Comparative genomics and functional characterization of 11 newly isolated non-equol-producing Adlercreutzia strains with anti-inflammatory properties

Comparative genomics and functional characterization of 11 newly isolated non-equol-producing Adlercreutzia strains with anti-inflammatory properties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative genomics and functional characterization of 11 newly isolated non-equol-producing Adlercreutzia strains with anti-inflammatory properties Célia Chamignon, Maxime Bredel, Amandine Lashermes, Benoit Quinquis, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8574898/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Adlercreutzia, a prevalent genus in the human gut microbiota, gains growing interest due to the potential beneficial effects on human health, particularly in the context of metabolic and inflammatory diseases. Previous work showed the A. equolifaciens type strain exhibits anti-inflammatory properties, but whether this effect is strain-specific or linked to equol production remained unclear. In this study, we isolated 11 novel Adlercreutzia strains from healthy European volunteers. Whole-genome sequencing classified most isolates closely related to A. rubneri or A. equolifaciens species. Phenotypic characterization revealed all isolates are obligate anaerobes, asaccharolytic, non-equol producers and exhibit strain-dependent tolerance to oxygen and bile salts, opening up possibilities for therapeutic options. Functional assays demonstrated that all strains exert significant anti-inflammatory effects in vitro by downregulating NF-κB activation in human intestinal epithelial and liver cells. These findings reveal potent anti-inflammatory activity independent of equol production, positioning these novel, well-characterized Adlercreutzia strains as promising candidates for microbiome-based therapies targeting metabolic diseases. Biological sciences/Biotechnology Biological sciences/Microbiology Adlercreutzia species inflammation metabolic diseases live biotherapeutic product gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The human gut microbiota plays a central role in host physiology, contributing to metabolic homeostasis, immune regulation, and protection against disease 1,2 . Disruptions to this complex ecosystem have been implicated in a wide range of chronic disorders, including metabolic, inflammatory, hepatic, and neurodegenerative diseases. As a result, increasing attention has been directed toward the identification of specific commensal bacteria that contribute to host health. Within this context, Adlercreutzia has emerged as a genus of particular interest. Adlercreutzia , a member of the family Eggerthellaceae within the phylum Actinomycetota (formerly Actinobacteria ), was first described following the isolation of A. equolifaciens from a healthy human donor in Japan and was named in honor of Professor H. Adlercreutz 3 . A. equolifaciens DSM 19450, the type strain of the genus, is best known for its ability to convert the soy isoflavone daidzein into equol, a metabolite with estrogenic and anti-inflammatory properties 4 . Since then, additional species closely related to A. equolifaciens , including A. hattorii 5 and A. rubneri 6 , have been described from healthy human donors. Notably, type strains of these species lack equol-producing capacity, highlighting metabolic and functional diversity within the genus. Furthermore, the type strain of A. rubneri has been shown to transform the stilbene resveratrol into dihydroresveratrol, a reaction with potential relevance for both host physiology and bio-based chemical production. Advances in anaerobic culturing techniques and high-throughput sequencing have substantially expanded our understanding of previously underexplored gut commensals, enabling detailed investigations of their prevalence, functional potential, and associations with health and disease 7 . Within this framework, metagenomic studies have identified A. equolifaciens as a prevalent health-associated taxon, contributing positively to composite metrics such as the Gut Microbiome Wellness Index 8 (GMWI) and the Health-Associated Core Keystone (HACK) index 9 . Notably, an in vivo study using a murine model demonstrated that increased abundance of A. equolifaciens enhances GMWI, in part by promoting the production of palmitoyl serinol 10 . These findings suggest a broader role for Adlercreutzia species in maintaining host health, extending beyond the production of polyphenol-derived metabolites. Consistent with these observations, we recently reported that both the prevalence and abundance of A. equolifaciens are markedly reduced in patients with metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD), with progressively lower levels observed as disease severity increases 11 . Importantly, we also demonstrated that A. equolifaciens DSM 19450 exerts anti-inflammatory effects in vitro and in vivo , supporting a potential protective role of this bacterium in metabolic liver disease. However, it remains unclear whether these beneficial effects are strain-specific and restricted to equol-producers or represent a conserved functional trait across the genus. Therapeutic management of MASLD remains a significant clinical challenge. While lifestyle interventions such as caloric restriction and regular physical activity are the cornerstone of treatment for achieving weight loss, their long-term efficacy is often limited by low patient adherence. Consequently, pharmacological strategies have emerged to target MASLD-associated risk factors, aiming to mitigate liver inflammation by modulating glucose and lipid metabolism 12 . Current therapeutic options include antidiabetic agents, such as glucagon-like peptide-1 receptor agonists (GLP-1 RA), sodium-glucose cotransporter 2 inhibitors (SGLT2i), biguanides, and thiazolidinediones, as well as lipid-lowering drugs like statins and peroxisome proliferator-activated receptor (PPAR) agonists 13 . The absence of a universally accepted standard and the limited efficacy of drugs for MASLD have prompted researchers to explore alternative treatment options. One such option that has gained more attention is the use of commensal bacteria as therapeutic agent 14 . This is due, in large part, to the growing body of evidence demonstrating the symbiotic relationship between the bacteria and their hosts, which make them a safe choice for therapeutic use 15 . Furthermore, their ability to colonize the intestinal niche offers the potential for sustained, long-term health benefits 16 through a more personalized and physiologically integrated approach 17 . However, transitioning from a commensal isolate to a regulated therapeutic product is complex. Candidates must satisfy rigorous criteria established by regulatory authorities, such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), which require comprehensive documentation of a positive benefit-risk balance through evidence of quality, safety, and efficacy 18 . A critical component of this process is the precise identification and characterization of strains using robust preclinical assays to elucidate their functional properties and mechanisms of action 19 , 20 . Moreover, to ensure successful delivery and therapeutic activity, candidates must demonstrate high viability and resilience against the various physiological stressors encountered during gastrointestinal transit, such as acidic pH and bile salt exposure 21 . In this study, we aimed at isolating and comprehensively characterizing Adlercreutzia strains from healthy European volunteers, with the goal of identifying candidates for potential therapeutic applications. In accordance with regulatory guidelines, the isolates were subjected to an integrated analysis combining genomic, phenotypic, and functional approaches. These included in silico taxonomic annotation, assessment of physiological and biochemical traits, tolerance to intestinal stressors, antibiotic resistance profiling, and in vitro evaluation of anti-inflammatory activity. By comparing the newly isolated strains to the reference strain A. equolifaciens DSM 19450, we assessed whether anti-inflammatory properties are a conserved feature within the genus. Our findings provide novel insights into the functional diversity of Adlercreutzia and support the potential use of these isolates as next-generation microbiome-based therapeutics. Materials and Methods Bacterial Isolation A. equolifaciens DSM 19450 was used as reference strain. Both the reference strain and the isolates were cultured using Peptone Yeast Glucose broth (PYG, also referred as M104) supplemented with 0.5% of arginine (Sigma-Aldrich, St Louis, MO, USA) and 1.5% agar (Invitrogen, Carlsbad, CA, USA) for the solid form. All media and reagents were reduced for a minimum of 48 h in an anaerobic chamber before use, and cultures were incubated at 37°C in anaerobic conditions. Strain isolation was performed by Bioaster (Paris, France), following the protocol published by Bellais and colleagues 22 . Briefly, faecal samples were collected from healthy volunteers and shipped within 2 hours at ambient temperature. In anaerobic conditions, 1 g of faecal sample was diluted in PBS and homogenized with 2.4mm glass beads. Bacteria were then selected and enriched with live/dead staining (BacLight™ Bacterial Viability Kit, Thermo Fisher Scientific, Waltham, MA, USA), and rabbit polyclonal antibodies (protein A purified from IgGs) coupled with Alexa Fluor™ 647 or Alexa Fluor™ 405 dyes (Invitrogen, Carlsbad, CA, USA) using flow cytometry coupled with cell sorting. These antibodies were obtained after rabbit immunization with A. equolifaciens DSM 19450 as previously described 22 . The bacteria were sorted and plated on M104 agar plates, then incubated at 37°C for 5 days. Colonies were visually selected based on their morphology (tiny and transparent). Culture Growth The isolates and A. equolifaciens DSM 19450 were cultured using the Hungate culture method 23 in pre-reduced (100% CO 2 ) M104-based food grade medium supplemented with 0.5% arginine (AppliChem, Darmstadt, Germany) at 37 °C. Genome sequencing and bioinformatics Genomic DNA was extracted using the NucleoSpin® Microbial DNA kit (Macherey-Nagel, Hoerdt, France) following the manufacturer’s instructions. Genome sequencing was performed on DNBSEQ-G400 (MGITech, China) instruments as previously described 24 , generating at least 670,000 paired-end reads (2 × 150 bp) per sample, ensuring a minimum sequencing coverage of 70×. Sequencing data quality control was conducted using fastp 25 to remove adapters, trim low-quality reads, and discard reads shorter than 80 bp or those containing undetermined bases. Genome assembly was performed with SPAdes 26 (parameters: --isolate --cov-cutoff auto). Other genomes derived from isolates assigned to the Adlercreutzia genus were downloaded from GenBank, except for A. equolifaciens UC1_BHI_P, whose raw sequencing data was obtained from the European Nucleotide Archive (sample accession SRS254664) and assembled as described above. The quality of both newly assembled and publicly available genomes was assessed using CheckM 27 and taxonomic annotation was performed with GTDB-Tk 28 based on GTDB R220. Public genomes not assigned to A. equolifaciens or A. rubneri were filtered out. Additionally, we removed short contigs (<1000 bp), as we found that this substantially reduces contamination. Average Nucleotide Identity (ANI) between all genome pairs was computed with PyOrthoANI 29,30 A distance matrix was then constructed, defining distance as 1 - ANI. Finally, genomes were clustered via average linkage hierarchical clustering (UPGMA). A core genome alignment of all genomes was generated using Parsnp 2 31 , with the genome assembly GCF_000478885.1 ( A. equolifaciens DSM 19450) as a reference. The multiple sequence alignment was then provided to RAxML-NG 32 to construct a phylogenetic tree with the GTR+CAT model. Branch support was assessed using 100 bootstrap replicates, and the final tree was visualized in iTOL 33 . Gene prediction in the assembled genomes was performed with RAST-tk 34 from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), as Prodigal 35 missed some genes, including those in the operon involved in equol production. Protein sequences constituting the