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Early-life microbial colonization plays a key role in future health. In this issue of Cell Host & Microbe, Bogaert et al. unravel the complexities of mother-infant microbial seeding by examining multiple maternal and infant niches. Importantly, they describe “auxiliary” seeding pathways that may partially compensate when seeding patterns are perturbed.

Parenteral nutrition-associated cholestasis (PNAC) in the neonatal intensive care unit (NICU) causes significant morbidity and associated healthcare costs. Laboratory detection of PNAC currently relies on elevated serum conjugated bilirubin levels in the aftermath of impaired bile flow. Here, we sought to identify fecal biomarkers, which when integrated with clinical data, would better predict risk for developing PNAC.Using untargeted metabolomics in 200 serial stool samples from 60 infants, we applied statistical and machine learning approaches to identify clinical features and metabolic biomarkers with the greatest associative potential for risk of developing PNAC. Stools were collected prospectively from infants receiving PN with soybean oil-based lipid emulsion at a level IV NICU.Low birth weight, extreme prematurity, longer duration of PN, and greater number of antibiotic courses were all risk factors for PNAC (P 0.05). We identified 78 stool biomarkers with early predictive potential (P 0.05). From these 78 biomarkers, we further identified 12 sphingomyelin lipids with high association for the development of PNAC in precholestasis stool samples when combined with birth anthropometry.We demonstrate the potential for stool metabolomics to enhance early identification of PNAC risk. Earlier detection of high-risk infants would empower proactive mitigation with alterations to PN for at-risk infants and optimization of energy nutrition with PN for infants at lower risk.

Background and Aims Faecal microbiota transplant [FMT] is effective in treating recurrent Clostridioides difficile infection [CDI] and restores gut microbiota composition. This is unlikely to account for its entire mechanism of efficacy, as studies have shown that factors such as bile acids influence the risk of infection by affecting Clostridioides difficile germination. We therefore aimed to investigate longitudinal changes in the gut bile acid composition after FMT performed for recurrent CDI, in children with and without inflammatory bowel disease [IBD]. Methods Eight children received FMT; five had underlying IBD. Primary and secondary faecal bile acids were measured by liquid chromatography–mass spectrometry in recipients [pre-FMT and longitudinally post-FMT for up to 6 months] and donors. Results Pre-FMT, recipients had higher primary and lower secondary bile acid proportions compared with donors. Post-FMT, there was a gradual increase of secondary and decrease of primary bile acids. Whereas gut bacterial diversity had been shown to be restored in all children shortly after FMT, normalisation of bile acids to donor levels occurred only by 6 months. In children with IBD, although microbiota diversity returned to pre-FMT levels within 6 months, secondary bile acids remained at donor levels. Conclusions The differences in bile acid profiles compared with gut bacterial diversity post-FMT suggests that interactions between the two may be more complex than previously appreciated and may contribute to FMT efficacy in different ways. This initial finding demonstrates the need to further investigate gut metabolites in larger cohorts, with longitudinal sampling to understand the mechanisms of FMT effectiveness.

Background Children with inflammatory bowel disease [IBD] are disproportionally affected by recurrent Clostridioides difficile infection [rCDI]. Although faecal microbiota transplantation [FMT] has been used with good efficacy in adults with IBD, little is known about outcomes associated with FMT in paediatric IBD. Methods We performed a retrospective review of FMT at 20 paediatric centres in the USA from March 2012 to March 2020. Children with and without IBD were compared with determined differences in the efficacy of FMT for rCDI. In addition, children with IBD with and without a successful outcome were compared with determined predictors of success. Safety data and IBD-specific outcomes were obtained. Results A total of 396 paediatric patients, including 148 with IBD, were included. Children with IBD were no less likely to have a successful first FMT then the non-IBD affected cohort [76% vs 81%, p = 0.17]. Among children with IBD, patients were more likely to have a successful FMT if they received FMT with fresh stool [p = 0.03], were without diarrhoea prior to FMT [p = 0.03], or had a shorter time from rCDI diagnosis until FMT [p = 0.04]. Children with a failed FMT were more likely to have clinically active IBD post-FMT [p = 0.002] and 19 [13%] patients had an IBD-related hospitalisation in the 3-month follow-up. Conclusions Based on the findings from this large US multicentre cohort, the efficacy of FMT for the treatment of rCDI did not differ in children with IBD. Failed FMT among children with IBD was possibly related to the presence of clinically active IBD.

