Correlation between changes in intestinal microbiota and defecatory function before and after radical resection of right-sided colon cancer

Correlation between changes in intestinal microbiota and defecatory function before and after radical resection of right-sided colon cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Correlation between changes in intestinal microbiota and defecatory function before and after radical resection of right-sided colon cancer Sohei Akuta, Masataka Hirasaki, Yasumitsu Hirano, Naoe Akimoto, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9337728/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Postoperative bowel dysfunction after colorectal cancer surgery impairs quality of life, but its longitudinal course and relationship with the gut microbiota after right-sided colon cancer surgery remain unclear. We retrospectively analyzed 60 patients who underwent curative resection for right-sided colon cancer between November 2023 and November 2024. Bowel function was assessed preoperatively and at 1, 6, and 12 months postoperatively using the Bristol Stool Form Scale, Constipation Scoring System, and Wexner score. Gut microbiota was analyzed by full-length 16S rRNA sequencing of fecal samples collected preoperatively and at 1 and 6 months after surgery. At 1 month, stool softening, incomplete evacuation, and incontinence to liquid stool were observed in 42, 21, and 5 patients, respectively. These symptoms improved after 6 months and generally returned to preoperative levels by 12 months. Microbiota analysis showed decreased α-diversity and marked shifts in β-diversity during the early postoperative period, followed by gradual reconstruction with interindividual variability. Collectively, bowel dysfunction after right-sided colon cancer surgery was common but largely transient, whereas gut microbiota changes were predominantly time-dependent rather than symptom-specific, suggesting generalized ecological reconstruction rather than direct microbial correlates of individual bowel symptoms. Biological sciences/Cancer Health sciences/Gastroenterology Health sciences/Medical research Biological sciences/Microbiology Health sciences/Oncology Bowel function Colorectal cancer Gut microbiota Quality of life Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer is one of the most commonly diagnosed malignancies worldwide and the second leading cause of cancer-related mortality [ 1 ]. Surgical resection remains the cornerstone of curative treatment for patients with resectable advanced colorectal cancer. Owing to advances in multimodal therapy, the overall 5-year survival rate for colorectal cancer in the United States has reached approximately 65% ( https://seer.cancer.gov/statfacts/html/colorect.html ; accessed January 31, 2026). With improved survival outcomes, increasing attention has been directed toward long-term quality of life (QOL) and functional outcomes after treatment. Among postoperative functional disorders, bowel dysfunction is a particularly important concern that substantially impairs patients’ QOL. Traditionally, postoperative bowel dysfunction has been most prominently discussed in the context of rectal cancer surgery, wherein unavoidable injury to the pelvic autonomic nerves can result in low anterior resection syndrome (LARS). Approximately 40–60% of patients are classified as having major LARS at 1 year after surgery, with persistent symptoms, such as fecal incontinence, urgency, and frequent bowel movements [ 2 , 3 ]. Bowel dysfunction may occur even after colon cancer surgery, despite the absence of direct injury to the pelvic floor or pelvic autonomic nerves. Following colon resection, 24.1% of patients experienced incontinence to liquid stool , 33.3% reported a sensation of incomplete evacuation, and 31.4% experienced difficulty with defecation [ 4 ]. Furthermore, a prospective multicenter longitudinal study demonstrated that approximately 30% of patients undergoing right-sided colon cancer surgery had significantly higher frequency of loose stools at 1 year after surgery, with similar rates persisting at 3 years [ 5 ]. Symptoms, such as fecal incontinence, urgency, and incomplete evacuation, are strongly associated with patient distress, underscoring postoperative bowel dysfunction as a clinically relevant issue, even in right-sided colon cancer [ 5 ]. The standard surgery for right-sided colon cancer involves resection of the ileocecal region, including removal of the ileocecal valve, followed by ileocolic anastomosis. Ileocecal valve resection may permanently alter intestinal transit and luminal conditions. Loss of the physical barrier between the small and large intestines may disrupt compartmentalization of the gut microbiota and substantially modify the intestinal ecosystem. Previous studies have reported partial perturbation and recovery of the gut microbiota following gastrointestinal surgery, suggesting that surgical intervention influences microbial dynamics [ 6 , 7 ]. Moreover, several studies have demonstrated that probiotic administration improves bowel movement frequency, sensation of incomplete evacuation, and QOL in patients after colorectal cancer surgery. Meta-analyses have also shown a reduction in postoperative ileus and earlier recovery of flatus and defecation [ 8 – 11 ]. These findings support the hypothesis that the gut microbiota may play a role in maintaining postoperative bowel function and development of bowel dysfunction. In patients with rectal cancer, reduced microbial diversity and depletion of beneficial bacteria are associated with LARS severity [ 12 ]. However, molecular investigations of alterations in the gut microbiota following colon cancer surgery, particularly in right-sided colon cancer, remain limited. Although small intestinal bacterial overgrowth has been implicated in 73% of patients with chronic diarrhea after right hemicolectomy, gene-sequencing–based microbiota analyses have not been conducted in such studies, and molecular characterization of microbial changes after ileocecal valve resection remains obscure [ 13 ]. Recently, longitudinal studies have shown that the gut microbiota undergoes marked time-dependent alterations after colorectal surgery, with substantial perturbation in the early postoperative period followed by gradual reconstruction accompanied by interindividual variability [ 14 ]. These, microbial shifts may also affect functional outputs such as short-chain fatty acid production and could be associated with postoperative bowel symptoms, including stool consistency and diarrhea frequency [ 15 ]. However, studies simultaneously evaluating longitudinal bowel function and comprehensive gut microbiota dynamics within the same cohort, specifically in patients with right-sided colon cancer, remain limited. Against this background, in the present study, we aimed to assess the longitudinal changes in postoperative bowel dysfunction in patients undergoing right-sided colon cancer surgery and comprehensively evaluated time-dependent alterations in the gut microbiota, thereby deciphering the relationship between bowel function and microbial dynamics. Materials and Methods Study design and patients This single-center retrospective observational study was conducted at the Saitama Medical University International Medical Center. Patients who underwent curative resection (ileocecal resection or right hemicolectomy) for right-sided colon cancer between November 2023 and November 2024 were eligible for inclusion. Patients with pathological stage I–III disease according to the TNM classification were included. Right-sided colon cancer was defined as cancer arising in the cecum, ascending colon, or transverse colon. The exclusion criteria were as follows: synchronous or metachronous double cancer, history of inflammatory bowel disease, severe hepatic or renal dysfunction, regular antibiotic use, preoperative use of probiotics or intestinal regulators, and preoperative chemotherapy. The target sample size was set at 60 patients, based on previously published longitudinal gut microbiota studies with similar designs. Clinical data, including age, sex, body mass index (BMI), American Society of Anesthesiologists Physical Status (ASA-PS) score, history of abdominal surgery, comorbidities, tumor-related factors (tumor location, histological differentiation, and TNM stage), surgical factors (procedure, surgical approach, operative time, extent of lymph node dissection, and length of bowel resection), postoperative complications, and postoperative medication use (probiotics, antibiotics, and adjuvant chemotherapy), were retrospectively collected from electronic medical records. Postoperative complications were evaluated according to the Clavien–Dindo classification, and complications of grade II or higher were included in the analysis [ 12 , 16 ]. Assessment of bowel function Bowel function was assessed using self-administered questionnaires preoperatively and at 1, 6, and 12 months postoperatively. The following validated scoring systems were used: Bristol stool form scale (BSFS), constipation scoring system (CSS), and Wexner score [ 17 – 19 ]. Changes in scores between the preoperative period and 1 month postoperatively were calculated. Items showing statistically significant changes were selected for further analyses. For each selected item, patients were classified into two groups: the aggravation group, with patients whose score changed in the direction indicating worsening symptoms; and the lighthearted group, comprising patients with no worsening of the score. Fecal sample collection and DNA extraction Fecal samples were collected at three time points: preoperatively, and at 1 and 6 months postoperatively. The samples were collected at home using a fecal collection kit for intestinal environment analysis (TechnoSuruga Laboratory Co., Ltd.) and stored at 4°C for up to 10 days before processing. Total DNA was extracted from fecal samples using the ISOSPIN Fecal DNA Kit (Nippon Gene, Tokyo, Japan). Bacterial cell walls were mechanically disrupted by bead beating to release DNA, and the DNA was purified using a silica membrane spin column according to the manufacturer’s protocol. 