery was determined by LEfSe and LDA (all p 2, Supplementary Fig. S 28, Supplementary Table S45 ). Moreover, we analyzed gut microbial composition and alterations between boys and girls. A heatmap revealed a discriminatory gut microbiome boys and girls, and demonstrated that 17 key OTUs were significantly different between the two groups ( Supplementary Fig. S 29 ). The average composition of the microbial community at the phylum and genus levels between the two groups is shown in Supplementary Fig. S 19c-d ( Supplementary Table S 46-S47 ). We found no significant between two groups at phylum level (all p < 0.05, Supplementary Table S 48 ). At genus level, 2 genera including Alistipes and Anaeroglobus were increased, whereas 4 genera including Parasutterella , Eubacterium, Peptoniphilus and Anaerosporobacter reduced, in boys versus girls (all p < 0.05, Fig. 6f , Supplementary Table S 49 ). The predominant fecal microbiome for vaginal delivery and cesarean section were determined by LEfSe ( Supplementary Fig. S 30a ). Based on LDA, 3 bacteria including Veillonellaceae , Selenomonadales and Negativicutes were enriched, while 2 bacteria including Burkholderiales and Family XI , were reduced for boys compared with girls (all p 2, Supplementary Fig. S 30b, Supplementary Table S50 ). We further identified different bacterial taxa associated with gender under fixed vaginal delivery and feeding with exclusively milk conditions ( Supplementary Fig. S 31-S32, Supplementary Table S51-S53 ). Functional prediction of microbial genes associated with delivery mode and feeding mode at 12 months The gut microbial community metabolic function profiles and the predominant microbial functions at 12 months related to vaginal delivery and cesarean sectionare shown in a cladogram ( Supplementary Fig. S 33a ). Based on LDA, nitrogen metabolism was enriched, while 3 functions, including replication recombination and repair proteins , valine leucine and isoleucine biosynthesis, and basal transcription factors , were reduced for vaginal delivery compared with cesarean section (all p 2, Supplementary Fig. S 33b , Supplementary Table S 54 ). Moreover, the gut microbial community metabolic function profiles and the predominant microbial functions at 12 months for exclusively breastfed and combined feeding are also shown in a cladogram ( Supplementary Fig. S 34a ). Based on LDA, compared to the combined feeding group, 20 functions, mainly ABC transporters , ascorbate and aldarate metabolism, and nitrogen metabolism, were increased, whereas 9 functions, mainly amino sugar and nucleotide sugar metabolism , replication recombination and repair proteins, were decreased in the breastfed group ( all p 2, Supplementary Fig. S 34b , Supplementary Table S 55 ) . Discussion We characterized the gut microbiome of 55 infants at 0, 1, 3, 6, and 12 months after birth; analyzed the influence of delivery mode, feeding mode and gender on the gut microbiota; and evaluated the contributions of these factors to gut microbial development. Although delivery mode 20 and feeding mode 21 together influenced infant gut microbial composition and evolution, our study demonstrated the contribution degrees of these factors to gut microbial development. We found that delivery mode was the main contribution at birth and that feeding mode played the main role in infant gut microbiota development after 6 months. Interestingly, we found that gender accounted for the dominant contribution to infant gut microbial development at 1, 3 and 6 months after birth, which overturned our common theory. In our study, individual microbial variation dynamically changed with infant growth. Individual microbial variation in the infants decreased from 0 months to 1 month but then increased from 6 months to 12 months. Individual microbial variation at birth was initiated by differences in the maternal microbiota 22 and vaginal 23 , maternal skin 24 and hospital environments 25 . Vaginally delivered infants are exposed to the vaginal and fecal microbiota, leading to gut colonization by vagina-associated microbes such as Lactobacillus 23 . In contrast, cesarean section-delivered infants do not experience direct contact with maternal microbes and are thus more likely to become colonized by maternal skin, the hospital environment, or hospital staff 20 . With the development of infants, complementary foods, different living environments and different contact populations together contributed to the evolution of the infant gut microbiota, subsequently resulting in increased individual variation after 6 months. The shift in dietary patterns from exclusive breastfeeding to the consumption of solid foods induces the development of the mature gut microbiota 26 . Our study indicated that due to the complementary introduction of a variety of novel food substances, alpha diversity and individual variation increased. We observed individual differences between vaginally delivered infants and cesarean delivered infants. We found that the variation in the microbial communities of vaginally delivered infants was greater than that of cesarean delivered infants. In healthy infants, delivery is the initial encounter with bacteria capable of colonizing the gut. In a previous study of 24 infants aged 3 to 4 months in Canada, Bacteroides was depleted in cesarean delivered infants relative to those who were vaginally delivered 6 . This result was also underscored in a study of 24 infants in Sweden that reported that the depletion of Bacteroides in infants delivered by cesarean section persisted until 12 months 27 . Our findings indicated that Bacteroides levels were differentially abundant between vaginally and cesarean delivered infants, in line with the results of earlier studies. These high levels of Bacteroides in infancy are associated with increased risks of asthma and obesity later in life 28 , 29 . Consistent with a previous study on premature infants, we observed that delivery mode was the main contribution at birth 13 , 15 . Independent of whether prenatal colonization takes place in the fetus, delivery marks the moment of extensive exposure to bacterial communities of vaginal, fecal, skin and environmental origins 30 . During vaginal birth, specific microbial strains associated with the mother’s vaginal microbiota are vertically transmitted from mothers to infants 24 , 31 . Therefore, delivery mode has a profound impact on the earliest microbial colonization of the neonatal gut. Moreover, we observed gut microbial differences between combined feeding infants and exclusively breastfed infants. The microbial variation between the combined feeding infants was greater than that between exclusively breastfed infants. In a previous study of 102 infants aged 6 weeks, infants who were fed breast milk supplemented with formula had greater individual differences than exclusively breastfed infants 3 . The large individual difference in the gut microbiota in combined feeding infants may be caused by the use of different brands of formula. Feeding mode has the potential for lasting effects on the gut microbiota, and these effects may be associated with childhood and lifelong health 32 . Studies show that breastfeeding confers protection against gastrointestinal tract and respiratory infections and allergic diseases in addition to reducing the risk of chronic diseases, such as inflammatory bowel disease, obesity and diabetes 5 , 33 , 34 . Our study indicated that feeding mode played the main role after 6 months in the development of the infant gut microbiota. A previous study showed that bacteria from breast milk are most prominent in infants’ guts in the first month, accounting for nearly 40% of the gut bacteria in primarily breastfed infants 5 . Ding et al. recently reported that breastfeeding during infancy was a major life-history characteristic that affected microbial community composition in adults 35 . The breast milk microbiota that seeds the infant’s gut first influences and selects for the microbiota that follow, leaving a footprint that can be detected even in adulthood. Although the differences in breast milk bacteria increase between mothers during the first 6 months of infancy 5 , the other factors that affect the gut microbiota, including delivery mode and gender, still have greater impacts than the feeding mode on the gut microbiota at 0 months and at 1 month to 6 months, respectively. Interestingly, we found that gender accounts for the dominant contribution at 1, 3 and 6 months after birth. Gender differences in the gut microbiota are driven by gender hormones, which in turn affect gender differences in susceptibility to a large number of chronic diseases and infections 36-38 . Three populations were found to significantly increase over time in the boy group: Selenomonadales, Negativicutes and Veillonellaceae . A recent study suggests that lower abundances of Selenomonadales and Veillonellaceae are associated with autism spectrum disorder 39 , 40 . Dysregulation of Selenomonadales affects gut secretion and in turn affects brain function by modulating the efficacy of the vagal response 41 . A relative paucity of Negativicutes might predispose patients to necrotizing enterocolitis 42 . From another perspective, the difference between boys and girls begins with the gut microbiota in infancy. Children manifest secondary genderual characteristics after gender hormone stimulation at puberty 43 . We hypothesize that the gender-specific early gut microbiota development precedes the development of secondary genderual characteristics. These studies and our results hint that the gender of the newborn affect the gut microbiome and can be valuable for optimizing prevention and treatment strategies. There is currently a paucity of data addressing how the gender of the infant affects the gut microbiota profiles; thus, this study provides important insights into the impact of the gender of the infant on gut microbiota development. In conclusion, based on a gut microbial characterization during infant development, this study found an increase in gut microbiota diversity that increased over time and indicated that individual microbial variation in infants decreased from 0 to 1 month but then increased from 6 to 12 months. Importantly, we demonstrated the contribution degrees of delivery mode, gender and feeding mode to gut microbial establishment and evolution, which overturned our common theory and provided a novel concept. Understanding the patterns of infant gut microbial colonization and development is critical for both short- and long-term health and may provide a solid foundation for future health outcomes through microbiota intervention. Abbreviations PCoA, principal coordinates analysis; NMDS, nonmetric multidimensional scaling; OTUs, operational taxonomic units; LEfSe, linear discriminant analysis effect size; PICRUSt, phylogenetic investigation of communities by reconstruction of unobserved states. Declarations Acknowledgement We thank clinical doctors from the First Affiliated Hospital of Zhengzhou University. We also thank the generous volunteer subjects who enrolled in the study. Funding This study was sponsored by grants from China Postdoctoral Science Foundation (2020T130609 and 2020T130109ZX), Henan Provincial Medical Science and Technology Project (SBGJ2018004), the National Key Research and Development Program of China (2018YFC2000500), Henan Province Science and Technology Project (182102310404 and 202102310055) and Key Scientific Research Projects of Higher Education Institutions in Henan Province (21A320055 and 20A320056). The funding sources had no role in the design of this study nor any role during its execution, analyses, data interpretation, or decision to submit results. Availability of data and materials The raw Illumina read data for all samples were deposited in the European Bioinformatics Institute European Nucleotide Archive database under the accession number PRJNA665920. Author contributions: RZG and DJ designed the study. DJ, MX, XQ, LZ, HLP, ZXL, ZCY and ZZH collected clinical samples. RHY, LA and LC performed Miseq sequencing and data analysis; DJ, RZG, and MX wrote the manuscript. All authors reviewed and approved the manuscript. Compliance with ethics guidelines All author declare that they have no conflict of interest. