Structural and Functional Differences in Small Intestinal and Fecal Microbiota: 16S rRNA Gene Investigation in Rats

Structural and Functional Differences in Small Intestinal and Fecal Microbiota: 16S rRNA Gene Investigation in Rats | 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 Research Article Structural and Functional Differences in Small Intestinal and Fecal Microbiota: 16S rRNA Gene Investigation in Rats Xiao Wei Sun, Hua Zhang, Hong Rui Li, Peng Fei Xin, Xue Gao, Cai Yun Zhou, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1697881/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background: The occurrence of serious diseases, such as inflammatory diseases and cancer, in the small intestine is significantly lower than that in the colon. The differentiation of small-intestine microbiota from large-intestine microbiota might hold great significance. Materials and methods: To compare floral composition and functions between the two types of microbiota, ileal contents and feces were collected from Sprague Dawley (SD) rats, and the V3–V4 region of the 16S ribosomal ribonucleic acid (rRNA) gene in these rats was amplified and sequenced. We subjected the data to bioinformatics analyzing. Results: Compared with feces, about 50% of bacterial genera in the ileum were exclusive, with low abundance (operational taxonomic units [OTUs] <1000). Of bacteria shared between the ileum and feces, a few genera were highly abundant (dominant), whereas most had low abundance (less dominant). Dominant bacteria differed between the ileum and feces. Ileal bacteria showed greater β-diversity, and the distance between in-group samples was nearer than that between paired ileum–feces samples. Moreover, the ileum shared various biomarkers and functions with feces ( P < 0.05). A high-fat diet (HFD) and specific-pathogen–free (SPF) conditions had a profound influence on α-diversity and abundance but not on the exclusive/shared features or β-diversity of samples. Intestinal microbiota were composed of high-prevalence dominant, low-prevalence dominant, and less-dominant bacteria. Conclusions: The present findings suggested that ileal and fecal bacteria were different structurally and functionally. These differences might be key to the fundamental protection of the small intestine from diseases. Ileum Feces Microbiota 16S rRNA gene Sequencing Rats Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Messages What is the context? Feces are rich in bacteria, and the development of sequencing technology makes it possible to identify and classify gut microbiota. The fecal microbiota is closely related to various diseases of extraintestinal organs and the colon. There has been relatively limited research on bacteria in the small intestine. The small intestine and colon have completely different metabolic environments. Additionally, the microbial composition is significantly affected by the local metabolic environment. What is new? In this study, we compared the microbiota of the small intestine and feces. The key findings are as follows: Ileal and fecal bacteria were structurally and functionally different. Dominant bacteria differed between the ileum and feces. Ileal bacteria showed greater β-diversity than fecal bacteria. Ileal bacteria were extrinsic to the feces and not well transferred to the feces. About 50% of bacterial genera in the small intestine were exclusive to the feces. The distance between in-group samples was nearer than that between paired ileum–feces samples. What is the impact? This study provides further solid evidence that the microbiota along intestine segmentally distributed. Introduction Trillions of bacteria exist in the human gastrointestinal (GI) tract. 1 With the help of high-throughput sequencing of nucleotides, and the identification of the 16S ribosomal ribonucleic acid (rRNA) gene as highly conserved in most bacterial clades, 2 researchers have been able to identify and classify gut microbiota, which are distributed throughout the entire GI tract in diverse microbial communities. 1 Gut bacteria have been shown to take part in many physiological or pathological processes in humans, such as inflammatory bowel disease, cancer, nutritional obesity, diabetes, and cardiovascular disease. 3–11 However, based on the idea that the colon is the site of final GI content collection and the fact that colonic samples are easily acquired, most researchers have obtained samples from feces and regarded fecal flora as naturally synonymous with gut microbiota. 12–16 During transportation and digestion of food component through the gut, different biochemical inner environments are formed in the small intestine (SI) and large intestine (LI). Accordingly, it is highly possible that the microbiota of these two organs are distinct. 15 The most significant distinction between SI and LI microbiota might be the persistent clinical fact that the SI has a much lower incidence of various diseases, such as carcinoma, than LI. Therefore, identifying differences and relationships between SI and LI microbiota is crucial to clarifying the gut bacterial mechanisms of various disorders. Several clinical and laboratory animal observations have proven the marked differences between SI and stool microbiota, as well as between different segments of the SI. 17,18 A systematic observational comparison between ileum and feces, however, is still lacking. To address this lack, we collected ileal contents and feces of rats and compared their structural and functional features based on 16S rRNA gene sequencing and bioinformatics analysis. Materials And Methods Animals Male Sprague Dawley (SD) rats, ages 4–5 weeks (n = 6) and weighing 52.32 ± 8.48 g, were raised individually in a conventional setting for 3 months, with diet and water ad libitum to simulate the natural state. The rats’ chow (3.16 kcal/g) was produced by KeAo-XieLi Co., Ltd. (Beijing, China) and sterilized with cobalt-60 ( 60 Co). All methods were carried out in accordance with relevant guidelines and regulations of the Laboratory Animal—Guidelines for Ethical Review of Animal Welfare (GB/T 35892—2018), and all experimental protocols were approved by the Medical Ethics Committee of Lanzhou University (jcyxy20190302). All methods were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments. Sample collection Rats were anesthetized by intraperitoneal (i.p.) injection of pentobarbital sodium (5 mg/100 g body mass) and killed by portal-vein blood drainage. We expressed no less than 2.0 g content of 5.0–7.0 cm of ileum or colon into sterile Eppendorf tubes (Eppendorf, Hamburg, Germany). Samples were then stored in a −80°C refrigerator after quick freezing in liquid nitrogen. To avoid environmental contamination, we performed our experiments on a clean bench. DNA extraction, PCR, and sequencing We extracted total deoxyribonucleic acid (DNA) from 0.5 g of each sample using a magnetic soil and stool DNA kit (#DP812; TIANGEN Biotech Co., Ltd., Shanghai, China) per the kit protocol. Primers were designed according to the conserved V3–V4 region of the 16S rRNA gene. Sequences were as follows: 338F: 5′-ACTCCTACGGGAGGCAGCA-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′. 19 The target region was amplified using polymerase chain reaction (PCR) with the barcode at the end of the primer. The amplification procedure was as follows: 95°C for 5 min, with 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 40 s; and extension at 72°C for 7 min. The products were then purified, quantified, and homogenized to form a sequencing library and run on a 1.8% agarose gel. After quality inspection and preparation of a flow cell chip, we sequenced 500 ng of PCR products by PE 250 mode (2 × 250 bp paired ends), performed on an Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA; via BMK Biotechnology Co., Beijing, China). Sequencing length was 350–450 bp. We transformed original image data files into sequenced reads via base calling. The DNA extraction, PCR, sequencing, data preprocessing and outcome assessment were finished in BMK Biotechnology Co., Beijing, China, who has no vested interest in the experiment and was not aware of the group allocation. Data preprocessing According to overlap relationships, we spliced PE reads using Fast Length Adjustment of SHort reads (FLASH) software v1.2.11 (Johns Hopkins University Center for Computational Biology, Baltimore, MD, USA). 20 Raw tags were filtered for quality using Trimmomatic software v0.33 (https://USADELLAB.org). 21 We removed chimeras using UCHIME software v4.2 (Edgar et al. , 2011) 22 to form clean tags, which were then clustered into operational taxonomic units (OTUs) at 97% sequence similarity using USEARCH software v10.0 (https://www.drive5.com/usearch/). 23 Representative sequences of OTUs were compared with the Silva microbial reference database v128 (http://www.arb-silva.de), 24 and OTUs were annotated using RDP Classifier v2.2 (https://rdp.cme.msu.edu). 25 We generated species richness at different taxonomic levels using Quantitative Insights Into Microbial Ecology (QIIME) software v2.2 (http://qiime.org/). Alpha-diversity indices (Ace, Chao1, Shannon, Simpson) and community dissimilarity (β-diversity) were evaluated using QIIME2 ( https://qiime2.org/ ). We then used R software v3.1.1 (R Foundation for Statistical Computing, Vienna, Austria) for both principal-component analysis (PCA) and principal-coordinate analysis (PCoA). Data analysis was performed by one investigator (XWS) on the platform of BMKCloud (https://international.biocloud.net), who was not aware of the group allocation, and the results interpretation was finished by another investigator (JGZ). Statistics Data were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). We conducted statistical analysis using SPSS v22 (IBM Corp., Armonk, NY, USA). An independent-sample t -test was used for comparisons between two groups; data were transformed to a natural logarithm (Ln) when equal variances were not assumed. We analyzed community dissimilarities via permutational multivariate analysis of variance (PERMANOVA). Differences were considered statistically significant when P < 0.05. Results Large proportions of ileal flora were exclusive to the feces From the 12 samples taken from the six rats, we obtained 904,117 PE reads (50,171–80,391), 849,599 raw tags (48,254–77,009), 801,140 clean tags (46,087–73,370), and 770,289 effective tags (43,314–72,269) with average length 408–417 bp. Guanine and cytosine (GC) content (52.66–56.06%; the quality score 20 (Q20), 94.05–97.19%; Q30, 88.89–94.79%) and effectiveness (effective tags/PE reads, 66.64–90.23%) met the qualification demands. In total, we obtained 1050 kinds of OTUs (range, 198–874), 490.00 ± 268.20 from the ileum and 430.50 ± 31.97 from feces. The two kinds did not significantly differ ( P > 0.05). Ileal OTUs were about twice as numerous as those in feces (1008 vs . 