16S rRNA gene sequencing on the MinIONTM platform Full-length 16S rRNA gene sequencing (V1–V9 region) was performed using the MinION platform (Oxford Nanopore Technologies, ONT, UK). The 16S rRNA gene was amplified using the universal primers: 27F (5′-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5′-CGGYTACCTTGTTACGACTT-3′). PCR amplification was performed using the KAPA2G Robust HotStart ReadyMix PCR Kit (Kapa Biosystems, MA, USA) with 200 nM of each primer in a total reaction volume of 25 µL. PCR conditions were as follows: initial denaturation at 95°C for 3 min, 30 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 30 s. Amplicons (~ 1,600 bp) were confirmed via electrophoresis on a 1% agarose gel (in 1× TAE buffer). PCR products were purified using AMPure XP beads (Beckman Coulter). Library preparation was performed using the Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies). Libraries were loaded onto FLO-MIN114 (R10.4.1) flow cells. Up to nine samples were sequenced simultaneously per run using a MinION Mk1B device. Sequencing was conducted using the MinKNOW software (Oxford Nanopore Technologies). Bioinformatic analyses Raw nanopore reads were processed using the MinKNOW software. A high-accuracy base-calling mode was applied with a minimum Q-score threshold of 9. Barcode sequences were trimmed automatically and FASTQ files were generated for each sample. Read-length filtering was performed using SeqKit version 0.10.0 [ 20 – 22 ], and reads between 1,300 and 1,950 bp (corresponding to the V1–V9 region) were retained. To minimize the effect of differences in sequencing depth across samples, rarefaction was performed to the minimum read count of 15,000 reads per sample using SeqKit, with a fixed random seed (-s 123) to ensure reproducibility. Taxonomic classification was performed using the GenomeSync database ( http://genomesync.org ), which includes bacterial and archaeal genomes as well as the human genome for contamination filtering. The corresponding NCBI Taxonomy data were downloaded from GenomeSync. Reads were mapped to the reference genomes using minimap2 via an internal Perl-based Genome Search Toolkit (Genome Search Toolkit: https://genomesync.org/tools/gstk-with-tools-2022-04-19/gstk-with-tools-m1_mac-2022-04-19.zip ) [ 23 , 24 ]. Each read was assigned to the species with the highest alignment score, followed by hierarchical taxonomic annotation (genus, family, etc.) according to NCBI Taxonomy. Taxa with ≤ 1 read per sample were excluded from the analysis. The relative abundance (%) was calculated by dividing the read count of each taxon by the total number of reads per sample. Statistical analysis All statistical analyses were performed using R version 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria) and EZR version 1.63 (Saitama Medical Center, Jichi Medical University, Japan). Pre- and postoperative bowel function scores were compared using the Wilcoxon signed-rank test. Continuous variables were expressed as median (interquartile range [IQR]) or mean ± standard deviation (SD), as appropriate. Categorical variables are presented as counts and percentages. A two-sided p -value < 0.05 was considered to indicate statistical significance. α-diversity analysis The alpha diversity indices included the Shannon index, Simpson index, observed richness, and Hill number (q = 1). These indices were calculated based on the genus-level relative abundance data. Longitudinal changes were analyzed using linear mixed-effects models (LMMs). Sampling time (Pre, 1M, and 6M), phenotype group (aggravation vs. lighthearted), and their interaction (time × group) were included as fixed effects, with patient ID included as a random effect (random intercept only). The random slopes for time were not included. This model structure accounted for repeated measurements within patients and baseline interindividual variability. The normality of residuals was assessed visually using Pearson residual plots against fitted values, normal Q–Q plots (qqnorm and qqline), and histograms of residuals ( Supplementary Fig. S1 ). For α-diversity indices showing significant effects ( p < 0.05), estimated marginal means (EMMs) were calculated, and post-hoc pairwise comparisons (Pre–1M, Pre–6M, and 1M–6M) were performed. Holm’s method was used to adjust for multiple comparisons. Distributions and within-patient changes were visualized using violin plots. β-diversity analysis Beta diversity was evaluated using Bray–Curtis distances calculated from the genus-level relative abundance data. To assess longitudinal changes, within-patient Bray–Curtis distances were calculated for Pre–1M and 1M–6M intervals. Paired Wilcoxon signed-rank tests were used to compare the distances between intervals. The results are presented as medians and IQR, and samples from the same patient are connected graphically. Permutational multivariate analysis of variance (PERMANOVA) with 999 permutations was performed based on Bray–Curtis distance matrices to evaluate the effects of sampling time and phenotype groups on the overall microbial community structure. Patient ID was included as a stratification factor. A permutational analysis of multivariate dispersions (PERMDISP) was also conducted to confirm that the PERMANOVA results were not driven by differences in within-group dispersion. Ethical statement This study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Saitama Medical School International Medical Center (Approval No. 2023-082, UMIN000052095). Results Patient characteristics The patient characteristics are shown in Table 1. Sixty patients with right-sided colon cancer were included. The tumor location was most frequently observed in the ascending colon ( n = 30, 50.0%), followed by the cecum ( n = 18, 30.0%), transverse colon ( n = 10, 16.7%), and appendix ( n = 2, 3.3%). Regarding surgical procedures, 36 patients (60.0%) underwent ileocecal resection and 24 patients (40.0%) underwent right hemicolectomy. According to the pathological TNM classification, 22 (36.7%) patients had stage I disease, 34 (56.7%) had stage II disease, and 4 (6.7%) had stage III disease. None of the patients received any perioperative chemotherapy. Proton-pump inhibitors were administered to 11 (18.3%) patients. None of the patients had received probiotics or antibiotics at baseline. Assessment of bowel function The mean BSFS score significantly increased from 3.28 ± 1.08 preoperatively to 4.68 ± 1.03 at 1 month after surgery ( p < 0.01), indicating softening of stool consistency ( Fig. 1a ). Thereafter, the mean score decreased to 3.88 ± 0.81 at 6 months and to 3.73 ± 0.64 at 12 months, approaching preoperative values. No significant difference was observed in the total CSS scores between the preoperative and 1-month postoperative assessments ( p = 0.16) ( Fig. 1b ). However, item-level analysis revealed that the sensation of incomplete evacuation significantly increased from 0.48 ± 1.02 preoperatively to 1.12 ± 1.42 at 1 month after surgery ( p < 0.01), with subsequent improvement at 6 and 12 months. Similarly, no significant difference was observed in the total Wexner score between the preoperative and 1-month postoperative assessments ( p = 0.07) ( Fig. 1c ). However, incontinence to liquid stoolincontinence to liquid stool, which was absent preoperatively, transiently increased to 0.15 ± 0.48 at 1 month ( p = 0.05) and resolved by 12 months of surgery. Based on these findings, BSFS, sensation of incomplete evacuation, and incontinence to liquid stoolincontinence to liquid stool were defined as bowel function parameters that showed significant postoperative changes (Supplementary Tables S1–3). At 1 month after surgery, stool softening (increase in the BSFS score) was observed in 42 (70.0%) patients, whereas incomplete evacuation and incontinence to liquid stoolincontinence to liquid stool were observed in 21 (35.0%) and 5 (8.3%) patients, respectively. Sequencing read counts across longitudinal samples Full-length 16S rRNA gene sequencing using the MinION™ platform was performed for fecal samples collected at all time points (Pre, 1M, and 6M). The distribution of the filtered sequencing reads at each time point is shown in Supplementary Fig. S2a . The median (IQR) number of reads was 45,313 (26,856) for Pre, 50,322.5 (23,876) for 1M, and 46,349 (18,499) for 6M ( Supplementary Fig. S2b ) samples. A linear mixed-effects model analysis with patient ID as a random effect revealed a significant main effect of time ( p = 0.0278). Post-hoc comparisons using estimated marginal means with Holm correction revealed a significant decrease in read counts between 1M and 6M ( p = 0.0267) samples ( Supplementary Fig. S2c,d ). Because variability in total read counts could introduce bias in downstream analyses, microbial composition analysis was performed after rarefaction to 15,000 reads per sample. A total of 1,527 bacterial species were identified after filtering. Longitudinal changes in α-diversity Alpha diversity indices, including the Shannon index, Simpson index, observed richness, and Hill number (q = 1), were calculated for Pre, 1M, and 6M fecal samples. The distributions and within-patient trajectories are shown in Fig. 2a, b . The Shannon index, observed richness, and Hill number (q = 1) decreased at 1 month after surgery, and this reduction persisted when compared with the values for the Pre and 6M samples ( Fig. 2a, b ). Linear mixed-effects model analysis (with patient ID as a random effect) revealed a significant main effect of time for the Shannon index ( p = 5.20 × 10⁻⁷), Observed richness ( p = 2.90 × 10⁻¹¹), and Hill number (q = 1) ( p = 2.90 × 10⁻¹¹) ( Fig. 2c; Supplementary Table S4 ). Post-hoc pairwise comparisons with Holm adjustment showed significant differences in all three indices for Pre–1M and Pre–6M contrasts, whereas no significant difference was observed between 1M and 6M ( Supplementary Table S4 ). These findings indicate an early postoperative reduction in microbial diversity without a clear recovery by 6 months. In contrast, the Simpson index showed no significant main effect of time ( p = 0.112), and no significant differences were observed in the pairwise comparisons ( Supplementary Table S4 ). Association between bowel function and gut microbial α-diversity Based on the questionnaire assessments at 1 month after surgery, differences according to the presence of symptoms were observed for incomplete evacuationincomplete evacuation, BSFS, and incontinence to liquid stool. Therefore, longitudinal changes in each α-diversity index were evaluated using linear mixed-effects models with phenotype (aggravation/lighthearted) and sampling time point (Pre, 1M, and 6M) as fixed effects ( Fig. 3a–c, Supplementary Table S5 ). For incomplete evacuationincomplete evacuation, the main effect of time was significant for the Shannon index ( p = 7.65 × 10⁻⁶), observed richness ( p = 4.06 × 10⁻¹⁰), and Hill number (q = 1) ( p = 4.11 × 10⁻⁷). In contrast, neither the main effect of group nor the group × time interaction was significant ( Supplementary Table S5 ). Post-hoc comparisons revealed significant differences primarily in the lighthearted group between Pre and 1M and Pre–6M. For the BSFS, the main effect of time was also significant for the Shannon index, observed richness, and Hill number (q = 1), whereas neither the main effect of group nor the interaction term was statistically significant ( Supplementary Table S5 ). A decrease in α-diversity was observed in both the groups during the early postoperative period. For fecal incontinence, only the observed richness showed a significant main effect of time ( p = 0.0022). No statistically significant changes were detected in the Shannon index or Hill number (q = 1) ( Supplementary Table S5 ). In the post-hoc analyses, significant differences between Pre–1M and Pre–6M were identified in the lighthearted group. At all time points, no significant between-group differences (aggravation vs. lighthearted) were observed for any α-diversity index, and no significant group × time interactions were detected ( Supplementary Table S5 ). These findings indicate that the early postoperative decline in α-diversity observed in the overall analysis was not restricted to a specific symptom group but primarily reflected time-dependent changes. However, more pronounced alterations were observed in the lighthearted group for certain indices, suggesting a possible contribution of interindividual variability. Longitudinal changes in β-diversity The gut microbial β-diversity was