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University (2017-XY-012). The study was performed in accordance with the Helsinki Declaration and Rules of Good Clinical Practice. All participants signed written informed consents after the study protocol was fully explained. References 1 Clemente, J. C., Ursell, L. K., Parfrey, L. W. & Knight, R. 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FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Molecular biology and evolution 26 , 1641-1650, doi:10.1093/molbev/msp077 (2009). Tables Table 1. Characteristics of 55 participants Variables No. (%) Newborn sex Male 28 (50.9) Female 27 (49.1) Delivery mode Vaginal delivery 36 (65.5) Cesarean section delivery 19 (34.5) Feeding patterns Exclusive breastfeeding 36 (65.5) Combined feeding 19 (34.5) Birth weight at birth (kg), mean±SD 3.34±0.39 Birth weight at 12 month (kg), mean±SD 10.01±1 Gestational age (days), mean±SD 278±5 Additional Declarations No competing interests reported. Supplementary Files SupplementaryfigursS1S34.pdf Additional file 1: Supplementary Figures S1-S34 supplementarytableS1S55.zip Additional file 2: Supplementary Tables S1-S55 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-828854","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":47606373,"identity":"5c8e7c03-a09e-43ec-97d2-55fa24634a01","order_by":0,"name":"Juan Ding","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Ding","suffix":""},{"id":47606374,"identity":"0c80b7f7-7aec-46c6-adf3-95690d35d1f8","order_by":1,"name":"Xiao Ma","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Ma","suffix":""},{"id":47606375,"identity":"b422e533-49d0-4100-bc55-e323b95e34cd","order_by":2,"name":"Hongyan Ren","email":"","orcid":"","institution":"Shanghai Mobio Biomedical Technology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Ren","suffix":""},{"id":47606376,"identity":"c971565c-108f-4789-bb7c-b3d873e0be37","order_by":3,"name":"Chenyang Zhi","email":"","orcid":"","institution":"People's Hospital of Zhengzhou","correspondingAuthor":false,"prefix":"","firstName":"Chenyang","middleName":"","lastName":"Zhi","suffix":""},{"id":47606377,"identity":"d20fa219-1bd6-460e-9744-ca6250ba1028","order_by":4,"name":"Qi Xin","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Xin","suffix":""},{"id":47606378,"identity":"ffbc94e9-d9bc-4328-aa49-a9d3a9cfc04e","order_by":5,"name":"Zhen Li","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Li","suffix":""},{"id":47606379,"identity":"69e2be7a-b487-47e7-bf75-69702f58e022","order_by":6,"name":"Liping Han","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Han","suffix":""},{"id":47606380,"identity":"a5bde4f4-54cd-4878-b949-56d58ed6a684","order_by":7,"name":"Xianlan Zhao","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xianlan","middleName":"","lastName":"Zhao","suffix":""},{"id":47606381,"identity":"f1c80789-fa5f-450d-be7c-a8255ff99b73","order_by":8,"name":"Zhihong Zhuo","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Zhuo","suffix":""},{"id":47606382,"identity":"8dc9f67d-9339-49a6-8a21-242dda2502c9","order_by":9,"name":"Ang Li","email":"","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ang","middleName":"","lastName":"Li","suffix":""},{"id":47606383,"identity":"df47cfd3-3703-4442-9ba7-48ed4a1692d6","order_by":10,"name":"Chao Liu","email":"","orcid":"","institution":"Shanghai Mobio Biomedical Technology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Liu","suffix":""},{"id":47606384,"identity":"a991a7cc-21a5-492e-8bca-688cc78f5a1a","order_by":11,"name":"Zhigang Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBAC9gYogx9CMRPWwnMAypBsIFmLwQGitbAffvbh4446u83njz+TYKiwTmxgP3sAvxaeNOOZM8+wJW87cCBNguFMemIDT14CXi32EjzMzLxtPMlmBxuOSTC2HU5skOAxwG8LSMvfNolk42bGNgnGf8RqYWwzsDNgY2aTYGwgRgvQL4y9bQkJEmfYmC0SjqUbt/HkENDCfvgxw8+2Onv+/uMPb3yosZbtZz+DXwsMJDaAyAQgZiNKPRDYE6twFIyCUTAKRiAAADALOzw0+kNgAAAAAElFTkSuQmCC","orcid":"","institution":"the First Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2021-08-20 03:14:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-828854/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-828854/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12765611,"identity":"ae2a3407-9d82-4aa5-b42f-ce4be7625b51","added_by":"auto","created_at":"2021-08-25 23:21:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156717,"visible":true,"origin":"","legend":"Study design and flow diagram. A total of 82 infants (314 fecal samples) were recruited, and samples were collected at 0, 1, 3, 6 and 12 months postpartum. After an exclusion process, the remaining samples were used for DNA extraction, 16S rRNA gene sequencing and data quality control. Finally, 213 fecal samples from 55 infants at 0 months (n = 19), 1 month (n = 49), 3 months (n = 50), 6 months (n = 47) and 12 months (n = 48) were included for the final analysis.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/4666b4b7edf35f8c76b07654.png"},{"id":12765933,"identity":"619f0185-7fb8-4d96-9617-38d5d2fff65a","added_by":"auto","created_at":"2021-08-25 23:30:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":242057,"visible":true,"origin":"","legend":"Infant gut microbial diversity during the first year of life. Fecal microbial diversity, as estimated by the Shannon index (A) and the Simpson index (B), significantly increased over developmental time (p \u003c 0.001 and p \u003c 0.001, respectively). (C, D) Overall diversity in bacterial composition was calculated using unweighted UniFrac distances by PCoA, and it indicated a notable separation of samples at 0, 1, 3, 6 and 12 months (Adonis p \u003c 0.005, R2 = 0.08). (E) Individual microbial differences of the infants were assessed using average UniFrac distances. The greatest distance was observed among those infants at 0 months, and the distance significantly decreased at 1, 3, and 6 months versus 0 months. Notably, the distance within each group increased from 6 months to 12 months. PCoA: principal coordinates analysis; M0: 0 months; M1: 1 month; M3: 3 months; M6: 6 months; M12: 12 months.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/b6308c711d08d1125c973512.png"},{"id":12765639,"identity":"24015de0-c936-469a-88cc-60ebe34264e8","added_by":"auto","created_at":"2021-08-25 23:24:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":457313,"visible":true,"origin":"","legend":"Gut microbiota community structure during the first year of life. Fecal microbiota composition and at the phylum (A) and genus levels (B) during the first year of life. Alterations in the bacterial phyla Firmicutes (C), Proteobacteria (D) and Actinobacteria (E) at 0, 1, 3, 6 and 12 months postpartum. Alterations in the bacterial genera Bifidobacterium (F), Streptococcus (G) and Veillonella (H) at 0, 1, 3, 6 and 12 months postpartum. The box presents the 95% confidence intervals, and the line inside denotes the median. M0: 0 months; M1: 1 month; M3: 3 months; M6: 6 months; M12: 12 months.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/617dc01306e12e6d4121a9bf.png"},{"id":12765722,"identity":"53739ddd-30f4-4026-9185-8bf0fbe6e2ca","added_by":"auto","created_at":"2021-08-25 23:27:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84819,"visible":true,"origin":"","legend":"Individual microbial differences of the infants were analyzed within specific groups. (A) Microbial composition variation in vaginally delivered infants was greater than that in cesarean section infants; (B) differences in combined feeding infants were greater than exclusively breastfed infants; and (C) the distances revealed no significant difference between boys and girls. N: vaginally delivered; C: cesarean section. EB: exclusive breastfeeding; MF: combined feeding.","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/44c94f22b2b8f45b8bea00f6.png"},{"id":12765616,"identity":"c35b91d5-879b-46cb-83b7-6afa635bf161","added_by":"auto","created_at":"2021-08-25 23:21:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176187,"visible":true,"origin":"","legend":"The microbial compositional similarities of the infants were calculated between groups. (A) At 0 months, the UniFrac distance between vaginal delivery and cesarean section was the largest, followed by the distances between feeding modes and gender differences. (B, C, D) At 1, 3 and 6 months, the average distances were greater between boys and girls than those between vaginal delivery and cesarean section and those between exclusively breastfed and combined feeding. (E) At 12 months, the greatest distance was observed between exclusively breastfed and combined feeding, which was followed by that between vaginal delivery and cesarean section and that between boys and girls. N: vaginally delivered; C: cesarean section. EB: exclusive breastfeeding; MF: combined feeding. M0: 0 months; M1: 1 month; M3: 3 months; M6: 6 months; M12: 12 months.","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/9f72ff8122c6e2dc553a8197.png"},{"id":12765613,"identity":"7e8f9095-9978-4245-a58a-b6fd3e513604","added_by":"auto","created_at":"2021-08-25 23:21:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":384197,"visible":true,"origin":"","legend":"Differences in fecal microbial communities between groups. (A) Composition of the fecal microbiota at the phylum level between vaginal delivery and cesarean section groups. (B) Compared with their levels in cesarean section infants, 2 phyla were significantly increased, while 2 phyla were significantly decreased in vaginally delivered infants (all p \u003c 0.05). (C) Four genera were increased, whereas 10 genera were decreased in vaginally delivered versus cesarean section infants (all p \u003c 0.05). (D) Composition of the fecal microbiota at the genus level between exclusive breastfeeding and combined feeding infants. (E) Two genera were enriched, whereas 3 genera were decreased in exclusive breastfeeding versus combined feeding infants (all p \u003c 0.05). (F) Two genera were increased, whereas 4 were reduced in boys versus girls (all p \u003c 0.05). N: vaginally delivered; C: cesarean section. EB: exclusive breastfeeding; MF: combined feeding.","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/b4e1e89f9c0b63af616ce9b2.png"},{"id":14307928,"identity":"9afb5c1b-8cb5-44f1-8199-3f32dee54c54","added_by":"auto","created_at":"2021-10-07 07:44:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2476058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/8372f0c6-d04a-47bc-ab6a-3580ccd4aed9.pdf"},{"id":12765618,"identity":"847249aa-a1d3-4b24-a6d5-3ccf5abf14b0","added_by":"auto","created_at":"2021-08-25 23:21:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5313413,"visible":true,"origin":"","legend":"Additional file 1: Supplementary Figures S1-S34","description":"","filename":"SupplementaryfigursS1S34.pdf","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/740d897fbe0da4096d41bcfb.pdf"},{"id":12765641,"identity":"80a86d62-c48c-4198-8836-e29b6f873eb5","added_by":"auto","created_at":"2021-08-25 23:24:45","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":954887,"visible":true,"origin":"","legend":"Additional file 2: Supplementary Tables S1-S55","description":"","filename":"supplementarytableS1S55.zip","url":"https://assets-eu.researchsquare.com/files/rs-828854/v1/a782db80b1903c54fdcf6333.