Bacterial Microbiome Dynamics in Post Pull-Through Hirschsprung-Associated Enterocolitis (HAEC): An Experimental Study Employing the Endothelin Receptor B-Null Mouse Model.

Zhi Cheng,Philip K. Frykman,Deepti Dhall,James Borneman,Paul M. Ruegger,Lifu Zhao

doi:10.3389/fsurg.2018.00030

Zhi Cheng, Philip K. Frykman + Show 4 more

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https://doi.org/10.3389/fsurg.2018.00030

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Abstract

PurposeHirschsprung-associated enterocolitis (HAEC) is the most frequent potentially life-threatening complication in children with Hirschsprung disease (HSCR) even after definitive corrective surgery. Mounting evidence suggests that intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to use a mouse model of post pull-through HAEC to compare the fecal bacterial communities of animals which developed HAEC to those free of enterocolitis.MethodsTen Ednrb−/− and 8wild type mice underwent the microsurgical pull-through surgery, and stool was collected at the time of surgery, and then either at 2 and 4 weeks after the operation, or when the mice developed enterocolitis. The mid-colon of all animals was collected, prepared and histologically graded for enterocolitis. Fecal DNA was isolated and bacterial 16S rRNA genes analyzed using Illumina sequencing.ResultsSix Ednrb−/− mice developed HAEC with a mean enterocolitis score of 5.7, while the remaining 4 mutant and 8 WT mice remained free of enterocolitis by 4 weeks. The HAEC group had lower alpha diversity by Chao1 analysis compared with WT group, while the Ednrb−/− mice demonstrated distinct bacterial communities from WT mice on beta diversity analysis. The most striking finding was increased proportion of Akkermansia and reduced Bacteroidetes compared with the NO HAEC and WT groups, suggesting Akkermansia may contribute to development of enterocolitis while Bacteroidetes may be protective. Less abundant genera that were reduced in HAEC were Dysgonomas and Clostridium XIVa which may play a protective role.ConclusionsThis is the first study to identify Akkermansia as potentially playing a role in HAEC, either as a pathobiont taxa contributing to pathogenesis of enterocolitis, or possibly a protective commensal taxa expanded in response to inflammation. These findings characterized the dynamic shifts in the gut microbial communities through the onset of post pull-through HAEC, and suggests that there may be identifiable bacterial community differences in HSCR patients that are high risk for developing HAEC.

Highlights

Hirschsprung-associated enterocolitis (HAEC) is the most frequent potentially life-threatening complication of Hirschsprung disease (HSCR) patients [1]
We found a significant increase in proportion of phyla Verrucomicrobia and reduction in Bacteroidetes in animals that developed HAEC, (HAEC 2 compared with HAEC 0, Figure 5, Tables S2, S3) suggesting that Verrucomicrobia may be contributing to the onset of the HAEC, while Bacteroidetes may protect against enterocolitis
When we focused on the bacteria at the genera level, we were not surprised to find Akkermansia expanded in the animals that developed HAEC (HAEC 2 compared with HAEC 0), given that it falls under the phylum Verrucomicrobia, supporting that Akkermansia may be contributing to enterocolitis (Figure 6, Table S4)

Summary

Introduction

Hirschsprung-associated enterocolitis (HAEC) is the most frequent potentially life-threatening complication of Hirschsprung disease (HSCR) patients [1]. While the etiology of HAEC is not clear, recent clinical studies have identified marked fecal bacterial and fungal community differences in patients with HAEC compared with those free from enterocolitis [4,5,6]. Some of the challenges with interpreting these human studies is variability between individual patient diets, antibiotic exposure, type of operation for HSCR, as well as lack of clear definition of what constitutes HAEC. To address some of these shortcomings, we created an experimental system to investigate HAEC bacterial community dynamics from the time of pull-through surgery to 4 weeks after surgery. We studied Ednrb−/− and wild type littermate control mice at the time of colon pull-through surgery, 2 and 4 weeks after surgery. We compared the colonic bacterial populations of mice which developed enterocolitis with those free of enterocolitis

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