Evaluating the Alimentary and Respiratory Tracts in Health and disease (EARTH) research programme: a protocol for prospective, longitudinal, controlled, observational studies in children with chronic disease at an Australian tertiary paediatric hospital.

INTRODUCTION: Chronic gastrointestinal and respiratory conditions of childhood can have long-lasting physical, psychosocial and economic effects on children and their families. Alterations in diet and intestinal and respiratory microbiomes may have important implications for physical and psychosocial health. Diet influences the intestinal microbiome and should be considered when exploring disease-specific alterations. The concepts of gut-brain and gut-lung axes provide novel perspectives for examining chronic childhood disease(s). We established the 'Evaluating the Alimentary and Respiratory Tracts in Health and disease' (EARTH) research programme to provide a structured, holistic evaluation of children with chronic gastrointestinal and/or respiratory conditions. METHODS AND ANALYSIS: The EARTH programme provides a framework for a series of prospective, longitudinal, controlled, observational studies (comprised of individual substudies), conducted at an Australian tertiary paediatric hospital (the methodology is applicable to other settings). Children with a chronic gastrointestinal and/or respiratory condition will be compared with age and gender matched healthy controls (HC) across a 12-month period. The following will be collected at baseline, 6 and 12 months: (i) stool, (ii) oropharyngeal swab/sputum, (iii) semi-quantitative food frequency questionnaire, (iv) details of disease symptomatology, (v) health-related quality of life and (vi) psychosocial factors. Data on the intestinal and respiratory microbiomes and diet will be compared between children with a condition and HC. Correlations between dietary intake (energy, macro-nutrients and micro-nutrients), intestinal and respiratory microbiomes within each group will be explored. Data on disease symptomatology, quality of life and psychosocial factors will be compared between condition and HC cohorts.Results will be hypothesis-generating and direct future focussed studies. There is future potential for direct translation into clinical care, as diet is a highly modifiable factor. ETHICS AND DISSEMINATION: Ethics approval: Sydney Children's Hospitals Network Human Research Ethics Committee (HREC/18/SCHN/26). Results will be presented at international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04071314.

Gut microbiota-mediated Gene-Environment interaction in the TashT mouse model of Hirschsprung disease.

Based on the bilateral relationship between the gut microbiota and formation/function of the enteric nervous system (ENS), we sought to determine whether antibiotics-induced dysbiosis might impact the expressivity of genetically-induced ENS abnormalities. To address this, we took advantage of the TashT mouse model of Hirschsprung disease, in which colonic aganglionosis and hypoganglionosis are both much more severe in males. These defects result into two male-biased colon motility phenotypes: either megacolon that is lethal around weaning age or chronic constipation in adults, the latter being also associated with an increased proportion of nitrergic neurons in the distal ENS. Induction of dysbiosis using a cocktail of broad-spectrum antibiotics specifically impacted the colonic ENS of TashTTg/Tg mice in a stage-dependent manner. It further decreased the neuronal density at post-weaning age and differentially modulated the otherwise increased proportion of nitrergic neurons, which appeared normalized around weaning age and further increased at post-weaning age. These changes delayed the development of megacolon around weaning age but led to premature onset of severe constipation later on. Finally, local inhibition of nitric oxide signaling improved motility and prevented death by megacolon. We thus conclude that exposure to antibiotics can negatively influence the expressivity of a genetically-induced enteric neuropathy.

Presence of blaCTX-M antibiotic resistance gene in Lactobacillus spp. isolated from Hirschsprung diseased infants with stoma.

