Species-targeted sorting and cultivation of commensal bacteria from the gut microbiome using flow cytometry under anaerobic conditions

Samuel Bellais,Maryne Ania,Ger Van Den Engh,Wilfrid Mazier,Jan Baijer,Nicole Simone Treichel,Vincent Thomas,Mélanie Nehlich,Aurore Duquenoy,Ilia Belotserkovsky,Thomas Clavel

doi:10.1186/s40168-021-01206-7

Abstract

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 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.ResultsA 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.ConclusionsTargeted 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.BUx8pvnE8Zxeu2HSzKY8-eVideo abstract

Highlights

There is a growing interest in using gut commensal bacteria as “ generation” probiotics
Staining efficiency was more variable for antibodies directed against strain A2-165, with the proportion of stained bacteria ranging from 50% to > 90% depending on the experiment
We evaluated the effect of different staining methods (SYTO 9 and propidium iodide in the LIVE/ DEADTM BacLightTM Bacterial Viability Kit and the 4 strain specific antibodies) on cultivability compared to unstained bacteria

Summary

Introduction

There is a growing interest in using gut commensal bacteria as “ generation” probiotics. Bellais et al Microbiome (2022) 10:24 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. There is a growing interest in using cultured, well-characterized strains to complement deficiencies in the gut microbiota, referred to as “next-generation probiotics” (NGP) [5] This is the case for Faecalibacterium prausnitzii, which accounts for about 5‐10% of dominant microbial communities within the healthy gut microbiota [6], and has been associated with a number of favorable outcomes in various pathologies including lower risk of postoperative recurrence of ileal Crohn’s disease [7] and an improved response to immune check point blockers [8, 9]. It has been proposed to use corresponding relative abundances as disease biomarker [15]

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