Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

Area of Science:

• Microbiology
• Ecosystem science
• Single-cell analysis

Background:

• Microbial communities are vital for ecosystems and eukaryotes.
• Identifying active microbes in complex environments remains a significant challenge.
• Current methods often struggle to distinguish active from dormant cells in situ.

Purpose of the Study:

• To develop a new method for identifying and sorting active microbes at the single-cell level.
• To enable functional analysis of microbial communities in complex samples.
• To facilitate single-cell genomics of specifically sorted active microbial populations.

Main Methods:

• Stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy.
• Detection of deuterium (D) incorporation via C-D signature peaks in single-cell Raman spectra.
• Confirmation of labeling using nanoscale-resolution secondary ion mass spectrometry (MS).
• Combination with Fluorescence In Situ Hybridization (FISH) for active microbial identification.
• Raman-based cell sorting using optical tweezers for subsequent DNA sequencing.

Main Results:

• Unambiguous detection of D incorporation in active bacteria and archaea.
• Label detection in fast-growing Escherichia coli within 20 minutes.
• Distinct metabolic responses of Akkermansia muciniphila and Bacteroides acidifaciens to mucin and sugar amendments in mouse cecal microbiota.
• Identification of novel cecal microbes stimulated by mucin and/or glucosamine.

Conclusions:

• The D2O-Raman microspectroscopy approach is effective for identifying and sorting metabolically active single microbial cells.
• This nondestructive method allows for targeted isolation of active cells for downstream applications like single-cell genomics.
• The study successfully identified novel microbes within the mouse cecal environment, highlighting the method's potential for microbial ecology research.