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Background: Fecal shedding of SARS-CoV-2 occurs during infection, particularly in pediatric populations. The gut microbiota are associated with resistance to enteric pathogens. COVID-19 is associated with alterations to the gut microbiome. We hypothesized that the gut microbiome of infants born to SARS-CoV-2+ mothers differs between infants with and without fecal shedding of the virus., Methods: We enrolled 10 infants born to SARS-CoV-2+ mothers. We used qPCR on fecal RNA to test for SARS-CoV-2 and 16S rRNA gene sequencing of the V4 region to assess the gut microbiome. Infant SARS-CoV-2 status from nasal swabs was abstracted from medical records., Results: Of the 10 included infants, nine were tested for SARS-CoV-2 by nasal swab with 1 testing positive. Four infants, including the nasal swab positive infant, had at least one sample with detectable levels of SARS-CoV-2 fecal shedding. Detection of both SARS-CoV-2 genes in feces was associated with increased gut alpha diversity compared to no detection by a linear mixed effects model ( p  < 0.001). Detection of both SARS-CoV-2 genes was associated with increased levels Erysipelotrichaceae, Lactobacillaceae, and Ruminococceae by MaAsLin2., Conclusion: Fecal shedding of SARS-CoV-2 occurs in infants who test negative on nasal swabs and is associated with differences in the gut microbiome., Competing Interests: The authors declare there are no competing interests., (©2024 Blaufus et al.)

Members of the mammalian gut microbiota metabolize diverse complex carbohydrates that are not digested by the host, which are collectively labeled "dietary fiber." While the enzymes and transporters that each strain uses to establish a nutrient niche in the gut are often exquisitely specific, the relationship between carbohydrate structure and microbial ecology is imperfectly understood. The present study takes advantage of recent advances in complex carbohydrate structure determination to test the effects of fiber monosaccharide composition on microbial fermentation. Fifty-five fibers with varied monosaccharide composition were fermented by a pooled feline fecal inoculum in a modified MiniBioReactor array system over a period of 72 hours. The content of the monosaccharides glucose and xylose was significantly associated with the reduction of pH during fermentation, which was also predictable from the concentrations of the short-chain fatty acids lactic acid, propionic acid, and the signaling molecule indole-3-acetic acid. Microbiome diversity and composition were also predictable from monosaccharide content and SCFA concentration. In particular, the concentrations of lactic acid and propionic acid correlated with final alpha diversity and were significantly associated with the relative abundance of several of the genera, including Lactobacillus and Dubosiella . Our results suggest that monosaccharide composition offers a generalizable method to compare any dietary fiber of interest and uncover links between diet, gut microbiota, and metabolite production., Importance: The survival of a microbial species in the gut depends on the availability of the nutrients necessary for that species to survive. Carbohydrates in the form of non-host digestible fiber are of particular importance, and the set of genes possessed by each species for carbohydrate consumption can vary considerably. Here, differences in the monosaccharides that are the building blocks of fiber are considered for their impact on both the survival of different species of microbes and on the levels of microbial fermentation products produced. This work demonstrates that foods with similar monosaccharide content will have consistent effects on the survival of microbial species and on the production of microbial fermentation products., Competing Interests: D.A.M., J.B.G., and C.B.L. are cofounders of Infinant Health, a probiotic company, and One.Bio, a company advancing novel bioactive glycans. None of these companies had any role in the conceptualization, design, analysis, or preparation of the manuscript.

Objective: Obesity as assessed by body mass index (BMI) is associated with increased risk of chronic disease. Health fatalism, defined as the belief that health outcomes are outside of one's control, is also associated with chronic disease risk. The purpose of this cross-sectional study was to understand the relationship between health fatalism and BMI in healthy adults. Secondary outcomes assessed the relationships between health fatalism and diet quality and health fatalism and physical activity., Method: Healthy individuals aged 18 to 65 years were recruited via ResearchMatch, electronic mailing lists, and social media. Participants completed online questionnaires on demographic characteristics, diet quality, physical activity, and degree of health fatalism. Regression models were used to assess the primary and secondary outcomes. For the primary outcome, the model of health fatalism (predictor) and BMI (outcome) was also adjusted for diet quality, physical activity, and demographic characteristics., Results: Participants (n = 496) were 38.7 ± 14.3 years old and primarily female (76%) and White (81%), with a BMI of 25.1 ± 5.2 kg/m 2 . Most participants had a college or post-college education (74%), stated that they always had sufficient income to live comfortably (90%), and were moderately to highly active (91%). There was no relationship between health fatalism and BMI ( p  > 0.05) or health fatalism and physical activity ( p  > 0.05); however, there was a significant relationship between health fatalism and diet quality (beta coefficient: -0.046; 95% confidence interval, -0.086 to -0.0058; p  = 0.025), such that a higher degree of fatalism predicted a slight decrease in diet quality., Conclusions: Although health fatalism did not predict BMI in this population, fatalistic beliefs were associated with poorer diet quality.

Gut bacteria are thought to contribute to neurodevelopmental disorders, but whether they are causal or predictive of disease remains unclear. In a prospective longitudinal study of thousands of children, Ahrens et al. generate evidence for the role of the gut microbiome in neurodevelopmental disorders while highlighting important open questions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Objectives: Few U.S. women meet the public health recommendations to exclusively breastfeed for 6 months and continue breastfeeding for at least 1-2 years. We compared prenatally collected demographic, health, and breastfeeding support/intention variables to examine how these factors intersect to predict meeting breastfeeding recommendations. Methods: PREVAIL, a CDC-funded birth cohort in Cincinnati, OH, was approved by the IRB at CDC, Cincinnati Children's Hospital, and the hospitals where enrollment (third trimester, 2017-2018) occurred. The prenatal questionnaire captured sociodemographics, pre-pregnancy weight and height, breastfeeding environment, and breastfeeding intention, while health factors were obtained from obstetrical records. Body mass index (BMI) (kg/m 2 ) was categorized as healthy (18.5-24.9), overweight (25-29.9), obesity 1 (30-34.9), and obesity 2+ (≥35). Mothers self-reported date of exclusive and any breastfeeding cessation through quarterly postnatal questionnaires. Random forest was used for variable selection, cross-validated in multivariable logistic models. Results: Analysis included n  = 237 mothers with BMI ≥18.5. Random forest identified BMI category, prenatal intention, and insurance type as the most important predictors of meeting breastfeeding recommendations. The resulting logistic models explained >40% of the variance with an area under the curve of ≥0.89 for both recommendations. More than 73% of the risk of not meeting breastfeeding recommendations was attributable to having an elevated BMI or lacking strong breastfeeding intention. Conclusions: In PREVAIL, maternal BMI and prenatal intention explained most risks of not meeting breastfeeding exclusivity and duration recommendations. Our findings suggest efforts to improve breastfeeding exclusivity and duration should focus on strengthening prenatal breastfeeding intention and identifying effective interventions for supporting breastfeeding among mothers with higher BMI.

