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 there are few or no strains available for specific species that are difficult to cultivate. Our objective was to adapt flow cytometry and cell sorting to be able to detect, separate, isolate, and cultivate new strains of commensal species from fecal material. We focused on the extremely oxygen sensitive (EOS) species Faecalibacterium prausnitzii and the under-represented, health-associated keystone species Christensenella minuta as proof-of-concept. Results A BD Influx® cell sorter was equipped with a glovebox that covered the sorting area. This box was flushed with nitrogen to deplete oxygen in the enclosure. Anaerobic conditions were maintained during the whole process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii strains ATCC 27766, ATCC 27768, and DSM 17677. We then generated polyclonal antibodies against target species by immunizing rabbits with heat-inactivated bacteria. Two polyclonal antibodies were directed against F. prausnitzii type strains that belong to different phylogroups, whereas one was directed against C. minuta strain DSM 22607. The specificity of the antibodies was demonstrated by sorting and sequencing the stained bacterial fractions from fecal material. In addition, staining solutions including LIVE/DEAD™ BacLight™ Bacterial Viability staining and polyclonal antibodies did not severely impact bacterial viability while allowing discrimination between groups of strains. Finally, we combined these staining strategies as well as additional criteria based on bacterial shape for C. minuta and were able to detect, isolate, and cultivate new F. prausnitzii and C. minuta strains from healthy volunteer’s fecal samples. Conclusions 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, thus opening new avenues for culture experiments.

We report the isolation, culture, and genome sequencing of isolate POC01, a strictly anaerobic bacterium isolated from a healthy donor, representing a previously uncultured member of the Oscillospiraceae family.

An ever-increasing number of studies report the importance of the composition of the intestinal microbiota in the treatment of cancers. The mechanisms involved are varied. The reciprocal interaction of drugs with bacteria can have an effect on the efficacy as well as the toxicity of treatments. In addition, some species or strains are able to stimulate CD8+ T cell production and the effectiveness of immune checkpoint blockers (ICBs). As part of these treatments, there is therefore a potential interest in being able to isolate and cultivate bacterial species associated with the response to ICBs in different cancers. The objective is to better understand the mechanisms involved and possibly develop complementary therapeutic and/or diagnostic solutions. However, this approach is still limited by the fact that a large number of commensal bacterial species in the intestine are difficult to cultivate, due to specific nutritive requirements, extreme oxygen sensitivity (EOS), or under-representation in the whole bacterial population. In an attempt to circumvent these limitations, we developed flow cytometry (FCM) and cell sorting in anaerobic conditions. A series of polyclonal antibodies specific to commensal species known to be beneficial to the host such as Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Eubacterium hallii have been produced and used in combination with viability and Gram staining to rapidly detect, sort, and culture species of interest. This strategy has enabled a rapid collection of strains to be built up from the stools of healthy volunteers, demonstrating the usefulness of the approach. Developments are under way to apply reverse genomics strategies to identify sequences encoding immunogenic proteins in order to produce antibodies even in the absence of already cultivated strains, which is still the case for approximately 70% of the human gut microbiota species. In conclusion, our work confirms that FCM is well adapted for complex bacterial microbiota studies. When used in conjunction with appropriate staining, it gives a general overview of microbiota composition and variations in longitudinal studies, including bacterial load, which is an important piece of information. In addition to sorting and cultivating target species of interest, it can be used for a quick and easy assessment of microbiota composition, either before starting a drug treatment or for monitoring the effects of the treatment on commensal microbiota. Citation Format: Samuel Bellais, Mélanie Nehlich, Aurore Duquenoy, Maryne Ania, Jan Baijer, Ilia Belotserkovsky, Vincent Thomas. Flow cytometry for targeted culturomics of gut commensal species and rapid overview of microbiota composition [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B14.

BackgroundThere is a growing interest in using gut commensal bacteria as ‘next generation’ probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was therefore to adapt flow cytometry and cell sorting to be able to detect, separate, isolate and cultivate new strains of Extremely Oxygen Sensitive (EOS) species from fecal material, focusing on Faecalibacterium prausnitzii as a proof-of-concept.ResultsA BD Influx® cell sorter was equipped with a glovebox that covers the sorting area. This box is flushed with nitrogen to deplete oxygen in the enclosure. Several non-specific staining methods including Wheat Germ Agglutinin (WGA), Vancomycin BODIPY™ and LIVE/DEAD BacLight were evaluated with three different strains of the EOS species F. prausnitzii. In parallel, we generated polyclonal antibodies directed against this species by immunizing rabbits with heat-inactivated bacteria. Anaerobic conditions were maintained during the full process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii reference strains. In addition, staining solutions did not severely impact bacterial viability while allowing discrimination between groups of strains. Efficient detection was achieved using polyclonal antibodies directed against heat-fixed bacteria. Finally, we were able to detect, isolate and cultivate a variety of F. prausnitzii strains from healthy volunteer’s fecal samples using WGA staining and antibodies. These strains present markedly different phenotypes, thus confirming the heterogeneity of the species.ConclusionsCell-sorting in anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations and to sort strains of interest using specific antibodies, thus opening new avenues for targeted culturomics experiments.