assessed using Bray–Curtis distances calculated from genus-level relative abundance data. To examine longitudinal changes, paired comparisons were performed within the same patients for the Pre–1M and 1M–6M intervals. The Bray–Curtis distance for the 1M–6M interval was significantly lower than that for the Pre–1M interval (Wilcoxon signed-rank test, p = 1.24 × 10⁻⁷) ( Fig. 4a ). Visualization of individual trajectories revealed that most patients experienced marked shifts in microbial composition during the early postoperative period, followed by reduced changes thereafter. When the distributions were summarized as median and IQR, the Pre–1M interval (median 0.807) showed higher distances than the 1M–6M interval (median 0.594) ( Fig. 4b ). PERMANOVA based on the Bray–Curtis distance matrix revealed that the sampling time significantly contributed to the overall variation in microbial composition ( R² = 0.033, p = 0.001; Supplementary Table S6 ). Additionally, PERMDISP analysis revealed significant differences in dispersion over time, indicating that temporal changes were associated with shifts in the community structure ( Supplementary Table S6 ). Association between bowel function and gut microbial β-diversity PERMANOVA, accounting for repeated measures within patients, was conducted for each phenotype (incomplete evacuation, BSFS, and fecal incontinence). For all phenotypes, the main effect of time was significant (all p = 0.001), indicating that the gut microbial community structure changed over time ( Fig. 5a–c, Supplementary Table S7 ). In contrast, neither the main effect of group nor the time × group interaction was significant, suggesting that the temporal changes in the microbial community were not phenotype-specific ( Supplementary Table S7 ). In paired comparisons of Bray–Curtis distances between the Pre–1M and 1M–6M intervals, the Pre–1M distance was significantly greater than the 1M–6M distance in both the groups across all phenotypes (Wilcoxon signed-rank test, p < 0.05; Supplementary Table S7 ). These results indicated that the largest changes in the microbial composition occurred during the early postoperative period. Furthermore, Friedman tests across the three time points (Pre, 1M, and 6M) revealed significant temporal changes in both the groups for each phenotype ( Supplementary Table S7 ). Post-hoc analyses showed significant differences primarily between Pre and 1M and between Pre and 6M, whereas no significant differences were detected between 1M and 6M. PERMDISP analysis revealed that, for incomplete evacuation, dispersion differences were observed only for time, indicating that changes in β-diversity mainly reflected temporal shifts in the community structure. In contrast, for BSFS and fecal incontinence, significant differences in dispersion were observed for both group and time, indicating that for these symptoms, β-diversity differences may reflect not only shifts in community centroids but also interindividual variability in microbial responses ( Supplementary Table S7 ). It should be noted that the number of patients in the aggravation group for fecal incontinence was small, which may have limited the statistical power. Discussion In the present study, we longitudinally evaluated the postoperative changes in bowel function and gut microbiota in patients undergoing surgery for right-sided colon cancer. At 1 month after surgery, bowel dysfunction, including a softer stool consistency, incomplete evacuation, and incontinence to liquid stool, was frequently observed. However, these symptoms were transient and improved after 6 months. Microbiome analyses revealed marked alterations in both α- and β-diversity during the early postoperative period, followed by a time-dependent microbial reconstruction. Notably, these microbial changes were driven predominantly by postoperative time course rather than by individual bowel symptom phenotypes. With regard to stool consistency, the BSFS score increased significantly 1 month after surgery, indicating stool softening. This finding is consistent with previous reports of assessment after right hemicolectomy and may be attributable to shortened intestinal transit time and bile acid malabsorption associated with ileocecal valve resection [13]. Although no significant deterioration was observed in the overall CSS or Wexner total scores, item-level analyses revealed transient increases in incomplete evacuation and incontinence to liquid stool during the early postoperative period. These changes likely represent clinically meaningful symptoms as perceived by patients, even in the absence of significant changes in the composite scores. In the analysis of gut microbial α-diversity, the Shannon index, Hill number (q = 1), and observed richness decreased at 1 month after surgery, and this trend persisted through 6 months. In contrast, no significant changes in the Simpson index were observed. These findings suggest that while the overall microbial diversity and moderately abundant taxa decreased after surgery, the composition of the dominant taxa was relatively preserved, which is consistent with previous reports [25,26]. The reduction in observed richness further indicates a selective loss of rare taxa. Besides anatomical alterations associated with right-sided colectomy and ileocecal valve resection, perioperative factors, such as antibiotic exposure and dietary changes, may have collectively influenced microbial homeostasis [27]. Regarding the association between bowel function and gut microbiota, phenotype-stratified analyses for incomplete evacuation and fecal incontinence revealed that early postoperative decline in α-diversity was statistically significant only in the lighthearted group. However, neither between-group comparisons nor interaction effects were significant, making it difficult to conclude whether bowel dysfunction and microbial alterations are directly linked in a simple, one-to-one relationship. Rather, interindividual differences in responsiveness to postoperative environmental changes and possible temporal discrepancies between symptom perception and microbiota alterations may have contributed to these findings. β-diversity analyses revealed that changes in the microbial structure were most pronounced during the early postoperative period, with the magnitude of change decreasing thereafter. Phenotype-stratified analyses indicated that shifts in the microbial composition were primarily time-dependent and not specifically associated with individual bowel dysfunction phenotypes. However, in cases of stool softening (BSFS) and fecal incontinence, variability in dispersion indicates substantial interindividual differences in microbial responses. Taken together, these findings indicate that postoperative alterations in the gut microbiota following gastrointestinal surgery are characterized by marked early shifts in β-diversity, followed by gradual reconstruction, with considerable interindividual variability. Notably, the relationship between microbial changes and clinical symptoms appears to be heterogeneous rather than uniform. In the present study, postoperative microbial alterations were more strongly associated with postoperative time course than with individual bowel symptom phenotypes, suggesting that symptom-specific microbial signatures were not evident. Rather, these changes are likely influenced by multiple factors, including host characteristics, surgical stress, reconstruction type, and postoperative environmental conditions. These results are consistent with those of previous systematic reviews, suggesting that alterations in the microbiota after digestive tract surgery are multifactorial and are not solely determined by symptom expression or clinical outcomes [28,29]. This study had several limitations. First, this was a single-center observational study with a relatively small sample size. Second, bowel function was assessed using a questionnaire-based subjective measures. In addition, microbiome analyses were limited to structural evaluations based on diversity indices, and specific bacterial taxa or functional alterations were not investigated. A major strength of this study is the longitudinal evaluation of time-dependent changes using paired samples from the same patients. By analyzing within-patient trajectories, we were able to characterize postoperative microbial dynamics while minimizing interindividual variability. Future studies should include larger prospective cohorts and incorporate functional analyses, including metabolic profiling and detailed assessments of specific bacterial taxa, to further clarify the relationship between postoperative bowel dysfunction and alterations in the gut microbiota. The novelty of this study lies in demonstrating that postoperative bowel dysfunction symptoms after right-sided colectomy are common but largely transient, and that postoperative gut microbiota changes are predominantly time-dependent rather than symptom-specific. Although stool softening, incomplete evacuation, and incontinence to liquid stool were frequently observed during the early postoperative period, these symptoms were not accompanied by distinct microbial signatures. Instead, the gut microbiota showed a pattern of early disruption followed by gradual reconstruction, suggesting a generalized ecological adaptation to postoperative intestinal environmental change rather than persistent pathological dysbiosis. These findings provide a new framework for understanding postoperative bowel dysfunction after right-sided colectomy as a multifactorial and time-dependent adaptive process, and may help inform future individualized intervention strategies. Declarations Acknowledgments We thank the staff of the Division of Analytical Science, Hidaka Branch, Biomedical Research Center, Saitama Medical University, for providing the research equipment and valuable technical advice. English language editing was performed by Editage (www.editage.jp). Author Contributions SA contributed to patient questionnaire administration and sample collection, performed statistical analyses, and drafted the manuscript. MH prepared the library for nanopore sequencing, conducted data analysis, and drafted the manuscript. NA extracted DNA from the patient stool samples. YM supported the patient questionnaire administration and sample collection. MO, YS, and YK1 provided advice on statistical analyses. HN provided technical advice on nanopore sequencing. YH, YI, HS, CH, TH, and YK2 designed and supervised the study and revised the manuscript. All the authors have read and approved the final version of the manuscript. Data Availability Raw sequences were deposited in the DDBJ (https://www.ddbj.nig.ac.jp/dra/index-e.html) with the accession number PRJDB40334. However, a correspondence table linking patient identification codes to personal information is not publicly available due to privacy and ethical constraints. When an application for the secondary use of sequence data is submitted, we ask the applicant to present the purpose of the use and review the pros and cons of granting access before making a decision. Masataka Hirasaki (hirasaki@saitama-med. ac. jp) handled the applications. Ethics Approval and Consent to Participate This study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Saitama Medical School International Medical Center (Approval No. 2023-082, UMIN000052095). Informed consent was waived because of the retrospective design and opt-out options provided. Consent for Publication Not applicable. Declaration of Conflicting Interests The authors declare no competing interests. Funding Statement This study was supported by a Grant-in-Aid for Young Researchers from the Saitama Medical University International Medical Center (4-D-1-14) (SA). References Bray, F. et al. 