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDelivery, Feeding and Gender Differently Contribute to Infant Gut Microbiota Development\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe infant gut microbiota is critical to human health, playing significant roles in metabolism, immunity and development\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Colonization of the infant gut microbiota is a complex process dependent on multiple overlapping factors, such as delivery mode \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, feeding mode\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, gestational age\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and environmental exposures\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is growing evidence linking host health with the establishment of the infant gut microbiome. Cesarean delivery is associated with a number of human diseases, including obesity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, asthma\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, celiac disease\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and type 1 diabetes\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Infant gut microbiota colonization patterns differ between infants experiencing vaginal delivery and those delivered by cesarean Sect.\u0026nbsp;\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Cesarean-delivered infants have particularly low richness and diversity compared with vaginally delivered infants. Breastfeeding is related to fewer illnesses, such as obesity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, asthma\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and metabolic syndrome\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, than formula feeding. Feeding mode also influences the establishment of the infant gut microbiota. Breastfed infants are reportedly colonized with bacteria in breast milk and with greater numbers of \u003cem\u003eBifidobacterium\u003c/em\u003e than formula-fed infants\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Gender-associated differences are relevant to the prevalence, manifestations, and outcomes of malignancies and autoimmune and infectious diseases\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Gender-associated differences in the gut microbiota can influence susceptibility to disease\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. There is an undeniable need to explore gut microbiota characteristics caused by gender differences. However, the contribution degrees of these factors to the development of the infant gut microbiota remain unknown.\u003c/p\u003e \u003cp\u003eIn this study, a total of 82 infants were prospectively enrolled, and fecal samples were separately collected at 0 months (meconium) and 1, 3, 6 and 12 months postpartum. Finally, 55 healthy infants were enrolled, and their samples were subjected to 16S rRNA gene sequencing. We characterized the gut microbiome at 5 time points during the first year of life, analyzed the bacterial community, and reported differences at crucial time points of infant development. Furthermore, we assessed the different contributions of delivery mode, feeding mode and infant gender to gut microbial development and compared microbial differences induced by the different delivery, feeding and gender.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy Participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary objective of this study was to use longitudinal sampling to monitor changes in the infant gut microbiome over time. Specific variables were examined,\u0026nbsp;including delivery\u0026nbsp;mode, feeding\u0026nbsp;mode\u0026nbsp;and infant gender.\u0026nbsp;All study procedures were consistent with the ethical guidelines of the Helsinki Declaration.\u0026nbsp;The study was approved by\u0026nbsp;the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University\u0026nbsp;(2017-KY-12).\u0026nbsp;Recruitment took place in China between July 2017 and\u0026nbsp;August 2017. Infants were excluded if they had a health condition or received antibiotics. Written informed consent was obtained from all parents of the infants before collecting data and fecal samples.\u003c/p\u003e\n\u003cp\u003eBasic demographics, delivery\u0026nbsp;mode, feeding\u0026nbsp;mode, and information concerning current or recent medications (including antibiotics) were obtained from hospital electronic medical records and questionnaires. Infants who had been breastfed and never given formula prior to fecal collection were assigned exclusively breastfed status. Infants who were fed both breast\u0026nbsp;milk and formula prior to the time of stool collection were identified\u0026nbsp;as having received combined feeding (both\u0026nbsp;breast milk and formula milk).\u0026nbsp;If an infant received formula milk at any collection time point, the infant was considered to have received combined feeding\u0026nbsp;for the remainder of the study, as even short-term formula feeding causes profound and long-lasting shifts in the gut microbiome composition\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study population was recruited before mothers gave birth.\u0026nbsp;Meconium samples were collected in the hospital, and other fresh fecal samples were collected at their homes. Fecal samples were collected by the infant\u0026rsquo;s parents and stored in a household freezer (\u0026minus;20\u0026deg;C); samples were immediately shipped on dry ice to the laboratory, where they were placed at \u0026minus;80\u0026deg;C until DNA was extracted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from the fecal samples using the E.Z.N.A.\u003csup\u003e\u0026reg;\u003c/sup\u003e Stool DNA Kit (Omega Bio-tek, Inc., GA) according to the instructions provided by the manufacturer. DNA was quantified by a Qubit\u003csup\u003e\u0026reg;\u003c/sup\u003e 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States), and fragment sizes were determined using 1.5% agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR amplification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extracted DNA samples were amplified with a set of primers targeting the hypervariable V3-V4 region (341F/805R) of the 16S rRNA gene. The forward primer (341F) was 5\u0026rsquo;-CCTACGGGNGGCWGCAG-3\u0026rsquo;, and the reverse primer (805R) was 5\u0026rsquo;-GACTACHVGGGTATCTAATCC-3\u0026rsquo;. PCR was performed with\u0026nbsp;an\u0026nbsp;EasyCycler 96 PCR system (Analytik Jena Corp., AG) using the\u0026nbsp;following\u0026nbsp;program: 3 min of denaturation at 95\u0026nbsp;℃;\u0026nbsp;21 cycles of 0.5 min\u0026nbsp;at 94\u0026nbsp;℃\u0026nbsp;(denaturation), 0.5 min for annealing at 58℃, and 0.5 min\u0026nbsp;at 72\u0026nbsp;℃\u0026nbsp;(elongation); and a final extension at 72\u0026nbsp;℃\u0026nbsp;for\u0026nbsp;5 min. Paired-end sequencing was performed with the constructed sequencing libraries on the Illumina MiSeq platform\u0026nbsp;by Shanghai Mobio Biomedical Technology Co., Ltd., China.\u0026nbsp;The raw Illumina read data from all samples were stored in the European Bioinformatics Institute European Nucleotide Archive database (PRJNA665920).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOperational\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etaxonomic unit (OTU\u003c/strong\u003e\u003cstrong\u003e) clustering and taxonomy annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRandom reads were chosen from all fecal samples with equal\u0026nbsp;numbers,\u0026nbsp;OTUs were binned by\u0026nbsp;the\u0026nbsp;UPARSE pipeline, and OTUs were binned by the UPARSE pipeline with\u0026nbsp;the\u0026nbsp;following steps: (i) abundant sequences and singletons were\u0026nbsp;first\u0026nbsp;removed,\u0026nbsp;(ii) unique sequences were binned into OTUs with\u0026nbsp;the\u0026nbsp;command \u0026ldquo;usearch-cluster_ otus\u0026rdquo;,\u0026nbsp;and\u0026nbsp;(iii) randomly chosen sequences were aligned against OTU sequences with\u0026nbsp;the\u0026nbsp;command \u0026ldquo;usearch-usearch_global-id 0.97\u0026rdquo;. The identity threshold was set as 0.97, and then\u0026nbsp;an\u0026nbsp;OTU composition table was created. We annotated sequences by using RDP classifier version 2.6\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial diversity and taxonomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial diversity was determined by sampling-based analysis of OTUs and represented by\u0026nbsp;the\u0026nbsp;Shannon index, Simpson index and Chao1 index, which were calculated using the R package \u0026ldquo;vegan\u0026rdquo;\u003csup\u003e46\u003c/sup\u003e. Principal coordinate analysis (PCoA) and\u0026nbsp;nonmetric multidimensional scaling (NMDS)\u0026nbsp;based on OTUs\u0026nbsp;were\u0026nbsp;performed by the R package (http://www.r-project.org/) to analyze microbiome distances between samples. Unweighted\u0026nbsp;UniFrac\u0026nbsp;distances were calculated with\u0026nbsp;the\u0026nbsp;phyloseq package\u003csup\u003e47\u003c/sup\u003e. Taxonomic group comparisons, including comparisons of bacterial\u0026nbsp;phyla,\u0026nbsp;order, family and genera, were conducted using the Wilcoxon rank sum test.\u0026nbsp;A heatmap\u0026nbsp;that\u0026nbsp;identified discriminatory variables was\u0026nbsp;generated\u0026nbsp;by a\u0026nbsp;heatmap\u0026nbsp;builder.\u003c/p\u003e\n\u003cp\u003eThe linear discriminant analysis (LDA) effect size (LEfSe) method was used to distinguish taxonomic types in microbial communities related to specific characteristics (http://huttenhower.sph.harvard.edu/lefse/)\u003csup\u003e48\u003c/sup\u003e. With a normalized relative abundance matrix, LEfSe used the Kruskal-Wallis rank sum test to detect the features related to markedly different abundances between assigned bacterial taxa and used LDA to assess the effect size of each feature\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional annotation of the 16S rRNA gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ephylogenetic investigation of communities by reconstruction of unobserved states\u0026nbsp;(PICRUSt)\u0026nbsp;version 1.0.0 pipeline\u003csup\u003e50\u003c/sup\u003e and human version 0.99\u003csup\u003e51\u003c/sup\u003e were used to establish KEGG orthology (KO) and KEGG pathway/module profiles\u0026nbsp;and\u0026nbsp;predict the functional profiles of the gut microbiota based on 16S rRNA gene sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClean data were extracted from raw data using USEARCH 7.1 with\u0026nbsp;the\u0026nbsp;following criteria: (i) sequences shorter than 400 bp after the merge or containing a homopolymer longer than four bases were discarded; (ii)\u0026nbsp;sequences\u0026nbsp;from each sample were extracted using each index with fewer than two\u0026nbsp;mismatches, and (iii) sequences with an overlap shorter than 16 bp were discarded.