Colonoscopy is a commonly used method for screening and treating intestinal diseases in children. Achieving good preparation of the bowel is crucial for improving diagnostic and therapeutic outcomes. The drug of choice for bowel preparation is polyethylene glycol (PEG) due to its high efficacy, safety, and ease of use, as confirmed by several studies. Both adult and child guidelines recommend PEG as the preferred laxative for bowel preparation (Pashankar et al., 2004; Safder et al., 2008; Pall et al., 2014; ASGE Standards of Practice Committee et al., 2015; Tsunoda et al., 2017; Mytyk et al., 2018). Split-dose PEG is commonly used for bowel preparation in adults before colonoscopy (Samarasena et al., 2012; Zawaly et al., 2019). Research has shown that split-dose PEG is more effective than a single dose for bowel preparation in children (Tripathi et al., 2020).

The human microbiome is composed of various microorganisms, including bacteria, archaea, fungi, protozoa, and viruses, with more than 100 times the number of genes as the human body (Qin et al., 2010; Ursell et al., 2012). These genes encode pathways that produce biologically active molecules from diet and metabolism (Lynch and Pedersen, 2016). The gut microbiota plays a vital role in obtaining energy from dietary fiber and linking its metabolites to the occurrence of various diseases, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) (Shreiner et al., 2015). Although previous studies have shown that colonoscopy and bowel preparation affect gut microbiota, there is still limited research in children (Drago et al., 2019). Furthermore, the impact of split-dose PEG bowel preparation on changing gut microbiota in children has not been well studied, and there are no metagenomic studies in this area. To obtain a more comprehensive view of the microbial community, metagenomics was employed for investigating gut microbiota of children in this study. Metagenomics can detect both bacterial and non-bacterial microorganisms, providing information about the functional potential of the microbial community. In contrast, 16S rRNA sequencing only detects bacterial microorganisms and does not provide functional potential information. Thus, our study aimed to characterize the potential differences in gut microbiota composition and functional capacity in children undergoing colonoscopy with split-dose PEG bowel preparation.

The impact of bowel preparation on the composition of gut microbiota was first reported by Mai’s team in 2006 (Mai et al., 2006). Since then, there have been a total of studies on this subject, with only one study reported in children ( Table 1 ) (Mai et al., 2006; Harrell et al., 2012; Gorkiewicz et al., 2013; O'Brien et al., 2013; Jalanka et al., 2015; Drago et al., 2016; Shobar et al., 2016; Shaw et al., 2017; Powles et al., 2022). These studies comprised a sample size ranging from 4 to 23 and included both healthy and those with diseases such as IBD. Most of the studies utilized 16S rRNA, with no reported use of metagenomics. Thus, our study was the first to utilize metagenomics in investigating the effects of split-dose PEG bowel preparation and colonoscopy on gut microbiota in children.

Our study revealed a decrease in alpha diversity on the second day after bowel preparation, which gradually returned to the level before bowel preparation over the following two weeks. This finding is consistent with another study that reported a decrease in alpha diversity three days after bowel preparation that returned to pre-preparation levels at six weeks (Powles et al., 2022). Additionally, another study reported a reduction in the Shannon index only in biopsy samples from IBD patients (Shobar et al., 2016). These results suggest that bowel preparation does indeed affect alpha diversity, but its levels return to normal over time. However, beta diversity was not influenced by bowel preparation at the phylum, genus, and species levels. We observed distinct dots, which indicates that the composition of gut microbiota was altered after bowel preparation. To date, no studies have reported on the effect of bowel preparation and colonoscopy on beta diversity of gut microbiota.

Our study did not reveal any significant difference at the phylum level, which was consistent with previous research (Harrell et al., 2012). However, some studies have reported changes at the phylum level following bowel preparation (Gorkiewicz et al., 2013; Jalanka et al., 2015; Drago et al., 2016; Shaw et al., 2017). Notably, in these studies, the PEG dose was relatively high or a single dose was used. It should be noted that one of these studies reported that bowel preparation using a single dose led to more alterations in gut microbiota than two separate doses (Jalanka et al., 2015). This suggests that split-dose PEG bowel preparation only has a minor effect on gut microbiota at the phylum level.