555), which implied higher variability in the ileum of rats. We next compared OTUs and species in and between ileal and fecal samples. OTU similarity in fecal samples was higher than that in ileal samples (the number of shared OTU was 257 vs. 81), but numbers of shared or exclusive species were similar (Fig. 1A–1B). Large proportions of OTUs and species were shared between ileal and fecal samples. A great many ileal OTUs and species remained exclusive to the ileum: 49.11% of OTUs, 36.84% of phyla and 58.31% of genera (Fig. 1C). That is, a considerable number of ileal bacteria were not detected in feces. We then analyzed the compositions and abundances (in OTUs) of ileum-exclusive bacteria. After annotation, we found that the phyla Chloroflexi, Fusobacteria, Latescibacteria, Nitrospirae, Planctomycetes, Rokubacteria, and unassigned bacteria were specific to the ileum, with OTUs ranging from 3 to 532. At the genus level, 193 bacteria were ileum exclusive. All had low abundance, with 61.82 ± 113.95 OTUs (SEM: 8.22) and prevalence of 17–100% (Table S1, Fig. S1), except for Acetobacter (Proteobacteria), with 1798 OTUs. Shared bacteria were composed of distinct floral structures in the ileum and feces Although large proportions of ileal OTUs and bacteria were exclusive to the ileum, their abundances were low. Shared bacteria, which can be defined as those transferred from the ileum to the colon along with digesta, constituted the main body of intestinal flora by virtue of their high abundance. The top 10 phyla—namely Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia, Spirochaetes, Patescibacteria, Epsilonbacteraeota, Acidobacteria, and Tenericutes (OTUs range, 36–152,356)—as well as Cyanobacteria and Gemmatimonadetes (OTUs < 1000), were the phyla of bacteria shared between ileal and fecal flora. They showed obviously different ratios of abundance. High ratios of Patescibacteria, Verrucomicrobia, Spirochaetes, Bacteroidetes, and Tenericutes were in feces, while Firmicutes, Proteobacteria, Acidobacteria, Epsilonbacteraeota, and Actinobacteria thrived more in ileal flora. At the genus level, Romboutsia (138,023 OTUs), Turicibacter (63,554 OTUs), and Rothia (12,466 OTUs) dominated the constitution of ileal bacteria, with levels 6–14 times higher than in feces. Moreover, Streptococcus (OTUs in feces: 136; in ileum: 4771), Lactobacillus (OTUs in feces: 23; in ileum: 4598), and Escherichia-Shigella (OTUs in feces: 29; in ileum: 3251) were also dominant in the ileum (35–200 times higher than in feces; Fig. 2). An additional 47 shared ileum-dominant bacteria had low abundances (<2000 OTUs in ileum; Table S1). Differing diversity , biomarkers, and functions in ileal and fecal bacteria To further confirm the differences between ileal microbiota and fecal flora, we studied microbiotal features of variability and biomarkers of ileal flora using bioinformatics. Results showed that the species richness (Chao1 and Ace indices) and evenness (Shannon and Simpson indices) of ileal and fecal microbiota were identical ( P > 0.05; Table S2). According to PCA and PCoA analyses by the binary Jaccard method, however, the distance between samples was significantly higher in ileal than in fecal flora ( P < 0.05; PERMANOVA, r 2 = 0.336, P = 0.002; Fig. 3A–3B). Moreover, according to the unweighted pair group method with arithmetic mean (UPGMA) and heatmap analyses, the distance between in-group samples was shorter than that between paired ileal–fecal samples. That is to say, microbiota were more similar across individuals than across body sites from the same individual (Fig. 3C–3D). Linear discriminant analysis with effect size (LEfSe) analysis showed that ileal and fecal bacteria had distinct biomarkers (Fig. 3E). Taken together, these results indicated that ileal and fecal flora were different, despite the bulk of dominant bacteria being continuously transferred from the ileum to the colon along with dietary residues. Functionally, according to Tax4Fun (http://tax4fun.gobics.de/) predictions, ileal bacteria displayed higher levels of metabolism (74.52 ± 0.71% vs. 69.28 ± 1.07%) and organismal systems (1.53 ± 0.14% vs. 1.16 ± 0.10%) but a lower level of environmental information processing (10.69 ± 0.70% vs. 15.54 ± 1.02%) than fecal flora ( P < 0.05). Ileal and fecal microbiota under high-fat diet intervention To validate the structures and features of ileal flora and clarify the possible influence on them of dietary diversity, we established another group of rats for which intervention was a high-fat diet (HFD). After being fed the HFD for 60 days, the animals were then switched to chow for another 60 days ( n = 10). We collected the rats’ intestinal contents and subjected them to 16S rRNA gene V3–V4 region sequencing and analysis. Results showed obvious differences between the ileal and fecal bacteria mentioned earlier. Ileum-exclusive bacteria constituted 26.32% of phyla and 53.57% in genera (Fig. 4). Latescibacteria, Nitrospirae, Planctomycetes, Gemmatimonadetes, and unassigned bacteria were specific to the ileum (OTU range, 16–503). In addition, 180 genera of bacteria were ileum exclusive, with OTUs ranging from 6 to 725 (96.48 ± 115.04), except for uncultured bacterium c Subgroup 6 ( Acidobacteria ) with 1483 OTUs (Table S3). Romboutsia (200,712 OTUs), Rothia (69,437 OTUs), Turicibacter (28,073 OTUs, 5–24 times higher than in feces), Streptococcus (23,487 OTUs), Helicobacter (13,346 OTUs), and Candidatus Arthromitus (22,921 OTUs, 44–997 times higher than in feces) dominated the constitution of ileal bacteria. Another 53 shared ileum-dominant bacteria had low abundances (<5056 in SI; Table S3). Moreover, they showed the same functional differences as those of chow-fed rats (data not shown). However, the HFD did exert lasting influence on the dominant categories and α-diversity of ileal bacteria, which might be biomarkers of specific-diet ingestion. The richness, diversity, and evenness of SI flora were all elevated (Table S4). Ileal microbiota under specific-pathogen–free conditions We also investigated the features of ileal bacteria in germ-free conditions. Male SD rats (n = 6, body weight 304.10 ± 29.92 g) that had been maintained in a specific-pathogen–free (SPF) environment and fed a chow diet and water ad libitum were chosen randomly. Their ileal and fecal contents produced a total of 1254 kinds of OTUs (ileum: 1013 [393.67 ± 77.19]; feces: 1005 [482.83 ± 37.38]; P > 0.05). The SPF environment remarkably influenced intestinal bacterial structure. The variability of ileal bacteria and exclusive bacteria was nearly diminished. Only Dependentiae (1 OTU) and 89 bacteria genera were ileum exclusive, with very few OTUs (1.78 ± 1.16 [range, 1–6]). Lactobacillus , Romboutsia , and Candidatus Arthromitus were dominant. Abundances of less-dominant shared bacteria were also decreased (Table S5). Shannon and Simpson indices were lower for ileal than for fecal flora ( P < 0.05; Table S6). The differences between ileal and fecal bacteria, however, also were remarkable (Fig. 5). Functionally, ileal bacteria also had a higher level of genetic-information processing and fewer cellular processes than fecal flora. In bugbase phenotype prediction (https://bugbase.cs.umn.edu/), ileal bacteria had advantages in mobile genetic elements (MGEs), Gram positivity, and facultative anaerobism, whereas anaerobism, Gram negativity, and potentially pathogenic phenotype were significantly lower than fecal flora. In Functional Annotation of Prokaryotic Taxa (FAPROTAX; Louca et al. , 2016) analysis, ileal bacteria also showed lower nitrate reduction, nitrification, xylanolysis, and aerobic ammonia oxidation than fecal flora (Fig. 6). Constitution of ileal microecology We merged the annotated species data of rats maintained in conventional conditions with those of rats maintained in an SPF environment to clarify ileal microecology. Ileal microbiota could be classified into three groups: exclusive bacteria, which were in very low abundance; dominant bacteria shared between the ileum and feces, and low-abundance bacteria shared between the ileum and feces (Tables S7–S18). Exclusive bacteria were sensitive to diet and feeding environment. SPF conditions decreased exclusive-bacteria count intensively. Therefore, exclusive bacteria were further divisible into condition related and condition irrelevant. We identified 14 condition-irrelevant bacteria (Table S7), 152 bacteria related to conventional conditions (Table S8), 24 chow-related bacteria (Table S9), 12 HFD/chow-related bacteria (Table S10), and 70 SPF environment–related bacteria (Table S11). Feeding environment had a greater influence on exclusive-bacteria species and abundance than diet diversity. In bacteria shared between the ileum and feces, the abundances of 80 condition-irrelevant bacteria varied enormously. Only very small proportions of bacteria had >10,000 OTUs, so-called dominant bacteria (3/80 shared bacteria in chow rats, 8/80 in HFD/chow rats, and 3/80 in SPF rats). Romboutsia was the leading bacterium with >100,000 OTUs. Most others were in low abundance (Table S12); conditions influenced the abundances and domination of these bacteria. Rothia and Turicibacter were dominant in conventional conditions, whereas Lactobacillus was dominant in an SPF environment. All condition-related bacteria in the ileum that were shared between the ileum and feces were in low abundance (Tables S13–S18). Rats maintained in a conventional environment (Tables S13, S16, S17) had lower kinds of bacteria than rats maintained in an SPF environment (Table 18), 64 vs. 223. There were also very small proportions of bacteria shared between the ileum and feces that were specific to chow (Table S16) or HFD/chow (Table S17) diets. Bacterial prevalence across individuals Apart from differences in structure between ileal and fecal microbiota, bacterial prevalence and abundance varied extensively across individuals and was obviously host specific. In rats fed normal chow, only 69 of 337 ileal bacteria and 75 of 337 fecal bacteria were 100% prevalent. All less-prevalent bacteria were also less dominant, mostly with OTUs < 300. Among the 100% prevalent bacteria, only a few were highly dominant, making up the top 10 dominant bacteria of the ileal or fecal group. Dominant bacteria varied across individuals. Romboutsia was dominant in ileal samples from all six individual rats, whereas Turicibacter and Rothia were dominant in three individuals only. In fecal microbiota, uncultured bacterium f Muribaculaceae was dominant in all six individuals, Lachnospiraceae NK4A136 group in four, and Treponema 2 in only two. Discussion In this study, we systematically observed differences between ileal and fecal microbiota based on sequencing and bioinformatics analysis of the 16S rRNA V3–V4 region. Intestinal microbiota have increasingly become a popular research topic in recent years. They are important symbionts of the gut and are involved in at least three primary physiological or pathological processes: (1) dynamic changes in and complexity of the microbial ecosystem; (2) dietary digestion, absorption, nutrition, synthesis, detoxification, and immune functions of the host; 26 and (3) various diseases of the host. 27,28 However, three key obstacles hinder the clarification of those processes. First, traditional culturing methods and conditions are limited in their ability to identify the trillions of intestinal microorganisms and the complicated microbial ecosystem. Second, the structures and functions of intestinal microorganisms are tightly coupled with various factors, such as environment, diet, and disease. They change dynamically along the GI tract (diversity of location), fluctuate widely over time (diversity of stability), vary among individuals (diversity of prevalence), and are inconsistent with abundance (diversity of abundance). Even increasing the sample sizes cannot include all intestinal bacteria and their trajectories. Last but not least might be the most important factor: at any given point in time, most of these microorganisms remain in the long, dark mystery tube of the SI, which cannot be reached noninvasively, rendering sampling difficult. Before becoming part of the composition of feces, they have experienced a long series of complicated biochemical reactions within the tube. Researchers can obtain final fecal samples only after this complicated process. These obstacles raise several key questions: Do fecal bacteria represent all microbial populations throughout the gut? To what extent do they change? Moreover, in terms of diseases, why is the morbidity of LI carcinoma extremely high in developed populations, whereas the SI remains much less vulnerable? Can this phenomenon be attributed to distinct microbiota in the SI? Many studies have deduced distinctions between fecal microorganisms and other components of intestinal microbiota according to functional heterogeneity 29 and the complicated biochemical environment ( e.g. , oxygen, pH, mucous thickness, antimicrobials, bile acids, transit time). 