\u003c/p\u003e\n\u003cp\u003eWe evaluated the associations of delivery mode, feeding mode,\u0026nbsp;and\u0026nbsp;infant gender with microbiome community composition using generalized UniFrac analysis\u003csup\u003e52\u003c/sup\u003e. The phylogenetic tree required for UniFrac analysis was computed based on FastTree\u003csup\u003e53\u003c/sup\u003e and was midpoint rooted. Statistical comparisons of groups were conducted using ADONIS between groups with 10,000 permutations. We computed mean generalized UniFrac distances for both within and between delivery mode groups (vaginal delivery\u0026nbsp;and cesarean section), feeding mode groups (exclusive breastfeeding\u0026nbsp;and combined feeding) and infant gender. Statistical comparisons of mean UniFrac distances were performed using the Kruskal-Wallis rank sum test. The statistical significance threshold was set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStudy Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, a total of\u0026nbsp;82\u0026nbsp;infants\u0026nbsp;(314 fecal samples)\u0026nbsp;were\u0026nbsp;recruited, and infant fecal samples were collected at 0 months\u0026nbsp;(meconium), 1 month\u0026nbsp;(27 to 33 days), 3\u0026nbsp;months (87 to 93 days),\u0026nbsp;6 months (177 to 183 days) and 12 months (357 to 363 days) after birth.\u0026nbsp;After confirmation, 2 infants without fecal samples, 3 premature infants, 5 infants with incomplete information, and 17 infants with drug therapy were excluded. After DNA extraction, 16S rRNA gene sequencing and data quality control, 38 fecal samples were further discarded.\u0026nbsp;Finally, 55\u0026nbsp;healthy infants with\u0026nbsp;213\u0026nbsp;fecal samples\u0026nbsp;at 0\u0026nbsp;months\u0026nbsp;(n = 19), 1 month (n = 49), 3 months (n = 50), 6 months (n = 47) and 12 months (n = 48) were included\u0026nbsp;in\u0026nbsp;the final analysis.\u003c/p\u003e\n\u003cp\u003eIn total, 8,542,788 raw reads were obtained after sequencing. The mean read depth per sample was 40,107\u0026nbsp;\u0026plusmn;\u0026nbsp;10,818\u0026nbsp;sequences per sample. After initial quality filtering, 6,904,876 sequences passed with a mean of 32,417 \u0026plusmn; 7,111 sequences per sample. A flowchart describing the\u0026nbsp;sample\u0026nbsp;collection is shown in \u003cstrong\u003eFig. 1\u003c/strong\u003e. All infants included in the study were fed complementary food\u0026nbsp;after fecal samples were collected at 6 months. The complementary food mainly included vegetables, porridge and pasta.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical information\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of the participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComprehensive clinical information for each enrolled individual was recorded\u0026nbsp;(\u003cstrong\u003eTable 1).\u0026nbsp;\u003c/strong\u003eWe evaluated delivery and\u0026nbsp;feeding characteristics\u0026nbsp;in 55 infants (\u003cstrong\u003eTable 1)\u003c/strong\u003e. Samples showed that 36 (65.5%) of enrolled infants\u0026nbsp;had\u0026nbsp;vaginal\u0026nbsp;deliveries, and 19 (34.5%) infants\u0026nbsp;had\u0026nbsp;cesarean section\u0026nbsp;deliveries. Thirty-six\u0026nbsp;(65.5%) infants were exclusively breastfed,\u0026nbsp;and\u0026nbsp;19 (34.5%) infants received combined feeding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfant gut microbial diversity during the first year of life\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial diversity was determined by sampling-based analysis of OTUs as estimated by\u0026nbsp;the Shannon index (\u003cstrong\u003eFig. 2a\u003c/strong\u003e) and the Simpson index \u003cstrong\u003e(Fig. 2b\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;fecal\u0026nbsp;microbial diversity was significantly decreased\u0026nbsp;at\u0026nbsp;0\u0026nbsp;months\u0026nbsp;compared with the other 4 times (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001 and \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001, respectively, \u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e).\u0026nbsp;Microbial\u0026nbsp;diversity was markedly increased\u0026nbsp;at\u0026nbsp;12 months compared with\u0026nbsp;the\u0026nbsp;other 4 times (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001 and \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001, respectively, \u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e). The microbial diversity presented no significant difference between 1, 3 and 6 months (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.4 and \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.87, respectively, \u003cstrong\u003eSupplementary Table S2\u003c/strong\u003e). These data suggested that gut microbial diversity was consistently increased from 0\u0026nbsp;months\u0026nbsp;to 12 months with the development of the infant.\u003c/p\u003e\n\u003cp\u003eTo assess the overall diversity in bacterial composition, we performed PCoA\u0026nbsp;and NMDS\u0026nbsp;analysis\u0026nbsp;based on unweighted UniFrac\u0026nbsp;distances\u0026nbsp;\u003cstrong\u003e(Fig. 2c-d and\u003c/strong\u003e\u003cstrong\u003eSupplementary Fig. S1\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. The PCoA based on the unweighted UniFrac distances indicated that there was a notable separation\u0026nbsp;of\u0026nbsp;samples at 0, 1, 3, 6 and 12 months (Adonis\u0026nbsp;\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001, R\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 0.08,\u0026nbsp;\u003cstrong\u003eSupplementary Table S4\u003c/strong\u003e). There was an obvious separation in gut microbiome composition between 0\u0026nbsp;months\u0026nbsp;and 1 month (Adonis \u003cem\u003ep\u003c/em\u003e = 0.001,\u003cem\u003e\u0026nbsp;R\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 0.04). Meanwhile,\u0026nbsp;the\u0026nbsp;microbial community\u0026nbsp;at\u0026nbsp;12 months\u0026nbsp;was\u0026nbsp;clustered together and significantly separated from\u0026nbsp;that at\u0026nbsp;6 months in the PCoA (Adonis \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001,\u003cem\u003e\u0026nbsp;R\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 0.06). No obvious separation\u0026nbsp;was\u0026nbsp;observed\u0026nbsp;in the microbiota between 1,\u0026nbsp;3 and 6 months (Adonis\u003cem\u003e\u0026nbsp;p\u003c/em\u003e = 0.22,\u003cem\u003e\u0026nbsp;R\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 0.02). These results suggested that infants\u0026rsquo; gut microbiota was consistently altered from 0\u0026nbsp;months\u0026nbsp;to 12 months with the development of the infant.\u003c/p\u003e\n\u003cp\u003eIn addition,\u0026nbsp;we calculated the average UniFrac distances within each group to assess the microbial\u0026nbsp;differences\u0026nbsp;between the individual infants. We found the greatest average distance within each group between the infants at 0 months. The distance within the group was significantly decreased at 1, 3, and 6 months versus 0\u0026nbsp;months. Notably, the distance within the group remarkably increased from 6 months to 12 months \u003cstrong\u003e(Fig. 2e,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S5\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. These data demonstrated that individual microbial variation of the infants was decreased from 0\u0026nbsp;months\u0026nbsp;to 1 month\u0026nbsp;but then increased from 6 months to 12 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGut\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emicrobiota community structure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eduring the first year of life\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA heatmap from hierarchical clustering analysis revealed a discriminatory gut microbiome during the first year of life. Comparison of fecal microbiomes demonstrated that 60\u0026nbsp;key\u0026nbsp;OTUs were significantly different at 0, 1, 3, 6, and 12 months (\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e).\u0026nbsp;We further analyzed\u0026nbsp;the infant gut microbiota\u0026nbsp;composition\u0026nbsp;and alterations\u0026nbsp;during the first year of life.\u0026nbsp;The fecal bacterial composition\u0026nbsp;in\u0026nbsp;each sample\u0026nbsp;at the phylum and genus levels\u0026nbsp;is\u0026nbsp;shown in\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e-4\u003c/strong\u003e,\u0026nbsp;respectively (\u003cstrong\u003eSupplementary Table S6-S7\u003c/strong\u003e).\u0026nbsp;The average compositions of the microbial community\u0026nbsp;at the phylum and genus levels\u0026nbsp;during the first year of life\u0026nbsp;are\u0026nbsp;shown\u0026nbsp;in\u0026nbsp;\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3a-b\u003c/strong\u003e (\u003cstrong\u003eSupplementary Table S8\u003c/strong\u003e\u003cstrong\u003e-S9\u003c/strong\u003e).\u0026nbsp;Moreover, bacterial compositions at the class and order levels during the first year of life\u0026nbsp;are\u0026nbsp;shown in\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e5a-b\u003c/strong\u003e (\u003cstrong\u003eSupplementary Table S10-S11\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe bacterial phyla\u0026nbsp;\u003cem\u003eFirmicutes, Proteobacteria\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Bacteroidetes\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Actinobacteria\u003c/em\u003e, together accounting for up to 99% of sequences on average, were the four dominant phyla at 0 months.\u0026nbsp;\u003cem\u003eFirmicutes, Proteobacteria\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Actinobacteria\u003c/em\u003e, together accounting for greater than 85%, were the three dominant phyla at 1, 3, 6 and 12 months, respectively.\u0026nbsp;The average abundance\u0026nbsp;of the phylum \u003cem\u003eFirmicutes\u003c/em\u003e increased from 16.34% at 0\u0026nbsp;months\u0026nbsp;to 48.15% at 12 months (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001,\u0026nbsp;\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3c,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e12\u003c/strong\u003e), while the abundance\u0026nbsp;of the phylum\u003cem\u003e\u0026nbsp;Proteobacteria\u0026nbsp;\u003c/em\u003edecreased gradually from 67.43% at 0\u0026nbsp;months\u0026nbsp;to 15.82% at 12 months (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001,\u0026nbsp;\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3d\u003c/strong\u003e). Moreover, the average relative abundance of\u0026nbsp;the\u0026nbsp;phylum \u003cem\u003eActinobacteria\u003c/em\u003e consistently increased from 4.59% at 0\u0026nbsp;months\u0026nbsp;to 33.34% at 3 months (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and then presented no significant alterations from 3 months to 12 months (26.25%) (\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eEighty-nine\u0026nbsp;genera in\u0026nbsp;the\u0026nbsp;infant gut microbiota were detected during the first year of life.