Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of ganglion cells in the distal intestine, leading to functional obstruction. Surgical removal of the aganglionic segment and restoration of bowel continuity to the anus are the primary treatments [1]. Despite surgical advancements, approximately one-third of patients experience postoperative complications [1]. Hirschsprung-associated enterocolitis (HAEC) remains a common and life-threatening complication in patients with HSCR, with a reported incidence of 25%–37% following definitive treatment [2]. Several studies have proposed multiple mechanisms predisposing to HAEC, including the disturbance of gut microbiota [1,3-7].

The gut microbiota plays a crucial role in maintaining intestinal homeostasis, and its composition has been linked to various pediatric gastrointestinal disorders, including inflammatory bowel disease (IBD) and short bowel syndrome [8]. However, detailed investigations of the gut microbiota in HSCR remain limited. Studies in animal models have demonstrated an increase in microbial diversity, with a predominance of the phyla Proteobacteria and Bacteroidetes [9,10]. In patients with HSCR, studies reported distinct differences in the gut microbiome between patients with and without HAEC [4,6,11]. Several studies in patients with HAEC found an increase in proteobacteria [6,11,12], especially Escherichia [12,13] and Enterobacteriaceae [11,14]. with a decrease in Bifidobacterium [12]. However, a detailed understanding of the gut microbiota in HSCR remains limited. Only a few studies have examined the longitudinal changes of gut microbiome in patients with HSCR before and after definitive surgery [15]. Therefore, this study aimed to: (1) compare gut microbiota profiles between children with HSCR and controls; (2) characterize longitudinal changes in the gut microbiota following definitive surgery; and (3) identify specific microbial features predictive of postoperative HAEC in high-risk patients.

Following these aims, this study was divided into 3 parts. First, we compared the fecal microbiota in 20 patients with 20 matched controls. Second, we characterized temporal alterations in the gut microbiota by analyzing samples collected at diagnosis, during surgery, 1-month, and 6-month postsurgery. Third, we collected intestinal content specimens from both the proximal and distal colon during surgery and then monitored clinical presentations of HAEC over 1 year to identify microbiome signatures predictive of postoperative HAEC.

HSCR is a functional intestinal obstruction in a distal aganglionic intestine. Consistent with previous animal and clinical studies, our findings confirm that HSCR is associated with gut dysbiosis, with significantly reduced alpha diversity and distinct beta diversity clustering when compared with age, delivery mode, and diet-matched controls [3,15,19]. While some animal models have reported increased microbial richness in obstructed segments due to delayed transit time and luminal stasis [10,20], our cohort showed compositional separation even against symptomatic children with HSCR-like constipation, supporting a disease-specific microbial signature beyond stasis alone. Although prior reports have described distinct microbiota profiles between proximal ganglionic and distal aganglionic segments [13,14], we did not observe marked regional beta diversity differences. This finding may be attributable to the preoperative bowel preparation protocol used at our center, which likely reduced the local microbial load and minimized segment-specific differences. Differential abundance analysis revealed enrichment of potentially pathogenic taxa, including Citrobacter and Fusobacterium, along with depletion of key butyrate-producing groups (NK4A214 group, Lachnospiraceae XPB1014 group) and the acetate producer Acetitomaculum. Notably, Citrobacter showed the highest log fold change among enriched taxa and is characterized as an enteric pathogenic bacterium [21]. This genus can produce glycoside hydrolases that break down the intestinal mucosal barrier, facilitating penetration by opportunistic bacteria and increasing the risk of local damage and infection [22]. Both clinical and preclinical studies have shown that increased abundance of Fusobacterium is linked to intestinal diseases, including IBDs, colitis, and colorectal cancer by promoting mucosal inflammation, releasing reactive oxygen species, and impairing the intestinal barrier [23,24]. Moreover, the depletion of butyrate and acetate-producing bacteria, well recognized for their anti-inflammatory and protective barrier roles, may further exacerbate intestinal pathology in HSCR by weakening mucosal integrity and amplifying local inflammatory responses [3,25].

