A prospective study of the infant gut microbiome in relation to vaccine response.

BACKGROUND: The establishment of the gut microbiome plays a key symbiotic role in the developing immune system; however, its influence on vaccine response is yet uncertain. We prospectively investigated the composition and diversity of the early-life gut microbiome in relation to infant antibody response to two routinely administered vaccines. METHODS: Eighty-three infants enrolled in the New Hampshire Birth Cohort Study were included in the analysis. We collected blood samples at 12 months of age and assayed the isolated serum to quantify total IgG and measured antibody to pneumococcal capsular polysaccharide and tetanus toxoid. Stool samples were collected from infants at 6 weeks of age and sequenced using 16S rRNA, and a subset of 61 samples were sequenced using shotgun metagenomics sequencing. RESULTS: We observed differences in beta diversity for 16S 6-week stool microbiota and pneumococcal and tetanus IgG antibody responses. Metagenomics analyses identified species and metabolic pathways in 6-week stool associated with tetanus antibody response, in particular, negative associations with the relative abundance of Aeriscardovia aeriphila species and positive associations with the relative abundance of species associated with CDP-diacylglycerol biosynthesis pathways. CONCLUSIONS: The early gut microbiome composition may influence an infant's vaccine response. IMPACT: Early intestinal microbiome acquisition plays a critical role in immune maturation and in both adaptive and innate immune response in infancy. We identified associations between early life microbiome composition and response to two routinely administered vaccines-pneumococcal capsular polysaccharide and tetanus toxoid-measured at approximately 1 year of age. Our findings highlight the potential impact of the gut microbiome on infant immune response that may open up opportunities for future interventions.

Impact of antibiotics on off-target infant gut microbiota and resistance genes in cohort studies.

BACKGROUND: Young children are frequently exposed to antibiotics, with the potential for collateral consequences to the gut microbiome. The impact of antibiotic exposures to off-target microbes (i.e., bacteria not targeted by treatment) and antibiotic resistance genes (ARGs) is poorly understood. METHODS: We used metagenomic sequencing data from paired stool samples collected prior to antibiotic exposure and at 1 year from over 200 infants and a difference-in-differences approach to assess the relationship between subsequent exposures and the abundance or compositional diversity of microbes and ARGs while adjusting for covariates. RESULTS: By 1 year, the abundance of multiple species and ARGs differed by antibiotic exposure. Compared to infants never exposed to antibiotics, Bacteroides vulgatus relative abundance increased by 1.72% (95% CI: 0.19, 3.24) while Bacteroides fragilis decreased by 1.56% (95% CI: -4.32, 1.21). Bifidobacterium species also exhibited opposing trends. ARGs associated with exposure included class A beta-lactamase gene CfxA6. Among infants attending day care, Escherichia coli and ARG abundance were both positively associated with antibiotic use. CONCLUSION: Novel findings, including the importance of day care attendance, were identified through considering microbiome data at baseline and post-intervention. Thus, our study design and approach have important implications for future studies evaluating the unintended impacts of antibiotics. IMPACT: The impact of antibiotic exposure to off-target microbes and antibiotic resistance genes in the gut is poorly defined. We quantified these impacts in two cohort studies using a difference-in-differences approach. Novel to microbiome studies, we used pre/post-antibiotic data to emulate a randomized controlled trial. Compared to infants unexposed to antibiotics between baseline and 1 year, the relative abundance of multiple off-target species and antibiotic resistance genes was altered. Infants who attended day care and were exposed to antibiotics within the first year had a higher abundance of Escherichia coli and antibiotic resistance genes; a novel finding warranting further investigation.

Contribution of gut bacteria to arsenic metabolism in the first year of life in a prospective birth cohort.

Gut bacteria are at the interface of environmental exposures and their impact on human systems, and may alter host absorption, metabolism, and excretion of toxic chemicals. We investigated whether arsenic-metabolizing bacterial gene pathways related to urinary arsenic concentrations. In the New Hampshire Birth Cohort Study, urine and stool samples were obtained at six weeks (n = 186) and one year (n = 190) of age. Inorganic arsenic (iAs), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) were quantified in infant urine samples using high-performance liquid chromatography with inductively coupled plasma mass spectrometry. Total arsenic exposure (tAs) was summarized as Σ(iAs, MMA, DMA) and log2-transformed. Fecal microbial DNA underwent metagenomic sequencing and the relative abundance of bacterial gene pathways were grouped as KEGG Orthologies (KOs) using BioBakery algorithms. Arsenic metabolism KOs with >80% prevalence were log2-transformed and modeled continuously using linear regression, those with <10% were not evaluated and those with 10-80% prevalence were analyzed dichotomously (detect/non-detect) using logistic regression. In the first set of models, tAs was regressed against KO relative abundance or detection adjusting for age at sample collection and child's sex. Effect modification by delivery mode was assessed in stratified models. In the second set of models, the association between the relative abundance/detection of the KOs and arsenic speciation (%iAs, %MMA, %DMA) was quantified with linear regression. Urinary tAs was associated with the increased relative abundance/detection odds of several arsenic-related KOs, including K16509, an arsenate reductase transcriptional regulator, with stronger associations among six-week-olds than one-year-olds. K16509 was also associated with decreased %MMA and increased %DMA at six weeks and one year. Notably, many associations were stronger among operatively-delivered than vaginally-delivered infants. Our findings suggest associations between arsenic-metabolizing bacteria in the infant gut microbiome and urinary arsenic excretion.