\u0026nbsp;The infant\u0026nbsp;gut microbiota\u0026nbsp;was\u0026nbsp;dominated by\u003cem\u003e\u0026nbsp;Bifidobacterium, Streptococcus,\u0026nbsp;\u003c/em\u003e\u003cem\u003eEscherichia\u0026minus;Shigella\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003cem\u003eKlebsiella\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003eBacteroides\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003eClostridium sensu stricto 1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003eVeillonella\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003eLactobacillus\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Lachnospiraceae incertae sedis\u003c/em\u003e,\u0026nbsp;and their total abundance\u0026nbsp;exceeded\u0026nbsp;70%.\u0026nbsp;The average relative abundance of\u003cem\u003e\u0026nbsp;Bifidobacterium\u003c/em\u003e significantly elevated during the period from 0\u0026nbsp;months\u0026nbsp;(2.66%) to 3 months (28.15) (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) and had no obvious increase from 3 months to 12 months (23.81%) (\u003cstrong\u003eFig. 3f,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u0026nbsp;The average\u0026nbsp;amount of \u003cem\u003eStreptococcus\u003c/em\u003e remarkably increased from 0\u0026nbsp;months\u0026nbsp;(6.19%) to 1 month (14.11%) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and presented no significant changes from 1 month to 12 months (11.91%) \u003cstrong\u003e(Fig. 3g)\u003c/strong\u003e. Additionally, \u003cem\u003eVeillonella\u003c/em\u003e consistently increased from 0\u0026nbsp;months\u0026nbsp;(2%)\u0026nbsp;and\u0026nbsp;1 month (3.22%) to 12 months (5.48%) \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003cstrong\u003eFig. 3h)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo identify specific bacterial taxa associated with the time points, we compared the fecal microbiota using\u0026nbsp;LEfSe. A cladogram representative of fecal microbial structures and their predominant bacteria displayed the greatest differences in taxa among different times\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e6a-b, Supplementary Table S14\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;gut microbial community metabolic function profiles\u0026nbsp;and the predominant\u0026nbsp;microbial functions\u0026nbsp;at\u0026nbsp;0, 1, 3, 6\u0026nbsp;and 12 months\u0026nbsp;were analyzed by a\u0026nbsp;cladogram\u0026nbsp;and LDA\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e7a\u003c/strong\u003e\u003cstrong\u003e-b, Supplementary Table S15\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGut microbial differences of the infants\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eat the crucial development time points\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with\u0026nbsp;that at\u0026nbsp;1 month, as estimated by\u0026nbsp;the\u0026nbsp;Shannon index, Simpson index and Chao1 index, fecal microbial diversity\u0026nbsp;at\u0026nbsp;0\u0026nbsp;months\u0026nbsp;was significantly decreased\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026nbsp;0.001, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.001 and \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026nbsp;0.05, respectively,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e8a-c,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S16\u003c/strong\u003e).\u0026nbsp;A heatmap revealed a discriminatory gut microbiome and demonstrated that 15\u0026nbsp;key\u0026nbsp;OTUs were significantly different\u0026nbsp;between 0\u0026nbsp;months\u0026nbsp;and 1 month (\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e9\u003c/strong\u003e)\u003cstrong\u003e.\u003c/strong\u003e We analyzed\u0026nbsp;differences in\u0026nbsp;bacterial taxonomic compositions at\u0026nbsp;the phylum and\u0026nbsp;genus levels between 0\u0026nbsp;months\u0026nbsp;and 1 month.(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e10a\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eb,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S17-S18\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;at\u0026nbsp;0\u0026nbsp;months\u0026nbsp;and 1 month\u0026nbsp;was\u0026nbsp;determined by\u0026nbsp;LEfSe\u0026nbsp;and LDA \u003cstrong\u003e(\u003c/strong\u003eall \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, LDA \u0026gt; 3,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e11a-b,\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003cstrong\u003e).\u0026nbsp;\u003c/strong\u003eThe\u0026nbsp;predominant\u0026nbsp;fecal microbial functions\u0026nbsp;at\u0026nbsp;0\u0026nbsp;and 1 month\u0026nbsp;were visualized by a\u0026nbsp;cladogram\u0026nbsp;and LDA\u0026nbsp;(all \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, LDA \u0026gt; 2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e12a-b,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S20\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCompared with\u0026nbsp;that at\u0026nbsp;12 months, fecal microbial diversity\u0026nbsp;at\u0026nbsp;6 months was significantly decreased (all\u003cem\u003e\u0026nbsp;p\u003c/em\u003e \u0026lt; 0.001,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003ea-c,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S21).\u003c/strong\u003e A\u0026nbsp;heatmap revealed a discriminatory gut microbiome and demonstrated that 35\u0026nbsp;key\u0026nbsp;OTUs were significantly different between 6 months and 12 months (\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e14\u003c/strong\u003e).\u0026nbsp;We analyzed\u0026nbsp;differences in\u0026nbsp;bacterial taxonomic compositions at\u0026nbsp;the phylum and\u0026nbsp;genus levels\u0026nbsp;(all \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05,\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e15a-b\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table S22-S23\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;at\u0026nbsp;6 months\u0026nbsp;and 12 months\u0026nbsp;was\u0026nbsp;determined by\u0026nbsp;LEfSe\u0026nbsp;and LDA (all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, LDA \u0026gt; 2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e16a-b, Supplementary Table S\u003c/strong\u003e\u003cstrong\u003e24\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. The\u0026nbsp;fecal\u0026nbsp;predominant\u0026nbsp;microbial functions\u0026nbsp;at\u0026nbsp;6 months\u0026nbsp;and 12 months\u0026nbsp;were visualized by a\u0026nbsp;cladogram\u0026nbsp;and LDA\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05\u003cstrong\u003e,\u003c/strong\u003e LDA \u0026gt; 2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e17a-b,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Supplementary Table S25\u003c/strong\u003e). These data revealed significant differences in the gut microbiota between\u0026nbsp;these\u0026nbsp;two groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDelivery, feeding and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egender\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econtribute differently to microbiota development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach infant has unique gut microbial characteristics. We analyzed gut microbial similarity within each group, compared the within-group\u0026nbsp;average UniFrac\u0026nbsp;distances between infants within specific delivery\u0026nbsp;mode, feeding\u0026nbsp;mode\u0026nbsp;and\u0026nbsp;gender\u0026nbsp;groups, and revealed\u0026nbsp;individual microbial\u0026nbsp;differences\u0026nbsp;of the infant. Greater average\u0026nbsp;UniFrac\u0026nbsp;distances were observed between infants who were born by vaginal delivery versus cesarean delivery\u0026nbsp;\u003cstrong\u003e(Fig. 4a,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. A greater average distance was observed between infants who received\u0026nbsp;combined feeding\u0026nbsp;versus those exclusively breastfed\u0026nbsp;\u003cstrong\u003e(Fig. 4b,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003cstrong\u003e).\u003c/strong\u003e Within-group distances revealed no significant difference between boys and girls\u0026nbsp;\u003cstrong\u003e(Fig. 4c,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. These data suggested that\u0026nbsp;vaginal delivery and\u0026nbsp;combined feeding played an important role in the development of the infant gut microbiota.\u003c/p\u003e\n\u003cp\u003eWe further calculated between-group average UniFrac distances to assess\u0026nbsp;the microbial compositional similarities.\u0026nbsp;After birth,\u0026nbsp;the UniFrac distance between\u0026nbsp;vaginal delivery and cesarean section was the largest, followed by the distances\u0026nbsp;between\u0026nbsp;feeding modes and gender differences\u0026nbsp;\u003cstrong\u003e(Fig. 5a,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u0026nbsp;At 1, 3 and 6 months, the\u0026nbsp;average distance was greater between boys and girls than between vaginal delivery and cesarean section, as well as exclusively breastfed and\u0026nbsp;combined feeding\u0026nbsp;\u003cstrong\u003e(Fig. 5b-d,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003cstrong\u003e).\u0026nbsp;\u003c/strong\u003eBy 12 months, the contributions to the development of gut microbiota had changed. The greatest distance was observed\u0026nbsp;between\u0026nbsp;exclusively breastfed and\u0026nbsp;combined feeding; then\u0026nbsp;between vaginal delivery and cesarean section, as well asbetweenboys and girls\u0026nbsp;\u003cstrong\u003e(Fig. 5e,\u003c/strong\u003e \u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003cstrong\u003e).\u003c/strong\u003e These data demonstrated that\u0026nbsp;for the development of the infant gut microbiota, the delivery mode was the main contribution at birth, gender accounted for the dominant contribution at 1, 3 and 6 months, and feeding mode played the main role after 12 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;delivery, feeding and gender\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on the gut microbiota\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed fecal bacterial composition and differences\u0026nbsp;between vaginal delivery and cesarean section. A heatmap revealed a discriminatory gut microbiome between vaginal delivery and cesarean section, and demonstrated that 17\u0026nbsp;key\u0026nbsp;OTUs were significantly different\u0026nbsp;between\u0026nbsp;the\u0026nbsp;two groups\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e18\u003c/strong\u003e).\u0026nbsp;The average composition of the microbial community at the phylum and genus levels between the two groups is shown\u0026nbsp;in \u003cstrong\u003eFig. 6a\u003c/strong\u003e and\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e19a\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e28-S29\u003c/strong\u003e).