Abstract
Elucidation of the mechanisms by which the microbiota-gut-brain axis influences behavior requires understanding the anatomical relationship of bacteria with mucosal elements. We herein report that microbes were mainly associated with food or fecal matter in the intestinal lumen. In the small intestine, bacterial density increased from proximal-to-distal levels and was much higher in the large intestine. A mucus layer was present between the mucosal epithelium and fecal boluses in the large intestine, but not between food and the mucosal epithelium in the small intestine. In contrast, in all intestinal regions lacking food or fecal boluses, the lumen was small, or absent, and contained little or no bacteria or mucus. The association of bacteria with food was tested in the small intestine by examining the effect of fasting on it. Bacterial density was equivalent in the ileum of fasted and fed mice, but fasting greatly reduced the amount of food containing bacteria, suggesting the amount of bacteria was reduced. Critically, this study provides evidence that the vast majority of the microbiota in the intestines are associated with the food matrix thereby raising questions regarding how the gut microbiota can potentially signal the brain and influence behavior. Given their spatial location within the lumen, which keeps them at a great distance from neuronal elements in the mucosa, combined with immune and mucus barriers, microbiota more likely to influence behavior through secretion of bacterial products that can traverse the spatial difference to interact with gut neurons and not through direct physical association.
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taken from the first 1 cm-long tissue block at the level of the proximal cecum (0 cm distal to the proximal cecum). a A large proportion of the lumen is filled with chyme that is stained fairly densely and evenly for bacteria. This staining extends into the folds between crypts. b A section adjacent to the one shown in a that was stained for mucus is shown. Some chyme was present in the lumen and a mucus layer lined the mucosal epithelium surrounding the lumen and the folds between crypts (arrows). The numerous dark-stained patches throughout the mucosa are clusters of presumptive goblet cells in the crypts stained with Alcian blue (individual stained goblet cells were visible at higher magnification, see panel d). c A portion of the image in a is shown at higher magnification to illustrate bacterial staining in the crypt lumens, which are shown cross-sectioned in some instances and sectioned obliquely or parallel to their long axis in others (arrowheads). Dense bacterial staining is also shown running along the mucosal epithelium around the crypts and extending into the folds between the crypts (arrows). d A higher magnification of a portion of the image shown in b stained for mucus. A mucus layer can be seen lining the mucosal epithelium along the folds between crypts (arrows). The dark-stained, grape-like clusters throughout the mucosa are composed of presumptive goblet cells in the crypts stained with Alcian blue. Scale bars in a,b = 200 μm, in c = 50 μm, and in d = 100 μm
taken from the 1 cm-long tissue block located 0 cm distal to the proximal small intestine, whereas, those in c and d from the block 20 cm distal to the proximal small intestine. a The low density of bacteria typically observed in the duodenum and the close association of these bacteria with food particles are shown. b A low magnification image of the section adjacent to the one shown in a that was stained for mucus is shown. A small amount of food is present in the lumen. c Increase in the density of bacterial staining in the lumen of the distal jejunum as compared with the duodenum is shown (compare c with a). d A lower magnification image of the section of the distal jejunum shown in c to illustrate the increased amount of food in the lumen relative to more proximal levels of the small intestine (compare d with b). Scale bars in a,c = 50 μm, and in b,d = 200 μm. F, food
source of these bacteria is that they extend from the dense bacteria in the chyme in the lumen of the cecum, which in fed mice may have expanded in volume and thus extended further distal into the proximal large intestine than it did in fasted mice (compare a with c). b A section adjacent to the one shown in a from a fed mouse that was stained for mucus is shown. The size of the lumen was greatly reduced. A large proportion of the lumen and folds between crypts in the top half of this section appeared to be filled with mucus (arrows). Also, in a portion of the bottom half of the section, a thin layer of mucus was present in the folds between crypts. The numerous dark patches throughout the mucosa are clusters of presumptive goblet cells in the crypts stained with Alcian blue. c In the proximal large intestine of a fasted mouse, little bacterial staining was present in the small lumen or the folds between crypts. d A section adjacent to the one shown in c from a fasted mouse that was stained for mucus is shown. The size of the lumen was also greatly reduced in fasted mice. In comparison with this same region from a fed mouse (compare d with b), there is no mucus along the surface of the mucosal epithelium in the lumen or in the folds between crypts. These observations suggest in some specimens there may have been an association between bacteria and mucus. For example, the possibility of greater spread of bacteria from the cecum into the proximal large intestine in fed mice shown in a may have mucus associated with its perimeter that extended into the proximal large intestine as shown in b. Scale bars in a–d = 200 μm
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Data is stored on the Fox lab computer and are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank Andrew Marquis for his technical assistance.