INTRODUCTION: Although antibiotics have revolutionized health care by saving lives, the evolution of both pathogenic and commensal antibiotic-resistant bacteria are emerging as a threat in the health sector. As for Lactobacillus spp., it is usually a non-pathogenic bacteria. However, it can cause infection in immunocompromised condition. In this study, Lactobacillus spp. has been isolated from the faeces of infants with Hirschsprung disease (HD), which is congenital aganglionosis of intestine, where surgical approach and antibiotics are frequently used as medical intervention. The aim of this study is to assess the antibiotic resistance pattern and determine the presence of resistance genes, if any, in Lactobacillus spp. isolated from HD infants with ileostomy. METHODOLOGY: Six Lactobacillus spp. were isolated from faeces of six HD infants and confirmed using both conventional and molecular methods. Antibiotic resistance pattern was checked through disc diffusion method and was further investigated for the presence of antibiotic resistance genes (blaTEM, blaCTX-M, blaOXA-2, blaIMP, blaVIM-2, blaNDM-1 and mcr-1). RESULTS: Antibiotic susceptibility of the isolates showed high level of resistance towards cephalosporins, oxacillin, aztreonam, meropenem and polymyxin group. However, four of the isolates showed the presence of blaCTX-M gene after PCR amplification. CONCLUSIONS: To our knowledge, this is the first report on the presence of antibiotic resistance gene blaCTX-M in Lactobacillus spp. and this presence may pose a serious threat in treatment regimen. As not much is known regarding the presence of blaCTX-M in Lactobacillus spp., this finding may provide new light to research on antibiotic resistance in gut microflora.

Intestinal Microbiota in Hirschsprung Disease.

OBJECTIVES: The aim of the study was to characterize the microbiota profiles of patients with Hirschsprung disease (HD) and to evaluate this in relation to postoperative bowel function and the incidence of Hirschsprung-associated enterocolitis (HAEC). METHODS: All patients operated on for HD at our center between 1987 and 2011 were invited to answer questionnaires on bowel function and to participate in a clinical follow-up for laboratory investigations, including fecal DNA extraction, fecal calprotectin (FC), and brush border lactase (LCT) genotyping. The microbiota compositions of patients with HD were compared with those of healthy controls aged between 2 and 7 years. RESULTS: The microbiota composition of eligible patients with HD (n = 34; median age 12 [range, 3-25] years) differed from the healthy controls (n = 141), showing decreased overall microbial richness (P < 0.005). Seventy-seven percent had experienced HAEC. Normal maturation of the intestinal flora was not observed, but patients had a significantly increased abundance of Proteobacteria among other taxa (P < 0.005) resulting in a reduced carbohydrate degradation potential, as predicted by the taxonomic composition. Genetic lactase deficiency was present in 17% and did not correlate with bowel symptoms. No patients reported active HAEC at the time of sampling and FC was within the normal range in all samples. CONCLUSIONS: Patients with HD and HAEC had a significantly altered intestinal microbiome compared to healthy individuals, characterized by a lack of richness and pathologic expansions of taxa, particularly Enterobacteria and Bacilli. Further evaluation is needed to identify whether these observations are intrinsic to HD or secondary to the recurrent use of antibiotics during early childhood.

Characterization of Intestinal Microbiomes of Hirschsprung's Disease Patients with or without Enterocolitis Using Illumina-MiSeq High-Throughput Sequencing.

Hirschsprung-associated enterocolitis (HAEC) is a life-threatening complication of Hirschsprung's disease (HD). Although the pathological mechanisms are still unclear, studies have shown that HAEC has a close relationship with the disturbance of intestinal microbiota. This study aimed to investigate the characteristics of the intestinal microbiome of HD patients with or without enterocolitis. During routine or emergency surgery, we collected 35 intestinal content samples from five patients with HAEC and eight HD patients, including three HD patients with a history of enterocolitis who were in a HAEC remission (HAEC-R) phase. Using Illumina-MiSeq high-throughput sequencing, we sequenced the V4 region of bacterial 16S rRNA, and operational taxonomic units (OTUs) were defined by 97% sequence similarity. Principal coordinate analysis (PCoA) of weighted UniFrac distances was performed to evaluate the diversity of each intestinal microbiome sample. The microbiota differed significantly between the HD patients (characterized by the prevalence of Bacteroidetes) and HAEC patients (characterized by the prevalence of Proteobacteria), while the microbiota of the HAEC-R patients was more similar to that of the HAEC patients. We also observed that the specimens from different intestinal sites of each HD patient differed significantly, while the specimens from different intestinal sites of each HAEC and HAEC-R patient were more similar. In conclusion, the microbiome pattern of the HAEC-R patients was more similar to that of the HAEC patients than to that of the HD patients. The HD patients had a relatively distinct, more stable community than the HAEC and HAEC-R patients, suggesting that enterocolitis may either be caused by or result in a disruption of the patient's uniquely adapted intestinal flora. The intestinal microbiota associated with enterocolitis may persist following symptom resolution and can be implicated in the symptom recurrence.