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Prophylactic effects of probiotics or synbiotics on postoperative ileus after gastrointestinal cancer surgery: A meta-analysis of randomized controlled trials. PLoS One 17, e0264759; 10.1371/journal.pone.0264759 (2022). D’Amico, F. et al. Tools for fecal incontinence assessment: lessons for inflammatory bowel disease trials based on a systematic review. United European Gasteroenterol J. 8, 886–922 (2020). Agachan, F., Chen, T., Pfeifer, J., Reissman, P. & Wexner, S. D. A constipation scoring system to simplify evaluation and management of constipated patients. Dis. Colon Rectum 39, 681–685 (1996). Blake, M. R., Raker, J. M. & Whelan, K. Validity and reliability of the Bristol Stool Form Scale in healthy adults and patients with diarrhoea-predominant irritable bowel syndrome. Aliment. Pharmacol. Ther. 44, 693–703 (2016). Shen, W., Le, S., Li, Y. & Hu, F. SeqKit: A cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS One 11, e0163962; 10.1371/journal.pone.0163962 (2016). Komiya, S. et al. MinION, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting. BMC Med. Genomics 15, 68; 10.1186/s12920-022-01218-8 (2022). Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590-D596 (2013). Kryukov, K., Nakagawa, S. & Imanishi, T. GenomeSync: a synchronizable database of genome sequences. iDarwin 4, 4–23 (2024). Kryukov, K., Imanishi, T. & Nakagawa, S. Nanopore sequencing data analysis of 16S rRNA genes using the GenomeSync-GSTK system. Methods Mol. Biol. 2632, 215–226 (2023). Suzuki, Y. et al. Comparison of pre- and postoperative gut microbiota diversity in patients with rectal cancer undergoing stoma creation and closure. Ann. Gastroenterol. Surg. 10.1002/ags3.70094 (2025). Png, C. W., Chua, Y. K., Law, J. H., Zhang, Y. & Tan, K. K. Alterations in co-abundant bacteriome in colorectal cancer and its persistence after surgery: a pilot study. Sci. Rep. 12, 9829 (2022). Park, S. S. et al. The effect of curative resection on fecal microbiota in patients with colorectal cancer: a prospective pilot study. Ann. Surg. Treat. Res. 99, 44–51 (2020). Ferrie, S., Webster, A., Wu, B., Tan, C. & Carey, S. Gastrointestinal surgery and the gut microbiome: a systematic literature review. Eur. J. Clin. Nutr. 75, 12–25 (2020). Tarazi, M., Jamel, S., Mullish, B. H., Markar, S. R. & Hanna, G. B. Impact of gastrointestinal surgery upon the gut microbiome: A systematic review. Surgery 171, 1331–1340 (2022). Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table20260311akuta.xlsx SupTable20260311.xlsx SupplementaryFigure20260303.pptx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 May, 2026 Reviews received at journal 06 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviews received at journal 26 Apr, 2026 Reviewers agreed at journal 26 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers invited by journal 24 Apr, 2026 Editor invited by journal 16 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 06 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9337728","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":634095526,"identity":"685b9970-010a-415d-adae-07bb9a5dc496","order_by":0,"name":"Sohei Akuta","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sohei","middleName":"","lastName":"Akuta","suffix":""},{"id":634095527,"identity":"ebcea05f-0f15-45bd-910f-e585ec48a212","order_by":1,"name":"Masataka Hirasaki","email":"data:image/png;base64,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","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Hirasaki","suffix":""},{"id":634095528,"identity":"4b2bf7e8-8da7-4fea-8a7b-ed54ce7d2c3c","order_by":2,"name":"Yasumitsu Hirano","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yasumitsu","middleName":"","lastName":"Hirano","suffix":""},{"id":634095529,"identity":"1eed599b-351b-4814-91e2-e488e74bfad8","order_by":3,"name":"Naoe Akimoto","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Naoe","middleName":"","lastName":"Akimoto","suffix":""},{"id":634095530,"identity":"6754a0b1-8015-4e39-8e57-acc0f156d796","order_by":4,"name":"Yume Minagawa","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yume","middleName":"","lastName":"Minagawa","suffix":""},{"id":634095531,"identity":"30196308-288b-4dec-a801-0b78a110efb9","order_by":5,"name":"Yasuhiro Ishiyama","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yasuhiro","middleName":"","lastName":"Ishiyama","suffix":""},{"id":634095532,"identity":"84de8ba5-edb8-446a-9ea7-bb07e9471d52","order_by":6,"name":"Hirofumi Sugita","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hirofumi","middleName":"","lastName":"Sugita","suffix":""},{"id":634095533,"identity":"dfa85c4b-d23b-40f3-89c3-13569de97df6","order_by":7,"name":"Chikashi Hiranuma","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Chikashi","middleName":"","lastName":"Hiranuma","suffix":""},{"id":634095534,"identity":"aac3b49d-eb0a-47ef-8f23-8109c58d2821","order_by":8,"name":"Maiko Osawa","email":"","orcid":"","institution":"Saitama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Maiko","middleName":"","lastName":"Osawa","suffix":""},{"id":634095535,"identity":"949d5f81-e489-40fa-b118-b64204cee3a3","order_by":9,"name":"Yuki Shiko","email":"","orcid":"","institution":"Saitama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Shiko","suffix":""},{"id":634095536,"identity":"3666e31e-8138-46fd-8384-459a86482198","order_by":10,"name":"Yohei Kawasaki","email":"","orcid":"","institution":"Saitama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yohei","middleName":"","lastName":"Kawasaki","suffix":""},{"id":634095537,"identity":"597927b2-fd47-4310-be22-ff2c7a3511d2","order_by":11,"name":"Hiroaki Nakanishi","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Nakanishi","suffix":""},{"id":634095538,"identity":"4df06b0f-30fa-4ddc-9ae1-0bf641f3eb8f","order_by":12,"name":"Tetsuya Hamaguchi","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Tetsuya","middleName":"","lastName":"Hamaguchi","suffix":""},{"id":634095539,"identity":"ca5710e6-1fea-4f78-acf4-a524e17e0b19","order_by":13,"name":"Yusuke Kinugasa","email":"","orcid":"","institution":"Saitama Medical University International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Kinugasa","suffix":""}],"badges":[],"createdAt":"2026-04-07 00:08:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9337728/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9337728/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109405243,"identity":"796e9d5e-2af7-409b-acbc-b4ce641dd36d","added_by":"auto","created_at":"2026-05-17 13:06:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal changes in bowel function after right hemicolectomy.\u003c/strong\u003e\u003cbr\u003e\nLongitudinal changes in bowel function among 60 patients undergoing right hemicolectomy are shown at preoperative baseline (Pre) and at 1 (1M), 6 (6M), and 12 (12M) months postoperatively. Changes in the (\u003cstrong\u003ea\u003c/strong\u003e) Bristol stool form scale (BSFS), (\u003cstrong\u003eb\u003c/strong\u003e) Constipation scoring system (CSS), and (\u003cstrong\u003ec\u003c/strong\u003e) Wexner score over time are presented. (\u003cstrong\u003ed\u003c/strong\u003e ) Summary of bowel function scores at each evaluation time point. Continuous variables are expressed as mean ± standard deviation (SD). \u003cem\u003eP\u003c/em\u003e-values represent comparisons between Pre and 1M calculated using the Wilcoxon signed-rank test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/c665aa78fd51a61d5c9eb2d8.png"},{"id":108976919,"identity":"39c92d02-43c8-4aa0-b433-c431309f0b64","added_by":"auto","created_at":"2026-05-11 11:29:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal changes in α-diversity indices before and after surgery.\u003c/strong\u003e\u003cbr\u003e\n (\u003cstrong\u003ea-b\u003c/strong\u003e) Longitudinal changes in α-diversity indices (Chao1, Simpson, and Shannon) at preoperative baseline (Pre), and at 1 (1M) and 6 (6M) months postoperatively in the overall cohort are shown. Each panel displays violin plots overlaid with individual patient data points and spaghetti plots illustrate within-patient longitudinal trajectories. (\u003cstrong\u003ec\u003c/strong\u003e) Statistical analyses were performed using linear mixed-effects models (LMMs) accounting for repeated measurements within patients to evaluate the main effect of time. For indices with a significant main effect of time, post hoc pairwise comparisons were conducted using estimated marginal means (emmeans) with Holm correction for multiple testing. The table in the lower panel presents adjusted \u003cem\u003eP\u003c/em\u003e values for comparisons between time points (Pre–1M, Pre–6M, and 1M–6M). Asterisks (*) indicate statistical significance (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/3f11abfe97d1d288a3dd640f.png"},{"id":108837697,"identity":"f151c5d4-f139-4d76-a6ce-0b0ca3dbdf63","added_by":"auto","created_at":"2026-05-09 00:14:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal changes in gut microbial α-diversity according to bowel function phenotype.\u003c/strong\u003e\u003cbr\u003e\nLongitudinal changes in gut microbial α-diversity are shown according to bowel function phenotype (Aggravation and Lighthearted groups) in patients undergoing right hemicolectomy. (a) Incomplete evacuation. (b) Bristol Stool Form Scale (BSFS). (c) Incontinence to liquid stool . Each panel presents α-diversity indices at preoperative baseline (Pre), and at 1 (1M) and 6 (6M) months postoperatively, evaluated using the Shannon index, Simpson index, observed richness, and Hill number (q = 1). Each dot represents an individual patient, and samples from the same patient are connected by lines to illustrate longitudinal changes. Violin plots depict the distribution at each time point. Within-group temporal changes were assessed using linear mixed-effects models. Asterisks (*) indicate statistically significant differences identified in post hoc comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/21638ae89ad0267d190f41dd.png"},{"id":108977171,"identity":"731652d8-0698-4252-b3c3-a944e3d00ddf","added_by":"auto","created_at":"2026-05-11 11:30:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal changes in β-diversity.\u003c/strong\u003e\u003cbr\u003e\nGut microbial β-diversity was evaluated using Bray–Curtis distances. (\u003cstrong\u003ea\u003c/strong\u003e) Comparison of Bray–Curtis distances within the same patients between the Pre–1M and 1M–6M intervals. Each dot represents an individual sample, and paired samples from the same patient are connected by lines. (\u003cstrong\u003eb\u003c/strong\u003e) Median and interquartile range (IQR) of Bray–Curtis distances for each interval. Compared with the Pre–1M interval, the Bray–Curtis distance was significantly lower in the 1M–6M interval (Wilcoxon signed-rank test, \u003cem\u003eP\u003c/em\u003e = 1.24 × 10⁻⁷). “N” in the figure indicates the number of analyzable samples in each interval. Paired analyses were performed in patients for whom data were available for both intervals.