1 But direct proof is still lacking. In humans, research on SI microbiota can be performed only in patients, 15,17 but the influences of the pathological environment (pathophysiology as well as medications) on microbiota are uncontrollable. The results might be limited in their ability to tell the real differences. The most acceptable methods are still those using laboratory animals. Mentula S. et al. compared microbiota between jejunal fluid and fecal samples of 22 beagles by means of organismal culture and proved the inability of fecal samples to represent the microbiota of the upper gut. 30 Also, systematic evidence and intensive study of the differences between ileal and fecal bacteria are still needed. Recently, evidence from the Human Microbiome Project has shown that human microbiota are more similar across individuals than across body sites. 12 Is that also the case for the continuously and transitionally connected contents of the ileum and feces? Metagenomic sequencing and bioinformatics analysis are powerful tools used to identify complicated intestinal microbiota. In this study, we used SD rats as research subjects, in which influencing factors, including diet, environment, and individual characteristics (sex, age, body weight) could be homogenized and controlled, and total ileal contents were easily sampled. Moreover, because of the rats’ stable long-term diets, sampling of the ileum and feces at the same point in time could represent the progressive relationship between ileal and colonic contents. To avoid possible influence from any procedure or medication, we achieved microbiotal stability by taking repeated samples from different individuals 31 rather than from the same individual at different timepoints. 30 Our results showed that, in genus-level annotation, a total 335 kinds of bacteria were in ileal and colonic contents sampled from normal rats ( n = 6). This number was far lower than trillions in terms of abundance. Even in this limited set of bacteria, 193 were ileum exclusive (58.30%) and could not be detected in feces. This means that fecal microbiota can represent only about 42% of ileal-bacteria species. Mentula S. et al reported that in beagles, about 25% of jejunal bacteria were not detectable in fecal samples, nor were 45% of fecal bacteria detectable in the jejunum. In this study, we found that in conventional conditions, ileum-exclusive bacteria were significantly more numerous than feces-exclusive bacteria, 58.30% or 53.57% vs. 2.82% or 0% in chow or HFD/chow rats, respectively; in the SPF environment, these values were respectively 20.51% vs. 16.87%. These differences might reflect the different detection method (organism culture vs. 16S rRNA gene sequencing and analysis), different locations (jejunum vs. ileum), different animals, or different environment, as well as different methods of sampling. Abundance is another important index for bacterial-structure evaluation. Large proportions of both ileal and fecal bacteria were in low abundance. Meanwhile, very small proportions thrived in extremely high abundance; these were the annotated “dominant” bacteria or biomarkers. These dominant bacteria included most of the trillions of intestinal bacteria that exist (the top 10 bacteria contributed 70–80% of abundance). Dominant bacteria were obviously distinct, however, between the ileum and feces. Escherichia-Shigella , Lactobacillus , Romboutsia, Rothia , Streptococcus , and Turicibacter were dominant in the ileum but not in feces, whereas the reverse was true of Lachnospiraceae NK4A136 group , uncultured bacterium f Muribaculaceae , Treponema 2 , uncultured bacterium f Lachnospiraceae , Akkermansia , Candidatus Saccharimonas , and Ruminococcaceae UCG-013 (Table S1). This result suggested that fecal microbiota missed the bacterial dominance information of ileal microbiota. Accordingly, their functions were distinct. Ileal bacteria displayed higher levels of metabolism and organismal systems but a lower level of environmental information processing than feces. Assessment of β-diversity also showed that ileal and fecal microbiota were more similar among individuals than among body sites of the same individual. These results suggested that fecal bacteria were limited in their ability to represent ileal bacteria and vice versa . According to our results, in a certain segment of the gut, bacteria could be classified into two groups: very few high-abundance bacteria (dominant) and a great many low-abundance bacteria (less dominant). Spatially, the very few dominant bacteria might mix with the gut contents, adapt to them, thrive with the components of digesta, and transition to the large intestine. They might be sensitive to and fluctuate with different digesta. For example, dominant bacteria changed significantly from the SI to the LI. The shared bacteria genera Escherichia-Shigella , Lactobacillus , Romboutsia, Rothia , Streptococcus , and Turicibacter were reduced by 7–200 times, while Akkermansia , Candidatus Saccharimonas , Lachnospiraceae NK4A136 group , Ruminococcaceae UCG-013 , Treponema 2 , uncultured bacterium f Lachnospiraceae , and uncultured bacterium f Muribaculaceae were increased by 5–63 times. The majority of less-dominant bacteria might be located in mucus 32 or mucosa. 15 They could be related to nutrients rather than digesta because their abundances are maintained at very low levels. These bacteria might or might not transition with gut content (exclusive or shared), maintain their abundance at a relatively constant level, and comprise the majority of the segment’s microbial ecosystem. We supposed that this ecosystem was interdependent with the host and played important roles in the stability of the intestinal environment. 26 The gut is notable in two aspects in terms of diseases. One is the diverse incidence rate of diseases in different parts of the tract. According to anatomical and histological structures and functions, the gut can be divided into seven segments: mouth, esophagus, stomach, duodenum, jejunum, ileum, and colon. Coincidently, this division also distinguishes diseases from one another in incidence. Carcinoma and some inflammatory diseases are significantly higher in the esophagus, stomach, and colon but rarely occur in the SI. Because of levels of ingested nutrients as well as those of possible toxins being very low in the colon after earlier absorption/degradation, the microbial ecosystem should be the key factor. Whether the SI microbial ecosystem is protective against injury or tumorigenesis of epithelial cells is an interesting topic. However, many visceral-organ diseases such as tumors and metabolic disorders have been linked with gut bacteria. Most relevant studies have derived their data from fecal samples. Bacteria or metabolites are assumed to possibly enter the circulatory system though gut leakage. As the main point of absorption, the SI has higher permeability than the colon, and therefore its microbial ecosystem might be more intimately connected with visceral-organ disorders, especially metabolic diseases. The relationship between diseases and exclusive bacteria in the SI needs to be established. In addition to the variation in structural characteristics from ileum to colon, diversity and dynamic change were additional primary features of and influenced the progression of intestinal microbiota. That is, the homogeneity and representativeness of samples are a topic of concern. In terms of microbiotal structure and bacterial abundance, no one individual can be represented by any other (prevalence), no one part of the intestine can be represented by any other (location), and no time point can be represented by other time point (stability). 33 In this study, we found that only about one-fifth of the bacteria at the genus level were 100% prevalent (69/337 in the SI vs. 75/337 in the LI). The less-prevalent bacteria (< 100%) were all less dominant, with abundance < 300 OTUs (with occasional exceptions). The top 10 dominant bacteria all had 100% prevalence. This direction of relevance was contrary to the finding in beagles that high prevalence reflected high proportions of total count. 23 However, dominant bacteria also varied across individuals. Of ileal bacteria, only Romboutsia was dominant in all six individuals (“Monarch”-dominant bacteria), while Turicibacter and Rothia were dominant in only three (“Minister”-dominant bacteria). In fecal microbiota, uncultured bacterium f Muribaculaceae was the “Monarch”-dominant bacterium, while Lachnospiraceae NK4A136 group and Treponema 2 were the “Minister”-dominant bacteria. The less prevalent bacteria and various dominant bacteria determined the microbiotal characteristics of unique individuals. This microbiotal imprinting should be addressed in investigations of the mechanisms of disease occurrence and different incidences among individuals and between the SI and LI. The main limitation of this study is the representativeness of the detected microbiota across species. Due to differences in living environment, diet structure, and health status, the domination and prevalence of bacteria may be extremely varied. Indeed, to date, it is nearly impossible to identify a typical bacterial inhabitant across species, or even across populations in the same species or across individuals in the same population. The most popular population studies aimed to identify diseases associated with bacteria by comparing bacteria in various types of patients with those in healthy people. Based on this background, this study aimed only to identify the features of bacterial distribution across the small intestine and the large intestine, rather than to identify specific bacteria. Another limitation is that this study did not identify the relationship between different distribution features and diseases. Although at the beginning of this study the authors aimed to address the interesting problem of the significantly lower occurrence of serious diseases, such as inflammatory diseases and cancer, in the small intestine compared to that in the colon, the identified distribution features cannot answer that question sufficiently. Whether or how the different distribution of bacteria between the small and large intestine are associated with varied disease occurrences still needs to be investigated in the future. Furthermore, failing to discover the source of intestinal bacteria, especially those in the small intestine, is the third limitation of this study. During ingestion, stomach acid may destroy most of the bacteria. It is still not clear how large numbers of bacteria survive the acid and reside prevalently or exclusively in the gut in a relative stable status. Conclusions Collectively, we proved that structure (dominant bacteria and β-diversity) and functions were markedly different between ileal and fecal microbiota in rats, although they were intimately transitional. The ileum had large proportions of exclusive bacteria with low abundances. HFD and SPF feeding environments had profound influences on ileal and fecal microbiota. Diversity was the basic characteristic of intestinal microbiota. In a specific location, intestinal