equol biosynthesis pathway were retrieved from NCBI, including daidzein reductase ( dzr ; WP_022741749.1), dihydrodaidzein reductase ( ddr ; WP_022741751.1), tetrahydrodaidzein reductase ( tdr ; WP_022741752.1), and a racemase (WP_022741755.1). These proteins were searched against all genomes using tblastn 36 , with thresholds of ≥ 95% amino acid identity and ≥ 90% query coverage. A strain was considered to harbor a complete equol biosynthesis pathway only when all four pathway genes were detected in its genome. Antimicrobial resistance genes were searched in genome sequences using AMRFinderPlus 37 . Microbial Characterization Oxidase activity was determined by using oxidase test strips (Sigma-Aldrich, St Louis, MO, USA) following the manufacturer’s instructions. A drop of an overnight bacterial culture was deposited on the strip, and the resulting colour change was observed. Catalase activity was measured using 3% and 12% hydrogen peroxide (Sigma-Aldrich, St Louis, MO, USA). A colony was deposited in a drop of hydrogen peroxide and the presence or absence of reaction was observed. Deoxyribonuclease (DNase) activity was determined by using a DNase agar plate supplemented with toluidine blue (Sigma-Aldrich, St Louis, MO, USA). An overnight bacterial culture was spotted on the DNase agar plate and incubated at 37°C for 24 h. The colour change was observed after incubation. Gram staining was performed by using the Gram coloration kit (Condalab, Madrid, Spain) following manufacturer’s instructions. The staining was observed under a light microscope (Olympus BX43, Rungis, France) with oil immersion at a magnification of 100. Spore formation assessment was performed using the Schaeffer-Fulton method 38 which utilises malachite green (Pro-lab Diagnostics, Richmond Hill, Canada). The staining was observed under a light microscope (Olympus BX43, Rungis, France) with oil immersion at a magnification of 100. The biochemical characterization was carried out using the API 20A anaerobic test kit (Biomérieux, Marcy-l’Etoile, France) following manufacturer’s instructions. The pellet of an overnight bacterial culture was resuspended in API20 medium and the strip was inoculated with 100 µL of the suspension. The strip was then incubated during 48 h at 37°C in anaerobic conditions (GenBag anaer, Biomérieux, Marcy-l’Etoile, France). After incubation, the strip was read under normal atmospheric conditions and interpreted by referring to the reading table provided by the manufacturer. Antibiotic resistance was determined by using the E-test® (Biomérieux, Marcy-l’Etoile, France and Liofilchem, Rosetto Degli Abruzzi, Italy for piperacillin-tazobactam) according to the manufacturer’s instructions. Briefly, a Petri dish containing pre-reduced (H 2 5%/CO 2 5%/N 2 90%) M104-based food-grade medium was inoculated with 100 µL of an overnight bacterial culture. The strip was positioned and the agar plate was inoculated under anaerobic conditions (H 2 5%/CO 2 5%/N 2 90%) for 48 h at 37°C. After incubation, the minimum inhibitory concentration (MIC, µg/mL) was determined by direct reading of the graduate scale found on the strip. The MIC was then interpreted as sensitive (S), intermediate (I) or resistant (R) based on the standards set by the Clinical and Laboratory Standards Institute (CLSI) 39 . The antibiotics tested were metronidazole (MZL), chloramphenicol (CL), vancomycin (VA), clindamycin (CM), imipenem (IP), piperacillin-tazobactam (P/T), and tetracycline (TC). Bile acid tolerance was assessed using M104-based food-grade medium with 1.5% agar (Invitrogen, Carlsbad, CA, USA). The agar medium was supplemented with Ox-bile (Sigma-Aldrich, St Louis, MO, USA) at concentrations of 0%, 0.25%, 2.5%, 5%, 7.5% and 10% w/v corresponding to 0%, 2%, 20%, 40%, 60% and 80% of fresh bile, respectively. The plates were reduced in anaerobic atmosphere (H 2 5%/CO 2 5%/N 2 90%) 48 h before their use. Overnight liquid cultures of the isolates were diluted from 10 -1 to 10 -10 . Next, 10 µL of each dilution were spotted on the pre-reduced agar plates containing varying percentages of bile. The agar plates were then incubated under anaerobic conditions (H 2 5%/CO 2 5%/N 2 90%) at 37°C for 72 h to 96 h. The growth was assessed by enumerating the colonies per dilution, and the bacterial concentration was calculated and expressed in CFU/mL. Finally, the results were compared to the control group, which was M104-based medium with 0% of bile. Oxygen tolerance was assessed using liquid M104-based food-grade medium and supplemented with 1.5% agar (Invitrogen, Carlsbad, CA, USA). Liquid medium was prepared following the Hungate method previously described and the agar plates were reduced in anaerobic atmosphere (H 2 5%/CO 2 5%/N 2 90%) for 48 h before their use. For the evaluation in liquid medium, overnight liquid cultures of the isolates were exposed to oxygen from ambient air for 0, 2, 4, 6 and 24 h at room temperature. After oxygen exposition, the bacterial cultures were subcultured in new pre-reduced liquid medium and incubated at 37°C for 24 h. The growth was revealed by reading the optical density (OD) at 600 nm. For the assessment on solid medium, overnight liquid cultures of the isolates were diluted from 10 -1 to 10 -10 and 10 µL of each dilution were spotted on the pre-reduced agar plates. Each plate was then exposed to oxygen for 0, 2, 4, 6 and 24 h at 37°C. After the exposition time had elapsed the agar plates were replaced under anaerobic conditions (H 2 5%/CO 2 5%/N 2 90%) and incubated at 37°C for a total of 72 h to 96 h. Colony enumeration was used to assess the growth, and the bacterial concentration was calculated and expressed in CFU/mL. Finally, the results were compared to the control group, which was M104-based medium with no oxygen exposure. The strain tolerance to basic, acid and neutral pH was evaluated using liquid M104-based food-grade medium buffered at pH 4, 7 and 12 for 24 h at 37°C. The medium was prepared following the previously described Hungate method. Overnight liquid cultures of the isolates were subcultured in the pH buffered media and incubated at 37°C for 24 h. The growth was assessed by reading the OD at 600 nm. In vitro assays Cell Culture and Reagents HT-29 human intestinal epithelial cells and HEK293 human renal epithelial cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco, Les Ulis, France) supplemented with 2 mM L-glutamine (Gibco, Les Ulis, France), 50 IU/mL penicillin (Gibco), 50 μg/mL streptomycin (Gibco), and 10% heat-inactivated fetal calf serum (Eurobio, Les Ulis, France). HepG2 human hepatocarcinoma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) and supplemented as RPMI medium with the addition of 1% nonessential amino acid (NEAA) 100X (Gibco, Les Ulis, France), 1% hepes 1M (Gibco, Les Ulis, France), and 1% sodium pyruvate 100 mM (Gibco, Les Ulis, France). The cells were cultured in a humidified 5% CO 2 atmosphere at 37°C. The absence of mycoplasma contamination was confirmed using a MycoAlert kit (Lonza, Levallois-Perret, France). The HT-29 NF-κB and HepG2 NF-κB reporter cell lines were constructed and validated as previously described by transfecting pNiFty2 plasmid (Invivogen, Toulouse, France) 11 , 40 , 41 . This reporter plasmid contains the Secreted Embryonic Alkaline Phosphatase (SEAP) gene under the control of NF-κB binding elements. HEK Blue™ reporter cell lines (HEK Blue™ Null1 and HEK Blue™ TLR2) were obtained from Invivogen, Toulouse, France. HEK Blue™ Null1 cells express the SEAP reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. Therefore, the activation of NF-κB leads to the expression of SEAP. HEK Blue™ TLR2 cells were generated from HEK Blue™ Null1 cells by transfection of human TLR2. Thus, stimulation via TLR2 ligands activates NF-κB and AP-1, inducing the production of SEAP. Commensal Strains and Preparation of Conditioned Media The isolates and A. equolifaciens DSM 19450 were cultured using the Hungate method in pre-reduced (100% CO 2 ) M104-based food-grade medium at 37 °C. After overnight incubation, the bacterial cultures were centrifuged at 11,000 rpm (or 13,663 x g ) for 10 min. The pellets and supernatants were collected separately, and the supernatant was filtered using 0.2 μm PES filters (Corning, NY, USA) before storage at −80°C. For the experiment, the cell pellets were resuspended in PBS 1X (Gibco). The non-inoculated bacterial culture medium was used as the control for the analysis of the supernatant effect, while PBS was used for the pellet effect analysis. Analyses of NF-κB Activation For each experiment, reporter cell lines were seeded at a density of 30,000 cells per well in 96-well plates and incubated for 24 h. Subsequently, cells were stimulated for 24 h with 10 μL of tested bacterial supernatant or resuspended bacterial pellet, for a final volume of 100 μL per well (i.e., 10% vol/vol), in the presence or absence of TNF-α (PeproTech, Cranbury, NJ, USA) to mimic inflammatory and non-inflammatory conditions, respectively. In case of inflammatory conditions, the cells were stimulated with 10 ng/mL of TNF-α (PeproTech), 1 h before sample deposit. The Quanti-Blue™ reagent (Invivogen, Toulouse, France) was used to reveal SEAP in the supernatant of reporter cell lines (HT-29 NF-κB, HepG2 NF-κB, HEK Blue™ Null1 and HEK Blue™ TLR2) and quantified at 655 nm OD. using a microplate reader (Infinite 200, Tecan, Lyon, France). All the measurements were performed according to the manufacturer’s protocol. After 24 h of incubation, the cell viability was controlled using a MTS assay (CellTiter 96 Aqueous One, Promega, Charbonnières-les-Bains, France) according to the manufacturer’s instructions. Statistical analysis Results were expressed as the mean ± the Standard Error of the Mean (SEM) from three independent experiments, each performed in triplicate. Statistical analysis was performed using GraphPad Prism version 8 (La Jolla, CA, USA). Differences between groups were assessed using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. P-values < 0.05 were considered statistically significant. Results Comparative genomics of Adlercreutzia reveals distinct phylogroups and atypical species boundaries Eleven new strains of bacteria from the Adlercreutzia genus were isolated from faecal samples of healthy volunteers using antibody driven- flow cytometry under anaerobic conditions. After short-read sequencing and de novo genome assembly, high-quality draft genomes were obtained (Supplementary Table 1), exhibiting high completeness (100% for all genomes), low contamination (≤0.81%), and high contiguity (N50 > 100 Kbp). Genome sizes ranged from 2.61 to 2.85 Mbp (mean: 2.75 ± 0.07 Mbp), with an average GC content of 63.2%, consistent with genome assemblies of the type strains A. rubneri ResAG-91 (2.78 Mbp, GC content 63.3%) and A. equolifaciens DSM 19450 (2.86 Mbp, GC content 63.5%). The number of coding sequences (CDS) ranged from 2,279 to 2,537 (mean: 2,420 ± 68), again consistent with values reported for A. rubneri ResAG-91 (2,438 CDS) and A. equolifaciens DSM 19450 (2,365 CDS). The pairwise average nucleotide identity (ANI) between the 11 newly assembled genomes and 13 publicly available genomes of A. rubneri , A. hattorii , and A. equolifaciens was computed. Analysis of the ANI distribution revealed that the conventional 95% species boundary 42 was not clearly defined for these genomes (Figure S1). Unlike the distinct gaps often seen at 95% in other bacterial species, high density was observed spanning this threshold. To better align with the observed local minima in the density distribution and provide a more biologically relevant separation of the genomic clusters, a 96% ANI cutoff was adopted. Hierarchical clustering with a 96% ANI cutoff revealed three distinct genome groups (Figure 1). The first group (labeled “ A. equolifaciens ”) comprised five genomes, including the type strains A. equolifaciens DSM 19450 and A. equolifaciens subsp. celata DSM 18795, but no newly assembled genomes from this study. All genomes in this group were taxonomically annotated as A. equolifaciens according to the ANI-based assignment method implemented in GTDB-Tk. The second group (“ A. rubneri / A. hattorii ”) included twelve genomes, comprising the type strains A. hattorii 8CFCBH1 and A. rubneri ResAG-91, as well as most of the newly assembled