\u0026nbsp;At phylum level, 2 phyla including Actinobacteria and Bacteroidetes were increased, whereas 2 phyla including Firmicutes and Proteobacteria reduced, in vaginal delivery versus cesarean section (all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cstrong\u003eFig. 6b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e30\u003c/strong\u003e). At genus level, 4 genera including \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eParabacteroides\u003c/em\u003e and \u003cem\u003ePhascolarctobacterium\u003c/em\u003e were increased, whereas 10 genera mainly including \u003cem\u003eClostridium sensu stricto 1,\u003c/em\u003e \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eEnterobacter\u003c/em\u003e reduced, in vaginal delivery versus cesarean section (all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cstrong\u003eFig. 6c\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e31\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;for\u0026nbsp;vaginal delivery and cesarean section\u0026nbsp;were determined by\u0026nbsp;LEfSe\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e20a\u003c/strong\u003e).\u0026nbsp;Based on LDA, 14 bacteria mainly including \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eBacteroidales\u003c/em\u003e, \u003cem\u003eBacteroidia\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e were enriched, while 14 bacteria mainly including \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eClostridiales\u003c/em\u003e, were reduced\u0026nbsp;for vaginal delivery compared with\u0026nbsp;cesarean\u0026nbsp;section\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA\u0026nbsp;\u0026gt;\u0026nbsp;2.5,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e20b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S32\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbial functions\u0026nbsp;for\u0026nbsp;vaginal delivery and cesarean section\u0026nbsp;were analyzed by a\u0026nbsp;cladogram (\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e21a\u003c/strong\u003e).\u0026nbsp;Based on LDA, 81 functions mainly including Genetic Information Processing, Replication and Repair, Metabolism and Translation were enriched, while 40 functions mainly including Environmental Information Processing, Membrane Transport and Transporters, were reduced for vaginal delivery compared with\u0026nbsp;cesarean\u0026nbsp;section\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA\u0026nbsp;\u0026gt;\u0026nbsp;2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e21b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S33\u003c/strong\u003e).\u0026nbsp;We further\u0026nbsp;analyzed\u0026nbsp;bacterial taxonomic compositions and alterations\u0026nbsp;at the phylum level\u0026nbsp;between vaginal delivery and cesarean section infants under fixed exclusive breastfeeding\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e22a-b,\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e34-S35\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;associated with delivery mode under fixed exclusive breastfeeding\u0026nbsp;was\u0026nbsp;determined by\u0026nbsp;LEfSe\u0026nbsp;and LDA\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA \u0026gt; 2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e23a-b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S36\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe also analyzed gut microbial composition and alterations between exclusive breastfeeding and combined feeding. A\u0026nbsp;heatmap revealed a discriminatory gut microbiome between exclusive breastfeeding and combined feeding, and\u0026nbsp;demonstrated that 17\u0026nbsp;key\u0026nbsp;OTUs were significantly different\u0026nbsp;between\u0026nbsp;the\u0026nbsp;two groups\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e24\u003c/strong\u003e).\u0026nbsp;The average composition of the microbial community at the phylum and genus levels between the two groups is shown\u0026nbsp;in\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e19b\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eFig. 6d\u003c/strong\u003e (\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e37-S38\u003c/strong\u003e).\u0026nbsp;We found no significant between two groups at phylum level\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e39\u003c/strong\u003e). At genus level, 2 genera including \u003cem\u003ePeptostreptococcaceae incertae sedis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAnaerococcus\u0026nbsp;\u003c/em\u003ewere increased, whereas 3 genera including \u003cem\u003eLachnospiraceae incertae sedis\u003c/em\u003e, \u003cem\u003eCoriobacteriaceae uncultured\u0026nbsp;\u003c/em\u003eand \u003cem\u003eErysipelotrichaceae norank\u0026nbsp;\u003c/em\u003ereduced, in\u0026nbsp;exclusive breastfeeding\u0026nbsp;versus\u0026nbsp;combined feeding\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cstrong\u003eFig. 6e\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e40\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;for\u0026nbsp;vaginal delivery and cesarean section\u0026nbsp;were determined by\u0026nbsp;LEfSe\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e25a\u003c/strong\u003e).\u0026nbsp;Based on LDA, 3 bacteria including \u003cem\u003eAnaerococcus\u003c/em\u003e, \u003cem\u003eSerratia\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePeptostreptococcaceae\u0026nbsp;\u003c/em\u003ewere enriched, while 2 bacteria including \u003cem\u003eLachnospiraceae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eIncertae Sedis\u003c/em\u003e, were reduced\u0026nbsp;for\u0026nbsp;exclusive breastfeeding\u0026nbsp;compared with\u0026nbsp;combined feeding\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA\u0026nbsp;\u0026gt;\u0026nbsp;2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e25b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S41\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbial functions\u0026nbsp;for\u0026nbsp;exclusive breastfeeding\u0026nbsp;compared with\u0026nbsp;combined feeding\u0026nbsp;were analyzed by a\u0026nbsp;cladogram (\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e26a\u003c/strong\u003e).\u0026nbsp;Based on LDA, 2 functions including Fatty acid metabolism and Glycine serine and threonine metabolism were enriched, while 2 functions including Sporulation and Methane metabolism, were reduced for\u0026nbsp;exclusive breastfeeding\u0026nbsp;compared with\u0026nbsp;combined feeding\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA\u0026nbsp;\u0026gt;\u0026nbsp;2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e26b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S42\u003c/strong\u003e).\u0026nbsp;We further\u0026nbsp;analyzed\u0026nbsp;bacterial taxonomic compositions and alterations\u0026nbsp;at the phylum level\u0026nbsp;between\u0026nbsp;exclusive breastfeeding and combined feeding\u0026nbsp;infants under fixed\u0026nbsp;vaginal delivery (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e27,\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e43-S44\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;associated with feeding mode under fixed\u0026nbsp;vaginal delivery\u0026nbsp;was\u0026nbsp;determined by\u0026nbsp;LEfSe\u0026nbsp;and LDA\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA \u0026gt; 2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e28,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S45\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eMoreover, we analyzed gut\u0026nbsp;microbial composition and alterations between boys and girls. A\u0026nbsp;heatmap revealed a discriminatory gut microbiome\u0026nbsp;boys and girls, and\u0026nbsp;demonstrated that 17\u0026nbsp;key\u0026nbsp;OTUs were significantly different\u0026nbsp;between\u0026nbsp;the\u0026nbsp;two groups\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e29\u003c/strong\u003e).\u0026nbsp;The average composition of the microbial community at the phylum and genus levels between the two groups is shown\u0026nbsp;in\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e19c-d\u003c/strong\u003e (\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e46-S47\u003c/strong\u003e).\u0026nbsp;We found no significant between two groups at phylum level\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e48\u003c/strong\u003e). At genus level, 2 genera including \u003cem\u003eAlistipes\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAnaeroglobus\u0026nbsp;\u003c/em\u003ewere increased, whereas 4 genera including \u003cem\u003eParasutterella\u003c/em\u003e, \u003cem\u003eEubacterium, Peptoniphilus\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAnaerosporobacter\u0026nbsp;\u003c/em\u003ereduced, in\u0026nbsp;boys\u0026nbsp;versus\u0026nbsp;girls\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cstrong\u003eFig. 6f\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e49\u003c/strong\u003e).\u0026nbsp;The\u0026nbsp;predominant\u0026nbsp;fecal microbiome\u0026nbsp;for\u0026nbsp;vaginal delivery and cesarean section\u0026nbsp;were determined by\u0026nbsp;LEfSe\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e30a\u003c/strong\u003e).\u0026nbsp;Based on LDA, 3 bacteria including \u003cem\u003eVeillonellaceae\u003c/em\u003e, \u003cem\u003eSelenomonadales\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNegativicutes\u0026nbsp;\u003c/em\u003ewere enriched, while 2 bacteria including \u003cem\u003eBurkholderiales\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFamily XI\u003c/em\u003e, were reduced\u0026nbsp;for\u0026nbsp;boys\u0026nbsp;compared with\u0026nbsp;girls\u0026nbsp;(all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA\u0026nbsp;\u0026gt;\u0026nbsp;2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e30b,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Table S50\u003c/strong\u003e).\u0026nbsp;We further identified different bacterial taxa associated with gender under fixed vaginal delivery and feeding with exclusively milk conditions (\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e31-S32, Supplementary Table S51-S53\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional prediction of microbial genes associated with delivery mode and feeding mode at 12 months\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;gut microbial community metabolic function profiles\u0026nbsp;and the predominant\u0026nbsp;microbial functions\u0026nbsp;at 12 months\u0026nbsp;related to\u0026nbsp;vaginal delivery and\u0026nbsp;cesarean\u0026nbsp;sectionare\u0026nbsp;shown in a\u0026nbsp;cladogram\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e33a\u003c/strong\u003e). Based on LDA,\u0026nbsp;\u003cem\u003enitrogen\u003c/em\u003e\u003cem\u003e\u0026nbsp;metabolism\u003c/em\u003e was enriched, while 3 functions,\u0026nbsp;including\u003cem\u003ereplication\u003c/em\u003e\u003cem\u003e\u0026nbsp;recombination and repair proteins\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003cem\u003evaline\u003c/em\u003e\u003cem\u003e\u0026nbsp;leucine and isoleucine biosynthesis,\u003c/em\u003e and\u003cem\u003ebasal\u003c/em\u003e\u003cem\u003e\u0026nbsp;transcription factors\u003c/em\u003e, were reduced\u0026nbsp;for vaginal delivery compared with\u0026nbsp;cesarean\u0026nbsp;section (all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA \u0026gt;2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e33b\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e54\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eMoreover, the\u0026nbsp;gut microbial community metabolic function