bioRxiv preprint doi: https://doi.org/10.1101/2019.12.30.890848; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Gut-derived Flavonifractor species variants are 2 differentially enriched during in vitro incubation with 3 quercetin 4 Short Title: Flavonifractor species variants are differentially enriched during incubation with 5 quercetin 6 Gina Paola Rodriguez-Castaño 1*, Federico E. Rey2, Alejandro Caro-Quintero3, Alejandro 7 Acosta-González1 8 1 Engineering Department, La Sabana University, Chia, Colombia 9 2 Department of Bacteriology, University of Wisconsin-Madison, USA 10 3 AGROSAVIA, Centro de Investigación Tibaitatá, Mosquera, Colombia. 11 * Correspondence: 12 Gina Paola Rodriguez-Castaño 13 [email protected] (GRC) 14 15 Abstract 16 Flavonoids are a common component of the human diet with widely reported health- 17 promoting properties. The gut microbiota transforms these compounds affecting the overall 18 metabolic outcome of their consumption. Flavonoid-degrading bacteria are often studied in 19 isolation under culture conditions that do not resemble the conditions in the colon and that bioRxiv preprint doi: https://doi.org/10.1101/2019.12.30.890848; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 20 eliminate the multiple interactions that take place in complex communities. In this study, a 21 comparative metataxonomic analysis of fecal communities supplemented with the flavonoid 22 quercetin led us to identify a potential competitive exclusion interaction between two sequence 23 variants related to the flavonoid-degrading species, Flavonifractor plautii, that belong to the same 24 genus but different species. During incubation of fecal slurries with quercetin, the relative 25 abundance of these two variants was inversely correlated; one variant, ASV_65f4, increased in 26 relative abundance in half of the libraries and the other variant, ASV_a45d, in the other half. This 27 pattern was also observed with 6 additional fecal samples that were transplanted into germ-free 28 mice fed two different diets. Mouse’s diet did not change the pattern of dominance of either 29 variant, and initial relative abundances did not predict which one ended up dominating. Potential 30 distinct metabolic capabilities of these two Flavonifractor-related species were evidenced, as only 31 one variant, ASV_65f4, became consistently enriched in complex communities supplemented with 32 acetate but no quercetin. Genomic comparison analysis of the close relatives of each variant 33 revealed that ASV_65f4 may be an efficient ethanolamine-utilizing bacterium which may increase 34 its fitness in media with no quercetin compared to ASV_a45d. Other discordant features between 35 ASV_65f4- and ASV_a45d-related groups may be the presence of flagellar and galactose- 36 utilization genes, respectively. Overall, we showed that the Flavonifractor genus harbors variants 37 that present a pattern of negative co-occurrence and that may have different metabolic and 38 structural traits, whether these differences affect the dynamic of quercetin degradation warrants 39 further investigation. 40 41 Introduction 42 Flavonoids are 3-ring phenolic compounds found in fruits and vegetables, their regular 43 consumption is associated with health benefits (1,2). Among these, quercetin is one of the most bioRxiv preprint doi: https://doi.org/10.1101/2019.12.30.890848; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 44 abundant in the human diet. It exerts effects on the immune, digestive, endocrine, nervous, and 45 cardiovascular systems (3–6). Some gut bacteria can cleave the central ring in the flavonoid 46 skeleton by a process known as C-ring fission which generates smaller phenolic products. In the 47 case of quercetin, phloroglucinol and 3,4-dihydroxyphenylacetic acid (DOPAC) are formed (7). It 48 is not clear to what extent do the health effects of flavonoids depend on their transformation to 49 biologically active compounds by the gut microbiota. Vissiennon and collaborators showed that 50 the anxiolytic activity of quercetin is induced by DOPAC and not by the parent compound, 51 evidencing a case in which the microbial metabolite exerts the beneficial effect (8). DOPAC has 52 also antiproliferative activity in colon cancer cells (9) and anti-platelet aggregation activity (10). 53 Additionally, the well-recognized quercetin-degraders, F. plautii (formerly Clostridium 54 orbiscindens) and E. ramulus, are also butyrate-producers, a short-chain fatty acid that is the 55 preferred source of energy of colonocytes and essential for colon health (11–14). 