genomes (9/11). All genomes in this group were assigned to A. rubneri by GTDB-Tk. The third group (labeled “Other”) consisted of seven genomes, including the remaining two newly assembled genomes. Although all genomes in this group were assigned to A. rubneri by GTDB-Tk, those available in GenBank were submitted as A. equolifaciens . Functional genome annotation further revealed that none of the newly isolated strains harbor genes involved in the daidzein-to-equol biosynthesis pathway (Figure 1). This pathway was complete in only 3 of the 24 analyzed genomes, all of which belonged to the A. equolifaciens group. In addition, six of the newly isolated strains carried a tetracycline resistance gene ( tetW or tetO ). A phylogenetic tree was then inferred from core-genome alignments of the 24 genomes (total alignment length: 1.08 Mbp, including 86,601 SNPs; Figure 2). The phylogenetic relationships were generally well supported by high bootstrap values, with most internal nodes showing strong support (≥90%), indicating a robust core-genome signal across the dataset. Three main phylogroups were recovered, broadly corresponding to the clusters identified by ANI analysis. However, the third (“Other”) group appeared more closely related to the A. equolifaciens clade, in contrast to the GTDB-Tk assignment and ANI-based analysis, which placed it closer to the A. rubneri group. In addition, strains CNCM I-6215 and CNCM I-6216 clustered closer to the A. rubneri clade but occupied relatively long and distinct branches, suggesting a more divergent evolutionary history. Phenotypic characterization: growth, antibiotic susceptibility, and stress tolerance of new Adlercreutzia isolates Physiological Properties The new isolates were obligate anaerobes, non-motile, and appeared as single or chained coccobacilli, with no evidence of spore formation. Their growth was enhanced by supplementation of arginine in the medium. Colonies cultivated on M104-based agar were small, transparent, circular, slightly convex, and smooth. Biochemical testing revealed that the isolates were negative for catalase, oxidase, and DNase activities (Table 1) Biochemical Properties The biochemical characteristics of the isolates were evaluated using API20A galleries. All isolates tested negative for the enzymes included in the panel (Table 2) and were classified as asaccharolytic, as they did not metabolize any of the sugars provided as carbon sources. These findings are consistent with previous reports on A. equolifaciens , A. rubneri and A. hattorii type strains 3,5,6 . Antibiotic Resistance The sensitivity of the isolates to a panel of antibiotics was evaluated (Table 3). Resistance to tetracycline was observed in strains CNCM I-6209, CNCM I-6210, CNCM I-6212, and CNCM I-6214, whereas strains CNCM I-6207 and CNCM I-6217 exhibited mild resistance. These phenotypic results were largely consistent with the genomic analysis, except for strain CNCM I-6211, which was sensitive to tetracycline despite the presence of a resistance gene in its genome. Conversely, strain CNCM I-6214 displayed tetracycline resistance, although no known resistance gene was detected in its genome. In addition, resistance to clindamycin was observed in strains CNCM I-6207 and CNCM I-6214, although no corresponding gene was identified in the respective genomes, suggesting the presence of uncharacterized resistance mechanisms. Stress Tolerance The tolerance of the Adlercreutzia isolates to multiple environmental stressors, including bile salts, pH, and oxygen, was evaluated. pH tolerance was assessed using M104-based liquid medium adjusted to acidic (pH 4), neutral (pH 7), and basic (pH 12) conditions. No growth was observed under acidic or basic conditions (data not shown). Optimal growth was observed at neutral pH (pH 7). Bile salt tolerance was evaluated on M104-based agar medium supplemented with increasing concentrations of bile (0%, 2%, 20%, 40%, 60%, and 80%). Two distinct growth profiles were identified (Figure 3). The first profile exhibited a conventional dose-dependent response, characterized by a progressive decrease in bacterial growth with increasing bile concentrations. Within this group, isolates CNCM I-6213 and CNCM I-6216 displayed greater tolerance to high bile concentrations compared with isolates CNCM I-6207, CNCM I-6209, CNCM I-6211, and CNCM I-6215, which showed marked growth reduction at bile concentrations as low as 2%. The second profile showed an atypical growth pattern and included isolates CNCM I-6208, CNCM I-6210, CNCM I-6212, CNCM I-6214, CNCM I-6217, as well as A. equolifaciens DSM 19450. In this group, growth decreased at 2% bile (and up to 20% for some isolates) but partially recovered at higher bile concentrations (20–60%). This profile was also associated with distinct colony morphologies on agar plates. Growth inhibition was accompanied by very small, translucent colonies, whereas growth recovery at higher bile concentrations resulted in larger, creamy colonies (data not shown). These observations may suggest bile-induced phenotypic adaptation, potentially involving biofilm formation. In both profiles, no bacterial growth was observed at 80% bile, with the exception of strain CNCM I-6213, which exhibited marked bile salt resistance. Oxygen tolerance was assessed by incubating agar plates under aerobic conditions for increasing durations (0, 2, 4, 6, and 24 h) on M104-based medium. Oxygen exposure negatively affected bacterial growth (Figure 4); however, viable counts remained above 1 × 10⁸ CFU/mL up to 6 h for strains CNCM I-6209, CNCM I-6210, CNCM I-6211, CNCM I-6212, and CNCM I-6213, up to 4 h for strains CNCM I-6207, CNCM I-6208, CNCM I-6214, and CNCM I-6216, and up to 2 h for strain CNCM I-6217. In contrast, the reference strain A. equolifaciens DSM 19450 was the most sensitive, with a marked decrease in viable counts observed before 2 h of oxygen exposure (Figure 4). After 24 h of aerobic exposure, no bacterial growth was detected. However, when these oxygen-exposed liquid cultures were subcultured under anaerobic conditions, bacterial growth resumed after 96 h of incubation. In vitro evaluation of anti-inflammatory effects of Adlercreutzia isolates The effects of the isolates on NF-κB and TLR2 inflammatory pathways were evaluated using stably transfected reporter cell lines (Figure 5 and Supplementary Figures S2-S5). The impact of the eleven isolates on NF-κB activation was assessed in intestinal epithelial cells (HT-29 NF-κB reporter) and hepatic cells (HepG2 NF-κB reporter). Furthermore, HEK Null1 and HEK TLR2 cell lines were used to determine the implication of TLR2 in the modulatory effects observed on the NF-κB signalling pathway. The results were then compared to those obtained with A. equolifaciens DSM 19450, for which anti-inflammatory properties had previously been demonstrated. Both bacterial pellets and culture supernatants were tested under non-inflammatory and inflammatory conditions. In non-inflammatory conditions, neither pellets nor supernatants induced NF-κB activation in HT-29 NF-κB or HEK Null1 cells. Similarly, no NF-κB activation was observed in HepG2 cells for most isolates, with the exception of the pellet of strain CNCM I-6029, which induced a limited response. Overall, these results indicate that the isolates exert little to no basal activation of the NF-κB pathway. In contrast, several isolates significantly activated the TLR2 pathway. The supernatants of five isolates (CNCM I-6207, CNCM I-6028, CNCM I-6209, CNCM I-6210, and CNCM I-6216), as well as A. equolifaciens DSM 19450, and the pellets of seven isolates (CNCM I-6207, CNCM I-6208, CNCM I-6209, CNCM I-6210, CNCM I-6213, CNCM I-6215, and CNCM I-6216) induced a strong activation of TLR2 in HEK TLR2 cells, with fold changes exceeding 3 compared to the M104 or PBS controls. Under inflammatory conditions, following stimulation with TNF-α, most bacterial pellets significantly reduced NF-κB activation in HT-29 cells, with decreases exceeding 40% in the most pronounced cases compared to the PBS control. Notably, the inhibitory effect of strain CNCM I-6216 was comparable to that observed for A. equolifaciens DSM 19450. Both the pellet and supernatant of CNCM I-6216 reduced NF-κB activation by more than 30% relative to the PBS or M104 controls (Figure S2). In HepG2 cells, the pellets of eight isolates (CNCM I-6208, CNCM I-6209, CNCM I-6210, CNCM I-6211, CNCM I-6213, CNCM I-6214, CNCM I-6215, and CNCM I-6216), as well as A. equolifaciens DSM 19450, strongly inhibited NF-κB activation, with reductions exceeding 70% in the most significant cases compared to the PBS control. Furthermore, the supernatants of most isolates (CNCM I-6212, CNCM I-6213, CNCM I-6214, CNCM I-6215, CNCM I-6216, and CNCM I-6217) and of A. equolifaciens DSM 19450 reduced NF-κB activation by approximately 40% compared to the M104 control. For isolates CNCM I-6213, CNCM I-6214, CNCM I-6215, and CNCM I-6216, both pellets and supernatants elicited inhibitory effects comparable to those of the reference strain (Figure S3). Consistent with these observations, several pellets and supernatants also significantly inhibited NF-κB and TLR2/NF-κB signaling in HEK embryonic kidney cells. The most pronounced reductions reached approximately 40% for bacterial pellets and 30% for supernatants relative to PBS or M104 controls. Again, isolates CNCM I-6213, CNCM I-6214, CNCM I-6215, and CNCM I-6216 exhibited effects comparable to those observed with A. equolifaciens DSM 19450 (Figures S4 and S5). Discussion In the present study, we described eleven new isolates of the genus Adlercreutzia from healthy European volunteers, nine of which were consistently assigned to A. rubneri based on both ANI-based and phylogenetic analyses. However, taxonomic annotation proved more challenging for the remaining strains. This complexity is largely driven by the atypical distribution of genomic identity within the genus. Whereas many bacterial groups exhibit a distinct gap at 95% ANI, the Adlercreutzia genomes analyzed here showed high density spanning this threshold. In particular, strain CNCM I-6207 showed inconsistent placement depending on the method used, clustering with A. rubneri according to ANI and GTDB-Tk, while appearing more closely related to A. equolifaciens in the core-genome phylogeny. Such discordances likely reflect methodological limitations, including the effects of recombination and the influence of accessory genome content on phylogenetic inference. Similarly, strains CNCM I-6215 and CNCM I-6216, although positioned within the broader A. rubneri lineage, formed long and well-supported branches that were clearly distinct from established A. rubneri reference genomes. Their divergent placement, together with the absence of clear genomic thresholds supporting a confident species-level assignment, suggests that these strains represent divergent lineages and potentially undescribed taxa. In the absence of additional discriminating phenotypic, genomic, or ecological markers, we therefore chose to adopt a conservative taxonomic approach. Accordingly, CNCM I-6207, CNCM I-6215, and CNCM I-6216 were designated as Adlercreutzia sp. strains, and the corresponding genomes were submitted to GenBank under this designation, pending further taxonomic resolution. Our comparative genomic analyses also revealed a high degree of relatedness between the type strains of A. rubneri and A. hattorii . The pairwise ANI value between these genomes reached 96.72%, exceeding the commonly accepted 95-96% threshold for bacterial species delineation. This observation was further supported by full-length 16S rRNA gene sequence alignments, which showed a nucleotide identity of 98.4%. Notably, A. rubneri and A. hattorii were described only a few months apart, in September 2021 and December 2021, respectively. According to nomenclatural conventions, when two names are proposed for the same species, the earliest published name takes precedence. On this basis, and supported by robust genomic evidence, we propose that A. hattorii should be considered a later heterotypic synonym of A. rubneri . The physiological and biochemical characteristics of the newly isolated strains were consistent with those reported for type strains of A. equolifaciens , A. hattorii , and A. rubneri . The isolates were asaccharolytic, as they