bacteria could be classified into two groups: large proportions of less-dominant bacteria with low abundances, and small proportions of dominant bacteria with high abundances. Dominant bacteria constituted 70–80% of intestinal-microbiota abundance. Dominant bacteria varied among individuals. In addition, “Monarch”-dominant bacteria were identical across individuals, while “Minister”-dominant bacteria varied across individuals. The less-dominant bacteria and varied dominant bacteria determined the characteristics of unique individuals’ microbiota. We expect microbiotal structural specificity and variation accompanied by pathogenic factors to become an important direction in etiological studies, and regulating and balancing the structure of the intestinal flora to become a new therapeutic strategy in the future. Declarations Supplementary Materials: Figure S1: Prevalence of ileum-exclusive bacteria. Tables S1, S3, S5: Fecal and ileal bacteria. Tables S2, S4, S6: Alpha-diversity in ileal and fecal samples. Tables S7–S18: Constitution of ileal microecology. Abbreviations SD: Sprague Dawley; 16S rRNA: 16S ribosomal ribonucleic acid; DNA: deoxyribonucleic acid; GI: gastrointestinal; SI: small intestine; LI: large intestine. Acknowledgments This work was supported by the National Natural Science Foundation of China under Grant 81670776 to J.G.Z. We thank Accdon (www.accdon.com) for its linguistic assistance during the preparation of this manuscript. A preprint has previously been published [34]. Declaration of Interest The authors report no conflict of interest. Author Contributions J.G.Z. contributed to the study concept and design. X.W.S. and J.G.Z. contributed to the analysis and interpretation of data and drafted the manuscript. H.R.L., P.F.X., X.G., C.Y.Z., W.M.G., X.X.K., X.L.J., X.T., and D.W.W. completed the animal experiment and sample collection. X.Z. revised the manuscript critically for intellectual content. All authors contributed to the acquisition of data and critical revisions of the manuscript. All authors approved the final manuscript prior to submission and agree to be accountable for all aspects of the work. X.W.S. and J.G.Z. shared equal co- authorship. Data availability statement The raw datasets generated during the current study are available in the NCBI repository, BioProject: PRJNA820028 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA820028) and PRJNA831335 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA831335). J.G.Z. should be contacted for data. References Kennedy MS, Chang EB. The microbiome: Composition and locations. Prog Mol Biol Transl Sci. 2020;176:1–42. Morgan XC, Huttenhower C. Meta'omic analytic techniques for studying the intestinal microbiome. Gastroenterology. 2014;146:1437–1448.e1. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444:1027–1031. 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Mentula S, Harmoinen J, Heikkilä M, Westermarck E, Rautio M, Huovinen P, Könönen E. Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs. Appl Environ Microbiol. 2005;71:4169–4175. Nejman D, Livyatan I, Fuks G, Gavert N, Zwang Y, Geller LT, Rotter-Maskowitz A, Weiser R, Mallel G, Gigi E, et al. The human tumor microbiome is composed of tumor type-specific intracellular bacteria. Science. 2020;368:973–980. Swidsinski A, Sydora BC, Doerffel Y, Loening-Baucke V, Vaneechoutte M, Lupicki M, Scholze J, Lochs H, Dieleman LA. Viscosity gradient within the mucus layer determines the mucosal barrier function and the spatial organization of the intestinal microbiota. Inflamm Bowel Dis. 2007;13:963–970. Caporaso JG, Lauber CL, Costello EK, Berg-Lyons D, Gonzalez A, Stombaugh J, Knights D, Gajer P, Ravel J, Fierer N, et al. Moving pictures of the human microbiome. Genome Biol. 2011;12:R50. https://doi.org/10.21203/rs.3.rs-1697881/v2. Supplementary Files AuthorChecklistFull.pdf FigureS1.doc Figure S1: Prevalence of ileum-exclusive bacteria. Supplementarytable1.doc Tables S1, S3, S5: Fecal and ileal bacteria. SupplementaryTable246.doc Tables S2, S4, S6: Alpha-diversity in ileal and fecal samples. Supplementarytable3.doc Tables S1, S3, S5: Fecal and ileal bacteria. Supplementarytable5.doc Tables S1, S3, S5: Fecal and ileal bacteria. SupplementaryTable718.doc Tables S7–S18: Constitution of ileal microecology. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-1697881","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":122069361,"identity":"04aff8d2-db6b-41f7-98ec-3fb2c3d0fceb","order_by":0,"name":"Xiao Wei Sun","email":"","orcid":"","institution":"School of Basic Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"Wei","lastName":"Sun","suffix":""},{"id":122069362,"identity":"9d82f86f-8151-4ba4-a25f-db73dc890aa1","order_by":1,"name":"Hua Zhang","email":"","orcid":"","institution":"School of Basic Medical 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00:58:43","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1697881/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1697881/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28192326,"identity":"37488845-b0a4-4caf-9250-9d2b68cacdef","added_by":"auto","created_at":"2022-10-24 17:56:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":502188,"visible":true,"origin":"","legend":"\u003cp\u003eOTU and species relationships in and between ileal and fecal samples. Most OTUs and species were shared among individuals, but the ileum (A) shared fewer OTUs with feces (B). C: Compared with fecal samples, large proportions of ileal OTUs and species were ileum exclusive.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/21f93f05fb6c8ae3a239e92e.jpg"},{"id":28192321,"identity":"cf0b4dfd-6b0f-4287-bb41-c9ca7cf57285","added_by":"auto","created_at":"2022-10-24 17:56:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":568289,"visible":true,"origin":"","legend":"\u003cp\u003eTop 10 abundant bacteria in feces and the ileum, and top 20 dominant bacteria in individuals. Bacteria exhibited obviously different abundances and domination levels between feces and the ileum. A: phyla, B: genera; C: heatmap of individuals, genus level, top 20, distance was calculated by the Euclidean method, abundance ratio \u0026gt;1%.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/dc7ac0f28def391cd769938a.jpg"},{"id":28192511,"identity":"a0a6ba77-e112-449e-81eb-b62625a868ff","added_by":"auto","created_at":"2022-10-24 18:01:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":550330,"visible":true,"origin":"","legend":"\u003cp\u003eBeta-diversity and sample distances and biomarkers of ileal and fecal bacteria. Ileal bacteria showed higher β-diversity and sample distance than fecal bacteria and shared different biomarkers. A: PCA, B: PCoA, C: UPGMA, D: distance heatmap, E: LeFSe biomarkers (linear discriminant analysis [LDA] threshold = 4.0). Distances were calculated using the binary Jaccard method.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/192948a51cd17ab0ccd50399.jpg"},{"id":28192510,"identity":"c55cbcef-5aa8-43f9-9395-70452650bded","added_by":"auto","created_at":"2022-10-24 18:01:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":961848,"visible":true,"origin":"","legend":"\u003cp\u003eIleal and fecal flora under HFD intervention. A: Genera of ileum-exclusive bacteria. Top, Venn diagram; middle, bar plot showing the different top 10 dominant bacteria between feces and ileum; bottom, heatmap showing the different distributions of dominant bacteria. B: Analysis of distance between samples showing that sample distance in fecal bacteria was shorter than that in ileal bacteria. Top, PCA and PCoA analyses; left bottom, UPGMA plot; right bottom, heatmap of sample distance. C: Cladogram showing different biomarkers between fecal and ileal bacteria.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/fd4137a5350ae5e535e29eaa.jpg"},{"id":28192325,"identity":"f3b17793-fad4-4026-ba26-ee7a000a3ca0","added_by":"auto","created_at":"2022-10-24 17:56:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":919725,"visible":true,"origin":"","legend":"\u003cp\u003eIleal and fecal flora under SPF conditions. A: Genera of ileum-exclusive bacteria. Top, Venn diagram; middle, bar plot showing the different top 10 dominant bacteria between feces and ileum; bottom, heatmap showing the different distributions of dominant bacteria. B: Analysis of distance between samples showing that sample distance in fecal bacteria was shorter than that in ileal bacteria. Top, PCA and PCoA analyses; left bottom, UPGMA plot; right bottom, heatmap of sample distance. C: Cladogram showing different biomarkers between fecal and ileal bacteria.\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/a2d83c40c033fae89a0518ca.jpg"},{"id":28192323,"identity":"734a526f-756d-4957-8101-4ed8a1881cc9","added_by":"auto","created_at":"2022-10-24 17:56:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":415152,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional prediction of fecal and ileal bacteria. A: PICRUSt 2 analysis. B: Bugbase phenotype prediction. C: FAPROTAX function analysis.\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/ef97fdba08dc1929fa52ca71.jpg"},{"id":28193409,"identity":"5647d951-74cf-41ae-b1cd-22bbf18e614e","added_by":"auto","created_at":"2022-10-24 18:06:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1114089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/a1f477f6-5297-4404-b677-aab5b477aba0.pdf"},{"id":28193402,"identity":"ebf6184b-c3a8-43fc-992b-f5b54705a92b","added_by":"auto","created_at":"2022-10-24 18:06:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7175160,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/d8e3f4ccfb839f19d703e630.pdf"},{"id":28193404,"identity":"26c0939c-7a0e-4156-89c6-5cba39bbe0c3","added_by":"auto","created_at":"2022-10-24 18:06:03","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":214528,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1: Prevalence of ileum-exclusive bacteria.\u003c/p\u003e","description":"","filename":"FigureS1.doc","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/24341517c2df6dc96bf43184.doc"},{"id":28192512,"identity":"da997a72-025e-4ab9-ab36-4e3f5a14a351","added_by":"auto","created_at":"2022-10-24 18:01:02","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":496128,"visible":true,"origin":"","legend":"\u003cp\u003eTables S1, S3, S5: Fecal and ileal bacteria.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Supplementarytable1.doc","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/2b8445cd947de9a79dff654c.doc"},{"id":28192328,"identity":"85bee3af-700d-4b73-9e48-ee9816f6502d","added_by":"auto","created_at":"2022-10-24 17:56:02","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":143360,"visible":true,"origin":"","legend":"\u003cp\u003eTables S2, S4, S6: Alpha-diversity in ileal and fecal samples.\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryTable246.doc","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/5f4150a06c43299248fc5d44.doc"},{"id":28192517,"identity":"9790b39a-36db-4898-83f4-a6ec7752f59d","added_by":"auto","created_at":"2022-10-24 18:01:03","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":383488,"visible":true,"origin":"","legend":"\u003cp\u003eTables S1, S3, S5: Fecal and ileal bacteria.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Supplementarytable3.doc","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/370753fc10d29538fa57e330.doc"},{"id":28192329,"identity":"ff9c0e5f-1b80-4083-9c53-2363e65fb9cf","added_by":"auto","created_at":"2022-10-24 17:56:02","extension":"doc","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":680960,"visible":true,"origin":"","legend":"\u003cp\u003eTables S1, S3, S5: Fecal and ileal bacteria.