Fecal microbiota transplantation (FMT) is currently the most effective but loosely regulated therapy, for recurrent Clostridioides difficile infection (rCDI) in pediatrics. Over the last 2 years, there have been mounting challenges in the ability to provide FMT to pediatric patients. Firstly, an Food and Drug Administration (FDA) safety alert in 2019 reported transmission of a multidrug resistant organism from FMT donor to recipient resulting in the death of 1 patient. Secondly, the coronavirus disease 2019 (COVID-19) pandemic induced further safety and regulatory challenges. Biotherapeutics are promising and more readily regulated treatment options for rCDI, which may replace FMT in the near future for adults upon regulatory agency approvals. Such approvals, however, are expected to be significantly delayed for children, raising concerns for limited access to effective treatment for children with rCDI. In this commentary, we discuss the recent challenges and future directions of FMT and microbial therapeutics in children with rCDI.

Background/Objective: Understanding the influence of genetically determined ancestry may give insight into the disparities of obesity seen in different ethnic groups beginning at a very early age. ...

Clostridioides difficile infection (CDI) is the most common cause of antibiotic-associated diarrhea and has high rates of recurrent disease. As a disease associated with intestinal dysbiosis, gastrointestinal microbiome manipulation and fecal microbiota transplantation (FMT) have evolved as effective, although relatively unregulated therapeutics and not without safety concerns. FMT for the treatment of CDI has been well studied in adults with increasing data reported in children. In this review, we discuss the current body of literature on the use of FMT in children including effectiveness, safety, risk factors for a failed FMT, and the role of FMT in children with comorbidities. We also review emerging microbial therapeutics for the treatment of rCDI.

Objective Although COVID-19 is primarily a respiratory illness, there is mounting evidence suggesting that the GI tract is involved in this disease. We investigated whether the gut microbiome is linked to disease severity in patients with COVID-19, and whether perturbations in microbiome composition, if any, resolve with clearance of the SARS-CoV-2 virus. Methods In this two-hospital cohort study, we obtained blood, stool and patient records from 100 patients with laboratory-confirmed SARS-CoV-2 infection. Serial stool samples were collected from 27 of the 100 patients up to 30 days after clearance of SARS-CoV-2. Gut microbiome compositions were characterised by shotgun sequencing total DNA extracted from stools. Concentrations of inflammatory cytokines and blood markers were measured from plasma. Results Gut microbiome composition was significantly altered in patients with COVID-19 compared with non-COVID-19 individuals irrespective of whether patients had received medication (p

Introduction The impact of the 2019 US Food and Drug Administration safety alert involving transmission of multidrug resistant organisms through fecal microbiota transplantation (FMT), and the COVID-19 pandemic on the use of FMT in children, is unknown. Methods A survey of pediatric gastroenterologists performing FMT for Clostridioides difficile infection was conducted. Results Of 36 respondents, 17 (47%) and 30 (83%) changed their FMT practices related to the US Food and Drug Administration safety alert and COVID-19 pandemic, respectively, with 22 (61%) of programs halted. Discussion The US Food and Drug Administration safety alert and COVID-19 pandemic have substantially influenced the availability and access of FMT for children.

Hispanic children are disproportionately affected by obesity, with this disparity starting at a young age, and there is a paucity of data comparing factors associated with excess weight in the first year of life in Hispanic vs. non-Hispanic populations. Excess weight was defined as weight-for-length ≥95th percentile. The associations of potential risk factors were compared by ethnicity stratification. Of the 1009 children, 302 (30.0%) were Hispanic and 707 (70.0%) were non-Hispanic White. The rate of excess weight was 30.1% and 13.6% among Hispanic and non-Hispanic White children, respectively. Factors associated with excess weight for non-Hispanic White children were higher than recommended weight gain during pregnancy (odds ratio (OR) 1.8 (1.2–3.1)), higher paternal body mass index (BMI) (OR 1.1 (1.02–1.15)), higher birth weight (OR 1.001 (1.001–1.002)), and lower breast milk feedings at 6 months (OR 0.98 (0.96–0.98)). Factors associated with excess weight for Hispanic children were lower maternal education (OR 2.37 (1.1–4.5)) and lower breast milk feedings at 6 months (OR 0.98 (0.96–0.99)). There are differential risk factors associated with excess weight at 12 months between Hispanic and non-Hispanic White children. Identification of differential factors in different ethnicities may allow for more targeted anticipatory guidance reduce obesity in at-risk populations.

Several lines of evidence suggest that children born via Cesarean section (C-section) are at greater risk for adverse health outcomes including allergies, asthma and obesity. Vaginal seeding is a medical procedure in which infants born by C-section are swabbed immediately after birth with vaginal secretions from the mother. This procedure has been proposed as a way to transfer the mother's vaginal microbiome to the child, thereby restoring the natural exposure that occurs during vaginal birth that is interrupted in the case of babies born via C-section. Preliminary evidence indicates partial restoration of microbes. However, there is insufficient evidence to determine the health benefits of the procedure. Several studies, including trial, are currently underway. At the same time, in the clinic setting, doctors are increasingly being asked to by expectant mothers to have their babies seeded. This article reports on the current research on this procedure and the issues it raises for regulators, researchers, physicians, and patients.