did not utilize any of the sugars tested in the API20A panel. Consistent with previous observations for A. equolifaciens DSM 19450, growth was enhanced by supplementation of the culture medium with L-arginine. L-arginine is a key intestinal metabolite that serves as a substrate for both the gut microbial community and host intestinal cells, and its disrupted metabolism has been linked to inflammatory diseases and microbiota depletion. These findings suggest that L-arginine supplementation could be considered in formulations intended to support these bacteria 43 . The development of microbiome-based therapies for disease treatment is a complex and challenging process. Regulatory agencies, such as the FDA and EMA, require comprehensive documentation of quality, safety, and efficacy before approval. Consequently, the development of Live Biotherapeutic Products (LBPs) and Next Generation Probiotics (NGPs) must be thoroughly documented to meet these regulatory standards. A critical component of safety assessment is strain characterization, which includes evaluating antibiotic resistance through both genotypic and phenotypic approaches 18 . Antimicrobial resistance represents a significant public health concern, as gut microbiota can carry resistance genes that may be horizontally transferred to other bacteria via mobile genetic elements, such as plasmids. To address this, we determined the minimum inhibitory concentration (MIC) for a selected panel of antibiotics, chosen based on the characteristics of the strains and the target population 44 , 45 . In parallel, genomic analyses of our isolates identified tetracycline resistance genes (tetW or tetO), most of which were functionally validated through in vitro assays. Importantly, no mobile genetic elements were detected in the genomes of our isolates, suggesting a low risk of horizontal gene transfer. Orally administered microbial agents face numerous challenges in achieving clinical efficacy as they pass through the gastrointestinal tract. Their viability and cellular integrity can be hampered by the highly acidic conditions of the stomach and subsequent exposure to bile salts in the small intestine 46 . Furthermore, as most gut commensals, they are often highly sensitive or intolerant to oxygen 47 . To address these challenges, we evaluated the tolerance of our isolates to oxygen, bile salts, and pH, with the goal of optimizing production and administration methods to ensure that the bacteria reach their target site in the gut and exert their beneficial effects. Under our experimental conditions, the isolates exhibited relative tolerance to oxygen, which may facilitate production and encapsulation strategies. However, they were unable to grow under either acidic or basic conditions, indicating limited survival in the stomach. Encapsulation thus represents a promising delivery method, with the choice of encapsulation materials being critical for protecting the therapeutic agent. Extensive research will be required to identify processes that ensure host safety while allowing reliable and scalable production 48 , 49 , 50 . Regarding bile salt tolerance, some isolates were sensitive even at low concentrations (2%), highlighting the need for specific delivery strategies, as discussed above for pH and oxygen. In contrast, other isolates were able to grow at higher bile concentrations, accompanied by morphological changes suggestive of bile-induced biofilm formation. Biofilm formation is a known bacterial strategy to survive the bactericidal effects of bile during gastrointestinal transit 51 , 52 , 53 , 54 , although it has been primarily observed in pathogenic species. Further investigation using electron microscopy could clarify the morphological changes observed, as bile salts are known to directly affect the bacterial cell surface 55 . The bile resistance observed in several isolates may also be attributed to bile salt hydrolase (BSH) activity. BSH enzymes, which are widespread in the human gut microbiota, contribute to bacterial survival and persistence in the gastrointestinal tract by deconjugating bile salts 56 . Beyond microbial survival, BSH enzymes play a critical role in host metabolism as bile salt deconjugation has been linked to cholesterol reduction and decreased weight gain 57 . Bile acids, synthesized in the liver, are released into the duodenum and reabsorbed in the colon through enterohepatic circulation, influencing metabolic pathways via nuclear receptors such as the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 58 . Activation of these receptors by bile acids may modulates lipid, glucose, and cholesterol metabolism 59 , making them promising targets for drug development in metabolic diseases, including MASLD. Investigating whether our isolates possess BSH activity and the capacity to influence these pathways is therefore of significant interest. In vitro assays demonstrated that both the pellet and supernatant of our isolates exert strain-dependent inhibitory effects on the NF-κB pathway in intestinal epithelial cells and hepatocytes, indicating potential anti-inflammatory properties. These results are consistent with our previous findings using the type strain A. equolifaciens DSM 19450 11 . Notably, none of the isolates carry genes involved in equol production from daidzein, indicating that this function is not required for these anti-inflammatory effects. However, the strains may metabolize other polyphenols, such as resveratrol, as previously reported for non-equol-producing strains, including A. rubneri ResAG -91 6 . This raises the possibility that the anti-inflammatory activity of these isolates could be enhanced through polyphenol supplementation. To further investigate the mechanism of action on the NF-κB pathway, we examined the implication of Toll-Like-Receptor-2 (TLR2) using HEK293 reporter cell lines. TLR2 is a host receptor expressed on epithelial cell membrane and activated by cell-wall components of Gram positive bacteria. TLR2 interacts with the myeloid differentiation factor 88 (MyD88), an adaptator protein which leads to the activation of NF-κB and to the induction of pro-inflammatory cytokines 60 , 61 . In this study, we used the control cell line HEK Null1, which do not express TLR2, to determine whether the observed inhibition of the NF-κB pathway was dependent of TLR2. We found that both supernatant and bacterial cell-wall components (pellet) had a mild effect on control HEK cells, whereas they strongly activated TLR2-dependent NF-κB pathway, although variability exist between strains under basal conditions. We did not find evidence that the anti-inflammatory properties were specific to the TLR2 pathway in inflammatory conditions as an inhibition of the inflammatory pathway was observed similarly in both reporter cell lines (HEK Null1 and HEK TLR2). Therefore, the mechanism of action by which our isolates exert their beneficial effects needs to be further clarified. Notably, it would be interesting to investigate whether the anti-inflammatory properties of the strains are linked to our observations with bile acids. Indeed, receptors such as FXR and TGR5 are known to inhibit NF-κB activity, thus lowering inflammation 62 . However, in the in vitro set-up no bile salts were added. If we hypothesize that our strains possess BSH activity, they may be able to deconjugate bile salts. This, in turn, could activate FXR or TGR5 receptors, leading to the repression of NF-κB activity and providing further anti-inflammatory properties. Conclusion Within the Actinomycetota phylum, Bifidobacteria are the most extensively studied in relation to human health, but interest in Coriobacteria , particularly the genus Adlercreutzia , is rapidly increasing. In this study, we isolated and characterized eleven new non-equol-producing Adlercreutzia strains from the stool of healthy French volunteers. The anti-inflammatory properties previously reported for A. equolifaciens DSM 19450 were also observed in these isolates. Thorough in vitro assays revealed specific functional and physiological traits, providing important insights into their mechanisms of action and informing their potential clinical and industrial applications. Further studies are needed to ensure the safety of these strains. Additionally, validating their beneficial effects in more complex models, such as gut organoids or gut-on-chip systems, will allow a more accurate assessment of their therapeutic potential and generate data required for regulatory approval. Declarations Data availability All sequencing data generated in this study have been submitted to the EMBL-EBI’s European Nucleotide Archive (ENA) and are available under the BioProject accession number PRJEB97395. All genome assemblies have also been submitted under the same BioProject and are accessible via NCBI GenBank. Ethics declaration Stool samples from which the strains were isolated were anonymously collected from healthy volunteers. In accordance with French regulations, this collection was declared to the French Ministry of Higher Education, Research and Innovation (no. DC-2015-2513) and received approval from the Ethical committee (Comité de Protection des Personnes CPP EST I) in March 2016. All participants received written information and provided informed consent prior to participation. Authors contributions C.C. and F.P.O. wrote the manuscript. C.C. designed the study, performed the experiments, and analyzed the data. M.B. performed microbial characterization and conducted the in vitro assays. A.L. contributed to figure preparation, data interpretation, and manuscript drafting and revision. B.Q. performed genome sequencing. E.M. and N.L. contributed to data interpretation and manuscript writing and editing. F.P.O. performed the bioinformatics genome analyses. P.Y.M. and H.M.B. supervised the project, secured funding, and critically revised the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was funded by Novobiome. 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Tables Strains characteristics Morphology Coccobacilli ; single or chains Growth conditions Anaerobic ; 37°C ; pH 7 Gram stain Positive Spore formation Negative Catalase Negative Oxidase Negative DNase Negative Table 1 : Physiological characteristics of the newly isolated strains and A. equolifaciens DSM 1945 Substrates Reactions Indole (IND) - Urea (URE) - Glucose (GLU) - Mannitol (MAN) - Lactose (LAC) - Saccharose (SAC) - Maltose (MAL) - Salicin (SAL) - Xylose (XYL) - Arabinose (ARA) - Gelatin (GEL) - Esculin (ESC) - Glycerol (GLY) - Cellobiose (CEL) - Mannose (MNE) - Melezitose (MLZ) - Raffinose (RAF) - Sorbitol (SOR) - Rhamnose (RHA) - Trehalose (TRE) - Table 2: Biochemical properties of the newly isolated strains and A. equolifaciens DSM 19450 Bacterial strains Antibiotics MIC (µg/mL) Metronidazole Chloramphenicol Vancomycin Clindamycin Imipenem Piperacillin-tazobactam Tetracycline Ae DSM 19450 0,032 1 0,25 0,25 0,094 0,75 0,125-0,38 CNCM I-6207 0,19 1 1,5 <256 0,047 1,5 6-8 CNCM I-6208 0,064 0,5 0,75 0,25 0,016 0,75 0,047-0,064 CNCM I-6209 0,5 2 1,5 0,016 0,047 0,75 <256 CNCM I-6210 0,125 3 1,5 0,016 0,064 0,5 48 CNCM I-6211 0,19 2 0,5 0,016 0,023 0,5 0,15-0,19 CNCM I-6212 0,125 1,5 1,5 0,023 0,094 16 <256 CNCM I-6213 0,25 1,5 1 0,016 0,032 6 0,064-0,094 CNCM I-6214 0,125 4 1 <256 0,032 4 <256 CNCM I-6215 0,19 2 1 0,016 0,047 4-6 0,38 CNCM I-6216 0,094 1 1 0,016 0,016 2 0,125-0,19 CNCM I-6217 0,19 0,75 1 0,016 0,047 2 8-12 Table 3 : Antibiotic resistance of the newly isolated strains and A. equolifaciens DSM 19450. MIC is expressed in µg/mL and the results were interpreted as sensitive (not highlighted), intermediate (yellow) and resistant (red) according to the CLSI standards for anaerobes Additional Declarations Competing interest reported. P.Y.M. is co-founder and CEO of Novobiome. H.M.B. is co-founder and scientific advisor of Novobiome. C.C., M.B., and A.L. are employees of Novobiome. F.P.O. and H.M.B. are co-inventors of the patent “USE OF ADLERCREUTZIA BACTERIA FOR THE TREATMENT OF INFLAMMATORY DISEASES” (no. 20230310519). Supplementary Files SupplementaryTable1vf.xlsx Chamignonetal2026Supplfigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8574898","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":602260458,"identity":"736b4e07-dc85-4f89-a71b-09a6bc35e27f","order_by":0,"name":"Célia