Considering the changes in the gut microbiota of patients with HSCR from diagnosis to the postoperative period, we observed a significant decrease in alpha diversity at 1-month postsurgery compared to controls. This finding is consistent with a previous limited study comparing patients with HSCR and controls that revealed a decrease in microbiota richness in HSCR patients following surgery compared to controls [15]. Some studies have reported a reduction in the diversity of gut microbiota following rectal resection for rectal cancer, compared to the preoperative period and healthy adults [26]. This surgery is like the pull-through procedure in patients with HSCR, with a similar decrease in postoperative microbiota diversity. These findings suggest that surgery, particularly rectal resection, leads to significant alterations in bacterial communities. Additionally, 6 months after surgery, the gut microbiota differed from earlier time points, with an alpha diversity that had significantly increased compared to the pretreatment period, though it did not fully resemble the profiles of controls. This finding might indicate either a move towards equilibrium or persistent dysbiosis, as the gut microbiota at this stage still differed from that of the matched controls. These results are consistent with a previous report suggesting that persistent shift in gut microbiota after surgery may result from structural changes in the intestine, chronic inflammation and mucosal damage, the effects of perioperative antibiotics and other treatments, as well as dietary behaviors and environmental factors [15,27]. ANCOM-BC comparisons across time points revealed that significant differences in gut microbiota composition emerged only at 6 months postsurgery (visit 4), suggesting partial re-equilibration of the microbial community. This was characterized by enrichment of butyrate-producing genera, including Eubacterium within the order Eubacteriales, which gradually recovered by 6 months [28]. Prior studies have linked a reduction in butyrate-producing bacteria, such as Eubacterium, to increased intestinal inflammation and impaired gut barrier function in patients with IBD, as well as altered gut motility and stool consistency in children with functional constipation [29]. Therefore, the observed rebound in Eubacterium abundance may contribute to improved mucosal homeostasis and partial recovery of gut function in patients with HSCR. This is further supported by predictive functional analysis, which showed that at 6-month postsurgery (visit 4), there was a marked decrease in pathogenicity-related pathways, including enterobacterial toxin production, compared to diagnosis (visit 1) [11]. In parallel, pathways related to vitamin and metabolic biosynthesis increased, consistent with the recovery of butyrate-producing bacteria such as Eubacterium [25]. These results highlight that Eubacterium may act as a potential microbial biomarker to monitor postoperative gut re-equilibration and predict treatment success in HSCR.

HAEC is a serious complication. The incidence of postoperative HAEC in our study was 27.8%, aligned with previous reports (25%–37%) [2]. Several previous studies have proposed gut-microbial dysbiosis in patients with a history of HAEC and those predisposed to HAEC [3,13,14]. Nonetheless, the direct comparison of gut microbiota diversity across studies has been challenging due to variations in study design and specimen collection methods [3]. Focusing on studies with designs similar to ours, 2 studies investigated the microbiota of intestinal tissue during surgery and its association with the development of enterocolitis after surgery [5,11]. Due to differences in specimen collection and preparation, the results of these studies were discordant. Tang et al. [11] investigated fresh mucosal microbiota from the dilated colon in Chinese patients during surgery and discovered a reduction in alpha diversity among patients who later developed enterocolitis. In contrast, the study of Arbizu et al. [5] in American patients observed higher alpha diversity in aganglionic paraffin-fixed tissue at surgery in those who developed postoperative enterocolitis. In our study, which analyzed fecal gut microbiota during surgery, we observed a nonsignificant decrease in alpha diversity in the proximal ganglionic specimens and a nonsignificant increase in distal aganglionic specimens from patients who developed postoperative enterocolitis. These diversity patterns were in close alignment with previous research [5,11]. However, our results did not reach statistical significance. Beta diversity analysis revealed no significant differences in bacterial composition between the proximal and distal regions during surgery in patients with and without enterocolitis. Variations in gut microbiota diversity outcomes could be due to differences in specimen collection and preoperative bowel preparation protocols at our center. However, ANCOM-BC analysis identified significant taxa differences within the proximal and distal regions during surgery in HSCR patients who later developed enterocolitis and those who did not. We observed a significant increase in Olsenella in proximal ganglionic specimens. Elevated levels of Olsenella have been associated with inflammatory gut diseases, such as IBD, due to overproduction of lactic acid in the intestine [30]. In the distal aganglionic region, ANCOM-BC analysis showed a significant rise in the abundance of Holdemanella, Corynebacterium (family Corynebacteriaceae), Collinsella (family Coriobacteriaceae), and CAG-352. Consistent with previous studies on gastrointestinal inflammatory disorders, Holdemanella has been frequently detected in higher abundance among patients with IBD compared to the healthy controls [31]. Likewise, an increase in Collinsella has been observed in patients with IBD [30]. Mechanistically, Collinsella has been shown to alter bile acid metabolism and produce hydrogen sulfide, which can reduce colonic transit and increase gut permeability-factors that promote local stasis and mucosal inflammation [32,33]. Together, these features suggest that enrichment of these taxa may contribute to barrier dysfunction and an increased risk of postoperative HAEC. Comparison of ANCOM-BC results revealed that enriched taxa differed between the distal aganglionic and proximal ganglionic regions, reflecting niche-specific microbial dynamics. The distal colon, lacking enteric ganglion cells, exhibits impaired peristalsis, whereas the proximal colon maintains normal innervation and motility. These region-specific differences likely represent preoperative ecological variations that could influence host-microbe interactions beyond the resected segment. However, due to the scarcity of comparative studies, further large-scale prospective research is needed to validate these findings and investigate their potential implications for future therapeutic strategies.

