Species-targeted sorting and cultivation of commensal bacteria from the gut microbiome using flow cytometry under anaerobic conditions.
Anaerobic
Christensenella
Culturomics
Faecalibacterium
Flow cytometry
Microbiota
Sorting
Journal
Microbiome
ISSN: 2049-2618
Titre abrégé: Microbiome
Pays: England
ID NLM: 101615147
Informations de publication
Date de publication:
03 02 2022
03 02 2022
Historique:
received:
16
11
2021
accepted:
04
12
2021
entrez:
4
2
2022
pubmed:
5
2
2022
medline:
22
3
2022
Statut:
epublish
Résumé
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. 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. 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. Video abstract.
Sections du résumé
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. Video abstract.
Identifiants
pubmed: 35115054
doi: 10.1186/s40168-021-01206-7
pii: 10.1186/s40168-021-01206-7
pmc: PMC8812257
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Video-Audio Media
Langues
eng
Sous-ensembles de citation
IM
Pagination
24Informations de copyright
© 2022. The Author(s).
Références
J Microbiol Methods. 2015 Oct;117:74-7
pubmed: 26187776
Sci Rep. 2016 Sep 23;6:33721
pubmed: 27659943
Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16731-6
pubmed: 18936492
Bioinformatics. 2012 Jul 15;28(14):1823-9
pubmed: 22556368
PLoS One. 2015 Apr 24;10(4):e0123013
pubmed: 25910186
Cell. 2014 Nov 6;159(4):789-99
pubmed: 25417156
PLoS Biol. 2019 Jan 23;17(1):e3000102
pubmed: 30673701
Eur J Immunol. 2016 May;46(5):1300-3
pubmed: 26909672
Nat Biotechnol. 2019 Nov;37(11):1314-1321
pubmed: 31570900
Nature. 2017 Nov 23;551(7681):507-511
pubmed: 29143816
Nucleic Acids Res. 2013 Jan 7;41(1):e1
pubmed: 22933715
BMC Genomics. 2018 Dec 14;19(1):931
pubmed: 30547746
FEMS Microbiol Rev. 2010 Jul;34(4):554-87
pubmed: 20337722
Front Microbiol. 2017 Sep 22;8:1790
pubmed: 28970823
Cell. 2019 Aug 22;178(5):1041-1056
pubmed: 31442399
Sci Rep. 2021 Mar 8;11(1):5426
pubmed: 33686095
Nucleic Acids Res. 2020 Jan 8;48(D1):D545-D553
pubmed: 31504765
ISME J. 2017 Apr;11(4):841-852
pubmed: 28045459
Nature. 2016 May 04;533(7604):543-546
pubmed: 27144353
Front Microbiol. 2017 Jun 30;8:1226
pubmed: 28713353
Nat Rev Clin Oncol. 2018 Jun;15(6):382-396
pubmed: 29636538
Blood. 2016 Aug 18;128(7):993-1002
pubmed: 27402974
Nat Methods. 2013 Oct;10(10):996-8
pubmed: 23955772
Appl Environ Microbiol. 2019 Mar 22;85(7):
pubmed: 30709823
Front Microbiol. 2021 Feb 22;12:632361
pubmed: 33692769
Bioinformatics. 2010 Oct 1;26(19):2460-1
pubmed: 20709691
Ann Oncol. 2017 Jun 1;28(6):1368-1379
pubmed: 28368458
Nat Rev Microbiol. 2016 Aug;14(8):508-22
pubmed: 27396567
Appl Environ Microbiol. 1997 Jul;63(7):2802-13
pubmed: 9212428
Microb Cell Fact. 2017 Oct 30;16(1):180
pubmed: 29084543
mBio. 2020 Feb 4;11(1):
pubmed: 32019803
Science. 2018 Jan 5;359(6371):97-103
pubmed: 29097493
Nat Microbiol. 2017 Apr 25;2:17057
pubmed: 28440276
BMC Biol. 2019 Oct 28;17(1):83
pubmed: 31660948
Appl Environ Microbiol. 2011 Nov;77(21):7846-9
pubmed: 21890669
Int J Obes (Lond). 2012 Jun;36(6):817-25
pubmed: 21829158
PeerJ. 2017 Jan 11;5:e2836
pubmed: 28097056
Cells. 2021 Apr 06;10(4):
pubmed: 33917566
EMBO Mol Med. 2019 Feb;11(2):
pubmed: 30591521
Cytometry A. 2007 Aug;71(8):592-8
pubmed: 17421025
J Virol. 2018 Jan 17;92(3):
pubmed: 29167334
Nat Med. 2019 Sep;25(9):1442-1452
pubmed: 31477907
Mol Nutr Food Res. 2015 Aug;59(8):1614-28
pubmed: 25988339
Nucleic Acids Res. 1992 Oct 11;20(19):5137-42
pubmed: 1408828
Mol Biol Evol. 2016 Jul;33(7):1870-4
pubmed: 27004904
Cell Host Microbe. 2021 May 12;29(5):765-776.e3
pubmed: 33794185
Nat Commun. 2018 Jul 20;9(1):2873
pubmed: 30030445
Int J Obes (Lond). 2013 Nov;37(11):1460-6
pubmed: 23459324
Nucleic Acids Res. 2004 Mar 19;32(5):1792-7
pubmed: 15034147
Front Microbiol. 2020 Jun 26;11:1469
pubmed: 32676069
Appl Environ Microbiol. 2015 Nov;81(21):7582-92
pubmed: 26296733
Redox Biol. 2018 Jun;16:381-387
pubmed: 29627745
Nat Biotechnol. 2021 Jan;39(1):105-114
pubmed: 32690973
Bioinformatics. 2018 Jul 15;34(14):2371-2375
pubmed: 29506021