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/fb5ed965dd280a28d1523265.png"},{"id":108977391,"identity":"ff022f4e-cdcf-4db9-8fec-b42d7c85db5d","added_by":"auto","created_at":"2026-05-11 11:31:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal changes in gut microbial β-diversity according to bowel function phenotype.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLongitudinal changes in gut microbial β-diversity according to bowel function phenotype (Aggravation and Lighthearted groups) are shown in patients undergoing right hemicolectomy. (a) Incomplete evacuation. (b) Bristol Stool Form Scale (BSFS). (c) Incontinence to liquid stool . β-diversity was evaluated using Bray–Curtis distances based on genus-level relative abundance data. Each panel presents the Bray–Curtis distances for the Pre–1M and 1M–6M intervals. Each dot represents an individual patient, and paired samples from the same patient are connected by lines. Orange indicates the Pre–1M interval, and blue indicates the 1M–6M interval. Within-group differences between the intervals were assessed using the Wilcoxon signed-rank test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/ec5397495d8eb7c4f897e7d9.png"},{"id":108837694,"identity":"ae4c2934-025c-43d8-9a7f-e7196ae7ccba","added_by":"auto","created_at":"2026-05-09 00:14:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":262204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/ec731c08-4b49-4e3c-b71d-755bcc46da91.pdf"},{"id":108837695,"identity":"6e2a3a14-f319-481a-95bb-35a18a85e4da","added_by":"auto","created_at":"2026-05-09 00:14:58","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10726,"visible":true,"origin":"","legend":"","description":"","filename":"Table20260311akuta.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/e01bdad765d2e50ea30803ee.xlsx"},{"id":108837699,"identity":"738470da-e00a-47a8-8439-c6106b426489","added_by":"auto","created_at":"2026-05-09 00:14:58","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24035,"visible":true,"origin":"","legend":"","description":"","filename":"SupTable20260311.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/0af5c9540672580d3e421cf9.xlsx"},{"id":108837700,"identity":"860e1102-c704-4a27-94e9-9308c3d3402c","added_by":"auto","created_at":"2026-05-09 00:14:58","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":688994,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure20260303.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9337728/v1/87ede0e9472b2bc4cffd69a3.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Correlation between changes in intestinal microbiota and defecatory function before and after radical resection of right-sided colon cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer is one of the most commonly diagnosed malignancies worldwide and the second leading cause of cancer-related mortality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Surgical resection remains the cornerstone of curative treatment for patients with resectable advanced colorectal cancer. Owing to advances in multimodal therapy, the overall 5-year survival rate for colorectal cancer in the United States has reached approximately 65% (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://seer.cancer.gov/statfacts/html/colorect.html\u003c/span\u003e\u003cspan address=\"https://seer.cancer.gov/statfacts/html/colorect.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed January 31, 2026). With improved survival outcomes, increasing attention has been directed toward long-term quality of life (QOL) and functional outcomes after treatment.\u003c/p\u003e \u003cp\u003eAmong postoperative functional disorders, bowel dysfunction is a particularly important concern that substantially impairs patients\u0026rsquo; QOL. Traditionally, postoperative bowel dysfunction has been most prominently discussed in the context of rectal cancer surgery, wherein unavoidable injury to the pelvic autonomic nerves can result in low anterior resection syndrome (LARS). Approximately 40\u0026ndash;60% of patients are classified as having major LARS at 1 year after surgery, with persistent symptoms, such as fecal incontinence, urgency, and frequent bowel movements [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Bowel dysfunction may occur even after colon cancer surgery, despite the absence of direct injury to the pelvic floor or pelvic autonomic nerves. Following colon resection, 24.1% of patients experienced incontinence to liquid stool , 33.3% reported a sensation of incomplete evacuation, and 31.4% experienced difficulty with defecation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, a prospective multicenter longitudinal study demonstrated that approximately 30% of patients undergoing right-sided colon cancer surgery had significantly higher frequency of loose stools at 1 year after surgery, with similar rates persisting at 3 years [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Symptoms, such as fecal incontinence, urgency, and incomplete evacuation, are strongly associated with patient distress, underscoring postoperative bowel dysfunction as a clinically relevant issue, even in right-sided colon cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe standard surgery for right-sided colon cancer involves resection of the ileocecal region, including removal of the ileocecal valve, followed by ileocolic anastomosis. Ileocecal valve resection may permanently alter intestinal transit and luminal conditions. Loss of the physical barrier between the small and large intestines may disrupt compartmentalization of the gut microbiota and substantially modify the intestinal ecosystem. Previous studies have reported partial perturbation and recovery of the gut microbiota following gastrointestinal surgery, suggesting that surgical intervention influences microbial dynamics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, several studies have demonstrated that probiotic administration improves bowel movement frequency, sensation of incomplete evacuation, and QOL in patients after colorectal cancer surgery. Meta-analyses have also shown a reduction in postoperative ileus and earlier recovery of flatus and defecation [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings support the hypothesis that the gut microbiota may play a role in maintaining postoperative bowel function and development of bowel dysfunction. In patients with rectal cancer, reduced microbial diversity and depletion of beneficial bacteria are associated with LARS severity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, molecular investigations of alterations in the gut microbiota following colon cancer surgery, particularly in right-sided colon cancer, remain limited. Although small intestinal bacterial overgrowth has been implicated in 73% of patients with chronic diarrhea after right hemicolectomy, gene-sequencing\u0026ndash;based microbiota analyses have not been conducted in such studies, and molecular characterization of microbial changes after ileocecal valve resection remains obscure [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, longitudinal studies have shown that the gut microbiota undergoes marked time-dependent alterations after colorectal surgery, with substantial perturbation in the early postoperative period followed by gradual reconstruction accompanied by interindividual variability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These, microbial shifts may also affect functional outputs such as short-chain fatty acid production and could be associated with postoperative bowel symptoms, including stool consistency and diarrhea frequency [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, studies simultaneously evaluating longitudinal bowel function and comprehensive gut microbiota dynamics within the same cohort, specifically in patients with right-sided colon cancer, remain limited. Against this background, in the present study, we aimed to assess the longitudinal changes in postoperative bowel dysfunction in patients undergoing right-sided colon cancer surgery and comprehensively evaluated time-dependent alterations in the gut microbiota, thereby deciphering the relationship between bowel function and microbial dynamics.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy design and patients\u003c/h2\u003e\n \u003cp\u003eThis single-center retrospective observational study was conducted at the Saitama Medical University International Medical Center. Patients who underwent curative resection (ileocecal resection or right hemicolectomy) for right-sided colon cancer between November 2023 and November 2024 were eligible for inclusion. Patients with pathological stage I\u0026ndash;III disease according to the TNM classification were included. Right-sided colon cancer was defined as cancer arising in the cecum, ascending colon, or transverse colon. The exclusion criteria were as follows: synchronous or metachronous double cancer, history of inflammatory bowel disease, severe hepatic or renal dysfunction, regular antibiotic use, preoperative use of probiotics or intestinal regulators, and preoperative chemotherapy. The target sample size was set at 60 patients, based on previously published longitudinal gut microbiota studies with similar designs. Clinical data, including age, sex, body mass index (BMI), American Society of Anesthesiologists Physical Status (ASA-PS) score, history of abdominal surgery, comorbidities, tumor-related factors (tumor location, histological differentiation, and TNM stage), surgical factors (procedure, surgical approach, operative time, extent of lymph node dissection, and length of bowel resection), postoperative complications, and postoperative medication use (probiotics, antibiotics, and adjuvant chemotherapy), were retrospectively collected from electronic medical records. Postoperative complications were evaluated according to the Clavien\u0026ndash;Dindo classification, and complications of grade II or higher were included in the analysis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAssessment of bowel function\u003c/h3\u003e\n\u003cp\u003eBowel function was assessed using self-administered questionnaires preoperatively and at 1, 6, and 12 months postoperatively. The following validated scoring systems were used: Bristol stool form scale (BSFS), constipation scoring system (CSS), and Wexner score [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Changes in scores between the preoperative period and 1 month postoperatively were calculated. Items showing statistically significant changes were selected for further analyses. For each selected item, patients were classified into two groups: the aggravation group, with patients whose score changed in the direction indicating worsening symptoms; and the lighthearted group, comprising patients with no worsening of the score.