Technology

ANOXIC PLATFORM

Sorting and isolation of bacteria using a cytometer allowing aerobic and anaerobic conditions.

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ANOXIC PLATFORM

Objectif/Description technologique

The aim of the technology is to allow sorting and isolation of bacteria using a cytometer allowing aerobic and anaerobic conditions.

Such sorting and isolation are of great help for getting access to nextgeneration probiotics” (NGP). Different types of non-specific labelling as well as specific labelling using antibodies allow the targeting of specific bacteria, and in particular low abundant ones, in order to be able to cultivate them.
Antibodies can be produced specifically from heat-inactivated bacteria.

Technology Anoxic Platform

Background

There is a growing interest in using gut commensal bacteria as “next generation” probiotics.

However, this approach is still hampered by the fact that retrieving target species from clinical samples (usually fecal material) can be difficult.

Extreme oxygen sensitivity (EOS) or under-representation of the target species in the community can be important limitations, with the addition of specific nutritional requirements rendering target species difficult to cultivate in synthetic media.

Flow cytometry (FCM) coupled with cell-sorting has the potential to circumvent most if not all these limitations. With constantly increasing technological performances, FCM can be used for bacterial or even viral cell populations’ analysis with or without subsequent sorting.

With the objective to use FCM and cell-sorting to analyze, sort, and cultivate bacterial species of interest from fecal samples, we adapted a cell sorter and associated workflow to conduct sorting experiments under strictly anaerobic conditions.

Advantages

The advantage is for companies wishing to isolate microorganisms of interest from complex ecosystems (e.g. human and animal health biotech, environment and food industry).

The high sensitivity of the system allow to sort and isolate very low abundant bacteria (as low as 0.02% of relative abundance).
Targeted cell-sorting under anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations, and can be used to sort EOS and/or under-represented strains of interest using specific antibodies.
Through the reverse genomic approach, it also allows the isolation of bacteria that have not already been isolated.

Potential Applications

With the availability of next-generation sequencing technologies that allow high-throughput analysis of the composition and function of complex microbial ecosystems, the field of microbiome research has grown rapidly in recent years, and countless associations have been reported between microbiota composition and specific health conditions.

This is especially true for the human gut ecosystem, for which microbial signatures have been associated with metabolic syndrome, inflammatory bowel diseases (IBD), and response to cancer immunotherapy to mention just a few. This offers new fundamental and applied research avenues, with the ultimate goal to develop new, complementary tools for treating these conditions. There is thus a growing interest in using cultured, wellcharacterized strains to complement deficiencies in the gut microbiota,
referred to as “next-generation probiotics” (NGP)
The technology is currently applied for isolation of bacteria having the potential to be NGPs.

Next steps

The advent of « biomic » techniques has allowed a fine characterization of complex microbial communities. Their use on a large scale has led to the in silico identification of several thousands of species constituting the intestinal microbiota, a large fraction of which has not been isolated or cultured to date. Among these non-isolated species, some are identified as potential new generation probiotics.

To date, the challenges to access this reservoir of new generation probiotics, even before their exploitation, are mainly scientific and technological:

  • precise identification of bacterial species
  • potential therapeutic target(s)
  • and also, and maybe more critical, ability to specifically target and isolate viable representatives in complex ecosystems.

In a project with INRAE, BIOASTER has validated the proof of principle of metagenomics-guided culturomics to select and isolate species of interest using reverse genomics to generate antibodies to target the species of interest identified in silico.
After this first step, BIOASTER wishes to develop further this approach to render it more robust and replicable through a proof-of-concept phase.