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.

Time-varying associations of gestational and childhood triclosan with pubertal and adrenarchal outcomes in early adolescence.

Background: Triclosan is an endocrine-disrupting chemical, but associations with pubertal outcomes remain unclear. We examined associations of gestational and childhood triclosan with adolescent hormone concentrations and pubertal stage. Methods: We quantified urinary triclosan concentrations twice during pregnancy and seven times between birth and 12 years in participants recruited from Cincinnati, OH (2003-2006). We averaged concentrations across pregnancy and childhood and separately considered individual exposure periods in multiple informant models. At 12 years, we measured serum hormone concentrations (males [n = 72] and females [n = 84]-dehydroepiandrosterone-sulfate, luteinizing hormone, follicle-stimulating hormone; males-testosterone; females-estradiol). Also at age 12 years, participants self-reported physical development and menarchal timing. We estimated associations (95% confidence interval) of triclosan with hormone concentrations, more advanced physical development, and age at menarche. Results: For females, each doubling of childhood triclosan was associated with 16% lower estradiol concentrations (-29%, 0%), with stronger associations for measures closer to adolescence. We found suggestive evidence that higher triclosan at any age was associated with ~10% (for gestational triclosan: -18%, -2%) lower follicle-stimulating hormone concentrations among males and early postnatal (1-3 years) triclosan was associated with 63% (5%, 96%) lower odds of advanced pubic hair development in females. In multiple informant models, each doubling of gestational triclosan concentrations was associated with 5% (0%, 9%) earlier age at menarche, equivalent to 5.5 months. Conclusion: Gestational and childhood triclosan concentrations were related to some pubertal outcomes including hormone concentrations and age at menarche. Our findings highlight the relevance of elucidating potential sex-specific and time-dependent actions of triclosan.

Patterns of infant fecal metabolite concentrations and social behavioral development in toddlers.

BACKGROUND: Gut-derived metabolites, products of microbial and host co-metabolism, may inform mechanisms underlying children's neurodevelopment. We investigated whether infant fecal metabolites were related to toddler social behavior. METHODS: Stool samples collected from 6-week-olds (n = 86) and 1-year-olds (n = 209) in the New Hampshire Birth Cohort Study (NHBCS) were analyzed using nuclear magnetic resonance spectroscopy metabolomics. Autism-related behavior in 3-year-olds was assessed by caregivers using the Social Responsiveness Scale (SRS-2). To assess the association between metabolites and SRS-2 scores, we used a traditional single-metabolite approach, quantitative metabolite set enrichment (QEA), and self-organizing maps (SOMs). RESULTS: Using a single-metabolite approach and QEA, no individual fecal metabolite or metabolite set at either age was associated with SRS-2 scores. Using the SOM method, fecal metabolites of six-week-olds organized into four profiles, which were unrelated to SRS-2 scores. In 1-year-olds, one of twelve fecal metabolite profiles was associated with fewer autism-related behaviors, with SRS-2 scores 3.4 (95%CI: -7, 0.2) points lower than the referent group. This profile had higher concentrations of lactate and lower concentrations of short chain fatty acids than the reference. CONCLUSIONS: We uncovered metabolic profiles in infant stool associated with subsequent social behavior, highlighting one potential mechanism by which gut bacteria may influence neurobehavior. IMPACT: Differences in host and microbial metabolism may explain variability in neurobehavioral phenotypes, but prior studies do not have consistent results. We applied three statistical techniques to explore fecal metabolite differences related to social behavior, including self-organizing maps (SOMs), a novel machine learning algorithm. A 1-year-old fecal metabolite pattern characterized by high lactate and low short-chain fatty acid concentrations, identified using SOMs, was associated with social behavior less indicative of autism spectrum disorder. Our findings suggest that social behavior may be related to metabolite profiles and that future studies may uncover novel findings by applying the SOM algorithm.

Intestinal Bacteroides modulates inflammation, systemic cytokines, and microbial ecology via propionate in a mouse model of cystic fibrosis.

Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1ß-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.

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.