Chamignon","email":"","orcid":"","institution":"NovoBiome","correspondingAuthor":false,"prefix":"","firstName":"Célia","middleName":"","lastName":"Chamignon","suffix":""},{"id":602260459,"identity":"81aefc22-b35d-4d91-bd5a-e46f9d6167aa","order_by":1,"name":"Maxime Bredel","email":"","orcid":"","institution":"NovoBiome","correspondingAuthor":false,"prefix":"","firstName":"Maxime","middleName":"","lastName":"Bredel","suffix":""},{"id":602260460,"identity":"946e1ef7-e6a6-4f4e-91c0-681de68ba51c","order_by":2,"name":"Amandine Lashermes","email":"","orcid":"","institution":"NovoBiome","correspondingAuthor":false,"prefix":"","firstName":"Amandine","middleName":"","lastName":"Lashermes","suffix":""},{"id":602260461,"identity":"47f7b8b6-e8e5-4756-9758-2be1fd3e20c5","order_by":3,"name":"Benoit Quinquis","email":"","orcid":"","institution":"Université Paris-Saclay, INRAE, MGP","correspondingAuthor":false,"prefix":"","firstName":"Benoit","middleName":"","lastName":"Quinquis","suffix":""},{"id":602260462,"identity":"b22e266c-b9d7-454a-99c1-7ed9d1150f6a","order_by":4,"name":"Elliot Mathieu","email":"","orcid":"","institution":"Université Paris-Saclay, INRAE, MGP","correspondingAuthor":false,"prefix":"","firstName":"Elliot","middleName":"","lastName":"Mathieu","suffix":""},{"id":602260463,"identity":"fc610115-2e4f-49fb-9511-f91d1f00d7b9","order_by":5,"name":"Nicolas Lapaque","email":"","orcid":"","institution":"Université Paris-Saclay, INRAE, MGP","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Lapaque","suffix":""},{"id":602260464,"identity":"dd406917-3ec4-4231-abe8-b990b47cab8d","order_by":6,"name":"Florian Plaza Oñate","email":"","orcid":"","institution":"Université Paris-Saclay, INRAE, MGP","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"Plaza","lastName":"Oñate","suffix":""},{"id":602260465,"identity":"37bc6c98-3b1b-46f7-8436-d8512ff10fba","order_by":7,"name":"Pierre-Yves Mousset","email":"","orcid":"","institution":"NovoBiome","correspondingAuthor":false,"prefix":"","firstName":"Pierre-Yves","middleName":"","lastName":"Mousset","suffix":""},{"id":602260466,"identity":"ba634833-2891-47d4-9383-f75acdf7032c","order_by":8,"name":"Hervé M. Blottière","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3PvarCMBTA8SOBuARcK5X2Fc4loBTEZ1GEOMldHZ06VV37MA6nCHbxAToIIkLnuhUseOPXGKvbHfKHJHDgBzkANts/DB8Pde63Ph5/TLxaIoCzO5FPIj8jt0bzOtJrLnKngL1o+auEzuv+ZOkujkUB+GsiQZR22zHkoh0ySOJcTcNOKvUEg7npY5liUsBG4JbpmzbT0FHgCqjQuMshZ7J6kYquE+4odqkAzSTjjRO8CBANNeEuvCM71ThGeNtljElE4x/9MR5E+IakW6BytvdaLDkVJQ18P1YsK2dm8oQfTGw2m832RX+jilD9ck7onQAAAABJRU5ErkJggg==","orcid":"","institution":"Nantes-Université, INRAE","correspondingAuthor":true,"prefix":"","firstName":"Hervé","middleName":"M.","lastName":"Blottière","suffix":""}],"badges":[],"createdAt":"2026-01-11 16:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8574898/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8574898/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104287995,"identity":"9b1d8445-e443-4673-8098-277ca4a3c6e7","added_by":"auto","created_at":"2026-03-10 05:58:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap of pairwise Average Nucleotide Identity (ANI) among Adlercreutzia genomes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePairwise ANI values were calculated between the 11 newly isolated \u003cem\u003eAdlercreutzia\u003c/em\u003e strains (CNCM I-62XX) and publicly available genomes retrieved from GenBank, including type strains (shown in \u003cstrong\u003ebold\u003c/strong\u003e). ANI values are indicated in each cell and displayed as a color gradient (blue to red). Hierarchical clustering was performed using \u003cem\u003e1 − ANI\u003c/em\u003eas the distance metric, and the resulting dendrograms are shown on both axes. Based on a 96% ANI cutoff, genomes were grouped into three clusters corresponding to \u003cem\u003eA. equolifaciens\u003c/em\u003e, \u003cem\u003eA. rubneri/hattorii\u003c/em\u003e, and an “Other” group.\u003c/p\u003e\n\u003cp\u003eThe presence or absence of tetracycline resistance genes (\u003cem\u003etet(W)\u003c/em\u003eor \u003cem\u003etet(O)\u003c/em\u003e) is indicated in the “Tet” column (green, present; red, absent). The presence or absence of the gene responsible for daidzein-to-equol conversion is shown in the “Equol” column (green, present; red, absent).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/f03f235cc8ad251a9ed85ace.jpg"},{"id":104287998,"identity":"f31ac829-babd-4b24-8024-0f9aaf887994","added_by":"auto","created_at":"2026-03-10 05:58:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree based on core-genome alignment of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAdlercreutzia \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenomes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tree was constructed using core-genome alignments of 11 newly strains isolated in this study (CNCM I-62XX) together with publicly available \u003cem\u003eAdlercreutzia\u003c/em\u003egenomes retrieved from GenBank, including type strains (shown in bold). Bootstrap support values (%) are indicated at each node.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/4e44d19a8696a2ea7833878c.png"},{"id":104779742,"identity":"415660c0-2111-4ca4-9717-d27c2ff682c7","added_by":"auto","created_at":"2026-03-17 07:45:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":269718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of different bile concentrations on the growth of newly isolated strains and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. equolifaciens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e DSM 19450\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBacterial suspensions were plated on M104 agar containing 0 %, 2 %, 20 %, 40 %, 60 % or 80 % (v/v) bile and incubated for 72 h to 96 h. Bacterial concentration is expressed as CFU/mL. Statistical significance was assessed with a Kruskal–Wallis test followed by Dunn’s multiple‑comparison post‑hoc test. * p \u0026lt; 0.05; ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/314458e9ada5210d772defb4.jpg"},{"id":104405115,"identity":"b06722c2-7911-44b4-8a05-5a4ec10b7283","added_by":"auto","created_at":"2026-03-11 12:21:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of oxygen exposure on the growth of newly isolated strains and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. equolifaciens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e DSM 19450.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial cultures were plated on M104 agar and subjected to aerobic conditions for 0 h, 4 h, 6 h, 12 h, or 24 h. The bacterial concentration is expressed as CFU/mL. Statistical significance was assessed with a Kruskal–Wallis test followed by Dunn’s multiple‑comparison post‑hoc test. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/6675832aa2c76485580cd667.jpg"},{"id":104288000,"identity":"674c80c9-051c-4459-b95d-0960f93036ab","added_by":"auto","created_at":"2026-03-10 05:58:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of supernatant (_SN) and pellet (_P) of the newly isolated strains and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. equolifaciens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e DSM 19450 on NF-κB activity of HT-29, HepG2 and HEK cell lines and on TLR2/NF-κB activity of HEK cell line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivities were measured in non-inflammatory or inflammatory (_TNF) conditions repeated three times independently with triplicate determinations. Values are expressed as fold increase over group controls (M104 or PBS) and displayed as colour ranging from green to red. Statistical significance was assessed with a Kruskal–Wallis test followed by Dunn’s multiple‑comparison post‑hoc test. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0,05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0,0002, **** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/4c349e4a3b156300ee82644f.jpg"},{"id":104784097,"identity":"90efc0d2-64ed-4fef-a5c0-90d273327b8f","added_by":"auto","created_at":"2026-03-17 08:04:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2656301,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/7deea3bc-2afa-4909-9b14-86fe89f2a6a3.pdf"},{"id":104405139,"identity":"3a5a300c-891e-4802-9481-a9c5cf27c4e2","added_by":"auto","created_at":"2026-03-11 12:21:54","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17372,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1vf.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/84e8bfee89abaff293df9773.xlsx"},{"id":104288001,"identity":"7f71d5b7-e4e5-4a96-bee2-b3fc539725c2","added_by":"auto","created_at":"2026-03-10 05:58:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":922829,"visible":true,"origin":"","legend":"","description":"","filename":"Chamignonetal2026Supplfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8574898/v1/be3dbcc414e0b40c911c63ab.docx"}],"financialInterests":"Competing interest reported. P.Y.M. is co-founder and CEO of Novobiome. H.M.B. is co-founder and scientific advisor of Novobiome. C.C., M.B., and A.L. are employees of Novobiome. F.P.O. and H.M.B. are co-inventors of the patent “USE OF ADLERCREUTZIA BACTERIA FOR THE TREATMENT OF INFLAMMATORY DISEASES” (no. 20230310519).","formattedTitle":"Comparative genomics and functional characterization of 11 newly isolated non-equol-producing Adlercreutzia strains with anti-inflammatory properties","fulltext":[{"header":"Introduction ","content":"\u003cp\u003eThe human gut microbiota plays a central role in host physiology, contributing to metabolic homeostasis, immune regulation, and protection against disease\u003csup\u003e1,2\u003c/sup\u003e. Disruptions to this complex ecosystem have been implicated in a wide range of chronic disorders, including metabolic, inflammatory, hepatic, and neurodegenerative diseases. As a result, increasing attention has been directed toward the identification of specific commensal bacteria that contribute to host health. Within this context, \u003cem\u003eAdlercreutzia\u003c/em\u003e has emerged as a genus of particular interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAdlercreutzia\u003c/em\u003e, a member of the family \u003cem\u003eEggerthellaceae\u003c/em\u003e within the phylum \u003cem\u003eActinomycetota\u003c/em\u003e (formerly \u003cem\u003eActinobacteria\u003c/em\u003e), was first described following the isolation of \u003cem\u003eA. equolifaciens\u003c/em\u003e from a healthy human donor in Japan and was named in honor of Professor H. Adlercreutz\u003csup\u003e3\u003c/sup\u003e. \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450, the type strain of the genus, is best known for its ability to convert the soy isoflavone daidzein into equol, a metabolite with estrogenic and anti-inflammatory properties\u003csup\u003e4\u003c/sup\u003e. Since then, additional species closely related to \u003cem\u003eA. equolifaciens\u003c/em\u003e, including \u003cem\u003eA. hattorii\u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e and \u003cem\u003eA. rubneri\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e, have been described from healthy human donors. Notably, type strains of these species lack equol-producing capacity, highlighting metabolic and functional diversity within the genus. Furthermore, the type strain of \u003cem\u003eA. rubneri\u003c/em\u003e has been shown to transform the stilbene resveratrol into dihydroresveratrol, a reaction with potential relevance for both host physiology and bio-based chemical production.\u003c/p\u003e\n\u003cp\u003eAdvances in anaerobic culturing techniques and high-throughput sequencing have substantially expanded our understanding of previously underexplored gut commensals, enabling detailed investigations of their prevalence, functional potential, and associations with health and disease\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;Within this framework, metagenomic studies have identified \u003cem\u003eA. equolifaciens\u003c/em\u003e as a prevalent health-associated taxon, contributing positively to composite metrics such as the Gut Microbiome Wellness Index\u003csup\u003e8\u003c/sup\u003e (GMWI)\u0026nbsp;and the Health-Associated Core Keystone (HACK) index\u003csup\u003e9\u003c/sup\u003e. Notably, an \u003cem\u003ein vivo\u003c/em\u003e study using a murine model demonstrated that increased abundance of \u003cem\u003eA. equolifaciens\u003c/em\u003e enhances GMWI, in part by promoting the production of palmitoyl serinol\u003csup\u003e10\u003c/sup\u003e.