These ecological shifts in the distal region were further supported by correlation analyses, which demonstrated that enriched pathogenic taxa were significantly linked to clinical parameters predisposing patients to postoperative HAEC. We also detected a correlation between previously known risk factors of postoperative HAEC and the distribution of gut microbiota. Lower weight at the time of surgery was associated with an increased abundance of Sphingomonas, Roseburia, Lachnospiraceae NK4A136 group, and Coprobacillus, and a reduction in Robinsoniella. Previous studies have proposed a correlation between lower weight at the time of surgery and early development of postoperative HAEC [34]. A previous study revealed a high incidence of HAEC at a younger age. Supporting this, our analysis detected a reduction in the abundance of Pseudomonas, Leptotrichia, and Gemella in patients diagnosed at a younger age [4]. Furthermore, our study found that a younger age at the time of surgery was associated with an increase in Incertae Sedis and Collinsella. Interestingly, an elevation in Collinsella was significantly correlated with postoperative obstruction, potentially due to its production of hydrogen sulfide, which reduces colonic transit [33]. Some studies have proposed a significant rise in the incidence of postoperative HAEC among patients with postoperative obstruction, which was also observed in our results [7]. However, after adjustment using multivariable logistic regression analysis, no significant differences were found in clinical parameters previously known to be risk factors for HAEC between the 2 groups. These findings may have been limited by the small sample size.

Predictive analysis indicated that patients with HSCR with enterocolitis exhibited increased pathways associated with inflammation and oxidative stress, and microbial energy metabolism. This elevation is consistent with previous reports and supports the notion that the gut microbiota in HAEC may actively promote mucosal inflammation while simultaneously initiating a compensatory antioxidant response to preserve the integrity of the mucosal barrier [3,35].

This study has several limitations. First, the small sample size limited statistical power, rendering some results inconclusive and lacking statistical significance. Second, the analysis of gut microbiota changes was confined to a 6-month postoperative period. Although no consensus exists regarding an optimal follow-up duration, this point was chosen based on clinical experience, as most patients with HSCR typically regain normal bowel function by 6-month postsurgery. At this stage, we observed significant alterations in gut microbial composition, although differences from controls persisted. This may reflect either progression toward microbial equilibrium or ongoing dysbiosis. Therefore, extended longitudinal studies beyond 6 months are necessary to determine whether these changes represent restoration of microbial homeostasis or continued dysregulation. Finally, consistent with many gut microbiome studies, interpretation is constrained by variability in study designs and sample collections. Additionally, factors such as race, environment, and geography substantially influence microbial composition and diversity, potentially limiting the generalizability of our findings. Variations in diet, lifestyle, and genetic background—particularly in Southeast Asia, where microbiome data remain limited—may contribute to the distinct microbial signatures observed in our cohort. To mitigate this, our study focused on microbiota analysis at the time of surgery to identify predictive microbial markers of postoperative HAEC and guide future preventive strategies.

These findings support the potential utility of microbial biomarkers—particularly Eubacterium and Eubacteriales—in monitoring postoperative recovery and predicting HAEC. Although overall microbial diversity did not significantly differ between patients with and without postoperative HAEC, differential taxa in proximal and distal segments suggest region-specific microbial dysbiosis. Collectively, these results highlight the role of gut microbiota in HAEC pathogenesis and underscore the need for larger, longitudinal studies to guide microbiota-based preventive strategies.