Comprehensive microbial and clinical profiling of functional constipation: A stratified comparative study of age and constipation subtype

Abstract

BACKGROUND

Gut microbiota dysbiosis plays a central role in the pathogenesis of functional constipation (FC), but clinical treatment has shown uneven efficacy through methods of regulating intestinal flora. Most existing studies have concentrated on overall case-control comparisons, with limited stratification by age or constipation subtype. Knowledge of how these factors shape gut microbiota composition remains inadequate, constraining the development of effective personalized microbiota-based interventions.

AIM

To characterize gut microbiota profiles by age and constipation subtype and identify factors associated with microbial composition in FC.

METHODS

Ninety-two patients with FC completed questionnaires and underwent anorectal manometry and gastrointestinal transit tests; stool samples were collected for 16S rRNA gene sequencing. Stool samples alone were collected from 34 healthy controls. Patients with FC were categorized into age groups (young, middle-aged, and older) and classified into normal-transit constipation, slow-transit constipation (STC), defecatory disorder (DD), and mixed constipation subtypes. Gut microbial compositions across age groups and constipation subtypes were compared, and their correlations with clinical parameters were investigated.

RESULTS

The young group demonstrated significantly higher anal resting and squeeze pressures than the middle-aged and the older groups. Microbial richness and diversity were substantially lower in the older group than in middle-aged and young groups. Gut microbiota in the young group was predominantly enriched in taxa increasing sphincter tone and inhibiting intestinal peristalsis; the older group featured abundances of short-chain fatty acid-producing, beneficial taxa. The middle-aged group showed an enrichment of pro-inflammatory and pathogenic bacteria. Microbial richness and diversity were higher in STC than in the DD group. Moreover, STC group was enriched in taxa associated with slower peristalsis; DD group showed enrichment of motility-promoting taxa.

CONCLUSION

Significant differences in microbial composition and function were observed across age groups and constipation subtypes in FC, suggesting underlying pathophysiological heterogeneity and providing a basis for precision diagnosis and treatment.

Key Words: Gut microbiota; Functional constipation; Gastrointestinal disorder; Microbial diversity; Age-related differences

Core Tip: This study highlights the distinct gut microbiota profiles associated with age and constipation subtypes in patients with functional constipation (FC). Using 16S rRNA sequencing, we found significant age-related differences in microbial diversity and composition, with older adults exhibiting reduced richness and beneficial taxa. Constipation subtypes also showed distinct microbial patterns, with slow-transit constipation enriched in taxa linked to reduced motility and defecatory disorder characterized by motility-promoting microbes. These findings underscore the pathophysiological heterogeneity of FC and support the development of age- and subtype-specific microbiota-targeted therapies for precision diagnosis and treatment.

INTRODUCTION

Functional constipation (FC), as defined by the Rome IV criteria, is a functional bowel disorder characterized by impaired defecation in the absence of structural or biochemical abnormalities. The clinical features include reduced stool frequency, hard or lumpy stools, excessive straining, and a sensation of incomplete evacuation, with notable variation across age groups and populations. As a common functional gastrointestinal disorder, FC compromises quality of life and creates marked healthcare, economic, and societal burdens at both the individual and system levels in China. It represents one of the most prevalent functional gastrointestinal disorders worldwide, the global prevalence of FC reaches 11.6%[1]. In the United Kingdom, the National Health Service spends approximately Great Britain Pound 162 million annually on constipation management[2]. The average annual treatment cost per patient with FC is United States Dollar 7522[3]. These challenges have stimulated increasing interest in exploring novel pathophysiological mechanisms.

Although the pathophysiology of FC remains incompletely elucidated, visceral hypersensitivity, enteric nervous system abnormalities, and disordered gastrointestinal motility are implicated[4]. Recently, advances in microbiome profiling have highlighted gut microbiota aberrations in patients with FC. Ideally, gut microbes and their host exist in a dynamic, mutualistic equilibrium. Disruption of this balance may contribute to dysbacteriosis and consequently the symptomatology and progression of FC through metabolism of bile acids, short-chain fatty acids (SCFAs), serotonin, and other mediators[5]. For example, reductions in Bacteroides and Bifidobacterium have been observed in the feces of patients with FC[6]. Conversely, butyrate-producing genera such as Coprococcus, Roseburia, and Faecalibacterium are markedly enriched in this cohort[7]. SCFAs not only regulate the absorption and secretion of colonic electrolytes and mucin but also directly activate intrinsic primary afferent neurons to stimulate intestinal contraction[6,8,9]. Microbiota-targeted therapies, including probiotics, prebiotics, and fecal microbiota transplantation, have demonstrated efficacy in FC, effectively improving bowel frequency and alleviating abdominal discomfort[10-12].