The infant gut microbiome is increasingly recognized as a reservoir of antibiotic resistance genes, yet the assembly of gut resistome in infants and its influencing factors remain largely unknown. We characterized resistome in 4132 metagenomes from 963 infants in six countries and 4285 resistance genes were observed. The inherent resistome pattern of healthy infants ( N  = 272) could be distinguished by two stages: a multicompound resistance phase (Months 0-7) and a tetracycline-mupirocin-β-lactam-dominant phase (Months 8-14). Microbial taxonomy explained 40.7% of the gut resistome of healthy infants, with Escherichia (25.5%) harboring the most resistance genes. In a further analysis with all available infants ( N  = 963), we found age was the strongest influencer on the resistome and was negatively correlated with the overall resistance during the first 3 years ( p  < 0.001). Using a random-forest approach, a set of 34 resistance genes could be used to predict age ( R 2  = 68.0%). Leveraging microbial host inference analyses, we inferred the age-dependent assembly of infant resistome was a result of shifts in the gut microbiome, primarily driven by changes in taxa that disproportionately harbor resistance genes across taxa (e.g., Escherichia coli more frequently harbored resistance genes than other taxa). We performed metagenomic functional profiling and metagenomic assembled genome analyses whose results indicate that the development of gut resistome was driven by changes in microbial carbohydrate metabolism, with an increasing need for carbohydrate-active enzymes from Bacteroidota  and a decreasing need for Pseudomonadota during infancy. Importantly, we observed increased acquired resistance genes over time, which was related to increased horizontal gene transfer in the developing infant gut microbiome. In summary, infant age was negatively correlated with antimicrobial resistance gene levels, reflecting a composition shift in the gut microbiome, likely driven by the changing need for microbial carbohydrate metabolism during early life., Competing Interests: David A. Mills is a cofounder of Evolve Biosystems and BCD Biosciences. Evolve Biosystems and BCD Biosciences had no role in this manuscript's conceptualization, design, data collection, analysis, or preparation. The remaining authors declare no conflict of interest., (© 2024 The Authors. iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.)

Background: Bifidobacteria represent an important gut commensal in humans, particularly during initial microbiome assembly in the first year of life. Enrichment of Bifidobacterium is mediated though the utilization of human milk oligosaccharides (HMOs), as several human-adapted species have dedicated genomic loci for transport and metabolism of these glycans. This results in the release of fermentation products into the gut lumen which may offer physiological benefits to the host. Synbiotic pairing of probiotic species with a cognate prebiotic delivers a competitive advantage, as the prebiotic provides a nutrient niche., Methods: To determine the fitness advantage and metabolic characteristics of an HMO-catabolizing Bifidobacterium strain in the presence or absence of 2'-fucosyllactose (2'-FL), conventionally colonized mice were gavaged with either Bifidobacterium pseudocatenulatum MP80 (B.p. MP80) (as the probiotic) or saline during the first 3 days of the experiment and received water or water containing 2'-FL (as the prebiotic) throughout the study., Results: 16S rRNA gene sequencing revealed that mice provided only B.p. MP80 were observed to have a similar microbiota composition as control mice throughout the experiment with a consistently low proportion of Bifidobacteriaceae present. Using 1 H NMR spectroscopy, similar metabolic profiles of gut luminal contents and serum were observed between the control and B.p. MP80 group. Conversely, synbiotic supplemented mice exhibited dramatic shifts in their community structure across time with an overall increased, yet variable, proportion of Bifidobacteriaceae following oral inoculation. Parsing the synbiotic group into high and moderate bifidobacterial persistence based on the median proportion of Bifidobacteriaceae, significant differences in gut microbial diversity and metabolite profiles were observed. Notably, metabolites associated with the fermentation of 2'-FL by bifidobacteria were significantly greater in mice with a high proportion of Bifidobacteriaceae in the gut suggesting metabolite production scales with population density. Moreover, 1,2-propanediol, a fucose fermentation product, was only observed in the liver and brain of mice harboring high proportions of Bifidobacteriaceae., Conclusions: This study reinforces that the colonization of the gut with a commensal microorganism does not guarantee a specific functional output. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The spread of antimicrobial resistance genes (ARGs) is a major public health crisis, with the ongoing spread of ARGs leading to reduced efficacy of antibiotic treatments. The gut microbiome is a key reservoir for ARGs, and because diet shapes the gut microbiome, diet also has the potential to shape the resistome. This diet-gut microbiome-resistome relationship may also be important in infants and young children. This narrative review examines what is known about the interaction between the infant gut microbiome, the infant resistome, and infant nutrition, including exploring the potential of diet to mitigate infant ARG carriage. While more research is needed, diet has the potential to reduce infant and toddler carriage of ARGs, an important goal as part of maintaining the efficacy of available antibiotics and preserving infant and toddler health.

Necrotizing enterocolitis (NEC) is the leading cause of death caused by gastrointestinal disease in preterm infants. Major risk factors include prematurity, formula feeding, and gut microbial colonization. Microbes have been linked to NEC, yet there is no evidence of causal species, and select probiotics have been shown to reduce NEC incidence in infants. In this study, we evaluated the effect of the probiotic Bifidobacterium longum subsp. infantis ( BL. infantis ), alone and in combination with a human milk oligosaccharide (HMO)-sialylactose (3'SL)-on the microbiome, and the incidence of NEC in preterm piglets fed an infant formula diet. We studied 50 preterm piglets randomized between 5 treatments: (1) Preterm infant formula, (2) Donor human milk (DHM), (3) Infant formula + 3'SL, (4) Infant formula + BL. infantis , and (5) Infant formula and BL. infantis + 3'SL. NEC incidence and severity were assessed through the evaluation of tissue from all the segments of the GI tract. The gut microbiota composition was assessed both daily and terminally through 16S and whole-genome sequencing (WGS) of rectal stool samples and intestinal contents. Dietary BL. infantis and 3'SL supplementation had no effect, yet DHM significantly reduced the incidence of NEC. The abundance of BL. infantis in the gut contents negatively correlated with disease severity. Clostridium sensu stricto 1 and Clostridium perfringens were significantly more abundant in NEC and positively correlated with disease severity. Our results suggest that pre- and probiotics are not sufficient for protection from NEC in an exclusively formula-based diet. The results highlight the differences in microbial species positively associated with both diet and NEC incidence.

