High-throughput identification and quantification of single bacterial cells in the microbiota

Jianshi Jin,Reiko Yamamoto,Nozomi Hojo,Katsuyuki Shiroguchi,Guangwei Cui,Eiji Miyauchi,Hiroshi Ohno,Koichi Ikuta,Tadashi Takeuchi

doi:10.1038/s41467-022-28426-1

Abstract

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. To fully understand the function of bacterial microbiota, individual cells must be identified; however, it is difficult with current techniques. Here, we develop a method, Barcoding Bacteria for Identification and Quantification (BarBIQ), which classifies single bacterial cells into taxa–named herein cell-based operational taxonomy units (cOTUs)–based on cellularly barcoded 16S rRNA sequences with single-base accuracy, and quantifies the cell number for each cOTU in the microbiota in a high-throughput manner. We apply BarBIQ to murine cecal microbiotas and quantify in total 3.4 × 105 bacterial cells containing 810 cOTUs. Interestingly, we find location-dependent global differences in the cecal microbiota depending on the dietary vitamin A deficiency, and more differentially abundant cOTUs at the proximal location than the distal location. Importantly, these location differences are not clearly shown by conventional 16S rRNA gene-amplicon sequencing methods, which quantify the 16S rRNA genes, not the cells. Thus, BarBIQ enables microbiota characterization with the identification and quantification of individual constituent bacteria, which is a cornerstone for microbiota studies.

Highlights

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells
By the cOTUbased analysis provided by Barcoding Bacteria for Identification and Quantification (BarBIQ), we discovered that dietary vitamin A deficiency effects the microbiota at both the proximal and distal locations of the murine cecum, and the effect at the proximal location was larger after 3 weeks of feeding
Based on the structure of the murine intestinal tract, this observed phenomenon suggested that the content within the cecum was not completely homogenized, and the dietary effect on the microbiota at the proximal location was partially transformed to the distal location in 3 weeks

Summary

Introduction

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. It is difficult to confirm the existence of newly identified 16S rRNA sequences from unknown bacteria To overcome these limitations in conventional methods, we developed a method that identified and quantified individual bacteria in microbiota in a high-throughput manner by cellularly barcoded 16S rRNA sequencing with single-base accuracy, which is named Barcoding Bacteria for Identification and Quantification (BarBIQ) (Fig. 1a–c, Methods section, Supplementary Note 1 and 2). This method clarified both the global microbiota and individual bacterial members. This was not clearly shown by the two conventional methods we conducted

Methods

Results

Conclusion