56 Most commonly, these flavonoid-degrading bacteria are studied in pure culture, however, 57 they reside in the colon where more than 1013 bacterial cells inhabit (15). In vitro fecal incubation 58 systems provide a scenario where complex microbial communities can be studied. In vitro fecal 59 incubations with quercetin have revealed the metabolites formed during the transformation of this 60 flavonoid; however, ecological interactions between quercetin-degraders and the rest of the 61 community have been overlooked. Additionally, most studies have analyzed fecal samples from 62 one donor (16,17) or pooled fecal samples from different donors (18–20), dismissing the 63 importance of the gut microbiota at the individual level. To characterize key players in quercetin 64 degradation that are common or unique among subjects, the evaluation of in vitro fecal incubation 65 experiments with individual samples is needed. 66 In this study, microbial community dynamics from different subjects were analyzed 67 individually by in vitro incubations of feces with quercetin. Through this approach, we identified 68 variants related to flavonoid degraders that became enriched upon incubation with quercetin and bioRxiv preprint doi: https://doi.org/10.1101/2019.12.30.890848; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 69 that were consistently observed across 15 healthy subjects. Since it is known that differences in 70 a small set of genes between bacterial strains can have a profound impact on the host’s 71 physiology (21), we used a genomic comparison analysis of close relatives of these variants to 72 infer genetic differences between them. Potential metabolic and structural differences were 73 detected between these variants, thus, we propose that their role in the gut microbiome is 74 differentially affected by carbon sources and interactions with other members of the community 75 and may have distinct roles in the degradation of the flavonoid. 76 77 Materials and Methods 78 Sample collection and processing. Stool samples were collected from subjects 79 participating in the Wisconsin Longitudinal Study (WLS) between November 2014 and February 80 2015 as previously described (22). Briefly, participants collected stool samples directly in sterile 81 containers, then samples were kept at ~4 °C until arrival to the processing laboratory within 48 82 hours of collection. Upon arrival, sterile straws were filled with the fecal material and stored at - 83 80˚ C as previously described (23). The use of WLS fecal microbiota was approved by the 84 Institutional Review Board at the University of Wisconsin-Madison. 85 In vitro incubations with human fecal samples and quercetin. Human fecal samples 86 were used for in vitro incubations with quercetin. Samples from 9 subjects kept in frozen straws 87 were aliquoted (~50 mg) on dry ice, weighted, and resuspended at 0.5 mg ml-1 final concentration 88 with heavy vortexing in anaerobic 7N minimal medium supplemented with 20 mM sodium acetate 89 (filter-sterilized through a 0.22-m pore diameter) (24). Quercetin dihydrate, 97 % w/w (Alfa Aesar) 90 concentration was 0.125 mg ml-1 (0.4 mM) in the corresponding medium. Controls consisted of 91 the same medium plus fecal sample. Treatments with quercetin had three replicates and controls 92 one replicate. Anaerobic bottles were kept statically at 37° C. When quercetin disappearance was bioRxiv preprint doi: https://doi.org/10.1101/2019.12.30.890848; this version posted December 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 93 visible, 10 % of the culture was transferred to another anaerobic bottle with the same medium. 94 Sample processing was done in an anaerobic chamber under an atmosphere of nitrogen (75%), 95 carbon dioxide (20%), and hydrogen (5%). Sampling was done at the end of the first and second 96 incubation time once quercetin degradation was completed across all samples (72 h of 97 incubation). In a second experiment, combinations of fecal matter from different subjects were 98 tested. Two fecal samples used in the previous in vitro incubation experiment (from subjects #3 99 and #4) were selected based on their enrichment of one or the other Flavonifractor-related variant 100 (ASV_65f4 or ASV_a45d). Three enrichments of these fecal samples were done as follows 101 (#3/#4): 0/0.1, 0.1/0.1, 0.1/0 mg ml-1. Incubations were done again in anaerobic 7N minimal 102 medium supplemented with 20 mM sodium acetate with and without quercetin using three 103 replicates.