profiles\u0026nbsp;and the predominant\u0026nbsp;microbial functions\u0026nbsp;at 12 months\u0026nbsp;for\u0026nbsp;exclusively breastfed and combined feeding are also\u0026nbsp;shown in a\u0026nbsp;cladogram\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e34a\u003c/strong\u003e). Based on LDA, compared to\u0026nbsp;the\u0026nbsp;combined feeding group, 20 functions,\u0026nbsp;mainly\u0026nbsp;\u003cem\u003eABC transporters\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003cem\u003eascorbate\u003c/em\u003e\u003cem\u003e\u0026nbsp;and aldarate metabolism,\u0026nbsp;\u003c/em\u003eand\u003cem\u003enitrogen\u003c/em\u003e\u003cem\u003e\u0026nbsp;metabolism,\u003c/em\u003e were increased, whereas 9 functions,\u0026nbsp;mainly\u0026nbsp;\u003cem\u003eamino\u003c/em\u003e\u003cem\u003e\u0026nbsp;sugar and nucleotide sugar metabolism\u003c/em\u003e,\u003cem\u003ereplication\u003c/em\u003e\u003cem\u003e\u0026nbsp;recombination\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;repair proteins,\u003c/em\u003e were\u0026nbsp;decreased in\u0026nbsp;the\u0026nbsp;breastfed\u0026nbsp;group\u0026nbsp;\u003cstrong\u003e(\u003c/strong\u003eall \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;LDA \u0026gt;2,\u0026nbsp;\u003cstrong\u003eSupplementary Fig. S\u003c/strong\u003e\u003cstrong\u003e34b\u003c/strong\u003e,\u003cstrong\u003eSupplementary Table S\u003c/strong\u003e\u003cstrong\u003e55\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eWe characterized the gut microbiome of\u0026nbsp;55\u0026nbsp;infants\u0026nbsp;at 0,\u0026nbsp;1, 3, 6, and 12 months\u0026nbsp;after birth; analyzed the influence of delivery mode, feeding mode and gender on the gut\u0026nbsp;microbiota; and evaluated the contributions of these factors to gut microbial development.\u0026nbsp;Although\u0026nbsp;delivery mode\u003csup\u003e20\u003c/sup\u003e and\u0026nbsp;feeding mode\u003csup\u003e21\u003c/sup\u003e together influenced infant gut microbial composition and evolution,\u0026nbsp;our study\u0026nbsp;demonstrated\u0026nbsp;the contribution degrees of these factors to gut microbial development. We found\u0026nbsp;that\u0026nbsp;delivery mode was the main contribution at birth and that feeding mode played the main role in infant gut microbiota development after 6 months. Interestingly,\u0026nbsp;we found that\u0026nbsp;gender accounted for the dominant contribution to infant gut microbial development at 1, 3 and 6 months\u0026nbsp;after birth, which overturned our common theory.\u003c/p\u003e\n\u003cp\u003eIn our study,\u0026nbsp;individual microbial variation dynamically changed\u0026nbsp;with infant growth.\u0026nbsp;Individual microbial variation\u0026nbsp;in\u0026nbsp;the infants decreased from 0\u0026nbsp;months\u0026nbsp;to 1 month but then increased from 6 months to 12 months.\u0026nbsp;Individual microbial variation at birth was initiated by\u0026nbsp;differences in the\u0026nbsp;maternal microbiota\u003csup\u003e22\u003c/sup\u003e and\u0026nbsp;vaginal\u003csup\u003e23\u003c/sup\u003e, maternal skin\u003csup\u003e24\u003c/sup\u003e and hospital environments\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;Vaginally delivered infants\u0026nbsp;are exposed to the vaginal and fecal microbiota, leading to gut colonization by vagina-associated microbes such as Lactobacillus\u003csup\u003e23\u003c/sup\u003e. In contrast,\u0026nbsp;cesarean\u0026nbsp;section-delivered infants do not\u0026nbsp;experience direct\u0026nbsp;contact with maternal microbes and are thus more likely to become colonized by maternal skin, the hospital environment, or hospital staff\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;With\u0026nbsp;the development of infants, complementary foods, different living environments and different contact populations together contributed to the evolution of the infant gut microbiota, subsequently resulting in increased\u0026nbsp;individual variation after 6 months.\u0026nbsp;The shift in dietary patterns\u0026nbsp;from\u0026nbsp;exclusive breastfeeding\u0026nbsp;to the consumption of solid foods induces the development of the mature gut microbiota\u003csup\u003e26\u003c/sup\u003e.\u0026nbsp;Our\u0026nbsp;study\u0026nbsp;indicated that\u0026nbsp;due to the complementary introduction of a variety of novel food substances, alpha diversity and\u0026nbsp;individual variation\u0026nbsp;increased.\u003c/p\u003e\n\u003cp\u003eWe observed\u0026nbsp;individual\u0026nbsp;differences\u0026nbsp;between vaginally delivered\u0026nbsp;infants\u0026nbsp;and cesarean delivered infants. We found that the variation in the microbial communities of\u0026nbsp;vaginally delivered\u0026nbsp;infants\u0026nbsp;was greater than that of cesarean delivered infants.\u0026nbsp;In healthy infants, delivery is the initial encounter with bacteria capable of colonizing the gut. In a previous study of 24 infants aged 3 to 4 months in Canada,\u0026nbsp;\u003cem\u003eBacteroides\u003c/em\u003e was depleted in cesarean delivered infants relative to those who were vaginally delivered\u003csup\u003e6\u003c/sup\u003e.\u0026nbsp;This result was\u0026nbsp;also\u0026nbsp;underscored\u0026nbsp;in a study of 24 infants in Sweden that reported that the depletion of\u0026nbsp;\u003cem\u003eBacteroides\u003c/em\u003e in infants delivered by cesarean section persisted until 12 months\u003csup\u003e27\u003c/sup\u003e. Our findings indicated that\u0026nbsp;\u003cem\u003eBacteroides\u003c/em\u003e levels were differentially abundant between vaginally and cesarean delivered infants,\u0026nbsp;in line with the results of earlier studies.\u0026nbsp;These high levels of \u003cem\u003eBacteroides\u0026nbsp;\u003c/em\u003ein\u0026nbsp;infancy are associated with\u0026nbsp;increased risks of asthma and obesity later in life\u003csup\u003e28\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e29\u003c/sup\u003e. Consistent with a previous study on premature infants, we observed that\u0026nbsp;delivery mode was the main contribution at birth\u003csup\u003e13\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e15\u003c/sup\u003e. Independent of whether\u0026nbsp;prenatal colonization\u0026nbsp;takes place\u0026nbsp;in the\u0026nbsp;fetus, delivery marks the moment of extensive exposure to bacterial communities of vaginal, fecal, skin and environmental origins\u003csup\u003e30\u003c/sup\u003e. During vaginal birth, specific microbial strains associated with the mother\u0026rsquo;s vaginal microbiota are\u0026nbsp;vertically\u0026nbsp;transmitted from mothers to infants\u003csup\u003e24\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e31\u003c/sup\u003e.\u0026nbsp;Therefore,\u0026nbsp;delivery mode has a profound impact on the earliest microbial colonization of the neonatal gut.\u003c/p\u003e\n\u003cp\u003eMoreover, we observed\u0026nbsp;gut microbial\u0026nbsp;differences\u0026nbsp;between\u0026nbsp;combined feeding\u0026nbsp;infants\u0026nbsp;and\u0026nbsp;exclusively breastfed\u0026nbsp;infants.\u0026nbsp;The microbial\u0026nbsp;variation between the\u0026nbsp;combined feeding\u0026nbsp;infants was greater than that\u0026nbsp;between\u0026nbsp;exclusively breastfed infants.\u0026nbsp;In a previous study of\u0026nbsp;102 infants\u0026nbsp;aged 6 weeks, infants who were fed breast milk supplemented with formula had greater individual differences than exclusively breastfed infants\u003csup\u003e3\u003c/sup\u003e. The large individual difference in the gut microbiota in\u0026nbsp;combined feeding\u0026nbsp;infants may be caused by the use of different brands of formula. Feeding\u0026nbsp;mode\u0026nbsp;has the potential for lasting effects on the gut microbiota, and these effects may be associated with childhood and lifelong health\u003csup\u003e32\u003c/sup\u003e. Studies show that breastfeeding confers protection against gastrointestinal tract and respiratory infections and allergic diseases in addition to reducing the risk of chronic diseases, such as inflammatory bowel disease, obesity and diabetes\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e33\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;Our study indicated that\u0026nbsp;feeding mode played the main role after 6 months in the development of the infant gut microbiota.\u0026nbsp;A previous\u0026nbsp;study\u0026nbsp;showed that bacteria from\u0026nbsp;breast milk\u0026nbsp;are most prominent in infants\u0026rsquo; guts in the first month, accounting for nearly 40% of the gut bacteria in primarily breastfed infants\u003csup\u003e5\u003c/sup\u003e. Ding\u0026nbsp;et al. recently reported that\u0026nbsp;breastfeeding during infancy was a major life-history characteristic that affected\u0026nbsp;microbial community\u0026nbsp;composition in adults\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;The breast milk microbiota that seeds the infant\u0026rsquo;s gut first influences and selects for the microbiota that follow, leaving a footprint that can be detected even in adulthood. Although the differences in breast milk bacteria increase between mothers during the first 6 months of infancy\u003csup\u003e5\u003c/sup\u003e,\u0026nbsp;the other factors that affect the gut microbiota, including delivery\u0026nbsp;mode\u0026nbsp;and gender, still have greater impacts than the feeding\u0026nbsp;mode\u0026nbsp;on the gut microbiota at 0 months and at 1 month to 6 months, respectively.\u003c/p\u003e\n\u003cp\u003eInterestingly, we found that\u0026nbsp;gender accounts for the dominant contribution at 1, 3 and 6 months\u0026nbsp;after birth.\u0026nbsp;Gender differences in the gut microbiota are driven by gender hormones, which in turn\u0026nbsp;affect\u0026nbsp;gender differences in susceptibility to a large number of chronic diseases and infections\u003csup\u003e36-38\u003c/sup\u003e.\u0026nbsp;Three populations were found to significantly increase over time in the boy group: \u003cem\u003eSelenomonadales,\u003c/em\u003e\u003cem\u003eNegativicutes\u003c/em\u003e and \u003cem\u003eVeillonellaceae\u003c/em\u003e.\u0026nbsp;A recent study suggests that\u0026nbsp;lower abundances of \u003cem\u003eSelenomonadales\u003c/em\u003e and \u003cem\u003eVeillonellaceae\u003c/em\u003e are associated with autism spectrum disorder\u0026nbsp;\u003csup\u003e39\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e40\u003c/sup\u003e. Dysregulation of \u003cem\u003eSelenomonadales\u003c/em\u003e affects gut secretion and in turn affects brain function\u0026nbsp;by modulating the\u0026nbsp;efficacy of the vagal response\u003csup\u003e41\u003c/sup\u003e. A relative paucity of \u003cem\u003eNegativicutes\u003c/em\u003e might predispose\u0026nbsp;patients\u0026nbsp;to necrotizing enterocolitis\u003csup\u003e42\u003c/sup\u003e.\u0026nbsp;From another perspective,\u0026nbsp;the difference between\u0026nbsp;boys and girls\u0026nbsp;begins\u0026nbsp;with\u0026nbsp;the gut microbiota in infancy. Children manifest secondary genderual characteristics after\u0026nbsp;gender\u0026nbsp;hormone stimulation at puberty\u003csup\u003e43\u003c/sup\u003e.\u0026nbsp;We\u0026nbsp;hypothesize\u0026nbsp;that the gender-specific early gut microbiota development precedes the development of secondary genderual characteristics.\u0026nbsp;These studies and our results hint\u0026nbsp;that\u0026nbsp;the\u0026nbsp;gender\u0026nbsp;of the newborn affect the gut microbiome\u0026nbsp;and\u0026nbsp;can be valuable for optimizing prevention and treatment strategies.