Objective: Feeding tubes harbor microbial contaminants; studies to date have not explored differences between orogastric (OG) and nasogastric (NG) tube biofilms. We sought to extend a previous analysis by comparing bacterial colonization by location (OG v NG) and by evaluating clinical factors that may affect tube bacterial populations., Study Design: The pharyngeal segments of 41 infant feeding tubes (14 OG and 27 NG) from 41 infants were analyzed by next generation 16 S rRNA sequencing on the MiSeq platform., Results: At the phylum level, Proteobacteria had the highest relative abundance of both OG and NG tubes. At the genus/species level, nine taxa differed significantly between OG and NG tubes. Alpha and beta diversity analyses showed significant differences between OG and NG tubes with relatively little contribution from clinical factors., Conclusion: The route of feeding tube insertion (oral vs nasal) had a greater impact on bacterial colonization than the assessed clinical factors., (© 2022. The Author(s).)

Objectives: To compare the impact of two probiotic supplements on fecal microbiota and metabolites, as well as on gut inflammation in human milk-fed preterm infants., Methods: In this single-center observational cohort study, we assessed the effects of Bifidobacterium longum subsp. infantis or Lactobacillus reuteri supplementation on the infant gut microbiota by 16S rRNA gene sequencing and fecal metabolome by 1 H nuclear magnetic resonance spectroscopy. Fecal calprotectin was measured as a marker of enteric inflammation. Aliquots of human or donor milk provided to each infant were also assessed to determine human milk oligosaccharide (HMO) content., Results: As expected, each probiotic treatment was associated with increased proportions of the respective bacterial taxon. Fecal HMOs were significantly higher in L. reuteri fed babies despite similar HMO content in the milk consumed. Fecal metabolites associated with bifidobacteria fermentation products were significantly increased in B. infantis supplemented infants. Fecal calprotectin was lower in infants receiving B. infantis relative to L. reuteri ( P &lt; 0.01, Wilcoxon rank-sum test) and was negatively associated with the microbial metabolite indole-3-lactate (ILA)., Conclusions: This study demonstrates that supplementing an HMO-catabolizing Bifidobacterium probiotic results in increased microbial metabolism of milk oligosaccharides and reduced intestinal inflammation relative to a noncatabolizing Lactobacillus probiotic in human milk-fed preterm infants. In this context, Bifidobacterium may provide greater benefit in human milk-fed infants via activation of the microbiota-metabolite-immune axis., (Copyright © 2022 by European Society for European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Maternal antibiotic (ABx) exposure can significantly perturb the transfer of microbiota from mother to offspring, resulting in dysbiosis of potential relevance to neurodevelopmental disorders such as autism spectrum disorder (ASD). Studies in rodent models have found long-term neurobehavioral effects in offspring of ABx-treated dams, but ASD-relevant behavior during the early preweaning period has thus far been neglected. Here, we exposed C57BL/6J mouse dams to ABx (5 mg/ml neomycin, 1.25 μg/ml pimaricin, .075% v/v acetic acid) dissolved in drinking water from gestational day 12 through offspring postnatal day 14. A number of ASD-relevant behaviors were assayed in offspring, including ultrasonic vocalization (USV) production during maternal separation, group huddling in response to cold challenge, and olfactory-guided home orientation. In addition, we obtained measures of thermoregulatory competence in pups during and following behavioral testing. We found a number of behavioral differences in offspring of ABx-treated dams (e.g., modulation of USVs by pup weight, activity while huddling) and provide evidence that some of these behavioral effects can be related to thermoregulatory deficiencies, particularly at younger ages. Our results suggest not only that ABx can disrupt microbiomes, thermoregulation, and behavior, but that metabolic effects may confound the interpretation of behavioral differences observed after early-life ABx exposure., (© 2022 Wiley Periodicals LLC.)

Antimicrobial resistance (AMR) represents a significant source of morbidity and mortality worldwide, with expectations that AMR-associated consequences will continue to worsen throughout the coming decades. Since resistance to antibiotics is encoded in the microbiome, interventions aimed at altering the taxonomic composition of the gut might allow us to prophylactically engineer microbiomes that harbor fewer antibiotic resistant genes (ARGs). Diet is one method of intervention, and yet little is known about the association between diet and antimicrobial resistance. To address this knowledge gap, we examined diet using the food frequency questionnaire (FFQ; habitual diet) and 24-h dietary recalls (Automated Self-Administered 24-h [ASA24 ® ] tool) coupled with an analysis of the microbiome using shotgun metagenome sequencing in 290 healthy adult participants of the United States Department of Agriculture (USDA) Nutritional Phenotyping Study. We found that aminoglycosides were the most abundant and prevalent mechanism of AMR in these healthy adults and that aminoglycoside- O -phosphotransferases ( aph3-dprime ) correlated negatively with total calories and soluble fiber intake. Individuals in the lowest quartile of ARGs (low-ARG) consumed significantly more fiber in their diets than medium- and high-ARG individuals, which was concomitant with increased abundances of obligate anaerobes, especially from the family Clostridiaceae , in their gut microbiota. Finally, we applied machine learning to examine 387 dietary, physiological, and lifestyle features for associations with antimicrobial resistance, finding that increased phylogenetic diversity of diet was associated with low-ARG individuals. These data suggest diet may be a potential method for reducing the burden of AMR. IMPORTANCE Antimicrobial resistance (AMR) represents a considerable burden to health care systems, with the public health community largely in consensus that AMR will be a major cause of death worldwide in the coming decades. Humans carry antibiotic resistance in the microbes that live in and on us, collectively known as the human microbiome. Diet is a powerful method for shaping the human gut microbiome and may be a tractable method for lessening antibiotic resistance, and yet little is known about the relationship between diet and AMR. We examined this relationship in healthy individuals who contained various abundances of antibiotic resistance genes and found that individuals who consumed diverse diets that were high in fiber and low in animal protein had fewer antibiotic resistance genes. Dietary interventions may be useful for lessening the burden of antimicrobial resistance and might ultimately motivate dietary guidelines which will consider how nutrition can reduce the impact of infectious disease.