Supplemental digital content is available in the text., Objectives To determine if maternal confidence affects emergency department (ED) utilization in the first year of life. Methods This retrospective cohort study examined the Maternal Confidence Questionnaire responses from a longitudinal birth cohort study and ED visits for these subjects across all Inova hospitals from January 2012 to July 2017 for full-term children 12 months or younger at the time of visit. Using logistic regression, maternal confidence, maternal race/ethnicity, age, education, parity, and insurance were evaluated against Emergency Severity Index acuity levels and ED visit frequency. Results Of 2429 participants in the longitudinal study, 316 subjects visited the ED and met inclusion criteria. Medicaid status was the main factor associated with any ED visit. Low maternal confidence did not correlate with more frequent or nonurgent ED visits. Higher maternal confidence scores were seen in Hispanic or Latino mothers and mothers with parity greater than 1. Hispanic or Latino mothers were more likely to have Medicaid and more likely to bring their child to the ED. Mothers with college education had lower maternal confidence scores, were less likely to visit the ED, but had higher acuity level visits. Conclusions Low maternal confidence did not correlate with frequent ED visits or nonurgent visits. Medicaid status was the main factor associated with any ED visit. Hispanic or Latino mothers had higher maternal confidence scores, were more likely to have Medicaid and more likely to bring their child to the ED.

Background Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at − 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs. flash-freezing. To do this stool was collected from an infant’s diaper was divided into two aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3–4 days. Samples from the same infant were collected at 2 different time points to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Results Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 time points (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given time point clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p Conclusion Ambient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.

ObjectivesPediatric SARS-CoV-2 data remain limited and seropositivity rates in children were reported as MethodsChildren and adolescents ≤19 years were enrolled in a cross-sectional, observational study of SARS-CoV-2 seroprevalence from July-October 2020 in Northern Virginia, United States. Demographic, health, and COVID-19 exposure information was collected, and blood was analyzed for SARS-CoV-2 spike protein total antibody. Risk factors associated with SARS-CoV-2 seropositivity were analyzed. Orthogonal antibody testing was performed, and samples were evaluated for responses to different antigens.ResultsIn 1038 children, the anti-SARS-CoV-2 total antibody positivity rate was 8.5%. After multivariate logistic regression, significant risk factors included Hispanic ethnicity, public or absent insurance, a history of COVID-19 symptoms, exposure to person with COVID-19, a household member positive for SARS-CoV-2 and multi-family or apartment dwelling without a private entrance. 66% of seropositive children had no symptoms of COVID-19. Orthogonal antibody testing with a receptor binding domain specific antigen revealed a high concordance of 80.5%. Children also demonstrated a robust immune response to the nucleocapsid antigen.ConclusionsA much higher burden of SARS-CoV-2 infection, as determined by seropositivity, was found in children than previously reported; this was also higher compared to adults in the same region at a similar time. Contrary to prior reports, we determined children shoulder a significant burden of COVID-19 infection. The role of children’s disease transmission must be considered in COVID-19 mitigation strategies including vaccination.Article Summary8.5% of children had SARS-CoV-2 antibodies in Fall 2020, double the adult rate. The role of pediatric infection is important to consider in mitigation strategies.What’s Known on This SubjectSARS-CoV-2 pediatric seroepidemiologic data is limited. Reported viral rates underestimate the burden of infection in children due to mild or asymptomatic disease. Limited cohorts of children suggest low seropositivity rates compared to adults.What This Study AddsUS children in the largest SARS-CoV-2 seroepidemiology study to date had double the rate of antibodies compared to adults. Most children were asymptomatic. Risk factors include age, ethnicity and living conditions. Most children made antibodies to different antigens of SARS-CoV-2.