Prenatal arsenic exposure alters the placental expression of multiple epigenetic regulators in a sex-dependent manner.

BACKGROUND: Prenatal exposure to arsenic has been linked to a range of adverse health conditions in later life. Such fetal origins of disease are frequently the result of environmental effects on the epigenome, leading to long-term alterations in gene expression. Several studies have demonstrated effects of prenatal arsenic exposure on DNA methylation; however the impact of arsenic on the generation and decoding of post-translational histone modifications (PTHMs) is less well characterized, and has not been studied in the context of prenatal human exposures. METHODS: In the current study, we examined the effect of exposure to low-to-moderate levels of arsenic in a US birth cohort, on the expression of 138 genes encoding key epigenetic regulators in the fetal portion of the placenta. Our candidate genes included readers, writers and erasers of PTHMs, and chromatin remodelers. RESULTS: Arsenic exposure was associated with the expression of 27 of the 138 epigenetic genes analyzed. When the cohort was stratified by fetal sex, arsenic exposure was associated with the expression of 40 genes in male fetal placenta, and only 3 non-overlapping genes in female fetal placenta. In particular, we identified an inverse relationship between arsenic exposure and expression of the gene encoding the histone methyltransferase, PRDM6 (p < 0.001). Mutation of PRDM6 has been linked to the congenital heart defect, patent ductus arteriosus. CONCLUSIONS: Our findings suggest that prenatal arsenic exposure may have sex-specific effects on the fetal epigenome, which could plausibly contribute to its subsequent health impacts.

Prospective association of the infant gut microbiome with social behaviors in the ECHO consortium.

BACKGROUND: Identifying modifiable risk factors of autism spectrum disorders (ASDs) may inform interventions to reduce financial burden. The infant/toddler gut microbiome is one such feature that has been associated with social behaviors, but results vary between cohorts. We aimed to identify consistent overall and sex-specific associations between the early-life gut microbiome and autism-related behaviors. METHODS: Utilizing the Environmental influences on Children Health Outcomes (ECHO) consortium of United States (U.S.) pediatric cohorts, we gathered data on 304 participants with fecal metagenomic sequencing between 6-weeks to 2-years postpartum (481 samples). ASD-related social development was assessed with the Social Responsiveness Scale (SRS-2). Linear regression, PERMANOVA, and Microbiome Multivariable Association with Linear Models (MaAsLin2) were adjusted for sociodemographic factors. Stratified models estimated sex-specific effects. RESULTS: Genes encoding pathways for synthesis of short-chain fatty acids were associated with higher SRS-2 scores, indicative of ASDs. Fecal concentrations of butyrate were also positively associated with ASD-related SRS-2 scores, some of which may be explained by formula use. LIMITATIONS: The distribution of age at outcome assessment differed in the cohorts included, potentially limiting comparability between cohorts. Stool sample collection methods also differed between cohorts. Our study population reflects the general U.S. population, and thus includes few participants who met the criteria for being at high risk of developing ASD. CONCLUSIONS: Our study is among the first multicenter studies in the U.S. to describe prospective microbiome development from infancy in relation to neurodevelopment associated with ASDs. Our work contributes to clarifying which microbial features associate with subsequent diagnosis of neuropsychiatric outcomes. This will allow for future interventional research targeting the microbiome to change neurodevelopmental trajectories.

Bacterial Modification of the Association Between Arsenic and Autism-Related Social Behavior Scores.