The gut microbiota refers to the complex and dynamic community of microorganisms that inhabit the human gastrointestinal tract. Its composition is strongly shaped by environmental factors such as diet, lifestyle, geography, and external exposures, whereas host genetics appear to play a comparatively minor role. This microbial ecosystem is essential for host physiology, supporting digestion, metabolism, immune regulation, and gut–brain communication. Importantly, the microbiota remains highly dynamic and can be perturbed by both internal and external influences. Across the human lifespan, microbial diversity exhibits distinct trajectories, including rapid establishment during infancy, stabilization in adulthood, and community remodeling in old age[13]. Moreover, environmental factors, geographic residence, and antibiotic use also significantly influence gut ecosystem stability[14]. Recognizing this malleability is key to understanding disease mechanisms and formulating personalized therapeutic strategies[15].

In this study, the gut microbiota of patients with FC was analyzed with respect to age and disease subtype. The study aimed to identify factors associated with the gut microbiota profiles of patients with FC, thereby guiding the development of personalized microbial interventions.

MATERIALS AND METHODS

Study design and participants

Patients diagnosed with FC between January and December 2024 at the Department of Gastroenterology of the China-Japan Friendship Hospital were enrolled as the experimental group. Concurrently, age- and sex-matched healthy volunteers without gastrointestinal symptoms were recruited as the healthy control (HC) group. All participants provided written informed consent. The study protocol was approved by the Ethics Committee of the China-Japan Friendship Hospital. The inclusion criteria for the experimental group were: Age > 18 years, and diagnosis of FC according to Rome IV criteria [including: (1) > 25% of defecations meeting two or more of the following: Straining, lumpy or hard stools, sensation of incomplete evacuation, sensation of anorectal obstruction/blockage, manual maneuvers to facilitate defecation, or fewer than three spontaneous bowel movements per week; (2) Loose stools rarely present without the use of laxatives; and (3) Insufficient criteria for irritable bowel syndrome; Symptom onset occurred at least 6 months prior, with fulfillment of the above criteria during the preceding 3 months]. The exclusion criteria were: (1) Pregnancy or lactation; (2) Administration of antibiotics, probiotics, or prebiotics within 4 weeks before sample collection; (3) Use of laxative more than once per week within 4 weeks before enrollment; (4) Serious diseases, such as malignancy, hypothyroidism, and hepatic or renal failure; (5) Intestinal organic lesions detected via colonoscopy; and (6) Language barriers or psychiatric disorders.

Study procedures

Upon enrollment, under the guidance of a gastroenterologist, all patients with FC completed several questionnaire surveys: A demographic questionnaire, the patient assessment of constipation quality of life (PAC-QOL), the Knowles Eccersley Scott symptom (KESS) questionnaire, the self-rating anxiety scale (SAS), and the self-rating depression scale (SDS). Subsequently, all patients with FC underwent the gastrointestinal transit time (GITT) test, fresh stool collection, and high-resolution anorectal manometry (HR-ARM).

GITT: Participants ingested 20 radio-opaque barium sulfate markers with breakfast. After 48 hours, a standing abdominal X-ray was acquired, and the number of markers expelled via the anus was recorded. The colonic transit rate [(20 - total number of residual markers)/20 × 100%], proximal marker retention [(number of residual markers above the rectosigmoid)/20 × 100%], and distal marker retention rate [(number of residual markers in the rectosigmoid)/20 × 100%] were subsequently calculated. GITT was considered delayed if the colonic transit rate was < 90%.

Fresh stool collection: After the patient defecated onto sterile paper, at least 5 g of stool was transferred into a sterile container using a sterile scoop.

HR-ARM: Following rectal cleansing with a glycerin enema, HR-ARM was performed using a solid-state HR-ARM system (ManoScan AR 360, Given Imaging). With the patient in the left lateral position and hips flexed towards the abdomen, a catheter-mounted balloon and complete set of recording electrodes on the catheter was inserted to the rectum. Participants were instructed to perform voluntary contraction, simulated defecation, and other maneuvers to assess anorectal muscle function and rectal sensation. Recorded metrics included the following: Maximum anal resting pressure (MARP), mean anal resting pressure (MERP), anal high pressure zone length, maximum anal squeeze pressure (MSP), mean squeeze pressure, squeeze duration, residual anal pressure (RAP), anal relaxation rate (ARR), intrarectal pressure during evacuation, rectoanal pressure difference (RAPD), rectal threshold volume for first sensation (FST), desire to defecate (DDT), and maximum discomfort (MDT). HR-ARM was deemed abnormal if rectal propulsive force was considered inadequate and/or the anal ARR was < 20%. For the HC group, only fresh stool samples were collected.

Sample processing and microbiota analysis

All fresh stool samples were snap-frozen in liquid nitrogen immediately after collection and stored at -80 °C. Microbial DNA extraction and 16S rRNA gene sequencing were performed according to protocols provided by Thermo Fisher Scientific Biotech Co., Ltd. (Beijing, China).