Estimate of the incidence of PANDAS and PANS in 3 primary care populations.

Objective: Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal infection (PANDAS) and Pediatric Acute-Onset Neuropsychiatric syndrome (PANS) are presumed autoimmune complications of infection or other instigating events. To determine the incidence of these disorders, we performed a retrospective review for the years 2017-2019 at three academic medical centers. Methods: We identified the population of children receiving well-child care at each institution. Potential cases of PANS and PANDAS were identified by including children age 3-12 years at the time they received one of five new diagnoses: avoidant/restrictive food intake disorder, other specified eating disorder, separation anxiety disorder of childhood, obsessive-compulsive disorder, or other specified disorders involving an immune mechanism, not elsewhere classified. Tic disorders was not used as a diagnostic code to identify cases. Data were abstracted; cases were classified as PANDAS or PANS if standard definitions were met. Results: The combined study population consisted of 95,498 individuals. The majority were non-Hispanic Caucasian (85%), 48% were female and the mean age was 7.1 (SD 3.1) years. Of 357 potential cases, there were 13 actual cases [mean age was 6.0 (SD 1.8) years, 46% female and 100% non-Hispanic Caucasian]. The estimated annual incidence of PANDAS/PANS was 1/11,765 for children between 3 and 12 years with some variation between different geographic areas. Conclusion: Our results indicate that PANDAS/PANS is a rare disorder with substantial heterogeneity across geography and time. A prospective investigation of the same question is warranted.

An In Vitro Medium for Modeling Gut Dysbiosis Associated with Cystic Fibrosis.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting non-CF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of gut dysbiosis in CF.

Development of the intestinal microbiome in cystic fibrosis in early life.

Cystic fibrosis (CF) is a heritable disease that causes altered physiology at mucosal sites; these changes result in chronic infections in the lung, significant gastrointestinal complications as well as dysbiosis of the gut microbiome, although the latter has been less well explored. Here, we describe the longitudinal development of the gut microbiome in a cohort of children with CF (cwCF) from birth through early childhood (0-4 years of age) using 16S rRNA gene amplicon sequencing of stool samples as a surrogate for the gut microbiota. Similar to healthy populations, alpha diversity of the gut microbiome increases significantly with age, but diversity plateaus at ~2 years of age for this CF cohort. Several taxa that have been associated with dysbiosis in CF change with age toward a more healthy-like composition; notable exceptions include Akkermansia, which decreases with age, and Blautia, which increases with age. We also examined the relative abundance and prevalence of nine taxa associated with CF lung disease, several of which persist across early life, highlighting the possibility of the lung being seeded directly from the gut early in life. Finally, we applied the Crohn's Dysbiosis Index to each sample, and found that high Crohn's-associated dysbiosis early in life (<2 years) was associated with significantly lower Bacteroides in samples collected from 2 to 4 years of age. Together, these data comprise an observational study that describes the longitudinal development of the CF-associated gut microbiota and suggest that early markers associated with inflammatory bowel disease may shape the later gut microbiota of cwCF. IMPORTANCE Cystic fibrosis is a heritable disease that disrupts ion transport at mucosal surfaces, causing a buildup of mucus and dysregulation of microbial communities in both the lungs and the intestines. Persons with CF are known to have dysbiotic gut microbial communities, but the development of these communities over time beginning at birth has not been thoroughly studied. Here, we describe an observation study following the development of the gut microbiome of cwCF throughout the first 4 years of life, during the critical window of both gut microbiome and immune development. Our findings indicate the possibility of the gut microbiota as a reservoir of airway pathogens and a surprisingly early indication of a microbiota associated with inflammatory bowel disease.

Latent Class Analysis of Prenatal Substance Exposure and Child Behavioral Outcomes.

