Imaging the in vivo growth patterns of bacteria in human gut Microbiota

Abstract

How to study the unculturable bacteria in the laboratory is one of the major challenges in human gut microbiota research. The resulting lack of microbiology knowledge of this “dark matter” greatly hinders further understanding of our gut microbiota. Here, to characterize the in vivo growth and division of human gut bacteria, we report the integrative use of STAMP (sequential tagging with D-amino acid–based metabolic probes) and fluorescence in situ hybridization (FISH) in a human microbiota-associated mouse model. After stable colonization of the human fecal microbiotas in germ-free mice, two fluorescent D-amino acid probes were sequentially administered by gavage, and the dually labeled peptidoglycan of the bacteria provided a chronological recording of their cell wall syntheses. Following taxonomic identification with FISH staining, the growth patterns of 32 species, including 5 currently unculturables, were identified. Surprisingly, we found that many bacterial species in the human microbiota were significantly shorter than those in the mouse gut microbiota. An imaging database for gut bacteria – Microbiome Atlas was built for summarizing STAMP imaging of bacteria from different microbiotas, which can be contributed by the microbiota research community worldwide. This integrative imaging strategy and the database will promote our understanding of the bacterial cytology in gut microbiotas and facilitate communications among cellular microbiologists.

Chemoenzymatic Labeling Pathogens Containing Terminal N-Acetylneuraminic Acid−α(2–3)-Galactose Glycans

“…Especially, the metabolic method was recently exploited to label live bacterial monosaccharides, such as N-acetylgalactosamine (GalNAc), 24 3-deoxy-D-mannooctulosonic acid (KDO), 25 N-acetylglucosamine (GlcNAc), 26 and peptidoglycan. 27 Our results revealed that the chemoenzymatic probe is compatible with both live and fixed bacteria. In the future, combining metabolic and chemoenzymatic methods to label bacteria will assist in the development of bacteria imaging.…”

“…Furthermore, antibody labeling, lectin labeling, antibiotic labeling, and metabolic labeling were the main methods for labeling the oligosaccharide enveloped on bacteria, whereas our results enriched this territory. Especially, the metabolic method was recently exploited to label live bacterial monosaccharides, such as N-acetylgalactosamine (GalNAc), 3-deoxy- d -mannooctulosonic acid (KDO), N-acetylglucosamine (GlcNAc), and peptidoglycan . Our results revealed that the chemoenzymatic probe is compatible with both live and fixed bacteria.…”

Advances in Bio‐Optical Imaging Systems for Spatiotemporal Monitoring of Intestinal Bacteria

“…Wang et al. [ 42,43 ] invented sequential tagging with d ‐amino acid‐based metabolic probes (STAMP) to assess the microbiota activity in fecal microbiota transplantations. Gut microbiota from FDAA‐labeled donor mice were transplanted into recipient mice, which were then treated with another FDAA probe of a different color.…”

“…[39,40] Lin et al [41] combined FDAA probes with FISH to develop two FDAA probes that could image intestinal flora in two colors, TAMRA-amino-d-alanine (TADA) and Cy5-amino-d-alanine (Cy5ADA), thereby visualizing different division patterns and growth rates of bacteria in vivo. Wang et al [42,43] invented sequential tagging with d-amino acid-based metabolic probes (STAMP) to assess the microbiota activity in fecal microbiota transplantations. Gut microbiota from FDAAlabeled donor mice were transplanted into recipient mice, which were then treated with another FDAA probe of a different color.…”

Intestinal Microbiota and Serum Metabolic Profile Responded to Two Nutritional Different Diets in Mice

“…For example, The GF diet should be as low in fiber as possible and nutrients such as vitamins, minerals, and amino acids should be supplemented additionally compared to the SPF diet ( 24 , 25 ). Our laboratory has been studying the diets of GF animals and has formulated diets that meet the growth and reproductive needs of GF mice ( 26 ). The GF diet has a higher nutritional content than the SPF diet, both as previously reported and in the formulas we have created.…”