A Metagenomics Study on Hirschsprung's Disease Associated Enterocolitis: Biodiversity and Gut Microbial Homeostasis Depend on Resection Length and Patient's Clinical History

Abstract

Objectives: Since 2010, several researches demonstrated that microbiota dynamics correlate and can even predispose to Hirschsprung (HSCR) associated enterocolitis (HAEC). This study aims at assessing the structure of the microbiota of HSCR patients in relation to extent of aganglionosis and HAEC status. Methods: All consecutive HSCR patients admitted to Gaslini Institute (Genova, Italy) between May 2012 and November 2014 were enrolled. Institutional review board (IRB) approval was obtained. Stools were sampled and 16S rDNA V3-V4 regions were sequenced using the Illumina-MiSeq. Taxonomy assignments were performed using QIIME RDP. Alpha diversity indexes were analyzed by Shannon and Simpson Indexes, and Phylogenetic Diversity. Results: We enrolled 20 patients. Male to female ratio was 4:1. Six patients suffered from Total Colonic Aganglionosis (TCSA). Considering sample site (i.e., extent of aganglionosis), we confirmed the known relationship between sample site and both biodiversity and composition of intestinal microbiota. Patients with TCSA showed lower biodiversity and increased Proteobacteria/Bacteroidetes relative abundance ratio. When addressing biodiversity, composition and dynamics of TCSA patients we could not find any significant relationship with regard to HAEC occurrences. Conclusions: The composition of HAEC predisposing microbiota is specific to each patient. We could confirm that total colon resections can change the composition of intestinal microbiota and to dramatically reduce microbial diversity. The subsequent reduction of system robustness could expose TCSA patients to environmental microbes that might not be part of the normal microbiota. Future long-term studies should investigate both patients and their family environment, as well as their disease history.

Keywords: Hirschsprung; RET gene; aganglionosis; enterocolitis; metagenomics.

Figure 1

The diversity of gut microbial communities was assessed through four alpha diversity measures. Together, these results suggest that ileal stools belonging to TCSA carry lower microbiota diversity.

Figure 2

We assessed microbial composition by comparing the relative abundances of taxa at phylum level. When considering the communities at the phylum level, differences between RSA and TCSA microbiota were clearly evident. Bacteroidetes represented over 33% of all bacteria in RSA and where basically absent (<2%) in TCSA. Similarly, Proteobacteria accounted for nearly 40% of all bacteria in TCSA and for <5% in RSA. Conversely, Firmicutes and Actinobacteria were present both in RSA and TCSA without statistically significant differences.

Figure 3

Diversity and composition in TCSA before and after surgery. X-axis depicts timepoints of sample collection: pre-operative (“1”) and post-operative (“3”). Subjects A, K, and C either developed HAEC postoperatively (pre-HAEC—HAEC status at stool sampling) or preoperatively (HAEC—HAEC status at stool sampling). Subjects L, N, and Q never developed. The top graph of each panel represents alpha diversity, normalized to values of sample collected pre-operatively, with each color line representing a different metric. The dotted line on each graph is set to 1.0. The bottom panel illustrates relative abundance of taxa at genus level.