Altered fecal short chain fatty acid composition in children with a history of Hirschsprung-associated enterocolitis.

PURPOSE: Children with Hirschsprung disease (HD) who have a history of enterocolitis (HAEC) have a shift in colonic microbiota, many of which are necessary for short chain fatty acid (SCFA) production. As SCFAs play a critical role in colonic mucosal preservation, we hypothesized that fecal SCFA composition is altered in children with HAEC. METHODS: A multicenter study enrolled 18 HD children, abstracting for history of feeding, antibiotic/probiotic use, and enterocolitis symptoms. HAEC status was determined per Pastor et al. criteria (12). Fresh feces were collected for microbial community analysis via 16S sequencing as well as SCFA analysis by gas chromatography-mass spectrometry. RESULTS: Nine patients had a history of HAEC, and nine had never had HAEC. Fecal samples from HAEC children showed a 4-fold decline in total SCFA concentration vs. non-HAEC HD patients. We then compared the relative composition of individual SCFAs and found reduced acetate and increased butyrate in HAEC children. Finally, we measured relative abundance of SCFA-producing fecal microbiota. Interestingly, 10 of 12 butyrate-producing genera as well as 3 of 4 acetate-producing genera demonstrated multi-fold expansion. CONCLUSION: Children with HAEC history have reduced fecal SCFAs and altered SCFA profile. These findings suggest a complex interplay between the colonic metabolome and changes in microbiota, which may influence the pathogenesis of HAEC.

Characterization of Bacterial and Fungal Microbiome in Children with Hirschsprung Disease with and without a History of Enterocolitis: A Multicenter Study.

Development of potentially life-threatening enterocolitis is the most frequent complication in children with Hirschsprung disease (HSCR), even after definitive corrective surgery. Intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to compare the fecal bacterial and fungal communities of children who developed Hirschsprung-associated enterocolitis (HAEC) with HSCR patients who had never had enterocolitis. Eighteen Hirschsprung patients who had completed definitive surgery were enrolled: 9 had a history of HAEC and 9 did not. Fecal DNA was isolated and 16S and ITS-1 regions sequenced using Next Generation Sequencing and data analysis for species identification. The HAEC group bacterial composition showed a modest reduction in Firmicutes and Verrucomicrobia with increased Bacteroidetes and Proteobacteria compared with the HSCR group. In contrast, the fecal fungi composition of the HAEC group showed marked reduction in diversity with increased Candida sp., and reduced Malassezia and Saccharomyces sp. compared with the HSCR group. The most striking finding within the HAEC group is that the Candida genus segregated into "high burden" patients with 97.8% C. albicans and 2.2% C. tropicalis compared with "low burden" patients 26.8% C. albicans and 73% C. tropicalis. Interestingly even the low burden HAEC group had altered Candida community structure with just two species compared to more diverse Candida populations in the HSCR patients. This is the first study to identify Candida sp. as potentially playing a role in HAEC either as expanded commensal species as a consequence of enterocolitis (or treatment), or possibly as pathobioants contributing to the pathogenesis of HAEC. These findings suggest a dysbiosis in the gut microbial ecosystem of HAEC patients, such that there may be dominance of fungi and bacteria predisposing patients to development of HAEC.

Mucus Barriers to Microparticles and Microbes are Altered in Hirschsprung's Disease.

