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

Abstract

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.

Immunity against fungi

“…Although fungi comprise less than 1% of total microbial rDNA sequences at different anatomic sites, the size of fungal cells (~3-10 μm diameter for most yeast cells, 2-5 μm diameter for typical mold conidia, and 10 to hundreds μm length for hyphae, compared with < 1 μm diameter for bacterial cells) suggests that fungal rDNA quantification underestimates fungal biomass in the microbiota. In the past five years, researchers have analyzed endogenous fungal communities in the oral cavity, gastrointestinal tract, skin, and mucosal sites in healthy and diseased individuals (168)(169)(170)(171)(172). In most studies, between 15 and 70 fungal genera have been identified, with Malassezia spp.…”

Sampling of Candida albicans and Candida tropicalis by Langerin-positive dendritic cells in mouse Peyer’s patches

“…For example, during periods of host immune suppression, inflammation or microbiome dysbiosis, Candida albicans is able to breach the epithelial barrier and cause invasive inflammatory disease of the intestinal mucosa [2,3]. C. albicans is also recognized as one of the dominant colonizers associated with inflammatory bowel diseases (IBD) like Crohn's and Hirschprung-associated enterocolitis [4][5][6][7][8][9] . Another constitute of the normal flora, Candida tropicalis has also been associated with colonic infections in immune compromised patients [10,11].…”

The pediatric intestinal mucosal microbiome remains altered after clinical resolution of inflammatory and ischemic disease

“…But in clinical settings, dysbiosis is an important risk factor, which is perpetuated by prolonged antibiotic treatment. 3,5 A recent multicenter study of pediatric patients starting treatment for Crohn's disease demonstrated that inflammation, diet, and antibiotics all independently contribute to dysbiosis. 11 Differences were noted as early as 1 week and persisted for at least 8 weeks, similar to the chronic changes we noted in our patients.…”

“…1,2 Conversely, reduced bacterial diversity and altered bacterial composition have been found in children with diseases such as necrotizing enterocolitis (NEC) and Hirschsprung's enterocolitis. [3][4][5] Treatment of these diseases may only require antibiotics, bowel rest, and supportive care, but more severe cases require an operation with or without diversion of luminal flow. The effects of luminal diversion on the intestinal microbiome remain unknown.…”

“…Small patient sample sizes represent another challenge to comprehensive analyses of microbiota in multiple pediatric operative diseases. 4,5,17,18 To determine the effect of operative disease and treatment on the pediatric intestinal microbiome, we compared mucosal bacteria from 43 samples by age, breastfeeding status, disease, acuity, anatomic location, and presence or absence of luminal flow. We evaluated the alpha diversity, beta diversity, and taxonomic composition of each group.…”