OBJECTIVES: To predict behavioral disruptions in middle childhood, we identified latent classes of prenatal substance use. STUDY DESIGN: As part of the Environmental influences on Child Health Outcomes Program, we harmonized prenatal substance use data and child behavior outcomes from 2195 women and their 6- to 11-year-old children across 10 cohorts in the US and used latent class-adjusted regression models to predict parent-rated child behavior. RESULTS: Three latent classes fit the data: low use (90.5%; n = 1986), primarily using no substances; licit use (6.6%; n = 145), mainly using nicotine with a moderate likelihood of using alcohol and marijuana; and illicit use (2.9%; n = 64), predominantly using illicit substances along with a moderate likelihood of using licit substances. Children exposed to primarily licit substances in utero had greater levels of externalizing behavior than children exposed to low or no substances (P = .001, d = .64). Children exposed to illicit substances in utero showed small but significant elevations in internalizing behavior than children exposed to low or no substances (P < .001, d = .16). CONCLUSIONS: The differences in prenatal polysubstance use may increase risk for specific childhood problem behaviors; however, child outcomes appeared comparably adverse for both licit and illicit polysubstance exposure. We highlight the need for similar multicohort, large-scale studies to examine childhood outcomes based on prenatal substance use profiles.

Human milk-associated bacterial communities associate with the infant gut microbiome over the first year of life.

Introduction: Microbial communities inhabiting the human infant gut are important for immune system development and lifelong health. One critical exposure affecting the bacterial colonization of the infant gut is consumption of human milk, which contains diverse microbial communities and prebiotics. We hypothesized that human milk-associated microbial profiles are associated with those of the infant gut. Methods: Maternal-infant dyads enrolled in the New Hampshire Birth Cohort Study (n = 189 dyads) contributed breast milk and infant stool samples collected approximately at 6 weeks, 4 months, 6 months, 9 months, and 12 months postpartum (n = 572 samples). Microbial DNA was extracted from milk and stool and the V4-V5 region of the bacterial 16S rRNA gene was sequenced. Results: Clustering analysis identified three breast milk microbiome types (BMTs), characterized by differences in Streptococcus, Staphylococcus, Pseudomonas, Acinetobacter, and microbial diversity. Four 6-week infant gut microbiome types (6wIGMTs) were identified, differing in abundances of Bifidobacterium, Bacteroides, Clostridium, Streptococcus, and Escherichia/Shigella, while two 12-month IGMTs (12mIGMTs) differed primarily by Bacteroides presence. At 6 weeks, BMT was associated with 6wIGMT (Fisher's exact test value of p = 0.039); this association was strongest among infants delivered by Cesarean section (Fisher's exact test value of p = 0.0028). The strongest correlations between overall breast milk and infant stool microbial community structures were observed when comparing breast milk samples to infant stool samples collected at a subsequent time point, e.g., the 6-week breast milk microbiome associated with the 6-month infant gut microbiome (Mantel test Z-statistic = 0.53, value of p = 0.001). Streptoccous and Veillonella species abundance were correlated in 6-week milk and infant stool, and 4- and 6-month milk Pantoea species were associated with infant stool Lachnospiraceae genera at 9 and 12 months. Discussion: We identified clusters of human milk and infant stool microbial communities that were associated in maternal-infant dyads at 6 weeks of life and found that milk microbial communities were more strongly associated with infant gut microbial communities in infants delivered operatively and after a lag period. These results suggest that milk microbial communities have a long-term effect on the infant gut microbiome both through sharing of microbes and other molecular mechanisms.

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.

The association between early life antibiotic exposure and the gut resistome of young children: a systematic review.