Background Pediatric SARS-CoV-2 data remain limited and seropositivity rates in children were reported as Methods Children and adolescents ≤19 years were enrolled in a cross-sectional, observational study of SARS-CoV-2 seroprevalence from July-October 2020 in Northern Virginia, US. Demographic, health, and COVID-19 exposure information was collected, and blood analyzed for SARS-CoV-2 spike protein total antibody. Risk factors associated with SARS-CoV-2 seropositivity were analyzed. Orthogonal antibody testing was performed, and samples were evaluated for responses to different antigens. Results In 1038 children, the anti-SARS-CoV-2 total antibody positivity rate was 8.5%. After multivariate logistic regression, significant risk factors included Hispanic ethnicity, public or absent insurance, a history of COVID-19 symptoms, exposure to person with COVID-19, a household member positive for SARS-CoV-2 and multi-family or apartment dwelling without a private entrance. 66% of seropositive children had no symptoms of COVID-19. Secondary analysis included orthogonal antibody testing with assays for 1) a receptor binding domain specific antigen and 2) a nucleocapsid specific antigen had concordance rates of 80.5% and 79.3% respectively. Conclusions A much higher burden of SARS-CoV-2 infection, as determined by seropositivity, was found in children than previously reported; this was also higher compared to adults in the same region at a similar time. Contrary to prior reports, we determined children shoulder a significant burden of COVID-19 infection. The role of children’s disease transmission must be considered in COVID-19 mitigation strategies including vaccination.

Background: Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for collection and storage of stool samples for metabolomics is flash-freezing at -80°C which can be inconvenient and expensive. Ambient temperature collection of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature collection and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs. flash-freezing.Methods: Stool collected from an infant’s diaper was divided into two aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different time points to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Results: Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 time points (n= 64 samples). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between collection methods (median Spearman correlation Rs=0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given time point clustered closely, regardless of the collection method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (pConclusion: Ambient temperature collection and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) the biochemical signature of microbiome development over time. This method potentially provides a more convenient, less expensive home collection option for stool metabolomic analysis.

Cesarean section (CS) is recognized as being a shared environmental risk factor associated with chronic immune disease. A study of maternal gene expression changes between different delivery modes can add to our understanding of how CS contributes to disease patterns later in life. We evaluated the association of delivery mode with postpartum gene expression using a cross-sectional study of 324 mothers who delivered full-term (≥ 37 weeks) singletons. Of these, 181 mothers had a vaginal delivery and 143 had a CS delivery (60 with and 83 without labor). Antimicrobial peptides (AMP) were upregulated in vaginal delivery compared to CS with or without labor. Peptidase inhibitor 3 (PI3), a gene in the antimicrobial peptide pathway and known to be involved in antimicrobial and anti-inflammatory activities, showed a twofold increase in vaginal delivery compared to CS with or without labor (adjusted p-value 1.57 × 10–11 and 3.70 × 10–13, respectively). This study evaluates differences in gene expression by delivery mode and provides evidence of antimicrobial peptide upregulation in vaginal delivery compared to CS with or without labor. Further exploration is needed to determine if AMP upregulation provides protection against CS-associated diseases later in life.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with high cure rates leading to rising numbers of long-term survivors. Adult survivors of childhood ALL are at increased risk of obesity, cardiovascular disease, and other chronic illnesses. We hypothesize that ALL therapy is associated with long-term gut microbiome alterations that contribute to predisposition to chronic medical conditions. We conducted a pilot study to test whether differences can be detected between stool microbiota of pediatric ALL survivors and their siblings. Stool samples were collected from 38 individuals under age 19 who were at least 1 year after completion of therapy for ALL. Stool samples collected from 16 healthy siblings served as controls. 16S ribosomal RNA gene sequencing was performed on the stool samples. Comparing microbiota of survivors to sibling controls, no statistically significant differences were found in alpha or beta diversity. However, among the top 10 operational taxonomic units (OTUs) from component 1 in sparse partial least squares discriminant analysis (sPLS-DA) with different relative abundance in survivors versus siblings, OTUs mapping to the genus

Parenteral nutrition-associated cholestasis (PNAC) causes serious morbidity in the neonatal intensive care unit. Infection with gut-associated bacteria is associated with cholestasis, but the role of intestinal microbiota in PNAC is poorly understood. We examined the composition of stool microbiota from premature twins discordant for PNAC as a strategy to reduce confounding from variables associated with both microbiota and cholestasis. Eighty-four serial stool samples were included from 4 twin sets discordant for PNAC. Random Forests was utilized to determine genera most discriminatory in classifying samples from infants with and without PNAC. In infants with PNAC, we detected a significant increase in the relative abundance of Klebsiella, Veillonella, Enterobacter, and Enterococcus (P < 0.05). Bray-Curtis dissimilarities in infants with PNAC were significantly different (P < 0.05) from infants without PNAC. Our findings warrant further exploration in larger cohorts and experimental models of PNAC to determine if a microbiota signature predicts PNAC, as a basis for future interventions to mitigate liver injury.