\u0026nbsp;These findings suggest a broader role for \u003cstrong\u003e\u003cem\u003eAdlercreutzia\u003c/em\u003e\u003c/strong\u003e species in maintaining host health, extending beyond the production of polyphenol-derived metabolites.\u003c/p\u003e\n\u003cp\u003eConsistent with these observations, we recently reported that both the prevalence and abundance of \u003cem\u003eA. equolifaciens\u003c/em\u003e are markedly reduced in patients with metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD), with progressively lower levels observed as disease severity increases\u003csup\u003e11\u003c/sup\u003e. Importantly, we also demonstrated that \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 exerts anti-inflammatory effects \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, supporting a potential protective role of this bacterium in metabolic liver disease. However, it remains unclear whether these beneficial effects are strain-specific and restricted to equol-producers or represent a conserved functional trait across the genus.\u003c/p\u003e\n\u003cp\u003eTherapeutic management of MASLD remains a significant clinical challenge. While lifestyle interventions such as caloric restriction and regular physical activity are the cornerstone of treatment for achieving weight loss, their long-term efficacy is often limited by low patient adherence. Consequently, pharmacological strategies have emerged to target MASLD-associated risk factors, aiming to mitigate liver inflammation by modulating glucose and lipid metabolism\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;Current therapeutic options include antidiabetic agents, such as glucagon-like peptide-1 receptor agonists (GLP-1 RA), sodium-glucose cotransporter 2 inhibitors (SGLT2i), biguanides, and thiazolidinediones, as well as lipid-lowering drugs like statins and peroxisome proliferator-activated receptor (PPAR) agonists\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe absence of a universally accepted standard and the limited efficacy of drugs for MASLD have prompted researchers to explore alternative treatment options. One such option that has gained more attention is the use of commensal bacteria as therapeutic agent\u003csup\u003e14\u003c/sup\u003e. This is due, in large part, to the growing body of evidence demonstrating the symbiotic relationship between the bacteria and their hosts, which make them a safe choice for therapeutic use\u003csup\u003e15\u003c/sup\u003e.\u0026nbsp;Furthermore, their ability to colonize the intestinal niche offers the potential for sustained, long-term health benefits\u003csup\u003e16\u003c/sup\u003e through a more personalized and physiologically integrated approach\u003csup\u003e17\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, transitioning from a commensal isolate to a regulated therapeutic product is complex. Candidates must satisfy rigorous criteria established by regulatory authorities, such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), which require comprehensive documentation of a positive benefit-risk balance through evidence of quality, safety, and efficacy\u003csup\u003e18\u003c/sup\u003e. A critical component of this process is the precise identification and characterization of strains using robust preclinical assays to elucidate their functional properties and mechanisms of action\u003csup\u003e19\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;Moreover, to ensure successful delivery and therapeutic activity, candidates must demonstrate high viability and resilience against the various physiological stressors encountered during gastrointestinal transit, such as acidic pH and bile salt exposure\u003csup\u003e21\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we aimed at isolating and comprehensively characterizing \u003cstrong\u003e\u003cem\u003eAdlercreutzia\u003c/em\u003e\u003c/strong\u003e strains from healthy European volunteers, with the goal of identifying candidates for potential therapeutic applications. In accordance with regulatory guidelines, the isolates were subjected to an integrated analysis combining genomic, phenotypic, and functional approaches. These included \u003cem\u003ein silico\u003c/em\u003e taxonomic annotation, assessment of physiological and biochemical traits, tolerance to intestinal stressors, antibiotic resistance profiling, and \u003cem\u003ein vitro\u003c/em\u003e evaluation of anti-inflammatory activity. By comparing the newly isolated strains to the reference strain \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450, we assessed whether anti-inflammatory properties are a conserved feature within the genus. Our findings provide novel insights into the functional diversity of \u003cstrong\u003e\u003cem\u003eAdlercreutzia\u003c/em\u003e\u003c/strong\u003e and support the potential use of these isolates as next-generation microbiome-based therapeutics.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eBacterial Isolation\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 was used as reference strain. Both the reference strain and the isolates were cultured using Peptone Yeast Glucose broth (PYG, also referred as M104) supplemented with 0.5% of arginine (Sigma-Aldrich, St Louis, MO, USA) and 1.5% agar (Invitrogen, Carlsbad, CA, USA) for the solid form. All media and reagents were reduced for a minimum of 48 h in an anaerobic chamber before use, and cultures were incubated at 37\u0026deg;C in anaerobic conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStrain isolation was performed by Bioaster (Paris, France), following the protocol published by Bellais and colleagues\u003csup\u003e22\u003c/sup\u003e. Briefly, faecal samples were collected from healthy volunteers and shipped within 2 hours at ambient temperature. In anaerobic conditions, 1 g of faecal sample was diluted in PBS and homogenized with 2.4mm glass beads. Bacteria were then selected and enriched with live/dead staining (BacLight\u0026trade; Bacterial Viability Kit, Thermo Fisher Scientific, Waltham, MA, USA), and rabbit polyclonal antibodies (protein A purified from IgGs) coupled with Alexa Fluor\u0026trade; 647 or Alexa Fluor\u0026trade; 405 dyes (Invitrogen, Carlsbad, CA, USA) using flow cytometry coupled with cell sorting. These antibodies were obtained after rabbit immunization with \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 as previously described\u003csup\u003e22\u003c/sup\u003e. The bacteria were sorted and plated on M104 agar plates, then incubated at 37\u0026deg;C for 5 days. Colonies were visually selected based on their morphology (tiny and transparent).\u003c/p\u003e\n\u003ch2\u003eCulture Growth\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eThe isolates\u003c/em\u003e and \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 were cultured using the Hungate culture method\u003csup\u003e23\u003c/sup\u003e in pre-reduced (100% CO\u003csub\u003e2\u003c/sub\u003e) M104-based food grade medium supplemented with 0.5% arginine (AppliChem, Darmstadt, Germany) at 37 \u0026deg;C.\u003c/p\u003e\n\u003ch2\u003eGenome sequencing and bioinformatics\u003c/h2\u003e\n\u003cp\u003eGenomic DNA was extracted using the NucleoSpin\u0026reg; Microbial DNA kit (Macherey-Nagel, Hoerdt, France) following the manufacturer\u0026rsquo;s instructions. Genome sequencing was performed on DNBSEQ-G400 (MGITech, China) instruments as previously described\u003csup\u003e24\u003c/sup\u003e, generating at least 670,000 paired-end reads (2 \u0026times; 150 bp) per sample, ensuring a minimum sequencing coverage of 70\u0026times;.\u003c/p\u003e\n\u003cp\u003eSequencing data quality control was conducted using fastp\u003csup\u003e25\u003c/sup\u003e to remove adapters, trim low-quality reads, and discard reads shorter than 80 bp or those containing undetermined bases. Genome assembly was performed with SPAdes\u003csup\u003e26\u003c/sup\u003e (parameters: --isolate --cov-cutoff auto).\u003c/p\u003e\n\u003cp\u003eOther genomes derived from isolates assigned to the \u003cem\u003eAdlercreutzia\u003c/em\u003e genus were downloaded from GenBank, except for \u003cem\u003eA. equolifaciens\u003c/em\u003e UC1_BHI_P, whose raw sequencing data was obtained from the European Nucleotide Archive (sample accession SRS254664) and assembled as described above. The quality of both newly assembled and publicly available genomes was assessed using CheckM\u003csup\u003e27\u003c/sup\u003e and taxonomic annotation was performed with GTDB-Tk\u003csup\u003e28\u003c/sup\u003e based on GTDB R220. Public genomes not assigned to \u003cem\u003eA. equolifaciens\u003c/em\u003e or \u003cem\u003eA. rubneri\u003c/em\u003e were filtered out. Additionally, we removed short contigs (\u0026lt;1000 bp), as we found that this substantially reduces contamination.\u003c/p\u003e\n\u003cp\u003eAverage Nucleotide Identity (ANI) between all genome pairs was computed with PyOrthoANI\u003csup\u003e29,30\u003c/sup\u003e A distance matrix was then constructed, defining distance as 1 - ANI. Finally, genomes were clustered via average linkage hierarchical clustering (UPGMA).\u003c/p\u003e\n\u003cp\u003eA core genome alignment of all genomes was generated using Parsnp 2\u003csup\u003e31\u003c/sup\u003e, with the genome assembly GCF_000478885.1 (\u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450) as a reference. The multiple sequence alignment was then provided to RAxML-NG\u003csup\u003e32\u003c/sup\u003e to construct a phylogenetic tree with the GTR+CAT model. Branch support was assessed using 100 bootstrap replicates, and the final tree was visualized in iTOL\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGene\u0026nbsp;prediction in the assembled genomes was performed with RAST-tk\u003csup\u003e34\u003c/sup\u003e from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), as Prodigal\u003csup\u003e35\u003c/sup\u003e missed some genes, including those in the operon involved in equol production.\u003c/p\u003e\n\u003cp\u003eProtein sequences constituting the equol biosynthesis pathway were retrieved from NCBI, including daidzein reductase (\u003cem\u003edzr\u003c/em\u003e; WP_022741749.1), dihydrodaidzein reductase (\u003cem\u003eddr\u003c/em\u003e; WP_022741751.1), tetrahydrodaidzein reductase (\u003cem\u003etdr\u003c/em\u003e; WP_022741752.1), and a racemase (WP_022741755.1). These proteins were searched against all genomes using \u003cstrong\u003etblastn\u003c/strong\u003e\u003csup\u003e36\u003c/sup\u003e, with thresholds of \u0026ge; 95% amino acid identity and \u0026ge; 90% query coverage. A strain was considered to harbor a complete equol biosynthesis pathway only when all four pathway genes were detected in its genome. Antimicrobial resistance genes were searched in genome sequences using AMRFinderPlus\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eMicrobial Characterization\u003c/h2\u003e\n\u003cp\u003eOxidase activity was determined by using oxidase test strips (Sigma-Aldrich, St Louis, MO, USA) following the manufacturer\u0026rsquo;s instructions. A drop of an overnight bacterial culture was deposited on the strip, and the resulting colour change was observed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCatalase activity was measured using 3% and 12% hydrogen peroxide (Sigma-Aldrich, St Louis, MO, USA). A colony was deposited in a drop of hydrogen peroxide and the presence or absence of reaction was observed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeoxyribonuclease (DNase) activity was determined by using a DNase agar plate supplemented with toluidine blue (Sigma-Aldrich, St Louis, MO, USA). An overnight bacterial culture was spotted on the DNase agar plate and incubated at 37\u0026deg;C for 24 h. The colour change was observed after incubation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGram staining was performed by using the Gram coloration kit (Condalab, Madrid, Spain) following manufacturer\u0026rsquo;s instructions. The staining was observed under a light microscope (Olympus BX43, Rungis, France) with oil immersion at a magnification of 100.