Funding
This study was supported by Iowa State University College of Veterinary Medicine Startup Funds (M.L.) and Salary savings (E.F.).
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ML and EF designed the research. EF helped perform the research, ML and EF analyzed the data. EF wrote the first draft of the manuscript and ML and EF made critical revisions to the manuscript.
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All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Eighth edition, 2011) and American Association for Accreditation of Laboratory Animal Care guidelines and were approved by the Purdue University Institutional Animal Care and Use Committee.
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203_2022_2952_MOESM1_ESM.tif
Supplementary file1 Photomicrographs of bacterial staining in the lumen of the proximal large intestine (includes cecum) and the adjacent crypt lumens. a. A 30 μm-thick frozen section stained by FISH as described in Methods section for paraffin sections is shown. This section was taken from the first 1 cm-long tissue block at the level of the proximal cecum (0 cm distal to the proximal cecum). A bright band of bacterial staining immediately adjacent to the mucosal epithelium that was observed in some specimens runs diagonally from the upper middle to the lower right of the image. Additionally, bacterial staining was observed in crypt lumens running perpendicular to the mucosal epithelium (arrows). b. As shown here, when the density of bacteria was sufficiently low in a crypt lumen, individual bacteria, which were rod shaped, could be visualized. Scale bar in a = 50 μm, in b = 25 μm (TIF 5538 KB)
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Supplementary file2 Photomicrographs and photomontages that illustrate the pattern of bacterial (FISH) and mucus (Alcian blue) staining in large intestine regions in which the lumen contained a fecal bolus. The sections in a-d were taken from the 1 cm-long tissue block located 4 cm distal to the proximal cecum. The section in e was taken from the 1 cm-long tissue block located 7 cm distal to the proximal cecum. a. Fairly dense bacterial staining occurred throughout most of fecal matter in the lumen of the large intestine, although typically the density was slightly reduced compared to the chyme in the cecum lumen (compare a with Fig. 1a). b. A section adjacent to the one shown in a that was stained for mucus is shown. A large proportion of the lumen is filled with a fecal bolus and a mucus layer can be seen between the mucosal epithelium and perimeter of the fecal bolus (arrow). The numerous dark-stained patches throughout the mucosa are clusters of presumptive goblet cells in the crypts stained with Alcian blue (individual stained goblet cells are visible at higher magnification in d and f). c. A portion of the image in a is shown at higher magnification to illustrate bacterial staining in the lumen, and to emphasize that some specimens had relatively uniform staining density throughout the lumen, including at the perimeter of the fecal bolus adjacent to the mucosal epithelium. d. The portion of the image in b with an arrow is shown at higher magnification to illustrate the mucus layer between the mucosal epithelium and perimeter of the fecal bolus (arrows). The dark-stained, grape-like clusters throughout the mucosa are composed of presumptive goblet cells in the crypts stained with Alcian blue. e. Bacterial FISH staining of a different fecal bolus than the one shown in a-d is shown here to illustrate that some fecal boluses had increased density of bacteria around their perimeter relative to the density of bacteria in the body of the bolus. f. A portion of the large intestine stained with Alcian blue is shown at high magnification to illustrate what appears to be a double layer of mucus lining the mucosal epithelium (arrows). The dark-stained cells throughout the mucosa are presumptive goblet cells in the crypts stained with Alcian blue. Scale bars in a,b = 200 μm, and in c-f = 50 μm (TIF 14031 KB)