Bifidobacterium species are beneficial and dominant members of the breastfed infant gut microbiome; however, their health benefits are partially species-dependent. Here, we characterize the species and subspecies of Bifidobacterium in breastfed infants around the world to consider the potential impact of a historic dietary shift on the disappearance of B. longum subsp. infantis in some populations. Across populations, three distinct patterns of Bifidobacterium colonization emerged: (1) The dominance of Bifidobacterium longum subspecies infantis , (2) prevalent Bifidobacterium of multiple species, and (3) the frequent absence of any Bifidobacterium. These patterns appear related to a country's history of breastfeeding, with infants in countries with historically high rates of long-duration breastfeeding more likely to be colonized by B. longum subspecies infantis compared with infants in countries with histories of shorter-duration breastfeeding. In addition, the timing of infant colonization with B. longum subsp. infantis is consistent with horizontal transmission of this subspecies, rather than the vertical transmission previously reported for other Bifidobacterium species. These findings highlight the need to consider historical and cultural influences on the prevalence of gut commensals and the need to understand epidemiological transmission patterns of Bifidobacterium and other major commensals.

Rationale: In murine models, microbial exposures induce protection from experimental allergic asthma through innate immunity. Objectives: Our aim was to assess the association of early life innate immunity with the development of asthma in children at risk. Methods: In the PASTURE farm birth cohort, innate T-helper cell type 2 (Th2), Th1, and Th17 cytokine expression at age 1 year was measured after stimulation of peripheral blood mononuclear cells with LPS in n  = 445 children. Children at risk of asthma were defined based on single-nucleotide polymorphisms at the 17q21 asthma gene locus. Specifically, we used the SNP rs7216389 in the GSDMB gene. Wheeze in the first year of life was assessed by weekly diaries and asthma by questionnaire at age 6 years. Measurements and Main Results: Not all cytokines were detectable in all children after LPS stimulation. When classifying detectability of cytokines by latent class analysis, carrying the 17q21 risk allele rs7216389 was associated with risk of wheeze only in the class with the lowest level of LPS-induced activation: odds ratio (OR), 1.89; 95% confidence interval [CI], 1.13-3.16; P  = 0.015. In contrast, in children with high cytokine activation after LPS stimulation, no association of the 17q21 risk allele with wheeze (OR, 0.63; 95% CI, 0.29-1.40; P  = 0.258, P  = 0.034 for interaction) or school-age asthma was observed. In these children, consumption of unprocessed cow's milk was associated with higher cytokine activation (OR, 3.37; 95% CI, 1.56-7.30; P  = 0.002), which was in part mediated by the gut microbiome. Conclusions: These findings suggest that within the 17q21 genotype, asthma risk can be mitigated by activated immune responses after innate stimulation, which is partly mediated by a gut-immune axis.

The ongoing COVID-19 pandemic is causing significant morbidity and mortality across the US. In this ecological study, we identified county-level variables associated with the COVID-19 case-fatality rate (CFR) using publicly available datasets and a negative binomial generalized linear model. Variables associated with decreased CFR included a greater number of hospitals per 10,000 people, banning religious gatherings, a higher percentage of people living in mobile homes, and a higher percentage of uninsured people. Variables associated with increased CFR included a higher percentage of the population over age 65, a higher percentage of Black or African Americans, a higher asthma prevalence, and a greater number of hospitals in a county. By identifying factors that are associated with COVID-19 CFR in US counties, we hope to help officials target public health interventions and healthcare resources to locations that are at increased risk of COVID-19 fatalities., Competing Interests: The authors have declared that no competing interests exist.

Breastfeeding is the gold standard for feeding infants because of its long-term benefits to health and development, but most infants in the United States are not exclusively breastfed in the first six months. We enrolled 24 infants who were either exclusively breastfed or supplemented with formula by the age of one month. We collected diet information, stool samples for evaluation of microbiotas by 16S rRNA sequencing, and blood samples for assessment of immune development by flow cytometry from birth to 6 months of age. We further typed the Bifidobacterium strains in stool samples whose 16S rRNA sequencing showed the presence of Bifidobacteriaceae. Supplementation with formula during breastfeeding transiently changed the composition of the gut microbiome, but the impact dissipated by six months of age. For example, Bifidobacterium longum , a bacterial species highly correlated with human milk consumption, was found to be significantly different only at 1 month of age but not at later time points. No immunologic differences were found to be associated with supplementation, including the development of T-cell subsets, B cells, or monocytes. These data suggest that early formula supplementation, given in addition to breast milk, has minimal lasting impact on the gut microbiome or immunity.

The ongoing COVID-19 pandemic is causing significant morbidity and mortality across the US. In this ecological study, we identified county-level variables associated with the COVID-19 case-fatality rate (CFR) using publicly available datasets and a negative binomial generalized linear model. Variables associated with decreased CFR included a greater number of hospitals per 10,000 people, banning religious gatherings, a higher percentage of people living in mobile homes, and a higher percentage of uninsured people. Variables associated with increased CFR included a higher percentage of the population over age 65, a higher percentage of Black or African Americans, a higher asthma prevalence, and a greater number of hospitals in a county. By identifying factors that are associated with COVID-19 CFR in US counties, we hope to help officials target public health interventions and healthcare resources to locations that are at increased risk of COVID-19 fatalities.

Understanding how exogenous microbes stably colonize the animal gut is essential to reveal mechanisms of action and tailor effective probiotic treatments. Bifidobacterium species are naturally enriched in the gastrointestinal tract of breast-fed infants. Human milk oligosaccharides (HMOs) are associated with this enrichment. However, direct mechanistic proof of the importance of HMOs in this colonization is lacking given milk contains additional factors that impact the gut microbiota. This study examined mice supplemented with the HMO 2'fucosyllactose (2'FL) together with a 2'FL-consuming strain, Bifidobacterium pseudocatenulatum MP80. 2'FL supplementation creates a niche for high levels of B.p . MP80 persistence, similar to Bifidobacterium levels seen in breast-fed infants. This synergism impacted gut microbiota composition, activated anti-inflammatory pathways and protected against chemically-induced colitis. These results demonstrate that bacterial-milk glycan interactions alone drive enrichment of beneficial Bifidobacterium and provide a model for tunable colonization thus facilitating insight into mechanisms of health promotion by bifidobacteriain neonates.