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpore formation assessment was performed using the Schaeffer-Fulton method\u003csup\u003e38\u003c/sup\u003e which utilises malachite green (Pro-lab Diagnostics, Richmond Hill, Canada). The staining was observed under a light microscope (Olympus BX43, Rungis, France) with oil immersion at a magnification of 100.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe biochemical characterization was carried out using the API 20A anaerobic test kit (Biom\u0026eacute;rieux, Marcy-l\u0026rsquo;Etoile, France) following manufacturer\u0026rsquo;s instructions. The pellet of an overnight bacterial culture was resuspended in API20 medium and the strip was inoculated with 100 \u0026micro;L of the suspension. The strip was then incubated during 48 h at 37\u0026deg;C in anaerobic conditions (GenBag anaer, Biom\u0026eacute;rieux, Marcy-l\u0026rsquo;Etoile, France). After incubation, the strip was read under normal atmospheric conditions and interpreted by referring to the reading table provided by the manufacturer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntibiotic resistance was determined by using the E-test\u0026reg; (Biom\u0026eacute;rieux, Marcy-l\u0026rsquo;Etoile, France and Liofilchem, Rosetto Degli Abruzzi, Italy for piperacillin-tazobactam) according to the manufacturer\u0026rsquo;s instructions. Briefly, a Petri dish containing pre-reduced (H\u003csub\u003e2\u003c/sub\u003e 5%/CO\u003csub\u003e2\u003c/sub\u003e 5%/N\u003csub\u003e2\u003c/sub\u003e 90%) M104-based food-grade medium was inoculated with 100 \u0026micro;L of an overnight bacterial culture. The strip was positioned and the agar plate was inoculated under anaerobic conditions (H\u003csub\u003e2\u003c/sub\u003e 5%/CO\u003csub\u003e2\u003c/sub\u003e 5%/N\u003csub\u003e2\u003c/sub\u003e 90%) for 48 h at 37\u0026deg;C. After incubation, the minimum inhibitory concentration (MIC, \u0026micro;g/mL) was determined by direct reading of the graduate scale found on the strip. The MIC was then interpreted as sensitive (S), intermediate (I) or resistant (R) based on the standards set by the Clinical and Laboratory Standards Institute (CLSI)\u003csup\u003e39\u003c/sup\u003e. The antibiotics tested were\u0026nbsp;metronidazole (MZL), chloramphenicol (CL), vancomycin (VA), clindamycin (CM), imipenem (IP), piperacillin-tazobactam (P/T), and tetracycline (TC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBile acid tolerance was assessed using M104-based food-grade medium with 1.5% agar (Invitrogen, Carlsbad, CA, USA). The agar medium was supplemented with Ox-bile (Sigma-Aldrich, St Louis, MO, USA) at concentrations of 0%, 0.25%, 2.5%, 5%, 7.5% and 10% w/v corresponding to 0%, 2%, 20%, 40%, 60% and 80% of fresh bile, respectively. The plates were reduced in anaerobic atmosphere (H\u003csub\u003e2\u003c/sub\u003e 5%/CO\u003csub\u003e2\u003c/sub\u003e 5%/N\u003csub\u003e2\u003c/sub\u003e 90%) 48 h before their use. Overnight liquid cultures of the isolates were diluted from 10\u003csup\u003e-1\u003c/sup\u003e to 10\u003csup\u003e-10\u003c/sup\u003e. Next, 10 \u0026micro;L of each dilution were spotted on the pre-reduced agar plates containing varying percentages of bile. The agar plates were then incubated under anaerobic conditions (H\u003csub\u003e2\u003c/sub\u003e 5%/CO\u003csub\u003e2\u003c/sub\u003e 5%/N\u003csub\u003e2\u003c/sub\u003e 90%) at 37\u0026deg;C for 72 h to 96 h. The growth was assessed by enumerating the colonies per dilution, and the bacterial concentration was calculated and expressed in CFU/mL. Finally, the results were compared to the control group, which was M104-based medium with 0% of bile.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOxygen tolerance was assessed using liquid M104-based food-grade medium and supplemented with 1.5% agar (Invitrogen, Carlsbad, CA, USA). Liquid medium was prepared following the Hungate method previously described and the agar plates were reduced in anaerobic atmosphere (H\u003csub\u003e2\u003c/sub\u003e 5%/CO\u003csub\u003e2\u003c/sub\u003e 5%/N\u003csub\u003e2\u003c/sub\u003e 90%) for 48 h before their use. For the evaluation in liquid medium, overnight liquid cultures of the isolates were exposed to oxygen from ambient air for 0, 2, 4, 6 and 24 h at room temperature. After oxygen exposition, the bacterial cultures were subcultured in new pre-reduced liquid medium and incubated at 37\u0026deg;C for 24 h. The growth was revealed by reading the optical density (OD) at 600 nm. For the assessment on solid medium, overnight liquid cultures of the isolates were diluted from 10\u003csup\u003e-1\u003c/sup\u003e to 10\u003csup\u003e-10\u003c/sup\u003e and 10 \u0026micro;L of each dilution were spotted on the pre-reduced agar plates. Each plate was then exposed to oxygen for 0, 2, 4, 6 and 24 h at 37\u0026deg;C. After the exposition time had elapsed the agar plates were replaced under anaerobic conditions (H\u003csub\u003e2\u003c/sub\u003e 5%/CO\u003csub\u003e2\u003c/sub\u003e 5%/N\u003csub\u003e2\u003c/sub\u003e 90%) and incubated at 37\u0026deg;C for a total of 72 h to 96 h. Colony enumeration was used to assess the growth, and the bacterial concentration was calculated and expressed in CFU/mL. Finally, the results were compared to the control group, which was M104-based medium with no oxygen exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe strain tolerance to basic, acid and neutral pH was evaluated using liquid M104-based food-grade medium buffered at pH 4, 7 and 12 for 24 h at 37\u0026deg;C. The medium was prepared following the previously described Hungate method. Overnight liquid cultures of the isolates were subcultured in the pH buffered media and incubated at 37\u0026deg;C for 24 h. The growth was assessed by reading the OD at 600 nm.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vitro\u003c/em\u003e assays\u003c/h2\u003e\n\u003ch3\u003eCell Culture and Reagents\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eHT-29 human intestinal epithelial cells and HEK293 human renal epithelial cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco, Les Ulis, France) supplemented with 2 mM L-glutamine (Gibco, Les Ulis, France), 50 IU/mL penicillin (Gibco), 50 \u0026mu;g/mL streptomycin (Gibco), and 10% heat-inactivated fetal calf serum (Eurobio, Les Ulis, France). HepG2 human hepatocarcinoma cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, Gibco) and supplemented as RPMI medium with the addition of 1% nonessential amino acid (NEAA) 100X (Gibco, Les Ulis, France), 1% hepes 1M (Gibco, Les Ulis, France), and 1% sodium pyruvate 100 mM (Gibco, Les Ulis, France). The cells were cultured in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg;C. The absence of mycoplasma contamination was confirmed using a MycoAlert kit (Lonza, Levallois-Perret, France). The HT-29 NF-\u0026kappa;B and HepG2 NF-\u0026kappa;B reporter cell lines were constructed and validated as previously described by transfecting pNiFty2 plasmid (Invivogen, Toulouse, France)\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e40\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e41\u003c/sup\u003e. This reporter plasmid contains the Secreted Embryonic Alkaline Phosphatase (SEAP) gene under the control of NF-\u0026kappa;B binding elements.\u0026nbsp;HEK Blue\u0026trade; reporter cell lines (HEK Blue\u0026trade; Null1 and HEK Blue\u0026trade; TLR2) were obtained from Invivogen, Toulouse, France. HEK Blue\u0026trade; Null1 cells express the SEAP reporter gene under the control of the IFN-\u0026beta; minimal promoter fused to five NF-\u0026kappa;B and AP-1 binding sites. Therefore, the activation of NF-\u0026kappa;B leads to the expression of SEAP. HEK Blue\u0026trade; TLR2 cells were generated from HEK Blue\u0026trade; Null1 cells by transfection of human TLR2. Thus, stimulation via TLR2 ligands activates NF-\u0026kappa;B and AP-1, inducing the production of SEAP.\u003c/p\u003e\n\u003ch3\u003eCommensal Strains and Preparation of Conditioned Media\u003c/h3\u003e\n\u003cp\u003eThe isolates and \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 were cultured using the Hungate method in pre-reduced (100% CO\u003csub\u003e2\u003c/sub\u003e) M104-based food-grade medium at 37 \u0026deg;C. After overnight incubation, the bacterial cultures were centrifuged at 11,000 rpm (or 13,663 x \u003cem\u003eg\u003c/em\u003e) for 10 min. The pellets and supernatants were collected separately, and the supernatant was filtered using 0.2 \u0026mu;m PES filters (Corning, NY, USA) before storage at \u0026minus;80\u0026deg;C. For the experiment, the cell pellets were resuspended in PBS 1X (Gibco). The non-inoculated bacterial culture medium was used as the control for the analysis of the supernatant effect, while PBS was used for the pellet effect analysis.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;Analyses of NF-\u0026kappa;B Activation\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eFor each experiment, reporter cell lines were seeded at a density of 30,000 cells per well in 96-well plates and incubated for 24 h. Subsequently, cells were stimulated for 24 h with 10 \u0026mu;L of tested bacterial supernatant or resuspended bacterial pellet, for a final volume of 100 \u0026mu;L per well (i.e., 10% vol/vol), in the presence or absence of TNF-\u0026alpha; (PeproTech, Cranbury, NJ, USA) to mimic inflammatory and non-inflammatory conditions, respectively. In case of inflammatory conditions, the cells were stimulated with 10 ng/mL of TNF-\u0026alpha; (PeproTech), 1 h before sample deposit. The Quanti-Blue\u0026trade; reagent (Invivogen, Toulouse, France) was used to reveal SEAP in the supernatant of reporter cell lines (HT-29 NF-\u0026kappa;B, HepG2 NF-\u0026kappa;B, HEK Blue\u0026trade; Null1 and HEK Blue\u0026trade; TLR2) and quantified at 655 nm OD. using a microplate reader (Infinite 200, Tecan, Lyon, France). All the measurements were performed according to the manufacturer\u0026rsquo;s protocol. After 24 h of incubation, the cell viability was controlled using a MTS assay (CellTiter 96 Aqueous One, Promega, Charbonni\u0026egrave;res-les-Bains, France) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;Statistical analysis\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eResults were expressed as the mean \u0026plusmn; the Standard Error of the Mean (SEM) from three independent experiments, each performed in triplicate. Statistical analysis was performed using GraphPad Prism version 8 (La Jolla, CA, USA). Differences between groups were assessed using the Kruskal-Wallis test, followed by Dunn\u0026rsquo;s multiple comparisons test. P-values \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results ","content":"\u003ch2\u003eComparative genomics of \u003cem\u003eAdlercreutzia\u003c/em\u003e reveals distinct phylogroups and atypical species boundaries\u003c/h2\u003e\n\u003cp\u003eEleven new strains of bacteria from the \u003cem\u003eAdlercreutzia\u0026nbsp;\u003c/em\u003egenus were isolated from faecal samples of healthy volunteers using antibody driven- flow cytometry under anaerobic conditions. After short-read sequencing and \u003cem\u003ede novo\u003c/em\u003e genome assembly, high-quality draft genomes were obtained (Supplementary Table 1), exhibiting high completeness (100% for all genomes), low contamination (\u0026le;0.81%), and high contiguity (N50 \u0026gt; 100 Kbp). Genome sizes ranged from 2.61 to 2.85 Mbp (mean: 2.75 \u0026plusmn; 0.07 Mbp), with an average GC content of 63.2%, consistent with genome assemblies of the type strains \u003cem\u003eA. rubneri\u003c/em\u003e ResAG-91 (2.78 Mbp, GC content 63.3%) and \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 (2.86 Mbp, GC content 63.5%). The number of coding sequences (CDS) ranged from 2,279 to 2,537 (mean: 2,420 \u0026plusmn; 68), again consistent with values reported for \u003cem\u003eA. rubneri\u0026nbsp;\u003c/em\u003eResAG-91 (2,438 CDS) and \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 (2,365 CDS).\u003c/p\u003e\n\u003cp\u003eThe pairwise average nucleotide identity (ANI) between the 11 newly assembled genomes and 13 publicly available genomes of \u003cem\u003eA. rubneri\u003c/em\u003e, \u003cem\u003eA. hattorii\u003c/em\u003e, and \u003cem\u003eA. equolifaciens\u003c/em\u003e was computed. Analysis of the ANI distribution revealed that the conventional 95% species boundary\u003csup\u003e42\u003c/sup\u003e was not clearly defined for these genomes (Figure S1). Unlike the distinct gaps often seen at 95% in other bacterial species, high density was observed spanning this threshold. To better align with the observed local minima in the density distribution and provide a more biologically relevant separation of the genomic clusters, a 96% ANI cutoff was adopted.