\u0026nbsp;There is currently a paucity of data addressing how\u0026nbsp;the\u0026nbsp;gender of the infant\u0026nbsp;affects\u0026nbsp;the gut microbiota profiles;\u0026nbsp;thus,\u0026nbsp;this study provides important insights into the impact of\u0026nbsp;the\u0026nbsp;gender of the infant on gut microbiota development.\u003c/p\u003e\n\u003cp\u003eIn conclusion, based on a gut microbial characterization during infant development, this study found an increase in gut microbiota diversity that increased over time and indicated that individual microbial variation in infants decreased from 0 to 1 month but then increased from 6 to 12 months. Importantly, we demonstrated the contribution degrees of delivery mode, gender and feeding mode to gut microbial establishment and evolution, which overturned our common theory and provided a novel concept. Understanding the patterns of infant gut microbial colonization and development is critical for both short- and long-term health and may provide a solid foundation for future health outcomes through microbiota intervention.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePCoA, principal coordinates analysis;\u0026nbsp;NMDS,\u0026nbsp;nonmetric multidimensional scaling;\u0026nbsp;OTUs,\u0026nbsp;operational taxonomic units; LEfSe, linear discriminant analysis\u0026nbsp;effect size;\u003c/p\u003e\n\u003cp\u003ePICRUSt, phylogenetic investigation of communities by reconstruction of unobserved states.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank clinical doctors from the First Affiliated Hospital of Zhengzhou University. We also thank the generous volunteer subjects who enrolled in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was sponsored by grants from China Postdoctoral Science Foundation (2020T130609 and 2020T130109ZX), Henan Provincial Medical Science and Technology Project (SBGJ2018004), the National Key Research and Development Program of China (2018YFC2000500), Henan Province Science and Technology Project (182102310404 and 202102310055) and Key Scientific Research Projects of Higher Education Institutions in Henan Province (21A320055 and 20A320056). The funding sources had no role in the design of this study nor any role during its execution, analyses, data interpretation, or decision to submit results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw Illumina read data for all samples were deposited in the European Bioinformatics Institute European Nucleotide Archive\u0026nbsp;database\u0026nbsp;under the accession number\u0026nbsp;PRJNA665920.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eRZG and DJ designed the study. DJ, MX, XQ, LZ, HLP, ZXL, ZCY and ZZH collected clinical samples. RHY, LA and LC performed Miseq sequencing and data analysis; DJ, RZG, and MX wrote the manuscript. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethics guidelines\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u0026nbsp;author declare that they have no conflict of interest. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University (2017-XY-012). The study was performed in accordance with the Helsinki Declaration and Rules of Good Clinical Practice. All participants signed written informed consents after the study protocol was fully explained.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1 Clemente, J. C., Ursell, L. K., Parfrey, L. W. \u0026amp; Knight, R. 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B.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study. \u003cem\u003eLancet (London, England)\u003c/em\u003e\u003cstrong\u003e387\u003c/strong\u003e, 1928-1936, doi:10.1016/s0140-6736(16)00081-7 (2016).\u003c/p\u003e\n\u003cp\u003e43 Mahfouda, S., Moore, J. K., Siafarikas, A., Zepf, F. D. \u0026amp; Lin, A. Puberty suppression in transgender children and adolescents. \u003cem\u003eThe lancet. Diabetes \u0026amp; endocrinology\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, 816-826, doi:10.1016/s2213-8587(17)30099-2 (2017).\u003c/p\u003e\n\u003cp\u003e44 Guaraldi, F. \u0026amp; Salvatori, G. Effect of breast and formula feeding on gut microbiota shaping in newborns. \u003cem\u003eFrontiers in cellular and infection microbiology\u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e, 94, doi:10.3389/fcimb.2012.00094 (2012).\u003c/p\u003e\n\u003cp\u003e45 Wang, Q., Garrity, G. M., Tiedje, J. M. \u0026amp; Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. \u003cem\u003eApplied and environmental microbiology\u003c/em\u003e\u003cstrong\u003e73\u003c/strong\u003e, 5261-5267, doi:10.1128/aem.00062-07 (2007).\u003c/p\u003e\n\u003cp\u003e46 Liu, Y. X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A practical guide to amplicon and metagenomic analysis of microbiome data. \u003cem\u003eProtein \u0026amp; cell\u003c/em\u003e, doi:10.1007/s13238-020-00724-8 (2020).\u003c/p\u003e\n\u003cp\u003e47 McMurdie, P. J. \u0026amp; Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. \u003cem\u003ePloS one\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, e61217, doi:10.1371/journal.pone.0061217 (2013).\u003c/p\u003e\n\u003cp\u003e48 Segata, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Metagenomic biomarker discovery and explanation. \u003cem\u003eGenome biology\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, R60, doi:10.1186/gb-2011-12-6-r60 (2011).\u003c/p\u003e\n\u003cp\u003e49 Ling, Z.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Impacts of infection with different toxigenic Clostridium difficile strains on faecal microbiota in children. \u003cem\u003eScientific reports\u003c/em\u003e\u003cstrong\u003e4\u003c/strong\u003e, 7485, doi:10.1038/srep07485 (2014).\u003c/p\u003e\n\u003cp\u003e50 Langille, M. G.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. \u003cem\u003eNature biotechnology\u003c/em\u003e\u003cstrong\u003e31\u003c/strong\u003e, 814-821, doi:10.1038/nbt.2676 (2013).\u003c/p\u003e\n\u003cp\u003e51 Abubucker, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Metabolic reconstruction for metagenomic data and its application to the human microbiome. \u003cem\u003ePLoS computational biology\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, e1002358, doi:10.1371/journal.pcbi.1002358 (2012).\u003c/p\u003e\n\u003cp\u003e52 Chen, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Associating microbiome composition with environmental covariates using generalized UniFrac distances. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 2106-2113, doi:10.1093/bioinformatics/bts342 (2012).\u003c/p\u003e\n\u003cp\u003e53 Price, M. N., Dehal, P. S. \u0026amp; Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. \u003cem\u003eMolecular biology and evolution\u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e, 1641-1650, doi:10.1093/molbev/msp077 (2009).\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Characteristics of 55 participants\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"69%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003eNo. (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eNewborn sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e28 (50.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e27 (49.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eDelivery mode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eVaginal delivery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e36 (65.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eCesarean section delivery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e19 (34.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eFeeding patterns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eExclusive breastfeeding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e36 (65.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eCombined feeding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e19 (34.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eBirth weight at birth (kg),\u0026nbsp;mean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e3.34\u0026plusmn;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eBirth weight at 12 month (kg),\u0026nbsp;mean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e10.01\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"76.76767676767676%\"\u003e\n \u003cp\u003eGestational age (days), mean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.232323232323232%\"\u003e\n \u003cp\u003e278\u0026plusmn;5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"infant gut microbiota, microbiota development, delivery mode, feeding mode, infant gender.","lastPublishedDoi":"10.21203/rs.3.rs-828854/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-828854/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo characterize gut microbiome of the infant during the first year of life and assess the different contributions of delivery mode, feeding mode and infant gender to gut microbial development. We collected 314 faecal samples from 80 infants at 5 time points of 0, 1st, 3rd, 6\u003csup\u003eth\u003c/sup\u003e and 12\u003csup\u003eth\u003c/sup\u003e months prospectively, and finally 213 samples completed Miseq sequencing and analysis. We characterized gut microbiome of the infant at the different phases and evaluated the different contributions of delivery mode, feeding mode and gender to gut microbial development. Delivery mode, gender and feeding mode were the strongest factors determining gut microbiome colonization at 0 months, from 1 month to 6 months and 12 months, respectively. Four genera including \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eParabacteroides\u003c/em\u003e and \u003cem\u003ePhascolarctobacterium\u003c/em\u003e were increased, whereas 10 genera e.g. \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eEnterobacter\u003c/em\u003e were reduced, in vaginal delivery versus cesarean section. Two genera including \u003cem\u003ePeptostreptococcaceae incertae sedis\u003c/em\u003e and \u003cem\u003eAnaerococcus\u003c/em\u003e were increased, whereas 3 genera e.g. \u003cem\u003eCoriobacteriaceae\u003c/em\u003e \u003cem\u003euncultured\u003c/em\u003e were reduced, in exclusive breastfeeding versus combined feeding. This study indicated the contribution degrees of delivery mode, feeding mode and gender to gut microbial initiation and evolvement, and reported microbial differences induced by the different delivery mode, feeding mode and gender.\u003c/p\u003e","manuscriptTitle":"Delivery, Feeding and Gender Differently Contribute to Infant Gut Microbiota Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-25 23:21:42","doi":"10.21203/rs.3.rs-828854/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dab97b91-a50c-4736-9eac-3495d373c290","owner":[],"postedDate":"August 25th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":6685079,"name":"Biomedical Engineering"},{"id":6685080,"name":"Obstetrics \u0026 Gynecology"},{"id":6685081,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2021-10-07T07:44:14+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-25 23:21:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-828854","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-828854","identity":"rs-828854","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}