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

Abstract

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.

Fig 1. The intestinal microbiome pattern of the HAEC-R patients is more similar to that of the HAEC patients than to that of the HD patients, and the HD patients have a relatively distinct, stable community compared with those of the HAEC and HAEC-R patients.

(A) Principal coordinate analysis (PCoA) of unweighted UniFrac distances for patients with different diagnoses. PC1, first principal coordinate; PC2, second principal coordinate. Numbers represent the patient ID numbers. The phylogenetic composition of the fecal microbiota in the HAEC-R patients was more similar to that of the HAEC patients (ANOSIM, p > 0.05) than to that of the HD patients (ANOSIM, p < 0.01). (B) The weighted UniFrac distances between the most distant samples of each patient. The microbiota of each HAEC or HAEC-R patient is more similar between the sample sites (Student’s t-test, p > 0.05) than that of each HD patient (Student’s t-test, p < 0.01).

Fig 2. Relative abundance of phyla in intestinal microbiomes.

Relative abundance of phyla in the intestinal microbiomes of the HD, HAEC, and HAEC-R patients. (A) The pie charts show the average relative abundance of five major phyla. (B) The histograms demonstrate the bacterial composition of the stool samples at the phylum level. The X-axis shows the sample numbers. The colors assigned to each of the five detected phyla are shown at the right side.

Fig 3. Relative abundance of genera in intestinal microbiomes.

While Escherichia was the most prominent genus detected in the HAEC (47%) and HAEC-R (61%) patients, Bacteroides (38%) was the most prominent genus in the HD patients.

Fig 4. The cladogram generated by the LEfSe software illustrates the taxa with different abundance among the three groups (HD, HAEC, and HAEC-R).

Different taxa are colored according to the most abundant class, and the size of the circle corresponds to the relative abundance of the taxon.

Fig 5. Relative abundance of four species those are most different in the abundance in the guts of the HD, HAEC, and HAEC-R patients.

Relative abundance of Bacteroides fragilis (A), Faecalibacterium prausnitzii (B), Veillonella parvula (C), and Veillonella dispar (D) in the intestines of five HD patients, five HAEC patients, and three HAEC-R patients.