Total genomic DNA from stool samples was extracted using the hexadecyltrimethylammonium bromide/sodium dodecyl sulfate method. The V3-V4 region of the 16S rDNA gene was amplified with primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’)/806R (5’-GGACTACHVGGGTWTCTAAT-3’), and polymerase chain reaction (PCR) products were purified. Libraries were constructed using the TruSeq^® DNA PCR-free kit and sequenced on the Illumina NovaSeq 6000 platform (PE150). Raw reads were processed using Trimmomatic v0.39 for quality control and assembled with USEARCH v11.2.64 to obtain high-quality tags. Operational taxonomic units were clustered at 97% similarity using UPARSE and annotated via the ribosomal database project database with SINTAX (confidence ≥ 0.8).

Grouping

Patients with FC comprised the experimental (FC group), whereas healthy volunteers formed the HC group. According to their age, patients with FC were categorized into the young (Y) (18-45 years), middle-aged (M) (45-60 years), and older (O) (> 60 years) groups. Moreover, based on the GITT and ARM results, participants in the FC group were further classified into the normal-transit constipation (NTC) (normal GITT and ARM results), slow-transit constipation (STC) (abnormal GITT but normal ARM results), defecatory disorder (DD) (normal GITT but abnormal ARM results), and mixed constipation (MC) (abnormal GITT and ARM results) subtypes.

Statistical analysis

Clinical data were analyzed using SPSS version 26.0 (IBM, United States). Continuous variables conforming to a normal distribution are presented as, while non-normally distributed data are described by median and interquartile range. Intergroup comparisons of continuous variables were conducted using t-test, one-way analysis of variance, Mann-Whitney U test, or Kruskal-Wallis H test. Categorical variables were compared using the χ² test or Fisher’s exact test. P ≤ 0.05 was considered statistically significant. For microbiota analysis, α-diversity was assessed by Chao1, richness, Shannon_2, and Simpson indices to reflect microbial richness and diversity. Moreover, β-diversity metrics were calculated to evaluate differences in community composition between samples using principal coordinate analysis based on Bray-Curtis and weighted UniFrac distances. Linear discriminant analysis effect size (LEfSe) was employed to identify taxa with significant differential abundance across groups, facilitating the discovery of group-dependent microbial biomarkers.

RESULTS

General and pathophysiological characteristics of FC patients

In total, 92 patients with FC were enrolled, comprising 24 males and 68 females (mean age = 49.34 ± 16.05 years). Their baseline demographics, SAS and SDS scores, constipation subtypes, and findings from GITT, HR-ARM, and rectal sensory tests are summarized in Table 1. The HC group comprised 34 individuals (10 males and 24 females; mean age = 46.55 ± 15.06 years).

Table 1 General clinical characteristics and gastrointestinal function test results in patients with functional constipation, mean ± SD/n (%).