Mucus forms a protective hydrogel layer over the intestinal epithelium, presenting a selective and robust barrier to the uptake of particulates and microbe invasion. Disease can alter mucus production and composition, thus potentially modifying mucosal barrier properties. Hirschsprung's disease (HD) is a developmental abnormality of the nervous system often complicated by intestinal infection. An investigation of colonic mucus barrier properties in an HD animal model, endothelin receptor B mutant mice, revealed significantly reduced microsphere (passive) and microbe (active) transport rates (7-fold and 3.6-fold, respectively, in proximal colonic mucus) relative to wild-type. Transport differences were evident in both the ganglionic and aganglionic colon segments, in agreement with the risk of HD-associated enterocolitis after surgery to remove aganglionic colon segments. The development of therapies aimed at altering colonic mucus barrier properties could be explored towards preventing the onset of enterocolitis in HD.

Characterization of the intestinal microbiome of Hirschsprung's disease with and without enterocolitis.

Hirschsprung's disease (HD) is a congenital malformation of the gastrointestinal tract characterized by the absence of the distal enteric nervous system. Hirschsprung-associated enterocolitis (HAEC) is severe life threatening complication of HD. The disease pathogenesis is still unclear, but evidences suggest that the intestinal microbiota may play important role in the development of HD and HAEC. Because microbial abundance and diversity might differ in HD patients with enterocolitis, we sought to generate comparative metagenomic signatures to characterize the structure of the microbiome in HD patients with and without enterocolitis. Our experimental design is to enroll four HD patients (two with enterocolitis and two without enterocolitis). The microbiome was characterized by 16S rRNA gene, and the data obtained will be used to taxonomically classify and compare community structure among different samples. We found that the structure of the microbiome within HAEC patients are differ from those without enterocolitis. This study helps us to understand microbial contributions to the etiology of Hirschsprung-associated enterocolitis.

Recent developments in Hirschsprung's-associated enterocolitis.

Hirschsprung's-associated enterocolitis (HAEC) continues to be a significant source of morbidity for patients with Hirschsprung's disease (HD). New clinical and histologic classification systems for HAEC will improve consistency between reports and increase the ability to compare outcomes. A complete understanding of disease pathogenesis is lacking, but evidence suggests that the intestinal microbiota may play a role in the development of HD and HAEC. The benefits of adjunctive therapies, such as anal dilations and botulinum toxin to reduce the incidence of HAEC following corrective endorectal pull-through, remain controversial. Finally, new clinical data have identified an association between HAEC and inflammatory bowel disease and will likely lead to further genetic studies to elucidate the connection between these two disease processes.

Risk factors for small bowel bacterial overgrowth and diagnostic yield of duodenal aspirates in children with intestinal failure: a retrospective review.

BACKGROUND: Children with intestinal failure (IF) are at risk for small bowel bacterial overgrowth (SBBO) because of anatomical and other factors. We sought to identify risk factors for SBBO confirmed by quantitative duodenal culture. METHODS: A single-center retrospective record review of children who had undergone endoscopic evaluation for SBBO (defined as bacterial growth in duodenal fluid of >10(5) colony-forming unit per mL) was performed. RESULTS: We reviewed 57 children with median (25th-75th percentile) age 5.0 (2.0-9.2) years. Diagnoses included motility disorders (28%), necrotizing enterocolitis (16%), atresias (16%), gastroschisis (14%), and Hirschsprung disease (10.5%). Forty patients (70%) had confirmed SBBO. Univariate analysis showed no significant differences between patients with and without SBBO for the following variables: age, sex, diagnosis, presence of ileocecal valve, and antacid use. Patients receiving parenteral nutrition (PN) were more likely to have SBBO (70% vs 35%, P = .02). Multiple logistic regression analysis confirmed that PN administration was independently associated with SBBO (adjusted odds ratio, 5.1; adjusted 95% confidence interval, 1.4-18.3; P = .01). SBBO was not related to subsequent risk of catheter-related bloodstream infection (CRBSI). CONCLUSION: SBBO is strongly and independently associated with PN use. Larger prospective cohorts and more systematic sampling techniques are needed to better determine the relationship between SBBO and gastrointestinal function.

Intestinal aganglionosis is associated with early and sustained disruption of the colonic microbiome.

