Integrated identification of growth pattern and taxon of bacterium in gut microbiota via confocal fluorescence imaging‐oriented single‐cell sequencing

Abstract

Despite the fast progress in our understanding of the complex functions of gut microbiota, it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis. To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput, here, we propose an integrative strategy, which combines the use of fluorescent probe labeling, confocal imaging with single‐cell sorting, and sequencing. Mouse gut bacteria sequentially labeled by two fluorescent d‐amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns, which can be unveiled by the distribution of the two fluorescence signals on each bacterium. Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment, and the collected cells were then sequenced individually to identify their taxa. Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single‐cell basis, which should also have great potential in studying many other complex bacterial systems.

Isolation and Cultivation of Human Gut Microorganisms: A Review

“…Additionally, targeted strategies can also enhance access to gut microbial resources [32]. The development of molecular biology, bioinformatics, flow cytometry, and microfluidics techniques have provided the technical basis for the targeted sorting of gut microbes [24,[33][34][35][36][37][38]. Except for the same procedures with the nontargeted approach, researchers need specific information on microorganisms for targeting, which is usually obtained through literature research, metagenomics data, etc.…”

Bacterial imaging in tumour diagnosis

“…For example, (Lin et al., 2020 ) proposed a method based on sequential tagging with d ‐amino acid‐based metabolic probes (STAMP) supplemented by fluorescent in situ hybridization (FISH) to reveal the in vivo growth and division patterns of different species of gut bacteria. To dig deeply into various complex bacterial samples, (Gao, Sun, et al., 2022 ) proposed a FLCiSS strategy, which combined fluorescent probe labeling, confocal imaging and single cell sorting and sequencing to realize the combination of bacterial genome information and bacterial phenotype information observed by microscopic imaging, so that bacteria in complex samples can be directly selected and sequenced.…”