At the genus level, we found that six genera, including Eubacterium, Escherichia, Intertinibacter, Veillonella, Ruminococcaceae_unclassified and Coprobacillus, were significantly different. Some increased after bowel preparation, while some decreased, but most of them returned to normal after 2 weeks. Many studies have reported changes at the genus level after bowel preparation (Harrell et al., 2012; Jalanka et al., 2015; Shobar et al., 2016; Shaw et al., 2017). Each study reported that different genera are changed after bowel preparation. A pediatric study showed a significant increase in Faecalibacterium and significant decreases in Ruminococcus, Escherichia, Pseudobutyrivibrio and Subdoligranulum (Shaw et al., 2017). This was different from what we reported. This may be related to the origin of the patients. Although our two subjects were about the same age, we took more stool samples than that study. We collected 19 stool samples at each time period, but that study collected 11 pre-bowel preparation stool samples, 7 post-bowel preparation stool samples, seventeen biopsy samples, sixteen luminal content samples and eighteen swabs taken at colonoscopy.

At the species level, we discovered that the abundance of Escherichia coli, Bacteroides fragilis and Veillonella parvula increased significantly at two days after bowel preparation, gradually decreased two weeks later and gradually recovered. The abundance of Intertinibacter bartlettii decreased obviously at two days after bowel preparation, recovered at two weeks later, and approached the preoperative level at four weeks. To date, no reports have been published on the effects of bowel preparation and colonoscopy on gut microbiota at the species level.

In our final analysis, we evaluated the functional diversity of gut microbiota at time points during bowel preparation. Our findings revealed a distinct decrease in the pathway of biosynthesis of siderophore group nonribosomal peptide two days after bowel preparation. Additionally, four pathways (baseision repair, biosynthesis of ansamycins, flavonoid biosynthesis, and biosynthesis of type II polyketide products) increased two days after bowel preparation, but all pathways recovered their pre-preparation levels within two weeks and gradually continued to recover. Our findings indicate that bowel preparation can decrease the biosynthesis of siderophore group nonribosomal peptide, while enhancing base excision repair, biosynthesis of ansamycins, flavonoid biosynthesis, and biosynthesis of type II polyketide products. Such effects of bowel preparation on gut microbiota have not been reported previously.

The results of our study show that after bowel preparation, the composition and functional pathways of gut microbiota undergo a change, which then gradually returns to baseline, demonstrating the stability and resilience of gut microbiota. Such qualities are considered basic ecological characteristics of gut microbiota (Lozupone et al., 2012). The ability of gut microbiota to restore its balance after infection with a pathogen or antibiotic treatment is referred to as resilience (Sommer et al., 2017; Palleja et al., 2018). The gut microbiota’s stability and resilience are affected by factors such as unhealthy status, antibiotic use, and dietary factors (Fassarella et al., 2021). Our study represents the first utilization of metagenomics in children with split-dose PEG bowel preparation to understand the stability and resilience of gut microbiota. We also discovered that the primary outcome across studies in this field both in children and adults is the compositional alteration of gut microbiota after bowel preparation and colonoscopy, after which it recovers with time. Our findings are generally consistent with previous studies, indicating that split-dose PEG can be widely utilized for bowel preparation in children.

Our study had two limitations. Firstly, no detailed food frequency questionnaires were included which could account for the potential influence of diet on the observed differences in gut microbiota. Secondly the disease spectrum of the subjects was not consistent, which may have influenced the results.

In conclusion, our research indicates that split-dose PEG bowel preparation and colonoscopy induce changes in gut microbiota of children at the genus and species levels, as well as in functional pathways. However, the phylum level remains unaffected. It is possible that future bowel preparation research may target these compositional and functional changes in gut microbiota. Our study also exhibits the stability and resilience of gut microbiota, laying the foundation for future research.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: European Bioinformatic Institute (EBI, https://www.ebi.ac.uk/) database under accession code PRJEB56144.

The studies involving human participants were reviewed and approved by Ethics Committee of Shenzhen Children’s Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Conceptualization: YZ, SHZ, SMZ, JZ. Data collection: MC. Formal analysis: SL, QF. Funding acquisition: SMZ, JZ. Project administration: YZ, SHZ, SMZ, JZ. Writing original draft: YZ, SHZ, SMZ, JZ. Writing-review & editing: YZ, SHZ, SMZ, JZ. All authors have read and approved the manuscript. All authors contributed to the article and approved the submitted version.