BACKGROUND: Congenital aganglionosis (Hirschsprung's disease) results in colonic dysmotility and a risk for Hirschsprung's-associated enterocolitis (HAEC), whose cause is unknown. We hypothesized that aganglionosis leads to microbiome changes that may contribute to HAEC risk. METHODS: Colon and fecal samples were collected from endothelin receptor B-null (Ednrb(-/-) ) mice, an established model of colorectal aganglionosis, at postnatal day 7 (P7), P20, and P24. We determined microbiome composition by 16S ribosomal RNA gene pyrosequencing and fecal metabolite profile by nuclear magnetic resonance spectroscopy. KEY RESULTS: Wild-type (WT) mice exhibited increasing species diversity with age, with mutant mice possessing even greater diversity. WT and mutant microbiomes, both fecal and colonic, significantly segregated by principal coordinates analysis based on species composition at all ages examined. Importantly, mutant mice contained more Bacteroidetes and less Firmicutes than WT, with additional genus- and species-level differences observed. Notably, mutant P7 colon was dominated by coagulase-negative Staphylococcus species, which were rare in WT. Mutant fecal metabolite profiles also differed, particularly in the abundance of formate, a short-chain fatty acid product of microbial fermentation. CONCLUSIONS & INFERENCES: Colorectal aganglionosis is associated with early and sustained disruption of the normal colonic and fecal microbiome, supporting the enteric nervous system as a determinant of microbiome composition. Furthermore, the differences observed suggest a potential contributory role for the microbiome in the etiology of HAEC. These findings provide a basis for further studies to determine the causative role of specific bacterial communities in HAEC and the potential to restore the normal microbiome in Hirschsprung's disease.

Detection of intestinal bifidobacteria and lactobacilli in patients with Hirschsprung's disease associated enterocolitis.

BACKGROUND: The etiology of Hirschsprung's disease associated enterocolitis (HAEC) is unknown. Previous investigations have suggested that several factors such as dilation of proximal bowel, changes in colonic mucosal defence, and overgrowth of toxigenic bacteria may be related with it. This study was to quantify bifidobacteria and lactobacilli in the feces of Hirschsprung's disease (HD) patients with or without enterocolitis and those of normal children. METHODS: Fresh stool specimens were collected at the first three days of the admission from 30 HD patients (aged 2 weeks to 2 years) and 15 healthy age-matched non-HD patients in the morning once a day for at least three days. All of them have not been given probiotics or antibiotics at least 7 days before stool collection. Hematoxylin-eosin and acetylcholinesterase histochemical staining on rectal biopsies of patients with HD confirmed the diagnosis of HD in all 30 patients. The 30 HD patients were divided into two groups based on the clinical history of enterocolitis: the HAEC group (n=10) and HD group (n=20). Fecal bifidobacteria and lactobacilli were consecutively quantified by SYBR Green I-based real-time PCR assay. Data were analyzed using SAS v. 12.6 for Windows. All tests were two-tailed, and P values <0.05 were considered statistically significant. RESULTS: The mean levels of bifidobacteria were 7.35+/-0.59, 8.16+/-1.17, and 8.35+/-0.74 in the HAEC, HD and control groups, respectively. The bifidobacteria colonization levels were lower in the HAEC group than in the HD and control groups (P<0.05, P<0.001 respectively). The mean level of lactobacilli in the HAEC (5.51+/-0.65) and HD groups (5.87+/-0.78) was significantly lower than that in the control group (6.39+/-0.56) (P<0.05). But there was no difference in log numbers of lactobacilli between HAEC and HD groups (P>0.05). CONCLUSIONS: The scarcity of bifidobacteria and lactobacilli in HAEC patients may result in a decrease in epithelial barrier function and be a predisposing factor in the development of HAEC. This decline suggests that treatment with probiotics or prebiotics may be beneficial in these individuals. Further research will focus on whether probiotics can decrease the incidence of HAEC.

Prolonged carriage of Clostridium difficile in Hirschsprung's disease.