BACKGROUND The urinary tract was once thought to be sterile, and little is known about the urinary microbiome in children. This study aimed to examine the urinary microbiome of young children across demographic and clinical factors. METHODS Children

Background Preliminary data suggest that the urinary microbiome may play a role in bladder cancer. Information regarding the most suitable method of collecting urine specimens is needed for the large population studies needed to address this. To compare microbiome metrics resulting from 16S ribosomal RNA gene sequencing between midstream, voided specimens and those obtained at cystoscopy. Methods Adults, with a history of superficial urothelial cell carcinoma (non-muscle invasive bladder cancer) being followed with periodic surveillance cystoscopy had a urine sample collected by a mid-stream, voided technique and then from the bladder at cystoscopy. Urine samples underwent 16S ribosomal RNA gene sequencing on the Illumina MiSeq platform. Results 22 subjects (8 female, 14 male) were included. There was no significant difference in beta diversity (diversity between samples) in all samples between collection methods. However, analysis by sex revealed a difference between voided and cystoscopy samples from the same individual in males (p = 0.006, Adonis test) but not in females (p = 0.317, Adonis test). No differences were seen by collection method in any alpha diversity (diversity within a sample) measurement or differential abundance of taxa. Conclusions Beta diversity of the urine microbiome did differ by collection method for males only. This suggests that the urinary microbiomes of the two collection methods are not equivalent to each other, at least in males, which is the sex that bladder cancer occurs most frequently in. Therefore, the same collection method within a given study should be used.

The meconium microbiome may provide insight into intrauterine and peripartum exposures and the very earliest intestinal pioneering microbes. Prenatal antibiotics have been associated with later obesity in children, which is thought to be driven by microbiome dependent mechanisms. However, there is little data regarding associations of prenatal or peripartum antibiotic exposure, with or without cesarean section (CS), with the features of the meconium microbiome. In this study, 16S ribosomal RNA gene sequencing was performed on bacterial DNA of meconium samples from 105 infants in a birth cohort study. After multivariable adjustment, delivery mode (p = 0.044), prenatal antibiotic use (p = 0.005) and peripartum antibiotic use (p &lt, 0.001) were associated with beta diversity of the infant meconium microbiome. CS (vs. vaginal delivery) and peripartum antibiotics were also associated with greater alpha diversity of the meconium microbiome (Shannon and Simpson, p &lt, 0.05). Meconium from infants born by CS (vs. vaginal delivery) had lower relative abundance of the genus Escherichia (p &lt, 0.001). Prenatal antibiotic use and peripartum antibiotic use (both in the overall analytic sample and when restricting to vaginally delivered infants) were associated with differential abundance of several bacterial taxa in the meconium. Bacterial taxa in the meconium microbiome were also differentially associated with infant excess weight at 12 months of age, however, sample size was limited for this comparison. In conclusion, prenatal and peripartum antibiotic use along with CS delivery were associated with differences in the diversity and composition of the meconium microbiome. Whether or not these differences in the meconium microbiome portend risk for long-term health outcomes warrants further exploration.

BACKGROUND: Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridioides difficile infection (rCDI) in adults and children, but donor stool samples are currently screened for only a limited number of potential pathogens. We sought to determine whether putative procarcinogenic bacteria (enterotoxigenic Bacteroides fragilis, Fusobacterium nucleatum, and Escherichia coli harboring the colibactin toxin) could be durably transmitted from donors to patients during FMT. METHODS: Stool samples were collected from 11 pediatric rCDI patients and their respective FMT donors prior to FMT as well as from the patients at 2–10 weeks, 10–20 weeks, and 6 months after FMT. Bacterial virulence factors in stool DNA extracts and stool cultures were measured by quantitative PCR: Bacteroides fragilis toxin (bft), Fusobacterium adhesin A (fadA), and Escherichia coli colibactin (clbB). RESULTS: Four of 11 patients demonstrated sustained acquisition of a procarcinogenic bacteria. Whole genome sequencing was performed on colony isolates from one of these donor/recipient pairs and demonstrated that clbB(+) E. coli strains present in the recipient after FMT were identical to a strain present in the donor, confirming strain transmission. Conversely, 2 patients exhibited clearance of procarcinogenic bacteria following FMT from a negative donor. CONCLUSION: Both durable transmission and clearance of procarcinogenic bacteria occurred following FMT, suggesting that additional studies on appropriate screening measures for FMT donors and the long-term consequences and/or benefits of FMT are warranted. FUNDING: Crohn’s & Colitis Foundation, the Bloomberg~Kimmel Institute for Cancer Immunotherapy at Johns Hopkins University School of Medicine, the National Cancer Institute, and the Canadian Institutes of Health Research.