Growing up on a farm is associated with an asthma-protective effect, but the mechanisms underlying this effect are largely unknown. In the Protection against Allergy: Study in Rural Environments (PASTURE) birth cohort, we modeled maturation using 16S rRNA sequence data of the human gut microbiome in infants from 2 to 12 months of age. The estimated microbiome age (EMA) in 12-month-old infants was associated with previous farm exposure (β = 0.27 (0.12-0.43), P = 0.001, n = 618) and reduced risk of asthma at school age (odds ratio (OR) = 0.72 (0.56-0.93), P = 0.011). EMA mediated the protective farm effect by 19%. In a nested case-control sample (n = 138), we found inverse associations of asthma with the measured level of fecal butyrate (OR = 0.28 (0.09-0.91), P = 0.034), bacterial taxa that predict butyrate production (OR = 0.38 (0.17-0.84), P = 0.017) and the relative abundance of the gene encoding butyryl-coenzyme A (CoA):acetate-CoA-transferase, a major enzyme in butyrate metabolism (OR = 0.43 (0.19-0.97), P = 0.042). The gut microbiome may contribute to asthma protection through metabolites, supporting the concept of a gut-lung axis in humans.

Background: Shotgun metagenomes are often assembled prior to annotation of genes which biases the functional capacity of a community towards its most abundant members. For an unbiased assessment of community function, short reads need to be mapped directly to a gene or protein database. The ability to detect genes in short read sequences is dependent on pre- and post-sequencing decisions. The objective of the current study was to determine how library size selection, read length and format, protein database, e-value threshold, and sequencing depth impact gene-centric analysis of human fecal microbiomes when using DIAMOND, an alignment tool that is up to 20,000 times faster than BLASTX., Results: Using metagenomes simulated from a database of experimentally verified protein sequences, we find that read length, e-value threshold, and the choice of protein database dramatically impact detection of a known target, with best performance achieved with longer reads, stricter e-value thresholds, and a custom database. Using publicly available metagenomes, we evaluated library size selection, paired end read strategy, and sequencing depth. Longer read lengths were acheivable by merging paired ends when the sequencing library was size-selected to enable overlaps. When paired ends could not be merged, a congruent strategy in which both ends are independently mapped was acceptable. Sequencing depths of 5 million merged reads minimized the error of abundance estimates of specific target genes, including an antimicrobial resistance gene., Conclusions: Shotgun metagenomes of DNA extracted from human fecal samples sequenced using the Illumina platform should be size-selected to enable merging of paired end reads and should be sequenced in the PE150 format with a minimum sequencing depth of 5 million merge-able reads to enable detection of specific target genes. Expecting the merged reads to be 180-250 bp in length, the appropriate e-value threshold for DIAMOND would then need to be more strict than the default. Accurate and interpretable results for specific hypotheses will be best obtained using small databases customized for the research question.

Dedicated lactation rooms are a modern development as mothers return to work while still providing breastmilk to their absent infants. This study describes the built environment microbiome of lactation rooms and daycares, and explores the influence of temperature and humidity on the microbiome of lactation rooms. Sterile swabs were used to collect samples from five different sites in lactation rooms at University of California, Davis and from five different sites in daycares located in Davis, California. DNA from the swabs was extracted and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Temperature and relative humidity data were collected on a subset of the lactation rooms. Sampled lactation rooms could be either dedicated lactation rooms or could also serve other functions (e.g., combined lactation room and restroom lounge). The majority of sequence reads were identified as belonging to family Moraxellaceae, with 73% of all reads included in analysis identified as an unknown species of Acinetobacter . Alpha diversity was analyzed using the Shannon index, while beta diversity was analyzed using unweighted and weighted UniFrac distance. The Jaccard distance was used to measure amount of change at sampling locations between time points for analysis of the impact of temperature and humidity on the microbiome. There were significant differences in the beta diversity of the microbiome of lactation rooms by room type. There were also significant differences in the beta diversity of the microbiome by sample collection location. There were no significant differences in either alpha or beta diversity associated with room temperature or humidity. Additional studies are needed to understand if the differences in lactation room type may result in differences in the breastmilk microbiome of milk collected in those rooms, and to what extent any such differences may influence the infant microbiome., Competing Interests: Zachery T. Lewis is now an employee of The Clorox Company., (© 2019 Taft et al.)

Antimicrobial resistance is a global public health concern, and livestock play a significant role in selecting for resistance and maintaining such reservoirs. Here we study the succession of dairy cattle resistome during early life using metagenomic sequencing, as well as the relationship between resistome, gut microbiota, and diet. In our dataset, the gut of dairy calves serves as a reservoir of 329 antimicrobial resistance genes (ARGs) presumably conferring resistance to 17 classes of antibiotics, and the abundance of ARGs declines gradually during nursing. ARGs appear to co-occur with antibacterial biocide or metal resistance genes. Colostrum is a potential source of ARGs observed in calves at day 2. The dynamic changes in the resistome are likely a result of gut microbiota assembly, which is closely associated with diet transition in dairy calves. Modifications in the resistome may be possible via early-life dietary interventions to reduce overall antimicrobial resistance.

