Characterization Of The Intestinal Microbiome Of Hirschsprung’s Disease With Or Without Enterocolitis Using Illumina-mi Seq High-throughput Sequencing And Clinical Risk Factors Analysis Of Recurrent Enterocolitis

Objective: To investigate the characterization of intestinal microbiome variation between HD and HAEC patients,and among HD,HAEC and HAEC remission(HAEC-R)patients using Illumina-MiSeq high-throughput sequencing.Methods: 2 HD and 2 HAEC patients were enrolled into the first part of study,the intestine content were sampled from different sites within the gut of each patient during surgery and a total of 13 samples were collected.Besides,5 HD,5 HAEC and 3 HAEC-R patients were enrolled into the second part of study,a total of 35 samples were collected during surgery.Total DNA was isolated from each sample using the PowerSoil DNA isolation kit.The V4 region of bacterial 16 S rRNA was amplified from each total DNA sample using bacteria/archaeal primers: 515 F and 806 R.16S rRNA gene sequences were clustered into operational taxonomic units(OTUs)at 97%,analyzed using RDP classifier bayesian algorithm.Alpha diversity and Beta diversity were calculated using QIIME(version:1.8.0).Linear discriminant analysis coupled with effect size measurements(LefSe)was utilized for detecting differences in the abundance of bacterial species among groups.A retrospective analysis was conducted on 67 cases of HD treated by radical operation.Patients suffered 3 times or more episodes of enterocolitis were enrolled into the recurrent HAEC group(HAEC-Rt group).Patients with 1 or 2 times episodes were enrolled into the HAEC group.Patients without enterocolitis were enrolled into the control group.The clinical data were collected and risk factors analyzed by SPSS19.0,including gender and age at surgery,clinical types of HD,preoperative malnutrition,hypoalbuminemia,enterocolitis,upper respiratory tract infection and pneumonia.Results: The intestine microbiota differed significantly between the HD and HAEC patients(P < 0.05),and the microbiota of HAEC-R patients was more similar to that of HAEC patients(P > 0.05).The diversity of the microbiome of intestine with and without ganglion cells(Proximal and Distal)had no siginficant difference(P > 0.05).Besides,we also observed that specimens from different intestinal sites in each HD patient differed significantly(P < 0.05),whilst specimens from different intestinal sites in each HAEC and HAEC-R patients were more similar(P > 0.05).Furthermore,in the retrospective analysis,fisher exact test showed that gender and age at surgery had no statistical differences among the three groups(P > 0.05);Clinical types of HD and the incidence of hypoalbuminemia had no statistical differences between the HAEC-Rt and the HAEC group(P > 0.05).The incidences of preoperative malnutrition,enterocolitis,upper respiratory tract infection and pneumonia in the HAEC-Rt group were significantly higher than that in the HAEC group(P < 0.05),and no statistical differences between the HAEC group and the control group(P > 0.05).The multivariate Logistic regression analysis showed that the incidences of preoperative malnutrition and enterocolitis were independent risk factors of recurrent HAEC(OR = 9.000,95% CI = 1.355-59.783;OR = 8.667,95% CI = 1.526-49.220).Conclusions: The intestinal microbiome of HAEC and HD patients differed significantly,however,the microbiota of HAEC-R patients was more similar to that of HAEC patients.It implied that the intestinal microbiota associated with enterocolitis may persist following resolution of symptoms,and could be implicated in the symptom recurrence.Presence of ganglion cells maybe not a major determinant of the intestinal microbiome composition,however,the colonization of specific gut pathogen may be contributed to HAEC occurrence.Besides,the gender,age and clinical types of HD showed no correlation with recurrent enterocolitis attacks.The incidences of preoperative malnutrition and enterocolitis were the risk factors of recurrent HAEC.Furthermore,further larger longitudinal studies will be needed to reveal the role of the intestinal microbiome pattern in the course of HAEC(attack/ remission/ recurrence)and provide a new therapy for prevention and treatment of HAEC,and further larger prospective clinical RCT study will be conducted to explore the role of improving the intestinal microbiome pattern in prevention and treatment of HAEC recurrent attacks.