Arsenic is related to neurodevelopmental outcomes and is associated with the composition of the gut microbiome. Data on the modifying role of the microbiome are limited. We probed suggestive relationships between arsenic and social behaviors to quantify the modifying role of the infant gut microbiome. We followed children for whom arsenic concentrations were quantified in 6-week-old toenail clippings. Scores on the Social Responsiveness Scale (SRS-2), which measures autism-related social behaviors, were provided by caregivers when the child was approximately 3 years of age. Metagenomic sequencing was performed on infant stools collected at 6 weeks and 1 year of age. To evaluate modification by the top ten most abundant species and functional pathways, we modeled SRS-2 total T-scores as a function of arsenic concentrations, microbiome features dichotomized at their median, and an interaction between exposure and the microbiome, adjusting for other trace elements and sociodemographic characteristics. As compared to the standardized population (SRS-2 T-scores = 50), participants in our study had lower SRS-2 scores (n = 78, mean = 44, SD = 5).The relative abundances of several functional pathways identified in 6-week stool samples modified the arsenic-SRS-2 association, including the pathways of valine and isoleucine biosynthesis; we observed no association among those with high relative abundance of each pathway [ß = - 0.67 (95% CI - 1.46, 0.12)], and an adverse association [ß = 1.67 (95% CI 0.3, 3.04), pinteraction= 0.05] among infants with low relative abundance. Our findings indicate the infant gut microbiome may alter neurodevelopmental susceptibility to environmental exposures.

Early-life exposure to per- and polyfluoroalkyl substances and infant gut microbial composition.

Human milk is rich in essential nutrients and immune-activating compounds but is also a source of toxicants including per- and polyfluoroalkyl substances (PFAS). Evidence suggests that immune-related effects of PFAS may, in part, be due to alterations of the microbiome. We aimed to identify the association between milk PFAS exposure and the infant gut microbiome. Methods: PFAS [perfluorooctane sulfonic acid (PFOS) and perfluorooctanoate (PFOA)] were quantified in milk from ~6 weeks postpartum using high-performance liquid chromatography with tandem mass spectrometry. A molar sum (ΣPFAS) was calculated. Caregivers collected infant stool samples at 6 weeks (n = 116) and/or 1 year postpartum (n = 119). Stool DNA underwent metagenomic sequencing. We estimated the association of PFAS with diversity and relative abundances of species with linear regression. Single- and multi-PFAS models adjusted for potential confounders in complete case analyses and with imputed missing covariate data for 6-week and 1-year microbiomes separately. We assessed sensitive populations with stratification. Results: PFOS and PFOA were detected in 94% and 83% of milk samples, respectively. PFOS was associated with increased diversity at 6 weeks among infants fed exclusively human milk [ß = 0.24 per PFOS doubling, (95% CI = 0.03, 0.45), P = 0.03] and born to primiparous mothers [ß = 0.37 (0.06, 0.67), P = 0.02]. Estimates were strongest in multi-PFAS models and among complete cases. ΣPFAS was associated with Bacteroides vulgatus relative abundance at 1 year [(ß = -2.34% per doubling (-3.63, -1.05), FDR q = 0.099]. Conclusions: PFAS may increase infant gut microbiome diversity and alter the relative abundance of biologically relevant bacteria. Additional analyses may identify related health outcomes.

Umbilical cord blood immune cell profiles in relation to the infant gut microbiome.

During infancy, the interplay between the developing immune system and the microbiome is critical. We examined whether blood immune cell composition at birth in the umbilical cord (inferred by DNA methylation profiling) related to the early infant gut microbiome (assessed by 16S rRNA gene sequencing) among 73 infants in the New Hampshire Birth Cohort Study. We used generalized estimating equations and controlled for false discovery rate to select microbial taxa associated with immune cells. We found associations between the infant gut microbiome and immune cells, including a positive association between B cells and Enterobacter, a negative association between natural killer cells and Bifidobacterium, and a positive association between granulocytes and Bifidobacterium. Our findings give clues that immune profiles at the time of birth as measured in umbilical cord blood are associated with the development of the gut microbiome in early life.

The relationship between the gut microbiome and the risk of respiratory infections among newborns.

Background: Emerging evidence points to a critical role of the developing gut microbiome in immune maturation and infant health; however, prospective studies are lacking. Methods: We examined the occurrence of infections and associated symptoms during the first year of life in relation to the infant gut microbiome at six weeks of age using bacterial 16S rRNA V4-V5 gene sequencing (N = 465) and shotgun metagenomics (N = 185). We used generalized estimating equations to assess the associations between longitudinal outcomes and 16S alpha diversity and metagenomics species. Results: Here we show higher infant gut microbiota alpha diversity was associated with an increased risk of infections or respiratory symptoms treated with a prescription medicine, and specifically upper respiratory tract infections. Among vaginally delivered infants, a higher alpha diversity was associated with an increased risk of all-cause wheezing treated with a prescription medicine and diarrhea involving a visit to a health care provider. Positive associations were specifically observed with Veillonella species among all deliveries and Haemophilus influenzae among cesarean-delivered infants. Conclusion: Our findings suggest that intestinal microbial diversity and the relative abundance of key taxa in early infancy may influence susceptibility to respiratory infection, wheezing, and diarrhea.