Characteristic		FC total (n = 92)	Age groups
			Y (n = 31)	M (n = 35)	O (n = 26)	P value
Clinical characteristics	Sex (male)	24 (26.09)	5 (15.6)	7 (20.6)	12 (46.2)	0.02
	BMI (kg/m2)	22.20 ± 3.41	21.18 ± 4.15	22.16 ± 2.75	23.46 ± 2.97	0.04
	Constipation duration (months), median IQR	96 (36, 216)	120 (24, 216)	120 (48, 256)	660 (36, 120)	0.44
	PAC-QOL score	76.27 ± 19.66	79.06 ± 18.18	73.4 ± 20.78	76.8 ± 20.40	0.50
	KESS score, median IQR	19.37 ± 5.17	21.00 (14.00, 25.00)	19.00 (16.00, 22.00)	19.50 (14.00, 22.50)	0.57
Bowel movement frequency	1-2 times every 1-2 days	38 (41.30)	6 (19.35)	17 (48.57)	15 (57.69)	0.05
	Less than 2 times per week	31 (33.70)	16 (51.61)	11 (31.43)	4 (15.38)
	Less than once per week	20 (21.74)	7 (22.58)	7 (20)	6 (23.08)
	Less than once per fortnight	3 (3.26)	2 (6.45)	0 (0)	1 (3.85)
Psychological evaluation	SAS	45.95 ± 10.51	47.12 ± 10.54	43.63 ± 8.88	47.69 ± 12.38	0.65
	SDS	51.39 ± 13.56	53.13 ± 14.43	50.06 ± 13.42	51.09 ± 13.26	0.42
Clinical subtype	NTC	6 (6.52)	2 (6.45)	2 (5.71)	2 (7.69)	0.81
	STC	19 (20.65)	8 (25.81)	7 (20)	4 (15.38)
	DD	22 (23.91)	6 (19.35)	10 (28.57)	6 (23.08)
	MC	45 (48.91)	15 (48.39)	16 (45.71)	14 (53.85)
GITT	48-hour colonic transit rate (%), median IQR	50.00 (50.00, 80.00)	10.00 (0, 90.00)	60.00 (0, 90.00)	32.50 (0, 95.00)	0.74
	Proximal colonic marker retention (%), median IQR	90.00 (60.00, 110.00)	20.00 (0, 60.00)	5.00 (0, 25.00)	7.50 (0, 25.00)	0.27
	Distal colonic marker retention (%), median IQR	170.00 (140.00, 220.00)	25.00 (10.00, 55.00)	20.00 (5.00, 60.00)	20.00 (0, 65.00)	0.99
ARM	MARP (mmHg)	89.52 ± 25.32	98.81 ± 22.86	92.63 ± 21.95	74.27 ± 26.73	0.01
	MERP (mmHg)	79.77 ± 25.59	86.78 ± 21.61	83.92 ± 26.56	65.79 ± 24.54	0.01
	HPZ length (cm)	3.64 ± 0.72	3.63 ± 0.73	3.67 ± 0.71	3.63 ± 0.76	0.97
	MSP (mmHg)	219.61 ± 66.81	232.90 ± 78.51	230.66 ± 57.42	188.87 ± 56.34	0.02
	Squeeze duration (second), median IQR	11.89 ± 5.62	16.10 (8.40, 19.40)	11.50 (7.30, 15.70)	10.80 (4.53, 14.40)	0.04
	RAP (mmHg), median IQR	76.16 ± 30.42	78.20 (66.00, 91.80)	74.30 (57.40, 100.20)	71.35 (60.40, 89.75)	0.50
	ARR, median IQR	2.00 (-14.00, 22.00)	9.00 (-4.00, 13.00)	3.00 (-13.00, 22.00)	-10.50 (-26.75, 22.50)	0.14
	Intrarectal pressure during evacuation (mmHg), median IQR	36.60 (21.60, 46.70)	31.50 (19.50, 40.20)	28.00 (18.70, 51.80)	40.90 (28.15, 50.90)	0.32
	RAPD (mmHg), median IQR	-38.50 (-58.90, -18.40)	-46.40 (-67.50, -28.30)	-36.50 (-70.60, -22.10)	-22.90 (-55.85, -12.03)	0.05
Rectal sensory thresholds	FST (cm3), median IQR	50 (40.00, 80.00)	50 (40.00, 70.00)	50 (40.00, 80.00)	60.00 (40.00, 80.00)	0.78
	DDT (cm3), median IQR	90 (60.00, 110.00)	90.00 (60.00, 100.00)	80.00 (50.00, 100.00)	90.00 (70.00, 130.00)	0.06
	MDT (cm3)	179.39 ± 55.46	171.77 ± 47.89	175.42 ± 59.96	195.00 ± 58.09	0.27

Y: Young; M: Middle-aged; O: Older; FC: Functional constipation; BMI: Body mass index; KESS: Knowles Eccersley Scott symptom; PAC-QOL: Patient assessment of constipation quality of life; IQR: Interquartile range; SAS: Self-rating anxiety scale; SDS: Self-rating depression scale; NTC: Normal-transit constipation; STC: Slow-transit constipation; DD: Defecatory disorder; MC: Mixed constipation; GITT: Gastrointestinal transit time; ARM: Anorectal manometry; MARP: Maximum anal resting pressure; MERP: Mean anal resting pressure; HPZ: Anal high pressure zone; MSP: Maximum anal squeeze pressure; RAP: Residual anal pressure; ARR: Anal relaxation rate; RAPD: Rectoanal pressure difference; FST: First sensation; DDT: Desire to defecate; MDT: Maximum discomfort.

Based on their age, patients with FC were stratified into a Y group, comprising 31 participants; an M group, comprising 35 participants; and an O group, comprising 26 participants. The O group had a significantly higher proportion of males and a larger body mass index (BMI) compared with the M and Y groups. No significant differences were observed for constipation duration, KESS score, PAC-QOL scores, and bowel movement frequency among the three groups. ARM indicated that, at rest, the Y group exhibited substantially higher MARP and MERP than the M group, which in turn exceeded the O group, all with significant differences. During voluntary contraction, the Y group demonstrated higher MSP, squeeze duration, and a greater RAPD than the M group, which again exceeded the O group, all with significant differences. However, RAP, ARR, and rectal sensory thresholds (FST, DDT, and MDT) did not differ significantly among the three groups. In addition, no significant differences were noted in GITT, anxiety/depression scores, or the distribution of FC subtypes.

Based on constipation subtype, patients with FC were classified into four groups: The NTC (6 participants), STC (19 participants), DD (22 participants), and MC (45 participants) groups. No significant differences were observed in age, BMI, constipation duration, bowel movement frequency, clinical symptom scores, or anxiety/depression scores, except for sex distribution (Supplementary Table 1).