Human milk feeding is associated with lower rates of necrotizing enterocolitis (NEC), but an understanding of mechanism is lacking. In recent work, Gopalakrishna et al. report that human infants who develop NEC first experience an increase in Enterobacteriaceae in the portion of the microbiota not bound to IgA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Infancy is a crucial period for establishing the intestinal microbiome. This process may be influenced by vitamin A (VA) status because VA affects intestinal immunity and epithelial integrity, factors that can, in turn, modulate microbiome development., Objectives: The aim of this study was to determine if neonatal VA supplementation (VAS) affected the abundance of Bifidobacterium, a beneficial commensal, or of Proteobacteria, a phylum containing enteric pathogens, in early (6-15 wk) or late (2 y) infancy. Secondary objectives were to determine if VAS affected the abundance of other bacterial taxa, and to determine if VA status assessed by measuring plasma retinol was associated with bacterial abundance., Methods: Three hundred and six Bangladeshi infants were randomized by sex and birthweight status (above/below median) to receive 1 VA dose (50,000 IU) or placebo within 48 h of birth. Relative abundance at the genus level and above was assessed by 16S rRNA gene sequencing. A terminal restriction fragment-length polymorphism assay was used to identify Bifidobacterium species and subspecies at 6 wk., Results: Linear regression showed that Bifidobacterium abundance in early infancy was lower in boys (median, 1st/3rd quartiles; 0.67, 0.52/0.78) than girls (0.73, 0.60/0.80; P = 0.003) but that boys receiving VAS (0.69, 0.55/0.78) had higher abundance than boys receiving placebo (0.65, 0.44/0.77; P = 0.039). However this difference was not seen in girls (VAS 0.71, 0.54/0.80; placebo 0.75, 0.63/0.81; P = 0.25). VAS did not affect Proteobacteria abundance. Sex-specific associations were also seen for VA status, including positive associations of plasma retinol with Actinobacteria (the phylum containing Bifidobacterium) and Akkermansia, another commensal with possible health benefits, for girls in late infancy., Conclusions: Better VA status in infancy may influence health both in infancy and later in life by promoting the establishment of a healthy microbiota. This postulated effect of VA may differ between boys and girls. This trial was registered at clinicaltrials.gov as NCT02027610., (Published by Oxford University Press on behalf of the American Society for Nutrition 2019.)

Enteral feeding is a key component of care in neonatal intensive care units (NICUs); however, feeding tubes harbor microbes. These microbes have the potential to cause disease, yet their source remains controversial and clinical recommendations to reduce feeding tube colonization are lacking. This study aims to improve our understanding of the bacteria in neonatal feeding tubes and to evaluate factors that may affect these bacteria. 16S rRNA gene sequencing was used to characterize the bacteria present in pharyngeal, esophageal, and gastric portions of feeding tubes, residual fluid of the tubes, and infant stool using samples from 47 infants. Similar distributions of taxa were observed in all samples, although beta diversity differed by sample type. Feeding tube samples had lower alpha diversity than stool samples, and alpha diversity increased with gestational age, day of life, and tube dwell time. In a subset of samples from 6 infants analyzed by whole metagenome sequencing, there was greater overlap in transferable antimicrobial resistance genes between tube and fecal samples in breast milk fed infants than in formula fed infants. These findings develop our understanding of neonatal feeding tube colonization, laying a foundation for research into methods for minimizing NICU patients' exposure to antimicrobial resistant microbes., (© FEMS 2019.)

Background: The intestinal microbiome in early infancy affects immunologic development and thus may affect vaccine memory, though few prospective studies have examined such associations. We examined the association of Bifidobacterium levels in early infancy with memory responses to early vaccination measured at 2 years of age., Methods: In this prospective observational study, we examined the association of Bifidobacterium abundance in the stool of healthy infants at 6 to 15 weeks of age, near the time of vaccination, with T-cell and antibody responses measured at 6 weeks, 15 weeks, and 2 years of age. Infants were vaccinated with Bacillus Calmette-Guérin (BCG) (at birth), oral polio virus (at birth and at 6, 10, and 14 weeks), tetanus toxoid (TT) (at 6, 10, and 14 weeks), and hepatitis B virus (at 6, 10, and 14 weeks). Fecal Bifidobacterium was measured at 6, 11, and 15 weeks. Bifidobacterium species and subspecies were measured at 6 weeks., Results: Mean Bifidobacterium abundance in early infancy was positively associated with the CD4 T-cell responses to BCG, TT, and hepatitis B virus at 15 weeks, with CD4 responses to BCG and TT at 2 years, and with plasma TT-specific immunoglobulin G and stool polio-specific immunoglobulin A at 2 years. Similar associations were seen for the predominant subspecies, Bifidobacterium longum subspecies infantis ., Conclusions: Bifidobacterium abundance in early infancy may increase protective efficacy of vaccines by enhancing immunologic memory. This hypothesis could be tested in clinical trials of interventions to optimize Bifidobacterium abundance in appropriate populations., Competing Interests: POTENTIAL CONFLICT OF INTEREST: Dr Underwood’s institution (University of California, Davis) has received grant support from Evolve Biosystems to fund a clinical trial of a probiotic in term infants; he has consulted for Avexegen and received payment for travel and lectures from Abbott. Dr Mills is a cofounder of and consultant for Evolve Biosystems and has stock and stock options therein; he has received payment for lectures from Nestle and Abbott; the other authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2019 by the American Academy of Pediatrics.)

Bifidobacterium species are important commensals capable of dominating the infant gut microbiome, in part by producing acids that suppress growth of other taxa. Bifidobacterium species are less prone to possessing antimicrobial resistance (AMR) genes (ARGs) than other taxa that may colonize infants. Given that AMR is a growing public health crisis and ARGs are present in the gut microbiome of humans from early life, this study examines the correlation between a Bifidobacterium- dominated infant gut microbiome and AMR levels, measured by a culture-independent metagenomic approach both in early life and as infants become toddlers. In general, Bifidobacterium dominance is associated with a significant reduction in AMR in a Bangladeshi cohort, both in the number of acquired AMR genes present and in the abundance of AMR genes. However, by year 2, Bangladeshi infants had no significant differences in AMR related to their early-life Bifidobacterium levels. A generalized linear model including all infants in a previously published Swedish cohort found a significant negative association between log-transformed total AMR and Bifidobacterium levels, thus confirming the relationship between Bifidobacterium levels and AMR. In both cohorts, there was no change between early-life and later-life AMR abundance in high- Bifidobacterium infants but a significant reduction in AMR abundance in low- Bifidobacterium infants. These results support the hypothesis that early Bifidobacterium dominance of the infant gut microbiome may help reduce colonization by taxa containing ARGs. IMPORTANCE Infants are vulnerable to an array of infectious diseases, and as the gut microbiome may serve as a reservoir of AMR for pathogens, reducing the levels of AMR in infants is important to infant health. This study demonstrates that high levels of Bifidobacterium are associated with reduced levels of AMR in early life and suggests that probiotic interventions to increase infant Bifidobacterium levels have the potential to reduce AMR in infants. However, this effect is not sustained at year 2 of age in Bangladeshi infants, underscoring the need for more detailed studies of the biogeography and timing of infant AMR acquisition., (Copyright © 2018 Taft et al.)