BackgroundFecal microbiota transplantation (FMT) treats Clostridioides difficile infection (CDI). Little is known regarding the changes in antimicrobial resistance (AMR) genes and potential pathogen burden that occur in pediatric recipients of FMT. The aim of this study was to investigate changes in AMR genes, potential pathogens, species, and functional pathways with FMT in children.MethodsNine children with recurrent CDI underwent FMT. Stool was collected from donor and recipient pre-FMT and longitudinally post-FMT for up to 24 weeks. Shotgun metagenomic sequencing was performed. Reads were analyzed using PathoScope 2.0.ResultsAll children had resolution of CDI. AMR genes decreased post-FMT (P < .001), with a sustained decrease in multidrug resistance genes (P < .001). Tetracycline resistance genes increased post-FMT (P < .001). Very low levels of potential pathogens were identified in donors and recipients, with an overall decrease post-FMT (P < .001). Prevotella sp. 109 expanded in all recipients post-FMT, and no recipients had any clinical infection. Alpha diversity was lower in recipients vs donors pre-FMT (P < .001), with an increase post-FMT (P ≤ .002) that was sustained. Beta diversity differed significantly in pre- vs post-FMT recipient samples (P < .001). Bacterial species Faecalibacterium prausnitzii and Bacteroides ovatus showed higher abundance in donors than recipients (P = .008 and P = .040, respectively), with expansion post-FMT. Biosynthetic pathways predominated in the donor and increased in the recipient post-FMT.ConclusionsFMT for CDI in children decreases AMR genes and potential pathogens and changes microbiota composition and function. However, acquisition of certain AMR genes post-FMT combined with low levels of potential pathogens found in donors suggests that further study is warranted regarding screening donors using metagenomics sequencing before FMT.

Article first published online 18 December 2015., Children with inflammatory bowel disease (IBD) are disproportionately susceptible to Clostridium difficile infection (CDI) and the incidence is increasing. There has also been growing recognition of asymptomatic C. difficile colonization in pediatric IBD, which can sometimes be very difficult to distinguish from symptomatic C. difficile–associated disease in this population. In this study, we discuss the current knowledge of C. difficile infection in children with IBD, reviewing epidemiology, risk factors, and outcomes that often differ from the adult IBD population, and discuss the complexities and dilemmas of diagnosing and treating CDI in pediatric IBD.

There has been a growing interest in fecal microbiota transplantation (FMT) over recent years, in part due to the increasing prevalence of Clostridium difficile infection (CDI) and expanding association of intestinal dysbiosis with a wide range of human diseases. Many adult studies have shown that FMT is an effective treatment for recurrent CDI and may possibly have applications in other illnesses such as inflammatory bowel disease (IBD); however, there is a paucity of data available in children who may differ from adults for many reasons including having a dynamic developing microbiome compared to adults who have a relatively stable microbiome. Here, we review published studies looking at FMT in children, for CDI and IBD, and discuss special considerations needed when conducting FMT in children.

Background & Aims Fecal microbiota transplantation (FMT) is commonly used to treat Clostridium difficile infection (CDI). CDI is an increasing cause of diarrheal illness in pediatric patients, but the effects of FMT have not been well studied in children. We performed a multi-center retrospective cohort study of pediatric and young adult patients to evaluate the efficacy, safety, and factors associated with a successful FMT for the treatment of CDI. Methods We performed a retrospective study of 372 patients, 11 months to 23 years old, who underwent FMT at 18 pediatric centers, from February 1, 2004, to February 28, 2017; 2-month outcome data were available from 335 patients. Successful FMT was defined as no recurrence of CDI in the 2 months following FMT. We performed stepwise logistic regression to identify factors associated with successful FMT. Results Of 335 patients who underwent FMT and were followed for 2 months or more, 271 (81%) had a successful outcome following a single FMT and 86.6% had a successful outcome following a first or repeated FMT. Patients who received FMT with fresh donor stool (odds ratio [OR], 2.66; 95% CI, 1.39–5.08), underwent FMT via colonoscopy (OR, 2.41; 95% CI, 1.26–4.61), did not have a feeding tube (OR, 2.08; 95% CI, 1.05–4.11), or had 1 less episode of CDI before FMT (OR, 1.20; 95% CI, 1.04–1.39) had increased odds for successful FMT. Seventeen patients (4.7%) had a severe adverse event during the 3-month follow-up period, including 10 hospitalizations. Conclusions Based on the findings from a large multi-center retrospective cohort, FMT is effective and safe for the treatment of CDI in children and young adults. Further studies are required to optimize the timing and method of FMT for pediatric patients—factors associated with success differ from those of adult patients.