Gut microbiota profiles of patients with FC

Comparison of α-diversity revealed significantly lower gut microbiota richness and diversity in the FC group than in the HC group (Figure 1A). Moreover, β-diversity analysis indicated distinct community structures between FC and HC samples, with the HC group exhibiting more homogeneous microbiota profiles (Figure 1B). Differential abundance analysis identified several microbiota constituents with significant differences in the FC group at order, family and genus level (Figure 1C-E). Specifically, at the genus level, Methanobrevibacter, Stenotrophomonas, Intestinibacter, Sellimonas, and Eisenbergiella were markedly enriched in patients with FC, whereas Helicobacter, Fusobacterium, Duncaniella, Paramuribaculum, Megamonas, Muribaculum, Achromobacter, Faecalimonas, Mucispirillum, Porphyromonas, and Prevotella were significantly depleted (Figure 1E).

Figure 1 Comparison of gut microbiota profiles between functional constipation and healthy control groups. A: Comparison of α-diversity indices between the two groups, including Chao1, richness, Shannon, and Simpson indices; B: Β-diversity analysis of gut microbiota composition between the two groups; C: Volcano plot of differential gut microbiota at the order level between the two groups; D: Volcano plot of differential gut microbiota at the family level between the two groups; E: Volcano plot of differential gut microbiota at the genus level between the two groups. ^aP < 0.05. ^cP < 0.001. FC: Functional constipation; HC: Healthy control; PCo: Principal co-ordinates; FDR: False discovery rate; DE: Differentially enriched.

Comparison of gut microbiota profiles between patients with FC of different ages

Comparison of α-diversity demonstrated considerable differences among patients with FC in different age groups (Figure 2A). Specifically, the O group exhibited significantly lower microbial richness and diversity than the M and Y groups. Although the Y group showed slightly higher richness and diversity than the M group, this difference was not statistically significant. β-diversity analysis (Figure 2B) confirmed distinct community clustering among the three age groups (R² = 0.03, P = 0.05). Within-group dispersion was comparable between the Y and O cohorts (PERMDISP: F = 0.2, P = 0.70; mean distance to centroid: Y = 0.58, O = 0.59), indicating similar microbial heterogeneity. However, the older group still showed a trend toward greater interindividual variability.

Figure 2 Comparison of gut microbiota profiles among different age groups. A: Comparison of α-diversity among different age groups, including Chao1, richness, Shannon, and Simpson indices; B: Β-diversity analysis of gut microbiota composition; C: Linear discriminant analysis effect size analysis identifying taxa with significantly different relative abundances among groups. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. Y: Young; M: Middle-aged; O: Older; PCo: Principal co-ordinates; LDA: Linear discriminant analysis.

The LEfSe algorithm identified differential microbiota among patients with FC of different age groups (Figure 2C). The Y group showed enrichment of Rikenellaceae, Hungatella hathewayi, Phocaeicola coprocola, Bacteroides cellulosilyticus, Bacteroides caccae, and Alistipes. The M group showed enrichment of Ruminococcus, members of class Clostridia, Alistipes putredinis, Bacteroides xylanisolvens, and Phocaeicola plebeius. Conversely, the O group was characterized by elevated abundances of Prevotella buccae, Veillonella, Mediterraneibacter faecis, and Limosilactobacillus.

To elucidate the potential functional roles of the microbiota in FC, correlation analysis was performed between differential genera and clinical characteristics (Figure 3A). In the Y group, Rikenellaceae and Alistipes were negatively correlated with GITT and positively correlated with MARP, MERP, and bowel movement frequency. Conversely, in the O group, Mediterraneibacter faecis showed positive correlations with SAS and SDS scores.

Figure 3 Correlation analysis of age-specific differential genera with clinical features and microbial co-occurrence networks. A: Correlation analysis between age-specific differential genera and clinical parameters; B: Microbial co-occurrence network in young group; C: Microbial co-occurrence network in middle-aged group; D: Microbial co-occurrence network in older group. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. MARP: Maximum anal resting pressure; MERP: Mean anal resting pressure; ARR: Anal relaxation rate; KESS: Knowles Eccersley Scott symptom; PAC-QOL: Patient assessment of constipation quality of life; SAS: Self-rating anxiety scale; SDS: Self-rating depression scale.

Analysis of microbial co-occurrence networks revealed variations in the core network density, key taxa roles, and peripheral microbes co-occurrence patterns among patients with FC of different ages (Figure 3). In the Y group, the network core was densely interconnected, with concentrated co-occurrences among multiple taxa, such as Anaeroglobus, Butyrivibrio, Campylobacter, Caulobacter, and Lawsonella (Figure 3B). In comparison, the M group showed relatively loose inter-taxon connections and fewer evident core taxa (Figure 3C). Meanwhile, the O group also displayed a dense core, featuring not only tight clusters around Alistipes, Ruthenibacterium, Eisenbergiella, and Ruminococcus_2, but also enhanced connectivity among many peripheral taxa, resulting in a more complex overall network connection and a more uniform link distribution for some nodes (Figure 3D).