The gastrointestinal (GI) tract plays a central role in the absorption, distribution, metabolism, and excretion of flavonoids, which ultimately define the health effects of these bioactives. These aspects are modulated by the interactions of flavonoids with other dietary components, environmental factors, the host, and the GI microbiota. Flavonoid can target molecules in the luminal content, the different GI tract cell types, and the microbiota. Importantly, flavonoid actions at the GI tract can have an impact systemically, e.g. on glucose homeostasis, lipid and energy metabolism, or cardiovascular risk factors. The beneficial actions of flavonoids at the GI include their capacity to: i) protect the intestinal epithelium against pharmacological insults and food toxins; ii) modulate the activity of enzymes involved in lipid and carbohydrate absorption; iii) maintain the intestinal barrier integrity; iv) modulate the secretion of gut hormones; v) modulate the GI tract immune system; vi) exert potential anti-colorectal cancer activity; and vii) shape microbiota composition and function. Further understanding of the mechanisms mediating the effects of flavonoids on the intestine (and its microbiota) is of critical importance given the relevance of the GI tract on sustaining overall health and of the widespread recommendations of increasing the intake of plant bioactives., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Metagenomics is a rapidly emerging field aimed to analyze microbial diversity and dynamics by studying the genomic content of the microbiota. Metataxonomics tools analyze high-throughput sequencing data, primarily from 16S rRNA gene sequencing and DNAseq, to identify microorganisms and viruses within a complex mixture. With the growing demand for analysis of the functional microbiome, metatranscriptome studies attract more interest. To make metatranscriptomic data sufficient for metataxonomics, new analytical workflows are needed to deal with sparse and taxonomically less informative sequencing data., Results: We present a new protocol, IMSA+A, for accurate taxonomy classification based on metatranscriptome data of any read length that can efficiently and robustly identify bacteria, fungi, and viruses in the same sample. The new protocol improves accuracy by using a conservative reference database, employing a new counting scheme, and by assembling shotgun reads. Assembly also reduces analysis runtime. Simulated data were utilized to evaluate the protocol by permuting common experimental variables. When applied to the real metatranscriptome data for mouse intestines colonized by ASF, the protocol showed superior performance in detection of the microorganisms compared to the existing metataxonomics tools. IMSA+A is available at https://github.com/JeremyCoxBMI/IMSA-A ., Conclusions: The developed protocol addresses the need for taxonomy classification from RNAseq data. Previously not utilized, i.e., unmapped to a reference genome, RNAseq reads can now be used to gather taxonomic information about the microbiota present in a biological sample without conducting additional sequencing. Any metatranscriptome pipeline that includes assembly of reads can add this analysis with minimal additional cost of compute time. The new protocol also creates an opportunity to revisit old metatranscriptome data, where taxonomic content may be important but was not analyzed.

Objective: Chorioamnionitis (inflammation of the placenta and fetal membranes) and abnormal gastrointestinal colonization have been associated with an increased risk of sepsis and death in preterm infants, but whether chorioamnionitis causes abnormal pioneering gastrointestinal colonization in infants is not known. We determined the relationship between chorioamnionitis, altered infant fecal microbiome indicating abnormal gastrointestinal colonization, and adverse outcomes., Study Design: Preterm infants ≤ 28 weeks at birth were enrolled from 3 level III NICUs in Cincinnati, Ohio and Birmingham, Alabama. Sequencing for 16S microbial gene was performed on stool samples in the first 3 weeks of life. Chorioamnionitis was diagnosed by placental histology. Late onset sepsis and death outcomes were analyzed in relation to fecal microbiota and chorioamnionitis with or without funisitis (inflammation of the umbilical cord)., Results: Of the 106 enrolled infants, 48 infants had no chorioamnionitis, 32 infants had chorioamnionitis but no funisitis (AC), and 26 infants had chorioamnionitis with funisitis (ACF). The fecal samples from ACF infants collected by day of life 7 had higher relative abundance of family Mycoplasmataceae (phylum Tenericutes), genus Prevotella (phylum Bacteroidetes) and genus Sneathia (phylum Fusobacteria). Further, AC and ACF infants had higher incidence of late-onset sepsis/death as a combined outcome. Presence of specific clades in fecal samples, specifically, order Fusobacteria, genus Sneathia or family Mycoplasmataceae, were significantly associated with higher risk of sepsis or death., Conclusion: The results support the hypothesis that specific alterations in the pioneering infant gastrointestinal microbiota induced by chorioamnionitis predispose to neonatal sepsis or death., Competing Interests: The authors have declared that no competing interests exist.

Objective: More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjögren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls., Methods: All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction., Results: We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67-86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18-123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients., Conclusion: The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity., (© 2016, American College of Rheumatology.)

Necrotizing enterocolitis (NEC) afflicts approximately 10% of extremely preterm infants with high fatality. Inappropriate bacterial colonization with Enterobacteriaceae is implicated, but no specific pathogen has been identified. We identify uropathogenic E. coli (UPEC) colonization as a significant risk factor for the development of NEC and subsequent mortality. We describe a large-scale deep shotgun metagenomic sequence analysis of the early intestinal microbiome of 144 preterm and 22 term infants. Using a pan-genomic approach to functionally subtype the E. coli, we identify genes associated with NEC and mortality that indicate colonization by UPEC. Metagenomic multilocus sequence typing analysis further defined NEC-associated strains as sequence types often associated with urinary tract infections, including ST69, ST73, ST95, ST127, ST131, and ST144. Although other factors associated with prematurity may also contribute, this report suggests a link between UPEC and NEC and indicates that further attention to these sequence types as potential causal agents is needed., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Late onset sepsis (LOS) contributes to mortality and morbidity in preterm infants. We tested the hypotheses that microbes causing LOS originate from the gut, and that distortions in the gut microbial community increases subsequent risk of LOS., Study Design: We examined the gut microbial community in prospectively collected stool samples from preterm infants with LOS and an equal number of age-matched controls at two sites (Cincinnati, OH and Birmingham, AL), by sequencing the bacterial 16S rDNA. We confirmed our findings in a subset of infants by whole genome shotgun sequencing, and analyzed the data using R and LEfSe., Results: Infants with LOS in Cincinnati, as compared to controls, had less abundant Actinobacteria in the first samples after birth (median 18 days before sepsis onset), and less abundant Pseudomonadales in the last samples collected prior to LOS (median 8 days before sepsis onset). Infants with LOS in Birmingham, as compared to controls, had no differences identified in the first sample microbial communities, but Lactobacillales was less abundant in the last samples prior to LOS (median 4 days before sepsis onset). Sequencing identified detectable levels of the sepsis-causative organism in stool samples prior to disease onset for 82% of LOS cases., Conclusions: Translocation of gut microbes may account for the majority of LOS cases. Distortions in the fecal microbiota occur prior to LOS, but the form of distortion depends on timing and site. The microbial composition of fecal samples does not predict LOS onset in a generalizable fashion.