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Supplementarytable5.doc","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/6c3e867ae27d3189d6ccf6fe.doc"},{"id":28192333,"identity":"e4ae2d72-41c0-4666-bc40-d6984f196b85","added_by":"auto","created_at":"2022-10-24 17:56:03","extension":"doc","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":894464,"visible":true,"origin":"","legend":"\u003cp\u003eTables S7–S18: Constitution of ileal microecology.\u003c/p\u003e","description":"","filename":"SupplementaryTable718.doc","url":"https://assets-eu.researchsquare.com/files/rs-1697881/v2/5ebbb1a84554c206e8461e1e.doc"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cstrong\u003eStructural and Functional Differences in Small Intestinal and Fecal Microbiota: 16S rRNA Gene Investigation in Rats\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Key Messages","content":"\u003cp\u003e\u003cstrong\u003eWhat is the context?\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eFeces are rich in bacteria, and the development of sequencing technology makes it possible to\u0026nbsp;identify\u0026nbsp;and classify\u0026nbsp;gut\u0026nbsp;microbiota.\u003c/li\u003e\n \u003cli\u003eThe\u0026nbsp;fecal microbiota is closely related to various diseases of extraintestinal organs and\u0026nbsp;the\u0026nbsp;colon.\u003c/li\u003e\n \u003cli\u003eThere has been relatively limited research on bacteria in the small intestine.\u003c/li\u003e\n \u003cli\u003eThe small intestine and colon have completely different metabolic environments. Additionally,\u0026nbsp;the microbial\u0026nbsp;composition is significantly affected by the local metabolic environment.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eWhat is new?\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we compared\u0026nbsp;the microbiota of the small intestine and\u0026nbsp;feces.\u0026nbsp;The key findings are as follows:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eIleal and fecal bacteria were structurally and functionally different.\u003c/li\u003e\n \u003cli\u003eDominant bacteria differed between the ileum and feces.\u003c/li\u003e\n \u003cli\u003eIleal bacteria showed greater \u0026beta;-diversity than fecal bacteria.\u003c/li\u003e\n \u003cli\u003eIleal bacteria were\u0026nbsp;extrinsic\u0026nbsp;to the feces\u0026nbsp;and not well transferred to the feces.\u003c/li\u003e\n \u003cli\u003eAbout 50% of bacterial genera in the small intestine were exclusive to the feces.\u003c/li\u003e\n \u003cli\u003eThe distance between in-group samples was nearer than that between paired ileum\u0026ndash;feces\u0026nbsp;samples.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eWhat is the impact?\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eThis study provides further solid evidence that the microbiota along intestine segmentally distributed.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eTrillions of bacteria exist in the human gastrointestinal (GI) tract.\u003csup\u003e1\u003c/sup\u003e With the help of high-throughput sequencing of nucleotides, and the identification of the 16S ribosomal ribonucleic acid (rRNA) gene as highly conserved in most bacterial clades,\u003csup\u003e2\u003c/sup\u003e researchers have been able to identify and classify gut microbiota, which are distributed throughout the entire GI tract in diverse microbial communities.\u003csup\u003e1\u003c/sup\u003e Gut bacteria have been shown to take part in many physiological or pathological processes in humans, such as inflammatory bowel disease, cancer, nutritional obesity, diabetes, and cardiovascular disease.\u003csup\u003e3\u0026ndash;11\u003c/sup\u003e However, based on the idea that the colon is the site of final GI content collection and the fact that colonic samples are easily acquired, most researchers have obtained samples from feces and regarded fecal flora as naturally synonymous with gut microbiota.\u003csup\u003e12\u0026ndash;16\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eDuring transportation and digestion of food component through the gut, different biochemical inner environments are formed in the small intestine (SI) and large intestine (LI). Accordingly, it is highly possible that the microbiota of these two organs are distinct.\u003csup\u003e15\u003c/sup\u003e The most significant distinction between SI and LI microbiota might be the persistent clinical fact that the SI has a much lower incidence of various diseases, such as carcinoma, than LI. Therefore, identifying differences and relationships between SI and LI microbiota is crucial to clarifying the gut bacterial mechanisms of various disorders. Several clinical and laboratory animal observations have proven the marked differences between SI and stool microbiota, as well as between different segments of the SI.\u003csup\u003e17,18\u003c/sup\u003e A systematic observational comparison between ileum and feces, however, is still lacking. To address this lack, we collected ileal contents and feces of rats and compared their structural and functional features based on 16S rRNA gene sequencing and bioinformatics analysis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\n\u003cp\u003eMale Sprague Dawley (SD) rats, ages 4\u0026ndash;5 weeks (n = 6) and weighing 52.32 \u0026plusmn; 8.48 g, were raised individually in a conventional setting for 3 months, with diet and water \u003cem\u003ead libitum\u003c/em\u003e to simulate the natural state. The rats\u0026rsquo; chow (3.16 kcal/g) was produced by KeAo-XieLi Co., Ltd. (Beijing, China) and sterilized with cobalt-60 (\u003csup\u003e60\u003c/sup\u003eCo).\u0026nbsp;All methods were carried out in accordance with relevant guidelines and regulations of the Laboratory Animal\u0026mdash;Guidelines for Ethical Review of Animal Welfare (GB/T 35892\u0026mdash;2018), and all experimental protocols were approved by the Medical Ethics Committee of Lanzhou University (jcyxy20190302). All methods were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.\u003c/p\u003e\n\u003cp\u003eSample collection\u003c/p\u003e\n\u003cp\u003eRats were anesthetized by intraperitoneal (i.p.) injection of pentobarbital sodium (5 mg/100 g body mass) and killed by portal-vein blood drainage. We expressed no less than 2.0 g content of 5.0\u0026ndash;7.0 cm of ileum or colon into sterile Eppendorf tubes (Eppendorf, Hamburg, Germany). Samples were then stored in a \u0026minus;80\u0026deg;C refrigerator after quick freezing in liquid nitrogen. To avoid environmental contamination, we performed our experiments on a clean bench.\u003c/p\u003e\n\u003cp\u003eDNA extraction, PCR, and sequencing\u003c/p\u003e\n\u003cp\u003eWe extracted total deoxyribonucleic acid (DNA) from 0.5 g of each sample using a magnetic soil and stool DNA kit (#DP812; TIANGEN Biotech Co., Ltd., Shanghai, China) per the kit protocol. Primers were designed according to the conserved V3\u0026ndash;V4 region of the 16S rRNA gene. Sequences were as follows: 338F: 5\u0026prime;-ACTCCTACGGGAGGCAGCA-3\u0026prime;; 806R: 5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;.\u003csup\u003e19\u003c/sup\u003e The target region was amplified using polymerase chain reaction (PCR) with the barcode at the end of the primer. The amplification procedure was as follows: 95\u0026deg;C for 5 min, with 25 cycles of 95\u0026deg;C for 30 s, 50\u0026deg;C for 30 s, and 72\u0026deg;C for 40 s; and extension at 72\u0026deg;C for 7 min. The products were then purified, quantified, and homogenized to form a sequencing library and run on a 1.8% agarose gel. After quality inspection and preparation of a flow cell chip, we sequenced 500 ng of PCR products by PE 250 mode (2 \u0026times; 250 bp paired ends), performed on an Illumina\u0026nbsp;HiSeq 2500\u0026nbsp;platform (Illumina, Inc., San Diego, CA, USA; via BMK Biotechnology Co., Beijing, China). Sequencing length was 350\u0026ndash;450 bp. We transformed original image data files into sequenced reads via base calling. The\u0026nbsp;DNA extraction, PCR,\u0026nbsp;sequencing,\u0026nbsp;data preprocessing\u0026nbsp;and outcome assessment were finished in BMK Biotechnology Co., Beijing, China, who has no vested interest in the experiment and was not aware of the group allocation.\u003c/p\u003e\n\u003cp\u003eData preprocessing\u003c/p\u003e\n\u003cp\u003eAccording to overlap relationships, we spliced PE reads using Fast Length Adjustment of SHort reads (FLASH) software v1.2.11 (Johns Hopkins University Center for Computational Biology, Baltimore, MD, USA).\u003csup\u003e20\u003c/sup\u003e Raw tags were filtered for quality using Trimmomatic software v0.33 (https://USADELLAB.org).\u003csup\u003e21\u003c/sup\u003e We removed chimeras using UCHIME software v4.2 (Edgar \u003cem\u003eet al.\u003c/em\u003e, 2011)\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e to form clean tags, which were then clustered into operational taxonomic units (OTUs) at 97% sequence similarity using USEARCH software v10.0 (https://www.drive5.com/usearch/).\u003csup\u003e23\u003c/sup\u003e Representative sequences of OTUs were compared with the Silva microbial reference database v128 (http://www.arb-silva.de),\u003csup\u003e24\u003c/sup\u003e and OTUs were annotated using RDP Classifier v2.2 (https://rdp.cme.msu.edu).\u003csup\u003e25\u003c/sup\u003e We generated species richness at different taxonomic levels using Quantitative Insights Into Microbial Ecology (QIIME) software v2.2 (http://qiime.org/). Alpha-diversity indices (Ace, Chao1, Shannon, Simpson) and community dissimilarity (\u0026beta;-diversity) were evaluated using QIIME2 (\u003ca href=\"https://qiime2.org/\"\u003ehttps://qiime2.org/\u003c/a\u003e). We then used R software v3.1.1 (R Foundation for Statistical Computing, Vienna, Austria) for both principal-component analysis (PCA) and principal-coordinate analysis (PCoA).\u0026nbsp;Data analysis was performed by one investigator (XWS) on the platform of BMKCloud (https://international.biocloud.net), who was not aware of the group allocation, and the results interpretation was finished by another investigator (JGZ).\u003c/p\u003e\n\u003cp\u003eStatistics\u003c/p\u003e\n\u003cp\u003eData were expressed as mean \u0026plusmn; standard deviation (SD) or standard error of the mean (SEM). We conducted statistical analysis using SPSS v22 (IBM Corp., Armonk, NY, USA). An independent-sample \u003cem\u003et\u003c/em\u003e-test was used for comparisons between two groups; data were transformed to a natural logarithm (Ln) when equal variances were not assumed. We analyzed community dissimilarities via permutational multivariate analysis of variance (PERMANOVA). Differences were considered statistically significant when \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLarge proportions of ileal flora were exclusive to the feces\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the 12 samples taken from the six rats, we obtained 904,117 PE reads (50,171\u0026ndash;80,391), 849,599 raw tags (48,254\u0026ndash;77,009), 801,140 clean tags (46,087\u0026ndash;73,370), and 770,289 effective tags (43,314\u0026ndash;72,269) with average length 408\u0026ndash;417 bp. Guanine and cytosine (GC) content (52.66\u0026ndash;56.06%; the quality score 20 (Q20), 94.05\u0026ndash;97.19%; Q30, 88.89\u0026ndash;94.79%) and effectiveness (effective tags/PE reads, 66.64\u0026ndash;90.23%) met the qualification demands.\u003c/p\u003e\n\u003cp\u003eIn total, we obtained 1050 kinds of OTUs (range, 198\u0026ndash;874), 490.00 \u0026plusmn; 268.20 from the ileum and 430.50 \u0026plusmn; 31.97 from feces. The two kinds did not significantly differ (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Ileal OTUs were about twice as numerous as those in feces (1008 \u003cem\u003evs\u003c/em\u003e. 555), which implied higher variability in the ileum of rats.