\u003c/p\u003e\n\u003ch3\u003eFecal sample collection and DNA extraction\u003c/h3\u003e\n\u003cp\u003eFecal samples were collected at three time points: preoperatively, and at 1 and 6 months postoperatively. The samples were collected at home using a fecal collection kit for intestinal environment analysis (TechnoSuruga Laboratory Co., Ltd.) and stored at 4\u0026deg;C for up to 10 days before processing. Total DNA was extracted from fecal samples using the ISOSPIN Fecal DNA Kit (Nippon Gene, Tokyo, Japan). Bacterial cell walls were mechanically disrupted by bead beating to release DNA, and the DNA was purified using a silica membrane spin column according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e16S rRNA gene sequencing on the MinIONTM platform\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull-length 16S rRNA gene sequencing (V1\u0026ndash;V9 region) was performed using the MinION platform (Oxford Nanopore Technologies, ONT, UK). The 16S rRNA gene was amplified using the universal primers: 27F (5\u0026prime;-AGRGTTYGATYMTGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-CGGYTACCTTGTTACGACTT-3\u0026prime;). PCR amplification was performed using the KAPA2G Robust HotStart ReadyMix PCR Kit (Kapa Biosystems, MA, USA) with 200 nM of each primer in a total reaction volume of 25 \u0026micro;L. PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 3 min, 30 cycles of 95\u0026deg;C for 15 s, 55\u0026deg;C for 15 s, and 72\u0026deg;C for 30 s. Amplicons (~\u0026thinsp;1,600 bp) were confirmed via electrophoresis on a 1% agarose gel (in 1\u0026times; TAE buffer). PCR products were purified using AMPure XP beads (Beckman Coulter). Library preparation was performed using the Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies). Libraries were loaded onto FLO-MIN114 (R10.4.1) flow cells. Up to nine samples were sequenced simultaneously per run using a MinION Mk1B device. Sequencing was conducted using the MinKNOW software (Oxford Nanopore Technologies).\u003c/p\u003e\n\u003ch3\u003eBioinformatic analyses\u003c/h3\u003e\n\u003cp\u003eRaw nanopore reads were processed using the MinKNOW software. A high-accuracy base-calling mode was applied with a minimum Q-score threshold of 9. Barcode sequences were trimmed automatically and FASTQ files were generated for each sample. Read-length filtering was performed using SeqKit version 0.10.0 [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and reads between 1,300 and 1,950 bp (corresponding to the V1\u0026ndash;V9 region) were retained. To minimize the effect of differences in sequencing depth across samples, rarefaction was performed to the minimum read count of 15,000 reads per sample using SeqKit, with a fixed random seed (-s 123) to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003eTaxonomic classification was performed using the GenomeSync database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genomesync.org\u003c/span\u003e\u003c/span\u003e), which includes bacterial and archaeal genomes as well as the human genome for contamination filtering. The corresponding NCBI Taxonomy data were downloaded from GenomeSync.\u003c/p\u003e\n\u003cp\u003eReads were mapped to the reference genomes using minimap2 via an internal Perl-based Genome Search Toolkit (Genome Search Toolkit: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genomesync.org/tools/gstk-with-tools-2022-04-19/gstk-with-tools-m1_mac-2022-04-19.zip\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Each read was assigned to the species with the highest alignment score, followed by hierarchical taxonomic annotation (genus, family, etc.) according to NCBI Taxonomy.\u003c/p\u003e\n\u003cp\u003eTaxa with \u0026le;\u0026thinsp;1 read per sample were excluded from the analysis. The relative abundance (%) was calculated by dividing the read count of each taxon by the total number of reads per sample.\u003c/p\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll statistical analyses were performed using R version 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria) and EZR version 1.63 (Saitama Medical Center, Jichi Medical University, Japan). Pre- and postoperative bowel function scores were compared using the Wilcoxon signed-rank test. Continuous variables were expressed as median (interquartile range [IQR]) or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), as appropriate. Categorical variables are presented as counts and percentages. A two-sided \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e\u0026alpha;-diversity analysis\u003c/h2\u003e\n \u003cp\u003eThe alpha diversity indices included the Shannon index, Simpson index, observed richness, and Hill number (q\u0026thinsp;=\u0026thinsp;1). These indices were calculated based on the genus-level relative abundance data. Longitudinal changes were analyzed using linear mixed-effects models (LMMs). Sampling time (Pre, 1M, and 6M), phenotype group (aggravation vs. lighthearted), and their interaction (time \u0026times; group) were included as fixed effects, with patient ID included as a random effect (random intercept only). The random slopes for time were not included. This model structure accounted for repeated measurements within patients and baseline interindividual variability. The normality of residuals was assessed visually using Pearson residual plots against fitted values, normal Q\u0026ndash;Q plots (qqnorm and qqline), and histograms of residuals (\u003cstrong\u003eSupplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). For \u0026alpha;-diversity indices showing significant effects (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), estimated marginal means (EMMs) were calculated, and post-hoc pairwise comparisons (Pre\u0026ndash;1M, Pre\u0026ndash;6M, and 1M\u0026ndash;6M) were performed. Holm\u0026rsquo;s method was used to adjust for multiple comparisons. Distributions and within-patient changes were visualized using violin plots.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e\u0026beta;-diversity analysis\u003c/h3\u003e\n\u003cp\u003eBeta diversity was evaluated using Bray\u0026ndash;Curtis distances calculated from the genus-level relative abundance data. To assess longitudinal changes, within-patient Bray\u0026ndash;Curtis distances were calculated for Pre\u0026ndash;1M and 1M\u0026ndash;6M intervals. Paired Wilcoxon signed-rank tests were used to compare the distances between intervals. The results are presented as medians and IQR, and samples from the same patient are connected graphically. Permutational multivariate analysis of variance (PERMANOVA) with 999 permutations was performed based on Bray\u0026ndash;Curtis distance matrices to evaluate the effects of sampling time and phenotype groups on the overall microbial community structure. Patient ID was included as a stratification factor. A permutational analysis of multivariate dispersions (PERMDISP) was also conducted to confirm that the PERMANOVA results were not driven by differences in within-group dispersion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Saitama Medical School International Medical Center (Approval No. 2023-082, UMIN000052095).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatient characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient characteristics are shown in Table 1. Sixty patients with right-sided colon cancer were included. The tumor location was most frequently observed in the ascending colon (\u003cem\u003en\u003c/em\u003e = 30, 50.0%), followed by the cecum (\u003cem\u003en\u003c/em\u003e = 18, 30.0%), transverse colon (\u003cem\u003en\u003c/em\u003e = 10, 16.7%), and appendix (\u003cem\u003en\u003c/em\u003e = 2, 3.3%). Regarding surgical procedures, 36 patients (60.0%) underwent ileocecal resection and 24 patients (40.0%) underwent right hemicolectomy. According to the pathological TNM classification, 22 (36.7%) patients had stage I disease, 34 (56.7%) had stage II disease, and 4 (6.7%) had stage III disease. None of the patients received any perioperative chemotherapy. Proton-pump inhibitors were administered to 11 (18.3%) patients. None of the patients had received probiotics or antibiotics at baseline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of bowel function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mean BSFS score significantly increased from 3.28 \u0026plusmn; 1.08 preoperatively to 4.68 \u0026plusmn; 1.03 at 1 month after surgery (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), indicating softening of stool consistency (\u003cstrong\u003eFig. 1a\u003c/strong\u003e\u003ca id=\"_anchor_2\" onmouseover=\"msoCommentShow('_anchor_2','_com_2')\" onmouseout=\"msoCommentHide('_com_2')\" href=\"#_msocom_2\" language=\"JavaScript\" name=\"_msoanchor_2\"\u003e\u003c/a\u003e). Thereafter, the mean score decreased to 3.88 \u0026plusmn; 0.81 at 6 months and to 3.73 \u0026plusmn; 0.64 at 12 months, approaching preoperative values.\u003c/p\u003e\n\u003cp\u003eNo significant difference was observed in the total CSS scores between the preoperative and 1-month postoperative assessments (\u003cem\u003ep\u003c/em\u003e = 0.16) (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). However, item-level analysis revealed that the sensation of incomplete evacuation significantly increased from 0.48 \u0026plusmn; 1.02 preoperatively to 1.12 \u0026plusmn; 1.42 at 1 month after surgery (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), with subsequent improvement at 6 and 12 months. Similarly, no significant difference was observed in the total Wexner score between the preoperative and 1-month postoperative assessments (\u003cem\u003ep\u003c/em\u003e = 0.07) (\u003cstrong\u003eFig. 1c\u003c/strong\u003e). However, incontinence to liquid stoolincontinence to liquid stool, which was absent preoperatively, transiently increased to 0.15 \u0026plusmn; 0.48 at 1 month (\u003cem\u003ep\u003c/em\u003e = 0.05) and resolved by 12 months of surgery.\u003c/p\u003e\n\u003cp\u003eBased on these findings, BSFS, sensation of incomplete evacuation, and incontinence to liquid stoolincontinence to liquid stool were defined as bowel function parameters that showed significant postoperative changes (Supplementary Tables S1\u0026ndash;3). At 1 month after surgery, stool softening (increase in the BSFS score) was observed in 42 (70.0%) patients, whereas incomplete evacuation and incontinence to liquid stoolincontinence to liquid stool were observed in 21 (35.0%) and 5 (8.3%) patients, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing read counts across longitudinal samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull-length 16S rRNA gene sequencing using the MinION\u0026trade; platform was performed for fecal samples collected at all time points (Pre, 1M, and 6M). The distribution of the filtered sequencing reads at each time point is shown in \u003cstrong\u003eSupplementary Fig. S2a\u003c/strong\u003e. The median (IQR) number of reads was 45,313 (26,856) for Pre, 50,322.5 (23,876) for 1M, and 46,349 (18,499) for 6M (\u003cstrong\u003eSupplementary Fig. S2b\u003c/strong\u003e) samples.