OBJECTIVES The gut microbiome is believed to play a role in the susceptibility to and treatment of Clostridium difficile infections (CDIs). It is, however, unknown whether the gut microbiome is also affected by asymptomatic C difficile colonization. Our study aimed to evaluate the fecal microbiome of children based on C difficile colonization, and CDI risk factors, including antibiotic use and comorbid inflammatory bowel disease (IBD). METHODS Subjects with IBD and non-IBD controls were prospectively enrolled from pediatric clinics for a biobanking project (n = 113). A fecal sample was collected from each subject for research purposes only and was evaluated for asymptomatic toxigenic C difficile colonization. Fecal microbiome composition was determined by 16S rRNA sequencing. RESULTS We found reduced bacterial diversity and altered microbiome composition in subjects with C difficile colonization, concurrent antibiotic use, and/or concomitant IBD (all P

Background Effective methods are needed to collect fecal samples from children for large-scale microbiota studies. Stool collected on fecal occult blood test (FOBT) cards that can be mailed provides an effective solution; however, the quality of sequencing resulting from this method is unknown. The aim of this study is to compare microbiota metrics of 16S ribosomal RNA (rRNA) gene sequencing from stool and meconium collected on FOBT cards with stool collected in an Eppendorf tube (ET) under different conditions. Methods Eight stool samples from children in diapers aged 0 month–2 years and three meconium samples were collected and stored as follows: (1) ≤ 2 days at room temperature (RT) in an ET, (2) 7 days at − 80 °C in an ET, (3) 3–5 days at RT on a FOBT card, (4) 7 days at RT on a FOBT card, and (5) 7 days at − 80 °C on a FOBT card. Samples stored at − 80 °C were frozen immediately. Each specimen/condition underwent 16S rRNA gene sequencing with replicates on the Illumina MiSeq. Alpha and beta diversity measures and relative abundance of major phyla were compared between storage conditions and container (ET vs. FOBT card), with pairwise comparison between different storage conditions and the “standard” of 7 days at − 80 °C in an ET and fresh stool in an ET. Results Stool samples clustered mainly by individual diaper (P

We describe a case of an infant presenting with intractable diarrhea who subsequently developed dilated cardiomyopathy, for whom a diagnosis was not initially achieved despite extensive clinical testing, including panel-based genetic testing. Research-based whole-genome sequences of the proband and both parents were analyzed by the SAVANNA pipeline, a variant prioritization strategy integrating features of variants, genes, and phenotypes, which was implemented using publicly available tools. Although the intestinal morphological abnormalities characteristic of congenital tufting enteropathy (CTE) were not observed in the initial clinical gastrointestinal tract biopsies of the proband, an intronic variant, EPCAM c.556-14A>G, previously identified as pathogenic for CTE, was found in the homozygous state. A newborn cousin of the proband also presenting with intractable diarrhea was found to carry the same homozygous EPCAM variant, and clinical testing revealed intestinal tufting and loss of EPCAM staining. This variant, however, was considered nonexplanatory for the proband's dilated cardiomyopathy, which could be a sequela of the child's condition and/or related to other genetic variants, which include de novo mutations in the genes NEDD4L and GSK3A and a maternally inherited SCN5A variant. This study illustrates three ways in which genomic sequencing can aid in the diagnosis of clinically challenging patients: differential diagnosis despite atypical clinical presentation, distinguishing the possibilities of a syndromic condition versus multiple conditions, and generating hypotheses for novel contributory genes.

Background Childhood obesity studies rely on parentally reported anthropometrics. However, the accuracy of such data has not been evaluated for 12-month-old children. Moreover, methods to improve the accuracy of reported data have not been assessed in prior studies. Methods A total of 185 children enrolled in a northern Virginia childhood longitudinal cohort genomic study had parentally completed surveys at 12 months. Measured weights and lengths were recorded for the same children from their 12-month paediatrician visit. Weight for length percentiles were calculated using World Health Organization gender-specific growth charts. The agreement between reported and measured values was examined using Pearson's correlation, paired t-test and κ statistics. The interquartile outlier rule was used to detect and remove outliers. Results Parentally reported weight was strongly associated with measured weight at 12 months (r=0.90). There was only a moderate correlation between parentally reported and measured lengths (r=0.52) and calculated weight for length percentiles (r=0.65). After removing outliers from parentally reported data, there was an increase in correlation between parentally reported and measured data for weight (r=0.93), length (r=0.69) and weight for length percentiles (r=0.76). Outliers removed compared to all children included were more likely to have maternal education less than a bachelor's degree (p=0.007). Conclusions After removal of outliers from reported data, there is a strong correlation between calculated reported and measured weight for length percentiles suggesting that this may be an effective method to increase accuracy when conducting large-scale obesity studies in young children where study costs benefit from using parentally reported data.