\u003c/p\u003e\n\u003cp\u003eWe next compared OTUs and species in and between ileal and fecal samples. OTU similarity in fecal samples was higher than that in ileal samples (the number of shared OTU was 257\u003cem\u003e\u0026nbsp;vs.\u0026nbsp;\u003c/em\u003e81), but numbers of shared or exclusive species were similar (Fig. 1A\u0026ndash;1B). Large proportions of OTUs and species were shared between ileal and fecal samples. A great many ileal OTUs and species remained exclusive to the ileum: 49.11% of OTUs, 36.84% of phyla and 58.31% of genera (Fig. 1C). That is, a considerable number of ileal bacteria were not detected in feces.\u003c/p\u003e\n\u003cp\u003eWe then analyzed the compositions and abundances (in OTUs) of ileum-exclusive bacteria. After annotation, we found that the phyla Chloroflexi, Fusobacteria, Latescibacteria, Nitrospirae, Planctomycetes, Rokubacteria, and unassigned bacteria were specific to the ileum, with OTUs ranging from 3 to 532. At the genus level, 193 bacteria were ileum exclusive. All had low abundance, with 61.82 \u0026plusmn; 113.95 OTUs (SEM: 8.22) and prevalence of 17\u0026ndash;100% (Table S1, Fig. S1), except for \u003cem\u003eAcetobacter\u003c/em\u003e (Proteobacteria), with 1798 OTUs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShared bacteria were composed of distinct floral structures in the ileum and feces\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough large proportions of ileal OTUs and bacteria were exclusive to the ileum, their abundances were low. Shared bacteria, which can be defined as those transferred from the ileum to the colon along with digesta, constituted the main body of intestinal flora by virtue of their high abundance. The top 10 phyla\u0026mdash;namely Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia, Spirochaetes, Patescibacteria, Epsilonbacteraeota, Acidobacteria, and Tenericutes (OTUs range, 36\u0026ndash;152,356)\u0026mdash;as well as Cyanobacteria and Gemmatimonadetes (OTUs \u0026lt; 1000), were the phyla of bacteria shared between ileal and fecal flora. They showed obviously different ratios of abundance. High ratios of Patescibacteria, Verrucomicrobia, Spirochaetes, Bacteroidetes, and Tenericutes were in feces, while Firmicutes, Proteobacteria, Acidobacteria, Epsilonbacteraeota, and Actinobacteria thrived more in ileal flora. At the genus level, \u003cem\u003eRomboutsia\u003c/em\u003e (138,023 OTUs), \u003cem\u003eTuricibacter\u003c/em\u003e (63,554 OTUs), and \u003cem\u003eRothia\u003c/em\u003e (12,466 OTUs) dominated the constitution of ileal bacteria, with levels 6\u0026ndash;14 times higher than in feces. Moreover, \u003cem\u003eStreptococcus\u003c/em\u003e (OTUs in feces: 136; in ileum: 4771), \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003e(OTUs in feces: 23; in ileum: 4598), and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e (OTUs in feces: 29; in ileum: 3251) were also dominant in the ileum (35\u0026ndash;200 times higher than in feces; Fig. 2). An additional 47 shared ileum-dominant bacteria had low abundances (\u0026lt;2000 OTUs in ileum; Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiffering diversity\u003c/strong\u003e, \u003cstrong\u003ebiomarkers, and functions in ileal and fecal bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further confirm the differences between ileal microbiota and fecal flora, we studied microbiotal features of variability and biomarkers of ileal flora using bioinformatics. Results showed that the species richness (Chao1 and Ace indices) and evenness (Shannon and Simpson indices) of ileal and fecal microbiota were identical (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; Table S2). According to PCA and PCoA analyses by the binary Jaccard method, however, the distance between samples was significantly higher in ileal than in fecal flora (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05; PERMANOVA, \u003cem\u003er\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e= 0.336, \u003cem\u003eP\u003c/em\u003e = 0.002; Fig. 3A\u0026ndash;3B). Moreover, according to the unweighted pair group method with arithmetic mean (UPGMA) and heatmap analyses, the distance between in-group samples was shorter than that between paired ileal\u0026ndash;fecal samples. That is to say, microbiota were more similar across individuals than across body sites from the same individual (Fig. 3C\u0026ndash;3D). Linear discriminant analysis with effect size (LEfSe) analysis showed that ileal and fecal bacteria had distinct biomarkers (Fig. 3E). Taken together, these results indicated that ileal and fecal flora were different, despite the bulk of dominant bacteria being continuously transferred from the ileum to the colon along with dietary residues.\u003c/p\u003e\n\u003cp\u003eFunctionally, according to Tax4Fun (http://tax4fun.gobics.de/) predictions, ileal bacteria displayed higher levels of metabolism (74.52 \u0026plusmn; 0.71% \u003cem\u003evs.\u003c/em\u003e 69.28 \u0026plusmn; 1.07%) and organismal systems (1.53 \u0026plusmn; 0.14% \u003cem\u003evs.\u003c/em\u003e 1.16 \u0026plusmn; 0.10%) but a lower level of environmental information processing (10.69 \u0026plusmn; 0.70% \u003cem\u003evs.\u003c/em\u003e 15.54 \u0026plusmn; 1.02%) than fecal flora (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIleal and fecal microbiota under high-fat diet intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the structures and features of ileal flora and clarify the possible influence on them of dietary diversity, we established another group of rats for which intervention was a high-fat diet (HFD). After being fed the HFD for 60 days, the animals were then switched to chow for another 60 days (\u003cem\u003en\u003c/em\u003e = 10). We collected the rats\u0026rsquo; intestinal contents and subjected them to 16S rRNA gene V3\u0026ndash;V4 region sequencing and analysis. Results showed obvious differences between the ileal and fecal bacteria mentioned earlier. Ileum-exclusive bacteria constituted 26.32% of phyla and 53.57% in genera (Fig. 4). Latescibacteria, Nitrospirae, Planctomycetes, Gemmatimonadetes, and unassigned bacteria were specific to the ileum (OTU range, 16\u0026ndash;503). In addition, 180 genera of bacteria were ileum exclusive, with OTUs ranging from 6 to 725 (96.48 \u0026plusmn; 115.04), except for \u003cem\u003euncultured bacterium c Subgroup 6\u0026nbsp;\u003c/em\u003e(\u003cem\u003eAcidobacteria\u003c/em\u003e) with 1483 OTUs (Table S3). \u003cem\u003eRomboutsia\u003c/em\u003e (200,712 OTUs), \u003cem\u003eRothia\u003c/em\u003e (69,437 OTUs), \u003cem\u003eTuricibacter\u003c/em\u003e (28,073 OTUs, 5\u0026ndash;24 times higher than in feces), \u003cem\u003eStreptococcus\u003c/em\u003e (23,487 OTUs), \u003cem\u003eHelicobacter\u0026nbsp;\u003c/em\u003e(13,346 OTUs), and \u003cem\u003eCandidatus Arthromitus\u003c/em\u003e (22,921 OTUs, 44\u0026ndash;997 times higher than in feces) dominated the constitution of ileal bacteria. Another 53 shared ileum-dominant bacteria had low abundances (\u0026lt;5056 in SI; Table S3). Moreover, they showed the same functional differences as those of chow-fed rats (data not shown). However, the HFD did exert lasting influence on the dominant categories and \u0026alpha;-diversity of ileal bacteria, which might be biomarkers of specific-diet ingestion. The richness, diversity, and evenness of SI flora were all elevated (Table S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIleal microbiota under specific-pathogen\u0026ndash;free conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also investigated the features of ileal bacteria in germ-free conditions. Male SD rats (n = 6, body weight 304.10 \u0026plusmn; 29.92 g) that had been maintained in a specific-pathogen\u0026ndash;free (SPF) environment and fed a chow diet and water \u003cem\u003ead libitum\u003c/em\u003e were chosen randomly. Their ileal and fecal contents produced a total of 1254 kinds of OTUs (ileum: 1013 [393.67 \u0026plusmn; 77.19]; feces: 1005 [482.83 \u0026plusmn; 37.38]; \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). The SPF environment remarkably influenced intestinal bacterial structure. The variability of ileal bacteria and exclusive bacteria was nearly diminished. Only Dependentiae (1 OTU) and 89 bacteria genera were ileum exclusive, with very few OTUs (1.78 \u0026plusmn; 1.16 [range, 1\u0026ndash;6]). \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, and \u003cem\u003eCandidatus Arthromitus\u003c/em\u003e were dominant. Abundances of less-dominant shared bacteria were also decreased (Table S5). Shannon and Simpson indices were lower for ileal than for fecal flora (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Table S6). The differences between ileal and fecal bacteria, however, also were remarkable (Fig. 5).\u003c/p\u003e\n\u003cp\u003eFunctionally, ileal bacteria also had a higher level of genetic-information processing and fewer cellular processes than fecal flora. In bugbase phenotype prediction (https://bugbase.cs.umn.edu/), ileal bacteria had advantages in mobile genetic elements (MGEs), Gram positivity, and facultative anaerobism, whereas anaerobism, Gram negativity, and potentially pathogenic phenotype were significantly lower than fecal flora. In Functional Annotation of Prokaryotic Taxa (FAPROTAX; Louca \u003cem\u003eet al.\u003c/em\u003e, 2016) analysis, ileal bacteria also showed lower nitrate reduction, nitrification, xylanolysis, and aerobic ammonia oxidation than fecal flora (Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstitution of ileal microecology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe merged the annotated species data of rats maintained in conventional conditions with those of rats maintained in an SPF environment to clarify ileal microecology. Ileal microbiota could be classified into three groups: exclusive bacteria, which were in very low abundance; dominant bacteria shared between the ileum and feces, and low-abundance bacteria shared between the ileum and feces (Tables S7\u0026ndash;S18).\u003c/p\u003e\n\u003cp\u003eExclusive bacteria were sensitive to diet and feeding environment. SPF conditions decreased exclusive-bacteria count intensively. Therefore, exclusive bacteria were further divisible into condition related and condition irrelevant. We identified 14 condition-irrelevant bacteria (Table S7), 152 bacteria related to conventional conditions\u0026nbsp;(Table S8), 24 chow-related bacteria (Table S9), 12 HFD/chow-related bacteria (Table S10), and 70 SPF environment\u0026ndash;related bacteria (Table S11). Feeding environment had a greater influence on exclusive-bacteria species and abundance than diet diversity.\u003c/p\u003e\n\u003cp\u003eIn bacteria shared between the ileum and feces, the abundances of 80 condition-irrelevant bacteria varied enormously. Only very small proportions of bacteria had \u0026gt;10,000 OTUs, so-called dominant bacteria (3/80 shared bacteria in chow rats, 8/80 in HFD/chow rats, and 3/80 in SPF rats). \u003cem\u003eRomboutsia\u003c/em\u003e was the leading bacterium with \u0026gt;100,000 OTUs. Most others were in low abundance (Table S12); conditions influenced the abundances and domination of these bacteria. \u003cem\u003eRothia\u003c/em\u003e and \u003cem\u003eTuricibacter\u003c/em\u003e were dominant in conventional conditions, whereas \u003cem\u003eLactobacillus\u003c/em\u003e was dominant in an SPF environment. All condition-related bacteria in the ileum that were shared between the ileum and feces were in low abundance (Tables S13\u0026ndash;S18). Rats maintained in a conventional environment (Tables S13, S16, S17) had lower kinds of bacteria than rats maintained in an SPF environment (Table 18), 64 \u003cem\u003evs.