The role of Clostridium difficile in the aetiology of diarrhoea in children with Hirschsprung's disease was investigated in a prospective longitudinal study. In 64 children with Hirschsprung's disease no significant difference was found in the isolation rate of C difficile in patients with diarrhoea (32%) and without diarrhoea (26%). Comparable isolation rates were found in 47 control children with and without diarrhoea (27% and 16% respectively). The number of strains producing toxin B was similar in the four groups of children. In contrast to the disappearance of C difficile by 12 months of age in the control groups of children, C difficile could be repeatedly isolated from a proportion of children with Hirschsprung's disease over 12 months of age. These findings help to reconcile the existing contradictory reports on the incidence of C difficile in Hirschsprung's disease associated enterocolitis.

Hirschsprung's disease: a viral etiology?

This study investigates the hypothesis that Hirschsprung's disease (HD), congenital rectal aganglionosis, may be etiologically linked to antenatal cytomegalovirus (CMV) infection. Bowel specimens from 72 HD patients, 144 control I infants (Hirschsprung-like symptoms, normal histology), and 36 control II infants (deaths from nongastrointestinal causes) were analyzed for CMV genomes by polymerase chain reaction. Positive results were obtained in 6 HD patients (8.8%) and none of the controls. Our findings suggest that antenatal CMV infection, a potentially preventable condition, may be one of the etiological factors in HD.

[Study of large intestinal microflora in newborns with Hirschsprung disease]. / Issledovanie mikroflory tolstoi kishki pri bolezni girshprunga u novorozhdënnykh.

In 35 newborns and children at the age under 4 mos with Hirschsprung's disease, colonic microflora was studied. Disbacteriosis at phase 1 was revealed in 1 (2.9%) child, at phase 2--in 7 (20%), at phase 3--in 27 (77.1%). In acute form of Hirschsprung's disease, in 68.5% of cases, disbacteriosis at phase 3 with the signs of enterocolitis causing acute ileus was revealed. Intestinal perforation against background of enterocolitis developed in 8 cases.

Microbiological studies of the enterocolitis of Hirschsprung's disease.

The results of a prospective study of 20 cases of newly diagnosed Hirschsprung's disease (nine of whom developed enterocolitis) and 10 normal controls showed no variations in the bacterial flora (including Clostridium difficile) in the stools of the groups studied. Viral studies showed that rotavirus was present in the stools of seven of the nine cases of enterocolitis during the episode. We suggest that Hirschsprung's enterocolitis may have a complex infective aetiology and that rotavirus plays a part.

The piebald-lethal murine strain: investigation of the cause of early death.

Forty-eight piebald-lethal (PL) mice with distal aganglionosis and 42 normal littermates (LM) were studied to determine the cause of early death. PL mice were noted to be smaller than their LM and to have normal albumin and immunoglobulin levels for the first 30 days of age. As PL mice aged, a significant decline in albumin with a concomitant rise in immunoglobulin levels was demonstratable. Systemic sepsis with enteric organisms was found in 10% of sacrificed PL mice and in 38% of spontaneously dying PL animals. Histologic examination of PL aganglionic and ganglionic colon demonstrated no evidence of enterocolitis. Ganglionic colon of PL mice contained a flattened, thinned mucosa. The early death of PL mice is related to generalized debilitation from prolonged distal colonic obstruction resulting in a decrease in immunologic integrity and an increased susceptibility to sepsis.

Enterocolitis in Hirschsprung's disease: a controlled study of the etiologic role of Clostridium difficile.

Cytopathic toxin neutralized by Clostridium sordellii antiserum was found in the feces of seven out of 13 children with Hirschsprung's disease complicated by enterocolitis (54%). Clostridium difficile was isolated from ten of these 13 children (77%). The frequency of fecal toxin positivity, the magnitude of toxin titers, and the isolation rate of C difficile were all significantly greater in children with Hirschsprung's enterocolitis than in children whose Hirschsprung's disease was not complicated by enterocolitis or in children without Hirschsprung's disease. It is suggested that C difficile may be causally related to enterocolitis in Hirschsprung's disease, but the age distribution of positive results indicates that the major etiologic role is confined to children under three years of age. Vancomycin was found to be an effective form of treatment in the children with enterocolitis in whom it was employed.