Purpose Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood and has a high treatment success rate. However, survivors of childhood ALL have a high prevalence of chronic medical illnesses, such as metabolic syndrome. Dysbiosis in the gut microbiome is associated with metabolic derangement and disease risk. Chemotherapy and antibiotics increase gut microbiome dysbiosis. We tested the hypothesis that ALL treatment can cause alterations in the gut microbiome that persist in survivorship. Method Stool samples were collected on fecal occult blood test cards from 38 survivors between 2-18 years of age who were >1 year off therapy for ALL. 16 stool samples from healthy siblings age 2-18 years were collected from 14 families as controls. Clinical data including anthropometrics, antibiotic use, and probiotic use were collected. DNA was extracted and sequenced using a modified Illumina 16S Metagenomics Sequencing Library Preparation protocol for analysis of hypervariable region V4. A single rarefaction was performed at 4,897 removing 2 samples with low mapped read counts. Alpha and beta (Bray) diversities were calculated; Wilcoxon rank-sum test was used to compare the alpha diversities between groups, as well as the beta diversities within and between groups. Wilcoxon signed-rank test was used to compare the alpha diversity between survivors and their matched siblings. Finally, read counts for each Operational Taxonomic Unit (OTU) was normalized and tested for differential abundance using edgeR, assuming a negative binomial model. Results Significant differences in the abundance of taxa were found, with the most notable being the depletion of Faecalibacterium in survivors (FDR= 0.0004). Figure 1 shows a heatmap with the complete linkage clustering method on the Euclidean distance using the 13 OTUs with statistically significant (FDR Conclusion Differences in the relative abundance of certain taxa were found between survivors and siblings. Specifically, Fecalibacterium was depleted in survivors, which has also been previously observed in older populations of childhood ALL survivors. This study shows that these microbiome changes are present in ALL survivors during childhood, and should be evaluated longitudinally for association with the chronic medical illnesses seen in ALL survivors in adulthood. Early interventions to restore the gut microbiome in ALL patients may potentially ameliorate the risk of long-term adverse health outcomes. Disclosures No relevant conflicts of interest to declare.

OBJECTIVES/SPECIFIC AIMS: Clostridium difficile infection (CDI) is the most common cause of antibiotic-associated diarrhea and an increasingly common infection in children in both hospital and community settings. Between 20% and 30% of pediatric patients will have a recurrence of symptoms in the days to weeks following an initial infection. Multiple recurrences have been successfully treated with fecal microbiota transplantation (FMT), though the body of evidence in pediatric patients is limited primarily to case reports and case series. The goal of our study was to better understand practices, success, and safety of FMT in children as well as identify risk factors associated with a failed FMT in our pediatric patients. METHODS/STUDY POPULATION: This multicenter retrospective analysis included 373 patients who underwent FMT for CDI between January 1, 2006 and January 1, 2017 from 18 pediatric centers. Demographics, baseline characteristics, FMT practices, C. difficile outcomes, and post-FMT complications were collected through chart abstraction. Successful FMT was defined as no recurrence of CDI within 60 days after FMT. Of the 373 patients in the cohort, 342 had known outcome data at two months post-FMT and were included in the primary analysis evaluating risk factors for recurrence post-FMT. An additional six patients who underwent FMT for refractory CDI were excluded from the primary analysis. Unadjusted analysis was performed using Wilcoxon rank-sum test, Pearson χ2 test, or Fisher exact test where appropriate. Stepwise logistic regression was utilized to determine independent predictors of success. RESULTS/ANTICIPATED RESULTS: The median age of included patients was 10 years (IQR; 3.0, 15.0) and 50% of patients were female. The majority of the cohort was White (89.0%). Comorbidities included 120 patients with inflammatory bowel disease (IBD) and 14 patients who had undergone a solid organ or stem cell transplantation. Of the 336 patients with known outcomes at two months, 272 (81%) had a successful outcome. In the 64 (19%) patients that did have a recurrence, 35 underwent repeat FMT which was successful in 20 of the 35 (57%). The overall success rate of FMT in preventing further episodes of CDI in the cohort with known outcome data was 87%. Unadjusted predictors of a primary FMT response are summarized. Based on stepwise logistic regression modeling, the use of fresh stool, FMT delivery via colonoscopy, the lack of a feeding tube, and a lower number of CDI episodes before undergoing FMT were independently associated with a successful outcome. There were 20 adverse events in the cohort assessed to be related to FMT, 6 of which were felt to be severe. There were no deaths assessed to be related to FMT in the cohort. DISCUSSION/SIGNIFICANCE OF IMPACT: The overall success of FMT in pediatric patients with recurrent or severe CDI is 81% after a single FMT. Children without a feeding tube, who receive an early FMT, FMT with fresh stool, or FMT via colonoscopy are less likely to have a recurrence of CDI in the 2 months following FMT. This is the first large study of FMT for CDI in a pediatric cohort. These findings, if confirmed by additional prospective studies, will support alterations in the practice of FMT in children.