Background: Intestinal microbiota are implicated in risk of necrotizing enterocolitis (NEC) and sepsis, major diseases of preterm infants in neonatal intensive care units (NICUs). Rates of these diseases vary over time and between NICUs, but time and NICU comparisons of the intestinal microbiota of preterm infants are lacking., Methods: We included 66 singleton infants <29 weeks gestational age with stool samples collected between postnatal days 3 to 21 of life who survived free of NEC and sepsis. Infants were enrolled during 2010 and 2011. Twenty-six infants were enrolled at hospital 1 in Cincinnati, OH, and 40 infants were enrolled at hospital 2 in Birmingham, AL. Samples collected from days 3-9 ("week 1") and days 10-16 ("week 2") were compared between years and hospitals. Microbial succession was compared between hospitals in 28 infants with samples from the first 3 weeks of life. DNA extracted from stool was used to sequence the 16S rRNA gene by Illumina MiSeq using universal primers. Resulting operational taxonomic unit tables were analyzed for differences between years and hospitals using linear discriminant analysis effect size algorithm (LEfSe; significance, p < 0.05)., Results: Significant variation was observed in infant microbiota by year and hospital. Among hospital 1 infants, week 1 samples had more phylum Firmicutes (class Bacilli, families Clostridiaceae and Enterococcaceae) in 2010 and more phylum Proteobacteria (family Enterobacteriaceae) in 2011; week 2 samples did not significantly vary over time. However, among hospital 2 infants, the week 1 shift was nearly opposite, with more Proteobacteria (Enterobacteriaceae) in 2010 and more Firmicutes (Bacilli) in 2011; week 2 samples exhibited the same pattern. Regression analysis of clinical covariates found that antibiotic use had an important influence but did not explain these observed shifts in microbiota over time and between hospitals. Microbial succession also differed by hospital, with greater change in microbiota in hospital 1 than hospital 2 infants (p < 0.01, Jaccard distance)., Conclusion: Colonizing microbiota differ over time and between NICUs in ways that could be relevant to disease. Multi-site, longitudinal studies are needed to reliably define the impact of intestinal microbiota on adverse outcomes of preterm infants.

Objectives: To determine the impact of empiric ampicillin and gentamicin use in the first week of life on microbial colonization and diversity in preterm infants., Study Design: The 16s ribosomal DNA community profiling was used to compare the microbiota of 74 infants born ≤32 weeks gestational age by degree of antibiotic use in the first week of life. The degree of antibiotic use was classified as 0 days, 1-4 days, and 5-7 days of antibiotic administration. All of the antibiotic use was empiric, defined as treatment based solely on clinical suspicion of infection without a positive culture result., Results: Infants who received 5-7 days of empiric antimicrobial agents in the first week had increased relative abundance of Enterobacter (P = .016) and lower bacterial diversity in the second and third weeks of life. Infants receiving early antibiotics also experienced more cases of necrotizing enterocolitis, sepsis, or death than those not exposed to antibiotics., Conclusions: Early empiric antibiotics have sustained effects on the intestinal microbiota of preterm infants. Intestinal dysbiosis in this population has been found to be associated with elevated risk of necrotizing enterocolitis, sepsis, or death., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Necrotizing enterocolitis (NEC) is a devastating intestinal disease that afflicts 10% of extremely preterm infants. The contribution of early intestinal colonization to NEC onset is not understood, and predictive biomarkers to guide prevention are lacking. We analyzed banked stool and urine samples collected prior to disease onset from infants <29 weeks gestational age, including 11 infants who developed NEC and 21 matched controls who survived free of NEC. Stool bacterial communities were profiled by 16S rRNA gene sequencing. Urinary metabolomic profiles were assessed by NMR., Results: During postnatal days 4 to 9, samples from infants who later developed NEC tended towards lower alpha diversity (Chao1 index, P = 0.086) and lacked Propionibacterium (P = 0.009) compared to controls. Furthermore, NEC was preceded by distinct forms of dysbiosis. During days 4 to 9, samples from four NEC cases were dominated by members of the Firmicutes (median relative abundance >99% versus <17% in the remaining NEC and controls, P < 0.001). During postnatal days 10 to 16, samples from the remaining NEC cases were dominated by Proteobacteria, specifically Enterobacteriaceae (median relative abundance >99% versus 38% in the other NEC cases and 84% in controls, P = 0.01). NEC preceded by Firmicutes dysbiosis occurred earlier (onset, days 7 to 21) than NEC preceded by Proteobacteria dysbiosis (onset, days 19 to 39). All NEC cases lacked Propionibacterium and were preceded by either Firmicutes (≥98% relative abundance, days 4 to 9) or Proteobacteria (≥90% relative abundance, days 10 to 16) dysbiosis, while only 25% of controls had this phenotype (predictive value 88%, P = 0.001). Analysis of days 4 to 9 urine samples found no metabolites associated with all NEC cases, but alanine was positively associated with NEC cases that were preceded by Firmicutes dysbiosis (P < 0.001) and histidine was inversely associated with NEC cases preceded by Proteobacteria dysbiosis (P = 0.013). A high urinary alanine:histidine ratio was associated with microbial characteristics (P < 0.001) and provided good prediction of overall NEC (predictive value 78%, P = 0.007)., Conclusions: Early dysbiosis is strongly involved in the pathobiology of NEC. These striking findings require validation in larger studies but indicate that early microbial and metabolomic signatures may provide highly predictive biomarkers of NEC.

Background: Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples., Methods: In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs)., Results: We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system., Conclusion: This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.