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Supplementary file3 Photomicrographs and photomontages that illustrate the pattern of bacterial and mucus staining in large intestine regions that did not contain a fecal bolus. The sections in a-d were taken from the 1 cm-long tissue block located 6 cm distal to the proximal cecum. a. At some proximal-distal levels of the large intestine where no fecal bolus was present, little or no bacterial staining could be visualized in the lumen, which was absent or greatly reduced in size (arrow indicates the lumen). b. A section adjacent to the one shown in a that was stained for mucus is shown. Little or no fecal matter was present in the lumen. At the bottom of this cross-section through the large intestine, a thin mucus layer was present along the surface of the mucosal epithelium in the folds between crypts (arrows). Very little mucus was associated with the mucosal epithelium throughout the tissue in the remainder of the section. The numerous dark-stained patches throughout the mucosa are composed of presumptive goblet cells in the crypts stained with Alcian blue. c. A portion of the image in a is shown at higher magnification to illustrate the small lumen and complete lack of bacterial staining in the lumen. Note that in a subset of samples in which the lumen was absent or greatly reduced in size, a few bacteria were present in the lumen (not shown). d. A portion of the image in b is shown at higher magnification to illustrate the presence of a thin mucus layer running along the surface of the mucosal epithelium in the folds between crypts (arrows). This was the only region of the section that contained such a mucus layer. The numerous dark-stained patches throughout the mucosa are composed of presumptive goblet cells in the crypts stained with Alcian blue. Scale bars in a,b = 200 μm, in c = 50 μm, and in d = 100 μm. L, lumen (TIF 9800 KB)
203_2022_2952_MOESM4_ESM.tif
Supplementary file4 Photomicrographs of bacteria stained with FISH in the small intestine that illustrate the increasing density of bacteria at progressively more distal levels in fed and fasted mice. In this example, there was also a small reduction of bacterial density at each of these proximal-distal levels in fasted (b,d,f) compared to fed (a,c,e) mice. Examples from the duodenum (a,b; 0 cm distal to the proximal small intestine), distal jejunum (c,d; approximately 18 cm distal to the proximal small intestine) and ileum (e,f; approximately 28 cm distal to the proximal small intestine) are shown. a. In the duodenum, the density of bacteria in fed mice was low and bacteria were invariably associated with particles of food in the lumen. b. The density of bacteria in the lumen of the duodenum of fasted mice was lower than in fed mice (compare b with a). c. The distal jejunum of this fed mouse had slightly greater density of bacteria in the lumen than the duodenum (compare c with a) and the distal jejunum of the fasted mouse (compare c with d). d. The lumen of the distal jejunum of this fasted mouse was small and contained almost no food or bacteria. e. The ileum had the greatest density of bacteria in the small intestine compared to duodenum and jejunum (compare e with a and c). Similar to more proximal levels, bacteria appeared to consistently be associated with food. f. Interestingly, in fasted mice the ileum not only had the greatest density of bacteria in the small intestine compared to the duodenum and jejunum (compare f with b or d), but this density was similar to that observed in the ileum of fed mice (compare f with e). Again, bacteria appeared to consistently be associated with food. Scale bars in a-d = 50 μm, and in e-f = 25 μm (TIF 15972 KB)
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Supplementary file5 Photomontages that illustrate the pattern of bacterial (a,b) and Alcian blue (c,d) staining of chyme in the cecum lumen (0 cm distal to the proximal cecum). a. In fed mice, the lumen was filled with chyme that was stained densely and evenly for bacteria. b. In fasted mice, the lumen was also filled with chyme that was stained densely and evenly for bacteria, but this density was slightly less than in fed mice. c. A section adjacent to the one shown in a that was stained for mucus is shown. The lumen was completely filled with chyme. d. A section adjacent to the one shown in b from a fasted mouse that was stained for mucus is shown. The lumen was completely filled with chyme. Scale bars in a-d = 400 μm (TIF 13057 KB)
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Fox, E., Lyte, M. Variation in spatial organization of the gut microbiota along the longitudinal and transverse axes of the intestines. Arch Microbiol 204, 424 (2022). https://doi.org/10.1007/s00203-022-02952-4
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DOI: https://doi.org/10.1007/s00203-022-02952-4