Comparison of gut microbiota profiles between patients with FC of different subtypes

Because the diagnosis of NTC can overlap with constipation-predominant irritable bowel syndrome, the NTC cohort was small. To mitigate potential bias, microbiota analysis was restricted to the STC, DD, and MC groups.

Analysis of Chao1, richness, Shannon_2, and Simpson demonstrated significantly higher microbial richness and diversity in the STC group than in the DD group, whereas the MC group showed intermediate levels of diversity but did not differ significantly from either STC or DD (Figure 4A). β-diversity analysis revealed distinct community structures among the STC, DD, and MC groups (R² = 0.0747, P = 0.001) (Figure 4B). Notably, the STC and DD groups had substantially different microbial community structures (R² = 0.0666, P = 0.014) (Figure 4C), with the latter exhibiting more homogeneous microbiota profiles across its samples.

Figure 4 Comparison of gut microbiota profiles among different functional constipation subtypes. A: Comparison of α-diversity among functional constipation (FC) subtypes, including Chao1, richness, Shannon, and Simpson indices; B: Β-diversity analysis of gut microbiota composition among different FC subtypes; C: Β-diversity analysis of gut microbiota composition between slow-transit constipation group and defecatory disorder group; D: Linear discriminant analysis effect size analysis identifying taxa with significantly different relative abundances among groups. ^aP < 0.05. STC: Slow-transit constipation; DD: Defecatory disorder; MC: Mixed constipation; PCo: Principal co-ordinates; LDA: Linear discriminant analysis.

LEfSe identified significant differential taxa between the STC, DD and MC groups (Figure 4D). The STC group showed enrichment in Lawsonibacter, Rikenellaceae, Anaerotruncus, Parabacteroides, Alistipes, Lawsonibacter asaccharolyticus, Howardella, and Howardella ureilytica, while the DD group was characterized by enrichment of Prevotella rara and Megasphaera micronuciformis. In contrast, the MC group showed enrichment in taxa such as Flavonifractor plautii, Intestinimonas, Bacteroides cellulosilyticus, Bacteroides ovatus, Parasutterella, and Dysosmobacter.

DISCUSSION

Gut microbiota dysbiosis is implicated in the pathogenesis and progression of multiple diseases, such as inflammatory bowel disease, colorectal cancer, and diabetes. However, numerous factors influence microbiota diversity and composition, varying between individuals. In this study, we integrated the clinical characteristics, gut microbiota profiling, gastrointestinal motility assessments, and anorectal function tests in patients with FC, finding that advancing age was associated with declining colonic and rectal function, reduced microbial diversity, and aggravated community structure disorder. Furthermore, patients with different FC subtypes exhibited distinct microbiota profiles.

Our analysis of demographic variables also revealed that females exhibited a higher prevalence of FC. Recent data from the United States National Health and Nutrition Examination Survey indicate constipation prevalence is significantly higher in women (10.19%) than in men (4.82%)[16]. This aligns with our finding that the number of female patients with FC was approximately 2.8 times that of males. This disparity may reflect the effects of progesterone and estrogen in slowing intestinal transit, as well as sex-specific anatomical differences in pelvic floor musculature[17]. Analysis of different age groups revealed that in younger and middle-aged cohorts, the proportion of female participants was significantly higher than males. Conversely, in the older group, the sex ratio approached parity. Collectively, these findings further suggest the potential involvement of estrogen and progesterone in the pathogenesis of FC.

Significant dysbiosis was observed among the patients with FC enrolled in this study, marked by a considerable reduction in microbiota richness and diversity relative to HCs. Moreover, several genera exhibited differential abundances in patients with FC. Specifically, Methanobrevibacter, Intestinibacter, Sellimonas, and Eisenbergiella were enriched, whereas Megamonas, Faecalimonas, and Prevotella were depleted. Methanobrevibacter produces methane, which inhibits intestinal contractions and slows luminal transit, thereby exacerbating constipation symptoms[18]. SCFAs are key mediators of microbiota-host interactions, and their biosynthesis is closely linked to the abundance of specific taxa. Increased levels of Intestinibacter, Sellimonas, and Eisenbergiella activate SCFA production, whereas reductions in taxa such as Faecalimonas diminish acetate and butyrate synthesis. Notably, certain SCFAs, such as butyrate, exhibit concentration-dependent, bidirectional effects on colonic motility. At physiological concentrations, they promote peristalsis via G-protein–coupled receptors and by enhancing epithelial cell metabolism. Conversely, at abnormal concentrations, these SCFAs can inhibit colonic motility, disrupt normal function, and precipitate abdominal discomfort, including pain[19].

