Abstract
While the diversity of the human gut microbiota is becoming increasingly well characterized, bacterial physiology is still a critical missing link in understanding how the gut microbiota may be implicated in disease. The current best practice for studying bacterial physiology involves the immediate storage of fecal samples in an anaerobic chamber. This reliance on immediate access to anaerobic chambers greatly limits the scope of sample populations that can be studied. Here, we assess the effects of short-term oxygen exposure on gut bacterial physiology and diversity. We use relative nucleic acid content and membrane integrity as markers of bacterial physiology, and 16S rRNA gene amplicon sequencing to measure bacterial diversity. Samples were stored for up to 6 h in either ambient conditions or in anoxic environments created with gas packs or in an anaerobic chamber. Our data indicate that AnaeroGen sachets preserve bacterial membrane integrity and nucleic acid content over the course of 6 h similar to storage in an anaerobic chamber. Short-term oxygen exposure increases bacterial membrane permeability, without exceeding inter-individual differences. As oxygen exposure remains an important experimental consideration for bacterial metabolism, our data suggest that AnaeroGen sachets are a valid alternative limiting loss of membrane integrity for short-term storage of samples from harder-to-access populations.
Highlights
The human gut microbiota is increasingly studied due to its widespread implications in human health
Bacterial physiology varies across individuals but is stable over time within individuals We determined the proportions of higher nucleic acid content (HNA) and lower nucleic acid content counterparts (LNA) bacteria through SYBR Green I staining, and the proportions of cells with impaired membranes through propidium iodide (PI) staining from fecal samples of 19 healthy unrelated individuals
When considering only the 6-h timepoint, we report no significant difference in the proportions of HNA or PI+ bacteria, as well as total counts between samples stored in anoxic conditions in the chamber or under ambient aerobic conditions
Summary
The human gut microbiota is increasingly studied due to its widespread implications in human health. Extensive sequencing efforts have characterized gut bacterial diversity in a variety of human and non-human populations, describing how this bacterial diversity changes over time and in response to different perturbations such as dietary changes, antibiotics exposure, and disease (Gilbert et al, 2016; David et al, 2014; Dethlefsen et al, 2008). These studies typically highlight the high diversity of bacterial species or strains that stably colonize healthy. To characterize different states of bacteria being sampled, we turn to single-cell techniques such as physiological dyes and flow cytometry
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