Antimicrobial resistance is a growing public health burden, but little is known about the effects of antibiotic exposure on the gut resistome. As childhood (0-5 years) represents a sensitive window of microbiome development and a time of relatively high antibiotic use, the aims of this systematic review were to evaluate the effects of antibiotic exposure on the gut resistome of young children and identify knowledge gaps. We searched PubMed, Scopus, Web of Science, and the Cochrane Central Register of Controlled Trials. A PICO framework was developed to determine eligibility criteria. Our main outcomes were the mean or median difference in overall resistance gene load and resistome alpha diversity by antibiotic exposure groups. Bias assessment was completed using RoB 2 and ROBINS-I with quality of evidence assessed via the GRADE criteria. From 4885 records identified, 14 studies (3 randomized controlled trials and 11 observational studies) were included in the qualitative review. Eight studies that included information on antibiotic exposure and overall resistance gene load reported no or positive associations. Inconsistent associations were identified for the nine studies that assessed resistome alpha diversity. We identified three main groups of studies based on study design, location, participants, antibiotic exposures, and indication for antibiotics. Overall, the quality of evidence for our main outcomes was rated low or very low, mainly due to potential bias from the selective of reporting results and confounding. We found evidence that antibiotic exposure is associated with changes to the overall gut resistance gene load of children and may influence the diversity of antimicrobial resistance genes. Given the overall quality of the studies, more research is needed to assess how antibiotics impact the resistome of other populations. Nonetheless, this evidence indicates that the gut resistome is worthwhile to consider for antibiotic prescribing practices.

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.

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.

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.

MarZIC: A Marginal Mediation Model for Zero-Inflated Compositional Mediators with Applications to Microbiome Data.

BACKGROUND: The human microbiome can contribute to pathogeneses of many complex diseases by mediating disease-leading causal pathways. However, standard mediation analysis methods are not adequate to analyze the microbiome as a mediator due to the excessive number of zero-valued sequencing reads in the data and that the relative abundances have to sum to one. The two main challenges raised by the zero-inflated data structure are: (a) disentangling the mediation effect induced by the point mass at zero; and (b) identifying the observed zero-valued data points that are not zero (i.e., false zeros). METHODS: We develop a novel marginal mediation analysis method under the potential-outcomes framework to address the issues. We also show that the marginal model can account for the compositional structure of microbiome data. RESULTS: The mediation effect can be decomposed into two components that are inherent to the two-part nature of zero-inflated distributions. With probabilistic models to account for observing zeros, we also address the challenge with false zeros. A comprehensive simulation study and the application in a real microbiome study showcase our approach in comparison with existing approaches. CONCLUSIONS: When analyzing the zero-inflated microbiome composition as the mediators, MarZIC approach has better performance than standard causal mediation analysis approaches and existing competing approach.

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.

Correction: Racial and geographic variation in effects of maternal education and neighborhood-level measures of socioeconomic status on gestational age at birth: Findings from the ECHO cohorts.

[This corrects the article DOI: 10.1371/journal.pone.0245064.].

The Developing Microbiome From Birth to 3 Years: The Gut-Brain Axis and Neurodevelopmental Outcomes.

The volume and breadth of research on the role of the microbiome in neurodevelopmental and neuropsychiatric disorders has expanded greatly over the last decade, opening doors to new models of mechanisms of the gut-brain axis and therapeutic interventions to reduce the burden of these outcomes. Studies have highlighted the window of birth to 3 years as an especially sensitive window when interventions may be the most effective. Harnessing the powerful gut-brain axis during this critical developmental window clarifies important investigations into the microbe-human connection and the developing brain, affording opportunities to prevent rather than treat neurodevelopmental disorders and neuropsychiatric illness. In this review, we present an overview of the developing intestinal microbiome in the critical window of birth to age 3; and its prospective relationship with neurodevelopment, with particular emphasis on immunological mechanisms. Next, the role of the microbiome in neurobehavioral outcomes (such as autism, anxiety, and attention-deficit hyperactivity disorder) as well as cognitive development are described. In these sections, we highlight the importance of pairing mechanistic studies in murine models with large scale epidemiological studies that aim to clarify the typical health promoting microbiome in early life across varied populations in comparison to dysbiosis. The microbiome is an important focus in human studies because it is so readily alterable with simple interventions, and we briefly outline what is known about microbiome targeted interventions in neurodevelopmental outcomes. More novel examinations of known environmental chemicals that adversely impact neurodevelopmental outcomes and the potential role of the microbiome as a mediator or modifier are discussed. Finally, we look to the future and emphasize the need for additional research to identify populations that are sensitive to alterations in their gut microbiome and clarify how interventions might correct and optimize neurodevelopmental outcomes.