Studies examining the impact of age on anorectal function in patients with FC have demonstrated that young individuals exhibit higher anal resting and squeeze pressures, longer contraction durations, and a larger RAPD compared with middle-aged patients, with both groups outperforming older patients in these parameters. The underlying mechanisms likely relate to changes in the structural and functional anatomy of the neuromuscular apparatus. An animal study has demonstrated age-related atrophy of the external anal sphincter, with reduced muscle fiber number and an increased connective and collagenous tissue, resulting in diminished muscle tone and contractility[20]. Meanwhile, the intensity of mean total calcium ion current in colonic smooth muscle cells also declines with advancing age, impairing contractile function[21]. Concurrently, levels of neuronal nitric oxide synthase immunoreactivity and substance P immunoreactivity in the internal anal sphincter decrease significantly with age, further weakening local neural regulation[22].

Although studies in healthy adults showed minimal association between gut microbial diversity and age[23], our findings revealed a clear age-related decline in microbial ecosystem stability among patients with FC, primarily characterized by progressive reductions in microbiota richness and diversity. Moreover, while microbiota composition and structure are relatively homogeneous among young patients, they are markedly heterogeneous in the elderly, with distinct age-specific differential taxa identified. These findings suggest that age plays an essential role in microbiota composition and its variability in FC. Numerous studies have demonstrated the relationship between advancing age and a series of alterations in intestinal mucosal architecture and barrier function, including reduced proliferation of intestinal stem cells and T lymphocytes, decreased generation of B-cell-derived antibodies, increased apoptotic and proliferative turnover of absorptive enterocytes, and thickening of the goblet cell-derived mucus layer[24,25]. These changes not only compromise the self-regulatory capacity of the microbial barrier but also destabilize the intestinal microecology, creating conditions for pathogen overgrowth and loss of microbial diversity, thus establishing a mucosal foundation for dysbiosis and functional impairment. Similarly, Kashtanova et al[26] confirmed correlations between microbial α-diversity, age, and metabolic disease, aligning with our results. In older patients with FC, delayed fecal transit leads to prolonged stool retention, along with the pathogenic bacteria and metabolic byproducts, continuously challenging the mucosal barrier and precipitating further ecological imbalance.

Notably, in young patients with FC, we observed the enrichment of Rikenellaceae, Hungatella hathewayi, Phocaeicola coprocola, Bacteroides, and Alistipes. The enrichment of Rikenellaceae and Alistipes was particularly positively correlated with MERP but negatively correlated with weekly bowel movement frequency and colonic transit rate. This likely reflects the ability of metabolites produced by Rikenellaceae and Alistipes to promote sphincter tone and inhibit colonic peristalsis by modulating enteric neural or humoral pathways. Alistipes activates intestinal G protein-coupled receptor 43 receptors via its metabolic products, promoting glucagon-like peptide-1 and PYY secretion, thereby suppressing fasting and postprandial gastrointestinal transit[27]. The functional profile of enriched microbiota in young patients with FC appears focused on inhibiting intestinal motility and impeding defecation. Conversely, older patients with FC in this study harbored a distinct microbial composition, featuring significant enrichment of Prevotella buccae, Veillonella, Mediterraneibacter faecis, and Limosilactobacillus. Among these, the relative abundance of Mediterraneibacter faecis correlated positively with SAS and SDS scores. The elevated levels of these differential taxa may reflect interactions with mucosal barrier function and host immune-inflammatory responses, though their causal relationships remain to be elucidated. Increased Veillonella in murine colon models exacerbates inflammatory cell infiltration and correlates with higher colitis scores[28]. Conversely, Limosilactobacillus has been demonstrated to not only attenuate inflammation via the mitogen-activated protein kinase/signal transducer and activator of transcription 3 signaling pathway but also reduce anxiety-like behavior in stress models[29]. As a gram-positive member of the Lachnospiraceae, Mediterraneibacter faecis produces butyrate and other SCFAs, key energy substrates for the mucosa. By resisting pathogenic invasion and preventing colonic inflammatory infiltration, these SCFAs play an important role in maintaining intestinal homeostasis[30,31]. The prominent enrichment of protective taxa in elderly patients may represent a self-protective response. Consistently, Guo et al[32] also reported significant increases in Prevotella among older patients with FC. Notably, middle-aged patients with FC displayed a mixed microbial profile. First, these profiles feature enrichment of butyrate- and propionate-producing taxa such as Ruminococcus and Clostridia, which enhance mucosal barrier function, promote epithelial repair, and stimulate intestinal stem cell proliferation. Second, they also harbor abundances of Alistipes putredinis, Bacteroides xylanisolvens, and Phocaeicola plebeius, which promote the accumulation of pro-inflammatory metabolites, disrupt enteric neurotransmission, and inhibit intestinal motility[33-35].