\u003c/em\u003e 223. There were also very small proportions of bacteria shared between the ileum and feces that were specific to chow (Table S16) or HFD/chow (Table S17) diets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial prevalence across individuals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApart from differences in structure between ileal and fecal microbiota, bacterial prevalence and abundance varied extensively across individuals and was obviously host specific. In rats fed normal chow, only 69 of 337 ileal bacteria and 75 of 337 fecal bacteria were 100% prevalent. All less-prevalent bacteria were also less dominant, mostly with OTUs \u0026lt; 300. Among the 100% prevalent bacteria, only a few were highly dominant, making up the top 10 dominant bacteria of the ileal or fecal group. Dominant bacteria varied across individuals. \u003cem\u003eRomboutsia\u003c/em\u003e was dominant in ileal samples from all six individual rats, whereas \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eRothia\u0026nbsp;\u003c/em\u003ewere dominant in three individuals only. In fecal microbiota, \u003cem\u003euncultured bacterium f Muribaculaceae\u003c/em\u003e was dominant in all six individuals, \u003cem\u003eLachnospiraceae NK4A136 group\u003c/em\u003e in four, and \u003cem\u003eTreponema 2\u003c/em\u003e in only two.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we systematically observed differences between ileal and fecal microbiota based on sequencing and bioinformatics analysis of the 16S rRNA V3\u0026ndash;V4 region. Intestinal microbiota have increasingly become a popular research topic in recent years. They are important symbionts of the gut and are involved in at least three primary physiological or pathological processes: (1) dynamic changes in and complexity of the microbial ecosystem; (2) dietary digestion, absorption, nutrition, synthesis, detoxification, and immune functions of the host;\u003csup\u003e26\u003c/sup\u003e and (3) various diseases of the host.\u003csup\u003e27,28\u003c/sup\u003e However, three key obstacles hinder the clarification of those processes. First, traditional culturing methods and conditions are limited in their ability to identify the trillions of intestinal microorganisms and the complicated microbial ecosystem. Second, the structures and functions of intestinal microorganisms are tightly coupled with various factors, such as environment, diet, and disease. They change dynamically along the GI tract (diversity of location), fluctuate widely over time (diversity of stability), vary among individuals (diversity of prevalence), and are inconsistent with abundance (diversity of abundance). Even increasing the sample sizes cannot include all intestinal bacteria and their trajectories. Last but not least might be the most important factor: at any given point in time, most of these microorganisms remain in the long, dark mystery tube of the SI, which cannot be reached noninvasively, rendering sampling difficult. Before becoming part of the composition of feces, they have experienced a long series of complicated biochemical reactions within the tube. Researchers can obtain final fecal samples only after this complicated process. These obstacles raise several key questions: Do fecal bacteria represent all microbial populations throughout the gut? To what extent do they change? Moreover, in terms of diseases, why is the morbidity of LI carcinoma extremely high in developed populations, whereas the SI remains much less vulnerable? Can this phenomenon be attributed to distinct microbiota in the SI? Many studies have deduced distinctions between fecal microorganisms and other components of intestinal microbiota according to functional heterogeneity \u003csup\u003e29\u003c/sup\u003e and the complicated biochemical environment (\u003cem\u003ee.g.\u003c/em\u003e, oxygen, pH, mucous thickness, antimicrobials, bile acids, transit time).\u003csup\u003e1\u003c/sup\u003e But direct proof is still lacking. In humans, research on SI microbiota can be performed only in patients,\u003csup\u003e15,17\u003c/sup\u003e but the influences of the pathological environment (pathophysiology as well as medications) on microbiota are uncontrollable. The results might be limited in their ability to tell the real differences. The most acceptable methods are still those using laboratory animals. Mentula S.\u003cem\u003e\u0026nbsp;et al.\u0026nbsp;\u003c/em\u003ecompared microbiota between jejunal fluid and fecal samples of 22 beagles by means of organismal culture and proved the inability of fecal samples to represent the microbiota of the upper gut.\u003csup\u003e30\u003c/sup\u003e Also, systematic evidence and intensive study of the differences between ileal and fecal bacteria are still needed. Recently, evidence from the Human Microbiome Project has shown that human microbiota are more similar across individuals than across body sites.\u003csup\u003e12\u003c/sup\u003e Is that also the case for the continuously and transitionally connected contents of the ileum and feces? Metagenomic sequencing and bioinformatics analysis are powerful tools used to identify complicated intestinal microbiota. In this study, we used SD rats as research subjects, in which influencing factors, including diet, environment, and individual characteristics (sex, age, body weight) could be homogenized and controlled, and total ileal contents were easily sampled. Moreover, because of the rats\u0026rsquo; stable long-term diets, sampling of the ileum and feces at the same point in time could represent the progressive relationship between ileal and colonic contents. To avoid possible influence from any procedure or medication, we achieved microbiotal stability by taking repeated samples from different individuals\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e rather than from the same individual at different timepoints.\u003csup\u003e30\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOur results showed that, in genus-level annotation, a total 335 kinds of bacteria were in ileal and colonic contents sampled from normal rats (\u003cem\u003en\u003c/em\u003e = 6). This number was far lower than trillions in terms of abundance. Even in this limited set of bacteria, 193 were ileum exclusive (58.30%) and could not be detected in feces. This means that fecal microbiota can represent only about 42% of ileal-bacteria species. Mentula S.\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e reported that in beagles, about 25% of jejunal bacteria were not detectable in fecal samples, nor were 45% of fecal bacteria detectable in the jejunum. In this study, we found that in conventional conditions, ileum-exclusive bacteria were significantly more numerous than feces-exclusive bacteria, 58.30% or 53.57% \u003cem\u003evs.\u003c/em\u003e 2.82% or 0% in chow or HFD/chow rats, respectively; in the SPF environment, these values were respectively 20.51% \u003cem\u003evs.\u003c/em\u003e 16.87%. These differences might reflect the different detection method (organism culture \u003cem\u003evs.\u003c/em\u003e 16S rRNA gene sequencing and analysis), different locations (jejunum \u003cem\u003evs.\u003c/em\u003e ileum), different animals, or different environment, as well as different methods of sampling. Abundance is another important index for bacterial-structure evaluation. Large proportions of both ileal and fecal bacteria were in low abundance. Meanwhile, very small proportions thrived in extremely high abundance; these were the annotated \u0026ldquo;dominant\u0026rdquo; bacteria or biomarkers. These dominant bacteria included most of the trillions of intestinal bacteria that exist (the top 10 bacteria contributed 70\u0026ndash;80% of abundance). Dominant bacteria were obviously distinct, however, between the ileum and feces. \u003cem\u003eEscherichia-Shigella\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eRomboutsia,\u003c/em\u003e \u003cem\u003eRothia\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Turicibacter\u003c/em\u003e were dominant in the ileum but not in feces, whereas the reverse was true of \u003cem\u003eLachnospiraceae NK4A136 group\u003c/em\u003e, \u003cem\u003euncultured bacterium f Muribaculaceae\u003c/em\u003e, \u003cem\u003eTreponema 2\u003c/em\u003e, \u003cem\u003euncultured bacterium f Lachnospiraceae\u003c/em\u003e, \u003cem\u003eAkkermansia\u003c/em\u003e, \u003cem\u003eCandidatus Saccharimonas\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Ruminococcaceae UCG-013\u003c/em\u003e (Table S1). This result suggested that fecal microbiota missed the bacterial dominance information of ileal microbiota. Accordingly, their functions were distinct. Ileal bacteria displayed higher levels of metabolism and organismal systems but a lower level of environmental information processing than feces. Assessment of \u0026beta;-diversity also showed that ileal and fecal microbiota were more similar among individuals than among body sites of the same individual. These results suggested that fecal bacteria were limited in their ability to represent ileal bacteria and \u003cem\u003evice versa\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAccording to our results, in a certain segment of the gut, bacteria could be classified into two groups: very few high-abundance bacteria (dominant) and a great many low-abundance bacteria (less dominant). Spatially, the very few dominant bacteria might mix with the gut contents, adapt to them, thrive with the components of digesta, and transition to the large intestine. They might be sensitive to and fluctuate with different digesta. For example, dominant bacteria changed significantly from the SI to the LI. The shared bacteria genera \u003cem\u003eEscherichia-Shigella\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eRomboutsia,\u003c/em\u003e \u003cem\u003eRothia\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Turicibacter\u003c/em\u003e were reduced by 7\u0026ndash;200 times, while \u003cem\u003eAkkermansia\u003c/em\u003e, \u003cem\u003eCandidatus Saccharimonas\u003c/em\u003e, \u003cem\u003eLachnospiraceae NK4A136 group\u003c/em\u003e, \u003cem\u003eRuminococcaceae UCG-013\u003c/em\u003e, \u003cem\u003eTreponema 2\u003c/em\u003e, \u003cem\u003euncultured bacterium f Lachnospiraceae\u003c/em\u003e, and \u003cem\u003euncultured bacterium f Muribaculaceae\u003c/em\u003e were increased by 5\u0026ndash;63 times. The majority of less-dominant bacteria might be located in mucus\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e or mucosa.\u003csup\u003e15\u003c/sup\u003e They could be related to nutrients rather than digesta because their abundances are maintained at very low levels. These bacteria might or might not transition with gut content (exclusive or shared), maintain their abundance at a relatively constant level, and comprise the majority of the segment\u0026rsquo;s microbial ecosystem. We supposed that this ecosystem was interdependent with the host and played important roles in the stability of the intestinal environment.