Infant infections, respiratory symptoms, and allergy in relation to timing of rice cereal introduction in a United States cohort.

Rice products marketed in the USA, including baby rice cereal, contain inorganic arsenic, a putative immunotoxin. We sought to determine whether the timing of introduction of rice cereal in the first year of life influences occurrence of infections, respiratory symptoms, and allergy. Among 572 infants from the New Hampshire Birth Cohort Study, we used generalized estimating equation, adjusted for maternal smoking during pregnancy, marital status, education attainment, pre-pregnancy body mass index, maternal age at enrollment, infant birth weight, and breastfeeding history. Among 572 infants, each month earlier of introduction to rice cereal was associated with increased risks of subsequent upper respiratory tract infections (relative risk, RR = 1.04; 95% CI: 1.00-1.09); lower respiratory tract infections (RR = 1.19; 95% CI: 1.02-1.39); acute respiratory symptoms including wheeze, difficulty breathing, and cough (RR = 1.10; 95% CI: 1.00-1.22); fever requiring a prescription medicine (RR = 1.22; 95% CI: 1.02-1.45) and allergy diagnosed by a physician (RR = 1.20; 95% CI: 1.06-1.36). No clear associations were observed with gastrointestinal symptoms. Our findings suggest that introduction of rice cereal earlier may influence infants' susceptibility to respiratory infections and allergy.

Metagenomic analysis reveals associations between salivary microbiota and body composition in early childhood.

Several studies have shown that body mass index is strongly associated with differences in gut microbiota, but the relationship between body weight and oral microbiota is less clear especially in young children. We aimed to evaluate if there is an association between child growth and the saliva microbiome. We hypothesized that associations between growth and the saliva microbiome would be moderate, similarly to the association between growth and the gut microbiome. For 236 toddlers participating in the New Hampshire Birth Cohort Study, we characterized the association between multiple longitudinal anthropometric measures of body height, body weight and body mass. Body Mass Index (BMI) z-scores were calculated, and dual-energy x-ray absorptiometry (DXA) was used to estimate body composition. Shotgun metagenomic sequencing of saliva samples was performed to taxonomically and functionally profile the oral microbiome. We found that within-sample diversity was inversely related to body mass measurements while community composition was not associated. Although the magnitude of associations were small, some taxa were consistently associated with growth and modified by sex. Certain taxa were associated with decreased weight or growth (including Actinomyces odontolyticus and Prevotella melaninogenica) or increased growth (such as Streptococcus mitis and Corynebacterium matruchotii) across anthropometric measures. Further exploration of the functional significance of this relationship will enhance our understanding of the intersection between weight gain, microbiota, and energy metabolism and the potential role of these relationships on the onset of obesity-associated diseases in later life.

Nutrient-toxic element mixtures and the early postnatal gut microbiome in a United States longitudinal birth cohort.

BACKGROUND: The infant microbiome contributes to health status across the lifespan, but environmental factors affecting microbial communities are poorly understood, particularly when toxic and essential elements interact. OBJECTIVE: We aimed to identify the associations between a spectrum of other early-postnatal nutrient or toxic elemental exposures measured and the infant gut microbiome. METHODS: Our analysis included 179 six-week-old infants from the New Hampshire Birth Cohort Study. Eleven elements were measured in infant toenail clippings. The gut microbiome was assessed using 16S rRNA V4-V5 hypervariable region targeted sequencing. Multivariable zero-inflated logistic normal regression (MZILN) was used to model the association between element concentrations and taxon relative abundance. To explore interactive and nonlinear associations between the exposures and specific taxa we employed Bayesian Kernel Machine Regression (BKMR). Effect modification by delivery mode, feeding mode, peripartum antibiotic exposure, and infant sex was assessed with stratified models. RESULTS: We found a negative association between arsenic and microbial diversity in the full population that was accentuated among infants exposed to peripartum antibiotics. Arsenic, cadmium, copper, iron, lead, manganese, nickel, selenium, tin, and zinc were each associated with differences in at least one taxon in the full study population, with most of the related taxa belonging to the Bacteroides and Lactobacillales. In stratified analyses, mercury, in addition to the other elements, was associated with specific taxa. Bifidobacterium, which associated negatively with zinc in MZILN and BKMR models, had a quadratic association with arsenic concentrations. These associations varied with the concentration of the other element. CONCLUSIONS: Early postnatal toxic and nutrient elemental exposures are associated with differences in the infant microbiome. Further research is needed to clarify the whether these alterations are a biomarker of exposure or if they have implications for child and lifelong health.