Microbial network analysis further revealed densely interconnected core microbiota in young and older patients. Specifically, core microbiota in the young cohort were more centralized around taxa implicated in mucosal injury[36,37]. Notably, Campylobacter, as one of the core nodes in the young group, may impair mucosal integrity and promote inflammatory responses[36], thereby potentially contributing to gastrointestinal dysmotility and aggravation of constipation. In addition, Anaeroglobus, although not previously associated with constipation, emerged as a core node in the young-group network, suggesting a potential role in microbial interactions among younger patients; this should be regarded as an exploratory finding requiring further validation. Conversely, in the co-occurrence network of older patients, multiple peripheral species also formed tight clusters, encompassing taxa that either induce pro-inflammatory cytokine expression and impair mucosal repair, or are capable of fermenting dietary fiber to preserve mucosal integrity[38,39]. Alistipes, a core taxon in the older group has been linked to intestinal inflammation and bile-acid dysregulation, which may indirectly contribute to constipation by affecting host inflammatory responses and metabolic homeostasis. The microbial network of the middle-aged cohort, however, exhibited loose interconnections and fewer core microbiota. These findings underscore the existence of age-specific gut microbiota profiles and suggest corresponding differences in the underlying pathophysiological mechanisms in patients with FC of different ages.

According to the Rome IV criteria, FC can be classified into four subtypes. Two of the subtypes, STC and DD, are driven by distinct pathophysiological mechanisms. STC is characterized primarily by prolonged colonic transit time, whereas DD, which arises from the pelvic floor and/or anal sphincter dyssynergia, manifests as difficulty in defecation[40]. In this study, the STC group exhibited significantly higher microbiota richness and diversity compared to the DD group, with markedly different community structures. The MC group exhibited intermediate diversity levels, lower than those observed in the STC group but higher than in the DD group. Such differences may reflect distinct underlying etiologies. In STC, slowed gastrointestinal transit prolongs luminal retention throughout the gut, providing favorable temporal and spatial conditions for microbial proliferation, thereby enhancing overall microbial diversity. Conversely, DD leads to fecal retention in the rectum, where altered local conditions and substrate depletion, owing to prior absorption of water and nutrients in the colon, suppress microbial fermentative activity when combined with elevated potential of hydrogen (pH) and lack of segmental mixing. This environment is unfavorable for anaerobic proliferation and therefore suppresses microbial diversity. Notably, intraluminal pH in the human colon gradually increases along the physiological gradient, rising from approximately 5.7 in the ileocecal region to near-neutral values of 6.7-7.0 in the rectum[41]. pH directly influences microbial competition for substrates[42]. For example, butyrate producers, such as Faecalibacterium and Roseburia, grow better and generate more butyrate at mildly acidic pH = 5.5 than at near-neutral pH = 6.7[43]. Moreover, since the rectum lacks segmental contractions and propagated peristalsis featured in the colon, luminal contents mix poorly with the resident microbiota, leading to substrate scarcity for microbial growth and restricted intestinal microbial proliferation[44,45]. Consistent with these dynamics, differential analysis in this study identified Alistipes and Anaerotruncus as the most predominantly enriched taxa in the STC group, which have been implicated in slowed intestinal motility and host metabolic modulation[46]. Conversely, in the DD group, abundances of Prevotella rara and Megasphaera micronuciformis were observed instead, both possessing the potential to stimulate gut motility and modulate enteric neuronal activity[47,48]. This suggests that, in the face of defecation difficulties, the host may attempt compensatory modulation of the gut ecosystem and its metabolites to activate local peristalsis. Dysosmobacter has been suggested to exert potential anti-inflammatory effects, whereas Intestinimonas is a butyrate-producing genus that supports mucosal barrier integrity and modulates gut motility[34,35]. Given that patients with MC present with both delayed colonic transit and defecatory dysfunction, their microbiota may display a hybrid profile that combines features of both subtypes.

Nevertheless, given the observational design, causal relationships between microbial alterations and FC cannot be definitively established. This study also has limitations: Some subgroups had insufficient sample sizes and were therefore excluded, and no functional prediction was performed, with functional inferences relying solely on genus-level traits. These limitations highlight the need for larger multi-center cohorts with balanced subtype distributions and the integration of multi-omics approaches, including metagenomics, to clarify the determinants of gut microbiota in FC and their interactions with host physiology. Mechanistic investigations, such as fecal microbiota transplantation in animal models, will be essential to determine whether these alterations directly contribute to FC pathogenesis.

CONCLUSION

Patients with FC commonly experience gut microbiota dysbiosis. Age and constipation subtype are key factors influencing microbial composition, structure, and function. Therefore, clinical management of FC should incorporate age- and subtype-specific microbiota profiles when formulating personalized microbial interventions. Our findings offer novel insights and theoretical foundations for FC diagnosis and treatment.

ACKNOWLEDGEMENTS

We gratefully acknowledge the contributions of all medical staff from the Department of Gastroenterology and the volunteers who participated in this study.