\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe gut is notable in two aspects in terms of diseases. One is the diverse incidence rate of diseases in different parts of the tract. According to anatomical and histological structures and functions, the gut can be divided into seven segments: mouth, esophagus, stomach, duodenum, jejunum, ileum, and colon. Coincidently, this division also distinguishes diseases from one another in incidence. Carcinoma and some inflammatory diseases are significantly higher in the esophagus, stomach, and colon but rarely occur in the SI. Because of levels of ingested nutrients as well as those of possible toxins being very low in the colon after earlier absorption/degradation, the microbial ecosystem should be the key factor. Whether the SI microbial ecosystem is protective against injury or tumorigenesis of epithelial cells is an interesting topic. However, many visceral-organ diseases such as tumors and metabolic disorders have been linked with gut bacteria. Most relevant studies have derived their data from fecal samples. Bacteria or metabolites are assumed to possibly enter the circulatory system though gut leakage. As the main point of absorption, the SI has higher permeability than the colon, and therefore its microbial ecosystem might be more intimately connected with visceral-organ disorders, especially metabolic diseases. The relationship between diseases and exclusive bacteria in the SI needs to be established.\u003c/p\u003e\n\u003cp\u003eIn addition to the variation in structural characteristics from ileum to colon, diversity and dynamic change were additional primary features of and influenced the progression of intestinal microbiota. That is, the homogeneity and representativeness of samples are a topic of concern. In terms of microbiotal structure and bacterial abundance, no one individual can be represented by any other (prevalence), no one part of the intestine can be represented by any other (location), and no time point can be represented by other time point (stability).\u003csup\u003e33\u003c/sup\u003e In this study, we found that only about one-fifth of the bacteria at the genus level were 100% prevalent (69/337 in the SI \u003cem\u003evs.\u003c/em\u003e 75/337 in the LI). The less-prevalent bacteria (\u0026lt; 100%) were all less dominant, with abundance \u0026lt; 300 OTUs (with occasional exceptions). The top 10 dominant bacteria all had 100% prevalence. This direction of relevance was contrary to the finding in beagles that high prevalence reflected high proportions of total count.\u003csup\u003e23\u003c/sup\u003e However, dominant bacteria also varied across individuals. Of ileal bacteria, only \u003cem\u003eRomboutsia\u003c/em\u003e was dominant in all six individuals (\u0026ldquo;Monarch\u0026rdquo;-dominant bacteria), while \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eRothia\u0026nbsp;\u003c/em\u003ewere dominant in only three (\u0026ldquo;Minister\u0026rdquo;-dominant bacteria). In fecal microbiota, \u003cem\u003euncultured bacterium f Muribaculaceae\u003c/em\u003e was the \u0026ldquo;Monarch\u0026rdquo;-dominant bacterium, while\u003cem\u003e\u0026nbsp;Lachnospiraceae NK4A136 group\u003c/em\u003e and \u003cem\u003eTreponema 2\u003c/em\u003e were the \u0026ldquo;Minister\u0026rdquo;-dominant bacteria. The less prevalent bacteria and various dominant bacteria determined the microbiotal characteristics of unique individuals. This microbiotal imprinting should be addressed in investigations of the mechanisms of disease occurrence and different incidences among individuals and between the SI and LI.\u003c/p\u003e\n\u003cp\u003eThe main limitation of this study is the representativeness of the detected microbiota across species. Due to differences in living environment, diet structure, and health status, the domination and prevalence of bacteria may be extremely varied. Indeed, to date, it is nearly impossible to identify a typical bacterial inhabitant across species, or even across populations in the same species or across individuals in the same population. The most popular population studies aimed to identify diseases associated with bacteria by comparing bacteria in various types of patients with those in healthy people. Based on this background, this study aimed only to identify the features of bacterial distribution across the small intestine and the large intestine, rather than to identify specific bacteria. Another limitation is that this study did not identify the relationship between different distribution features and diseases. Although at the beginning of this study the authors aimed to address the interesting problem of the significantly lower occurrence of serious diseases, such as inflammatory diseases and cancer, in the small intestine compared to that in the colon, the identified distribution features cannot answer that question sufficiently. Whether or how the different distribution of bacteria between the small and large intestine are associated with varied disease occurrences still needs to be investigated in the future. Furthermore, failing to discover the source of intestinal bacteria, especially those in the small intestine, is the third limitation of this study. During ingestion, stomach acid may destroy most of the bacteria. It is still not clear how large numbers of bacteria survive the acid and reside prevalently or exclusively in the gut in a relative stable status.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCollectively, we proved that structure (dominant bacteria and \u0026beta;-diversity) and functions were markedly different between ileal and fecal microbiota in rats, although they were intimately transitional. The ileum had large proportions of exclusive bacteria with low abundances. HFD and SPF feeding environments had profound influences on ileal and fecal microbiota. Diversity was the basic characteristic of intestinal microbiota. In a specific location, intestinal bacteria could be classified into two groups: large proportions of less-dominant bacteria with low abundances, and small proportions of dominant bacteria with high abundances. Dominant bacteria constituted 70\u0026ndash;80% of intestinal-microbiota abundance. Dominant bacteria varied among individuals. In addition, \u0026ldquo;Monarch\u0026rdquo;-dominant bacteria were identical across individuals, while \u0026ldquo;Minister\u0026rdquo;-dominant bacteria varied across individuals. The less-dominant bacteria and varied dominant bacteria determined the characteristics of unique individuals\u0026rsquo; microbiota. We expect microbiotal structural specificity and variation accompanied by pathogenic factors to become an important direction in etiological studies, and regulating and balancing the structure of the intestinal flora to become a new therapeutic strategy in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u003c/strong\u003e Figure S1: Prevalence of ileum-exclusive bacteria. Tables S1, S3, S5: Fecal and ileal bacteria. Tables S2, S4, S6: Alpha-diversity in ileal and fecal samples. Tables S7\u0026ndash;S18: Constitution of ileal microecology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSD: Sprague Dawley; 16S rRNA: 16S ribosomal ribonucleic acid; DNA: deoxyribonucleic acid; GI: gastrointestinal; SI: small intestine; LI: large intestine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China\u0026nbsp;under Grant 81670776\u0026nbsp;to J.G.Z.\u0026nbsp;We thank\u0026nbsp;Accdon\u0026nbsp;(www.accdon.com) for its linguistic assistance during the preparation of this manuscript. A preprint has previously been published [34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.G.Z.\u0026nbsp;contributed to the study concept and design. X.W.S. and J.G.Z. contributed to the analysis and interpretation of data and drafted the manuscript. H.R.L.,\u0026nbsp;P.F.X.,\u0026nbsp;X.G.,\u0026nbsp;C.Y.Z., W.M.G., X.X.K.,\u0026nbsp;X.L.J.,\u0026nbsp;X.T., and\u0026nbsp;D.W.W.\u0026nbsp;completed the animal experiment and sample collection.\u0026nbsp;X.Z.\u0026nbsp;revised the manuscript critically for intellectual content. All authors contributed to the acquisition of data and critical revisions of the manuscript. All authors approved the final manuscript prior to submission and agree to be accountable for all aspects of the work. X.W.S. and J.G.Z. shared equal co- authorship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw datasets generated during the current study are available in the NCBI repository, BioProject: PRJNA820028 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA820028) and PRJNA831335 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA831335). J.G.Z. should be contacted for data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKennedy MS, Chang EB. The microbiome: Composition and locations. Prog Mol Biol Transl Sci. 2020;176:1\u0026ndash;42.\u003c/li\u003e\n \u003cli\u003eMorgan XC, Huttenhower C. Meta\u0026apos;omic analytic techniques for studying the intestinal microbiome. Gastroenterology. 2014;146:1437\u0026ndash;1448.e1.\u003c/li\u003e\n \u003cli\u003eTurnbaugh PJ, Ley RE, Mahowald MA,\u0026nbsp;Magrini V, Mardis ER, Gordon JI. 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Genome Biol. 2011;12:R50.\u003c/li\u003e\n \u003cli\u003ehttps://doi.org/10.21203/rs.3.rs-1697881/v2.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ileum, Feces, Microbiota, 16S rRNA gene, Sequencing, Rats","lastPublishedDoi":"10.21203/rs.3.rs-1697881/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1697881/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe occurrence of serious diseases, such as inflammatory diseases and cancer, in the small intestine is significantly lower than that in the colon. The differentiation of small-intestine microbiota from large-intestine microbiota might hold great significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods:\u003c/strong\u003e To compare floral composition and functions between the two types of microbiota, ileal contents and feces were collected from Sprague Dawley (SD) rats, and the V3–V4 region of the 16S ribosomal ribonucleic acid (rRNA) gene in these rats was amplified and sequenced. We subjected the data to bioinformatics analyzing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCompared with feces, about 50% of bacterial genera in the ileum were exclusive, with low abundance (operational taxonomic units [OTUs] \u0026lt;1000). Of bacteria shared between the ileum and feces, a few genera were highly abundant (dominant), whereas most had low abundance (less dominant). Dominant bacteria differed between the ileum and feces. Ileal bacteria showed greater β-diversity, and the distance between in-group samples was nearer than that between paired ileum–feces samples. Moreover, the ileum shared various biomarkers and functions with feces (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). A high-fat diet (HFD) and specific-pathogen–free (SPF) conditions had a profound influence on α-diversity and abundance but not on the exclusive/shared features or β-diversity of samples. Intestinal microbiota were composed of high-prevalence dominant, low-prevalence dominant, and less-dominant bacteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe present findings suggested that ileal and fecal bacteria were different structurally and functionally. These differences might be key to the fundamental protection of the small intestine from diseases.\u003c/p\u003e","manuscriptTitle":"Structural and Functional Differences in Small Intestinal and Fecal Microbiota: 16S rRNA Gene Investigation in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-10-24 17:56:00","doi":"10.21203/rs.3.rs-1697881/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2022-07-18 18:03:04","doi":"10.21203/rs.3.rs-1697881/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11c88601-8af9-4f21-932c-86e2195dfccd","owner":[],"postedDate":"October 24th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-12T03:55:27+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-24 17:56:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1697881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1697881","identity":"rs-1697881","version":["v2"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}