Getting heavy with microbes: Raman tweezes them apart

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  • Published: Jan 8, 2015
  • Author: David Bradley
  • Channels: Raman
thumbnail image: Getting heavy with microbes: Raman tweezes them apart

Heavy microbes

 Quantification of D incorporation in individual E. coli cells from the same experiment as detected by NanoSIMS and shown as the isotope fraction D/(H + D) given as at%. All isotope fraction images are on the same scale (0–100 at%). 31 P − signal intensity distribution is displayed to indicate the location of cellular biomass. Credit: Proc Natl Acad Sci USA)

The incorporation of heavy water into the biomolecules of bacteria has given an international research team the means to tease apart and identify the activities of different members of microbial communities using Raman microscopy.

The presence of microbial communities is essential to the existence of almost all ecosystems, including the human body. However, identifying all the individual flora and fauna at this level, under natural conditions, is difficult and so often stymies research into the roles played by particular species in health and disease. Now, a team comprising researchers from the University of Vienna, Austria, Chinese Academy of Sciences, Qingdao, Shandong, China, the University of Sheffield, UK, Agricultural Research Organization, Bet-Dagan, Israel, the University of Jena, Germany and the University of Oxford, UK, have delivered a possible solution.

FISH for mouse clues

The team has developed a novel approach to identifying and sorting active microbes at the single-cell level in complex samples by using heavy water (D2O) as a stable isotopic probe coupled with Raman microspectroscopy. They demonstrated that they could unambiguously detect incorporation of deuterium, by the presence of carbon-deuterium (C-D) bonds as signature peaks in single-cell Raman spectra corroborated by nanoscale- resolution secondary ion mass spectrometry. However, it was the use of FISH, fluorescence in situ hybridization, with Raman microspectroscopy that allowed them to then identify individual active microbes in a complex sample acting as a surrogate for a complete ecosystem.

A proof of principle was carried out using mouse cecal microbiota, the microbes in the intraperitoneal pouch at the beginning of the large intestine. This showed that host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens were present and exhibited distinctive response patterns to changes in levels of the mucus glycoprotein mucin and sugars. The team was also able to separate the deuterated cells based on their Raman signatures using optical tweezers and to then carry out amplification and DNA sequencing, which led to the identification of previously unknown microbes in the mouse cecum stimulated by mucin and/or glucosamine. Thus, the team says, "demonstrating the potential of the non-destructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics."

Microbial community spirit

Microbial communities are important in a wide range of systems, in human health and disease, in the catalysis of biotech processes, in global biogeochemical cycles and at the level of countless ecosystems from stagnant ponds to tropical rainforests. The new technique developed by the team side-steps many of the problems scientists face using conventional techniques to study such communities, not least the lack of spatial resolution of bulk analysis of a complex sample. The team points out that their approach not only allows the detailed microbial activity within a biofilm, for instance, to be examined, but even allows identification of single cells having different activity in what otherwise appears to be a clonal community of purportedly identical cells.

The team points out that, "Because Raman microspectroscopy and optical tweezing are non-destructive techniques that work in water, no pre-treatment of the cells (other than the isotope labelling) is required." They thus conclude that their approach facilitates single-cell genomics experiments that might be focused on individual cells within a defined microbial "guild". It could also prove useful for the targeted cultivation of members of particular microbial communities that have interesting or potentially useful ecophysiological properties.

"The ultimate goal of the research is to identify organisms with a particular activity of interest, such as degradation of a pollutant or toxin, and sort them in a high-throughput manner for cultivation and genome analysis," Berry told SpectroscopyNOW. "This will allow us to discover novel physiologies and organisms in a targeted and rapid way."

Related Links

Proc Natl Acad Sci, 2015, online: "Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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