\u003c/p\u003e\n\u003cp\u003eA linear mixed-effects model analysis with patient ID as a random effect revealed a significant main effect of time (\u003cem\u003ep\u003c/em\u003e = 0.0278). Post-hoc comparisons using estimated marginal means with Holm correction revealed a significant decrease in read counts between 1M and 6M (\u003cem\u003ep\u003c/em\u003e = 0.0267) samples (\u003cstrong\u003eSupplementary Fig. S2c,d\u003c/strong\u003e). Because variability in total read counts could introduce bias in downstream analyses, microbial composition analysis was performed after rarefaction to 15,000 reads per sample. A total of 1,527 bacterial species were identified after filtering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLongitudinal changes in \u0026alpha;-diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlpha diversity indices, including the Shannon index, Simpson index, observed richness, and Hill number (q = 1), were calculated for Pre, 1M, and 6M fecal samples. The distributions and within-patient trajectories are shown in \u003cstrong\u003eFig. 2a, b\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe Shannon index, observed richness, and Hill number (q = 1) decreased at 1 month after surgery, and this reduction persisted when compared with the values for the Pre and 6M samples (\u003cstrong\u003eFig. 2a, b\u003c/strong\u003e). Linear mixed-effects model analysis (with patient ID as a random effect) revealed a significant main effect of time for the Shannon index (\u003cem\u003ep\u003c/em\u003e = 5.20 \u0026times; 10⁻⁷), Observed richness (\u003cem\u003ep\u003c/em\u003e = 2.90 \u0026times; 10⁻\u0026sup1;\u0026sup1;), and Hill number (q = 1) (\u003cem\u003ep\u003c/em\u003e = 2.90 \u0026times; 10⁻\u0026sup1;\u0026sup1;) (\u003cstrong\u003eFig. 2c; Supplementary Table S4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003ePost-hoc pairwise comparisons with Holm adjustment showed significant differences in all three indices for Pre\u0026ndash;1M and Pre\u0026ndash;6M contrasts, whereas no significant difference was observed between 1M and 6M (\u003cstrong\u003eSupplementary Table S4\u003c/strong\u003e). These findings indicate an early postoperative reduction in microbial diversity without a clear recovery by 6 months. In contrast, the Simpson index showed no significant main effect of time (\u003cem\u003ep\u003c/em\u003e = 0.112), and no significant differences were observed in the pairwise comparisons (\u003cstrong\u003eSupplementary Table S4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociation between bowel function and gut microbial \u0026alpha;-diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the questionnaire assessments at 1 month after surgery, differences according to the presence of symptoms were observed for incomplete evacuationincomplete evacuation, BSFS, and incontinence to liquid stool. Therefore, longitudinal changes in each \u0026alpha;-diversity index were evaluated using linear mixed-effects models with phenotype (aggravation/lighthearted) and sampling time point (Pre, 1M, and 6M) as fixed effects (\u003cstrong\u003eFig. 3a\u0026ndash;c, Supplementary Table S5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFor incomplete evacuationincomplete evacuation, the main effect of time was significant for the Shannon index (\u003cem\u003ep\u003c/em\u003e = 7.65 \u0026times; 10⁻⁶), observed richness (\u003cem\u003ep\u003c/em\u003e = 4.06 \u0026times; 10⁻\u0026sup1;⁰), and Hill number (q = 1) (\u003cem\u003ep\u003c/em\u003e = 4.11 \u0026times; 10⁻⁷). In contrast, neither the main effect of group nor the group \u0026times; time interaction was significant (\u003cstrong\u003eSupplementary Table S5\u003c/strong\u003e). Post-hoc comparisons revealed significant differences primarily in the lighthearted group between Pre and 1M and Pre\u0026ndash;6M. For the BSFS, the main effect of time was also significant for the Shannon index, observed richness, and Hill number (q = 1), whereas neither the main effect of group nor the interaction term was statistically significant (\u003cstrong\u003eSupplementary Table S5\u003c/strong\u003e). A decrease in \u0026alpha;-diversity was observed in both the groups during the early postoperative period. For fecal incontinence, only the observed richness showed a significant main effect of time (\u003cem\u003ep\u003c/em\u003e = 0.0022). No statistically significant changes were detected in the Shannon index or Hill number (q = 1) (\u003cstrong\u003eSupplementary Table S5\u003c/strong\u003e). In the post-hoc analyses, significant differences between Pre\u0026ndash;1M and Pre\u0026ndash;6M were identified in the lighthearted group.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;At all time points, no significant between-group differences (aggravation vs. lighthearted) were observed for any \u0026alpha;-diversity index, and no significant group \u0026times; time interactions were detected (\u003cstrong\u003eSupplementary Table S5\u003c/strong\u003e). These findings indicate that the early postoperative decline in \u0026alpha;-diversity observed in the overall analysis was not restricted to a specific symptom group but primarily reflected time-dependent changes. However, more pronounced alterations were observed in the lighthearted group for certain indices, suggesting a possible contribution of interindividual variability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLongitudinal changes in \u0026beta;-diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gut microbial \u0026beta;-diversity was assessed using Bray\u0026ndash;Curtis distances calculated from genus-level relative abundance data. To examine longitudinal changes, paired comparisons were performed within the same patients for the Pre\u0026ndash;1M and 1M\u0026ndash;6M intervals. The Bray\u0026ndash;Curtis distance for the 1M\u0026ndash;6M interval was significantly lower than that for the Pre\u0026ndash;1M interval (Wilcoxon signed-rank test, \u003cem\u003ep\u003c/em\u003e = 1.24 \u0026times; 10⁻⁷) (\u003cstrong\u003eFig. 4a\u003c/strong\u003e). Visualization of individual trajectories revealed that most patients experienced marked shifts in microbial composition during the early postoperative period, followed by reduced changes thereafter.\u003c/p\u003e\n\u003cp\u003eWhen the distributions were summarized as median and IQR, the Pre\u0026ndash;1M interval (median 0.807) showed higher distances than the 1M\u0026ndash;6M interval (median 0.594) (\u003cstrong\u003eFig. 4b\u003c/strong\u003e). PERMANOVA based on the Bray\u0026ndash;Curtis distance matrix revealed that the sampling time significantly contributed to the overall variation in microbial composition (\u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.033, \u003cem\u003ep\u003c/em\u003e = 0.001; \u003cstrong\u003eSupplementary Table S6\u003c/strong\u003e). Additionally, PERMDISP analysis revealed significant differences in dispersion over time, indicating that temporal changes were associated with shifts in the community structure (\u003cstrong\u003eSupplementary Table S6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociation between bowel function and gut microbial \u0026beta;-diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePERMANOVA, accounting for repeated measures within patients, was conducted for each phenotype (incomplete evacuation, BSFS, and fecal incontinence). For all phenotypes, the main effect of time was significant (all \u003cem\u003ep\u003c/em\u003e = 0.001), indicating that the gut microbial community structure changed over time (\u003cstrong\u003eFig. 5a\u0026ndash;c, Supplementary Table S7\u003c/strong\u003e). In contrast, neither the main effect of group nor the time \u0026times; group interaction was significant, suggesting that the temporal changes in the microbial community were not phenotype-specific (\u003cstrong\u003eSupplementary Table S7\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn paired comparisons of Bray\u0026ndash;Curtis distances between the Pre\u0026ndash;1M and 1M\u0026ndash;6M intervals, the Pre\u0026ndash;1M distance was significantly greater than the 1M\u0026ndash;6M distance in both the groups across all phenotypes (Wilcoxon signed-rank test, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; \u003cstrong\u003eSupplementary Table S7\u003c/strong\u003e). These results indicated that the largest changes in the microbial composition occurred during the early postoperative period.\u003c/p\u003e\n\u003cp\u003eFurthermore, Friedman tests across the three time points (Pre, 1M, and 6M) revealed significant temporal changes in both the groups for each phenotype (\u003cstrong\u003eSupplementary Table S7\u003c/strong\u003e). Post-hoc analyses showed significant differences primarily between Pre and 1M and between Pre and 6M, whereas no significant differences were detected between 1M and 6M.\u003c/p\u003e\n\u003cp\u003ePERMDISP analysis revealed that, for incomplete evacuation, dispersion differences were observed only for time, indicating that changes in \u0026beta;-diversity mainly reflected temporal shifts in the community structure. In contrast, for BSFS and fecal incontinence, significant differences in dispersion were observed for both group and time, indicating that for these symptoms, \u0026beta;-diversity differences may reflect not only shifts in community centroids but also interindividual variability in microbial responses (\u003cstrong\u003eSupplementary Table S7\u003c/strong\u003e). It should be noted that the number of patients in the aggravation group for fecal incontinence was small, which may have limited the statistical power.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we longitudinally evaluated the postoperative changes in bowel function and gut microbiota in patients undergoing surgery for right-sided colon cancer. At 1 month after surgery, bowel dysfunction, including a softer stool consistency, incomplete evacuation, and incontinence to liquid stool, was frequently observed. However, these symptoms were transient and improved after 6 months. Microbiome analyses revealed marked alterations in both \u0026alpha;- and \u0026beta;-diversity during the early postoperative period, followed by a time-dependent microbial reconstruction. Notably, these microbial changes were driven predominantly by postoperative time course rather than by individual bowel symptom phenotypes.\u003c/p\u003e\n\u003cp\u003eWith regard to stool consistency, the BSFS score increased significantly 1 month after surgery, indicating stool softening. This finding is consistent with previous reports of assessment after right hemicolectomy and may be attributable to shortened intestinal transit time and bile acid malabsorption associated with ileocecal valve resection [13]. Although no significant deterioration was observed in the overall CSS or Wexner total scores, item-level analyses revealed transient increases in incomplete evacuation and incontinence to liquid stool during the early postoperative period. These changes likely represent clinically meaningful symptoms as perceived by patients, even in the absence of significant changes in the composite scores.\u003c/p\u003e\n\u003cp\u003eIn the analysis of gut microbial \u0026alpha;-diversity, the Shannon index, Hill number (q = 1), and observed richness decreased at 1 month after surgery, and this trend persisted through 6 months. In contrast, no significant changes in the Simpson index were observed. These findings suggest that while\u0026nbsp;the overall microbial diversity and moderately abundant taxa decreased after surgery, the composition of the dominant taxa was relatively preserved, which is consistent with previous reports\u0026nbsp;[25,26]. The reduction in observed richness further indicates a selective loss of rare taxa. Besides anatomical alterations associated with right-sided colectomy and ileocecal valve resection, perioperative factors, such as antibiotic exposure and dietary changes, may have collectively influenced microbial homeostasis [27].\u003c/p\u003e\n\u003cp\u003eRegarding the association between bowel function and gut microbiota, phenotype-stratified analyses for incomplete evacuation and fecal incontinence revealed that early postoperative decline in \u0026alpha;-diversity was statistically significant only in the lighthearted group. However, neither between-group comparisons nor interaction effects were significant, making it difficult to conclude whether bowel dysfunction and microbial alterations are directly linked in a simple, one-to-one relationship. Rather, interindividual differences in responsiveness to postoperative environmental changes and possible temporal discrepancies between symptom perception and microbiota alterations may have contributed to these findings. \u0026beta;-diversity analyses revealed that changes in the microbial structure were most pronounced during the early postoperative period, with the magnitude of change decreasing thereafter. Phenotype-stratified analyses indicated that shifts in the microbial composition were primarily time-dependent and not specifically associated with individual bowel dysfunction phenotypes. However, in cases of stool softening (BSFS) and fecal incontinence, variability in dispersion indicates substantial interindividual differences in microbial responses. Taken together, these findings indicate that postoperative alterations in the gut microbiota following gastrointestinal surgery are characterized by marked early shifts in \u0026beta;-diversity, followed by gradual reconstruction, with considerable interindividual variability. Notably, the relationship between microbial changes and clinical symptoms appears to be heterogeneous rather than uniform. In the present study, postoperative microbial alterations were more strongly associated with postoperative time course than with individual bowel symptom phenotypes, suggesting that symptom-specific microbial signatures were not evident. Rather, these changes are likely influenced by multiple factors, including host characteristics, surgical stress, reconstruction type, and postoperative environmental conditions. These results are consistent with those of previous systematic reviews, suggesting that alterations in the microbiota after digestive tract surgery are multifactorial and are not solely determined by symptom expression or clinical outcomes [28,29].\u003c/p\u003e\n\u003cp\u003eThis study had several limitations. First, this was a single-center observational study with a relatively small sample size. Second, bowel function was assessed using a questionnaire-based subjective measures. In addition, microbiome analyses were limited to structural evaluations based on diversity indices, and specific bacterial taxa or functional alterations were not investigated. A major strength of this study is the longitudinal evaluation of time-dependent changes using paired samples from the same patients. By analyzing within-patient trajectories, we were able to characterize postoperative microbial dynamics while minimizing interindividual variability. Future studies should include larger prospective cohorts and incorporate functional analyses, including metabolic profiling and detailed assessments of specific bacterial taxa, to further clarify the relationship between postoperative bowel dysfunction and alterations in the gut microbiota.\u003c/p\u003e\n\u003cp\u003eThe novelty of this study lies in demonstrating that postoperative bowel dysfunction symptoms after right-sided colectomy are common but largely transient, and that postoperative gut microbiota changes are predominantly time-dependent rather than symptom-specific. Although stool softening, incomplete evacuation, and incontinence to liquid stool were frequently observed during the early postoperative period, these symptoms were not accompanied by distinct microbial signatures. Instead, the gut microbiota showed a pattern of early disruption followed by gradual reconstruction, suggesting a generalized ecological adaptation to postoperative intestinal environmental change rather than persistent pathological dysbiosis. These findings provide a new framework for understanding postoperative bowel dysfunction after right-sided colectomy as a multifactorial and time-dependent adaptive process, and may help inform future individualized intervention strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staff of the Division of Analytical Science, Hidaka Branch, Biomedical Research Center, Saitama Medical University, for providing the research equipment and valuable technical advice. English language editing was performed by Editage (www.editage.jp).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA contributed to patient questionnaire administration and sample collection, performed statistical analyses, and drafted the manuscript. MH prepared the library for nanopore sequencing, conducted data analysis, and drafted the manuscript. NA extracted DNA from the patient stool samples. YM supported the patient questionnaire administration and sample collection. MO, YS, and YK1 provided advice on statistical analyses. HN provided technical advice on nanopore sequencing. YH, YI, HS, CH, TH, and YK2 designed and supervised the study and revised the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequences were deposited in the DDBJ (https://www.ddbj.nig.ac.jp/dra/index-e.html) with the accession number PRJDB40334. However, a correspondence table linking patient identification codes to personal information is not publicly available due to privacy and ethical constraints. When an application for the secondary use of sequence data is submitted, we ask the applicant to present the purpose of the use and review the pros and cons of granting access before making a decision. Masataka Hirasaki (hirasaki@saitama-med. ac. jp) handled the applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Saitama Medical School International Medical Center (Approval No. 2023-082, UMIN000052095). Informed consent was waived because of the retrospective design and opt-out options provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a Grant-in-Aid for Young Researchers from the Saitama Medical University International Medical Center (4-D-1-14) (SA).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries\u003cem\u003e. CA Cancer J. 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Rep.\u003c/em\u003e\u003cstrong\u003e 12,\u003c/strong\u003e 9829 (2022).\u003c/li\u003e\n\u003cli\u003ePark, S. S. et al. The effect of curative resection on fecal microbiota in patients with colorectal cancer: a prospective pilot study. \u003cem\u003eAnn. Surg. Treat. Res.\u003c/em\u003e \u003cstrong\u003e99,\u003c/strong\u003e 44\u0026ndash;51 (2020).\u003c/li\u003e\n\u003cli\u003eFerrie, S., Webster, A., Wu, B., Tan, C. \u0026amp; Carey, S. Gastrointestinal surgery and the gut microbiome: a systematic literature review. \u003cem\u003eEur. J. Clin. Nutr.\u003c/em\u003e \u003cstrong\u003e75,\u003c/strong\u003e 12\u0026ndash;25 (2020).\u003c/li\u003e\n\u003cli\u003eTarazi, M., Jamel, S., Mullish, B. H., Markar, S. R. \u0026amp; Hanna, G. B. Impact of gastrointestinal surgery upon the gut microbiome: A systematic review. \u003cem\u003eSurgery\u003c/em\u003e \u003cstrong\u003e171,\u003c/strong\u003e 1331\u0026ndash;1340 (2022).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bowel function, Colorectal cancer, Gut microbiota, Quality of life","lastPublishedDoi":"10.21203/rs.3.rs-9337728/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9337728/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePostoperative bowel dysfunction after colorectal cancer surgery impairs quality of life, but its longitudinal course and relationship with the gut microbiota after right-sided colon cancer surgery remain unclear. We retrospectively analyzed 60 patients who underwent curative resection for right-sided colon cancer between November 2023 and November 2024. Bowel function was assessed preoperatively and at 1, 6, and 12 months postoperatively using the Bristol Stool Form Scale, Constipation Scoring System, and Wexner score. Gut microbiota was analyzed by full-length 16S rRNA sequencing of fecal samples collected preoperatively and at 1 and 6 months after surgery. At 1 month, stool softening, incomplete evacuation, and incontinence to liquid stool were observed in 42, 21, and 5 patients, respectively. These symptoms improved after 6 months and generally returned to preoperative levels by 12 months. Microbiota analysis showed decreased α-diversity and marked shifts in β-diversity during the early postoperative period, followed by gradual reconstruction with interindividual variability. Collectively, bowel dysfunction after right-sided colon cancer surgery was common but largely transient, whereas gut microbiota changes were predominantly time-dependent rather than symptom-specific, suggesting generalized ecological reconstruction rather than direct microbial correlates of individual bowel symptoms.\u003c/p\u003e","manuscriptTitle":"Correlation between changes in intestinal microbiota and defecatory function before and after radical resection of right-sided colon cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-09 00:14:50","doi":"10.21203/rs.3.rs-9337728/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-14T08:47:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T11:56:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275353755801395667129860591512366259904","date":"2026-04-30T09:31:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48444252983625038269990364969972270586","date":"2026-04-28T09:27:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2873267292930325392232293080005198153","date":"2026-04-28T05:53:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T22:32:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332163898854794803259536722037209552924","date":"2026-04-26T22:28:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T21:55:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11724529732399512180120316031183307004","date":"2026-04-24T14:51:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T11:20:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-16T07:40:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T14:21:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-07T14:20:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-06T23:59:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f89570c0-4b5e-4167-97b7-f41a49736fbd","owner":[],"postedDate":"May 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-14T08:47:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T11:56:41+00:00","index":102,"fulltext":""},{"type":"reviewerAgreed","content":"275353755801395667129860591512366259904","date":"2026-04-30T09:31:16+00:00","index":101,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":67478736,"name":"Biological sciences/Cancer"},{"id":67478737,"name":"Health sciences/Gastroenterology"},{"id":67478738,"name":"Health sciences/Medical research"},{"id":67478739,"name":"Biological sciences/Microbiology"},{"id":67478740,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-14T09:10:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-09 00:14:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9337728","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9337728","identity":"rs-9337728","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}