\u003c/p\u003e\n\u003cp\u003eHierarchical clustering with a 96% ANI cutoff revealed three distinct genome groups (Figure 1). The first group (labeled \u0026ldquo;\u003cem\u003eA. equolifaciens\u003c/em\u003e\u0026rdquo;) comprised five genomes, including the type strains \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 and \u003cem\u003eA. equolifaciens\u003c/em\u003e \u003cem\u003esubsp. celata\u003c/em\u003e DSM 18795, but no newly assembled genomes from this study. All genomes in this group were taxonomically annotated as \u003cem\u003eA. equolifaciens\u003c/em\u003e according to the ANI-based assignment method implemented in GTDB-Tk. The second group (\u0026ldquo;\u003cem\u003eA. rubneri / A. hattorii\u003c/em\u003e\u0026rdquo;) included twelve genomes, comprising the type strains \u003cem\u003eA. hattorii\u003c/em\u003e 8CFCBH1 and \u003cem\u003eA. rubneri\u003c/em\u003e ResAG-91, as well as most of the newly assembled genomes (9/11). All genomes in this group were assigned to \u003cem\u003eA. rubneri\u003c/em\u003e by GTDB-Tk. The third group (labeled \u0026ldquo;Other\u0026rdquo;) consisted of seven genomes, including the remaining two newly assembled genomes. Although all genomes in this group were assigned to \u003cem\u003eA. rubneri\u003c/em\u003e by GTDB-Tk, those available in GenBank were submitted as \u003cem\u003eA. equolifaciens\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFunctional genome annotation further revealed that none of the newly isolated strains harbor genes involved in the daidzein-to-equol biosynthesis pathway (Figure 1). This pathway was complete in only 3 of the 24 analyzed genomes, all of which belonged to the \u003cem\u003eA. equolifaciens\u003c/em\u003e group. In addition, six of the newly isolated strains carried a tetracycline resistance gene (\u003cem\u003etetW\u003c/em\u003e or \u003cem\u003etetO\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eA phylogenetic tree was then inferred from core-genome alignments of the 24 genomes (total alignment length: 1.08 Mbp, including 86,601 SNPs; Figure 2). The phylogenetic relationships were generally well supported by high bootstrap values, with most internal nodes showing strong support (\u0026ge;90%), indicating a robust core-genome signal across the dataset. Three main phylogroups were recovered, broadly corresponding to the clusters identified by ANI analysis. However, the third (\u0026ldquo;Other\u0026rdquo;) group appeared more closely related to the \u003cem\u003eA. equolifaciens\u003c/em\u003e clade, in contrast to the GTDB-Tk assignment and ANI-based analysis, which placed it closer to the \u003cem\u003eA. rubneri\u003c/em\u003e group. In addition, strains CNCM I-6215 and CNCM I-6216 clustered closer to the \u003cem\u003eA. rubneri\u003c/em\u003e clade but occupied relatively long and distinct branches, suggesting a more divergent evolutionary history.\u003c/p\u003e\n\u003ch2\u003ePhenotypic characterization: growth, antibiotic susceptibility, and stress tolerance of new \u003cem\u003eAdlercreutzia\u003c/em\u003e isolates\u0026nbsp;\u003c/h2\u003e\n\u003ch3\u003ePhysiological Properties\u003c/h3\u003e\n\u003cp\u003eThe new isolates were obligate anaerobes, non-motile, and appeared as single or chained coccobacilli, with no evidence of spore formation. Their growth was enhanced by supplementation of arginine in the medium. Colonies cultivated on M104-based agar were small, transparent, circular, slightly convex, and smooth. Biochemical testing revealed that the isolates were negative for catalase, oxidase, and DNase activities (Table 1)\u003c/p\u003e\n\u003ch3\u003eBiochemical Properties\u003c/h3\u003e\n\u003cp\u003eThe biochemical characteristics of the isolates were evaluated using API20A galleries. All isolates tested negative for the enzymes included in the panel (Table 2) and were classified as asaccharolytic, as they did not metabolize any of the sugars provided as carbon sources. These findings are consistent with previous reports on \u003cem\u003eA. equolifaciens\u003c/em\u003e, \u003cem\u003eA. rubneri\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. hattorii\u003c/em\u003e type strains\u0026nbsp;\u003csup\u003e3,5,6\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAntibiotic Resistance\u003c/h3\u003e\n\u003cp\u003eThe sensitivity of the isolates to a panel of antibiotics was evaluated (Table 3). Resistance to tetracycline was observed in strains CNCM I-6209, CNCM I-6210, CNCM I-6212, and CNCM I-6214, whereas strains CNCM I-6207 and CNCM I-6217 exhibited mild resistance. These phenotypic results were largely consistent with the genomic analysis, except for strain CNCM I-6211, which was sensitive to tetracycline despite the presence of a resistance gene in its genome. Conversely, strain CNCM I-6214 displayed tetracycline resistance, although no known resistance gene was detected in its genome. In addition, resistance to clindamycin was observed in strains CNCM I-6207 and CNCM I-6214, although no corresponding gene was identified in the respective genomes, suggesting the presence of uncharacterized resistance mechanisms.\u003c/p\u003e\n\u003ch3\u003eStress Tolerance\u003c/h3\u003e\n\u003cp\u003eThe tolerance of the\u003cem\u003e\u0026nbsp;Adlercreutzia\u003c/em\u003e isolates to multiple environmental stressors, including bile salts, pH, and oxygen, was evaluated. pH tolerance was assessed using M104-based liquid medium adjusted to acidic (pH 4), neutral (pH 7), and basic (pH 12) conditions. No growth was observed under acidic or basic conditions (data not shown). Optimal growth was observed at neutral pH (pH 7).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBile salt tolerance was evaluated on M104-based agar medium supplemented with increasing concentrations of bile (0%, 2%, 20%, 40%, 60%, and 80%). Two distinct growth profiles were identified (Figure 3). The first profile exhibited a conventional dose-dependent response, characterized by a progressive decrease in bacterial growth with increasing bile concentrations. Within this group, isolates CNCM I-6213 and CNCM I-6216 displayed greater tolerance to high bile concentrations compared with isolates CNCM I-6207, CNCM I-6209, CNCM I-6211, and CNCM I-6215, which showed marked growth reduction at bile concentrations as low as 2%.\u003c/p\u003e\n\u003cp\u003eThe second profile showed an atypical growth pattern and included isolates CNCM I-6208, CNCM I-6210, CNCM I-6212, CNCM I-6214, CNCM I-6217, as well as \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450. In this group, growth decreased at 2% bile (and up to 20% for some isolates) but partially recovered at higher bile concentrations (20\u0026ndash;60%). This profile was also associated with distinct colony morphologies on agar plates. Growth inhibition was accompanied by very small, translucent colonies, whereas growth recovery at higher bile concentrations resulted in larger, creamy colonies (data not shown). These observations may suggest bile-induced phenotypic adaptation, potentially involving biofilm formation.\u003c/p\u003e\n\u003cp\u003eIn both profiles, no bacterial growth was observed at 80% bile, with the exception of strain CNCM I-6213, which exhibited marked bile salt resistance.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOxygen tolerance was assessed by incubating agar plates under aerobic conditions for increasing durations (0, 2, 4, 6, and 24 h) on M104-based medium. Oxygen exposure negatively affected bacterial growth (Figure 4); however, viable counts remained above 1 \u0026times; 10⁸ CFU/mL up to 6 h for strains CNCM I-6209, CNCM I-6210, CNCM I-6211, CNCM I-6212, and CNCM I-6213, up to 4 h for strains CNCM I-6207, CNCM I-6208, CNCM I-6214, and CNCM I-6216, and up to 2 h for strain CNCM I-6217. In contrast, the reference strain \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 was the most sensitive, with a marked decrease in viable counts observed before 2 h of oxygen exposure (Figure 4).\u003c/p\u003e\n\u003cp\u003eAfter 24 h of aerobic exposure, no bacterial growth was detected. However, when these oxygen-exposed liquid cultures were subcultured under anaerobic conditions, bacterial growth resumed after 96 h of incubation.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vitro\u003c/em\u003e evaluation of anti-inflammatory effects of \u003cem\u003eAdlercreutzia\u003c/em\u003e isolates\u003c/h2\u003e\n\u003cp\u003eThe effects of the isolates on NF-\u0026kappa;B and TLR2 inflammatory pathways were evaluated using stably transfected reporter cell lines (Figure 5 and Supplementary Figures S2-S5). The impact of the eleven isolates on NF-\u0026kappa;B activation was assessed in intestinal epithelial cells (HT-29 NF-\u0026kappa;B reporter) and hepatic cells (HepG2 NF-\u0026kappa;B reporter). Furthermore, HEK Null1 and HEK TLR2 cell lines were used to determine the implication of TLR2 in the modulatory effects observed on the NF-\u0026kappa;B signalling pathway. The results were then compared to those obtained with \u003cem\u003eA. equolifaciens\u0026nbsp;\u003c/em\u003eDSM 19450, for which anti-inflammatory properties had previously been demonstrated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth bacterial pellets and culture supernatants were tested under non-inflammatory and inflammatory conditions. In non-inflammatory conditions, neither pellets nor supernatants induced NF-\u0026kappa;B activation in HT-29 NF-\u0026kappa;B or HEK Null1 cells. Similarly, no NF-\u0026kappa;B activation was observed in HepG2 cells for most isolates, with the exception of the pellet of strain CNCM I-6029, which induced a limited response. Overall, these results indicate that the isolates exert little to no basal activation of the NF-\u0026kappa;B pathway.\u003c/p\u003e\n\u003cp\u003eIn contrast, several isolates significantly activated the TLR2 pathway. The supernatants of five isolates (CNCM I-6207, CNCM I-6028, CNCM I-6209, CNCM I-6210, and CNCM I-6216), as well as \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450, and the pellets of seven isolates (CNCM I-6207, CNCM I-6208, CNCM I-6209, CNCM I-6210, CNCM I-6213, CNCM I-6215, and CNCM I-6216) induced a strong activation of TLR2 in HEK TLR2 cells, with fold changes exceeding 3 compared to the M104 or PBS controls.\u003c/p\u003e\n\u003cp\u003eUnder inflammatory conditions, following stimulation with TNF-\u0026alpha;, most bacterial pellets significantly reduced NF-\u0026kappa;B activation in HT-29 cells, with decreases exceeding 40% in the most pronounced cases compared to the PBS control. Notably, the inhibitory effect of strain CNCM I-6216 was comparable to that observed for \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450. Both the pellet and supernatant of CNCM I-6216 reduced NF-\u0026kappa;B activation by more than 30% relative to the PBS or M104 controls (Figure S2).\u003c/p\u003e\n\u003cp\u003eIn HepG2 cells, the pellets of eight isolates (CNCM I-6208, CNCM I-6209, CNCM I-6210, CNCM I-6211, CNCM I-6213, CNCM I-6214, CNCM I-6215, and CNCM I-6216), as well as \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450, strongly inhibited NF-\u0026kappa;B activation, with reductions exceeding 70% in the most significant cases compared to the PBS control. Furthermore, the supernatants of most isolates (CNCM I-6212, CNCM I-6213, CNCM I-6214, CNCM I-6215, CNCM I-6216, and CNCM I-6217) and of \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 reduced NF-\u0026kappa;B activation by approximately 40% compared to the M104 control. For isolates CNCM I-6213, CNCM I-6214, CNCM I-6215, and CNCM I-6216, both pellets and supernatants elicited inhibitory effects comparable to those of the reference strain (Figure S3).\u003c/p\u003e\n\u003cp\u003eConsistent with these observations, several pellets and supernatants also significantly inhibited NF-\u0026kappa;B and TLR2/NF-\u0026kappa;B signaling in HEK embryonic kidney cells. The most pronounced reductions reached approximately 40% for bacterial pellets and 30% for supernatants relative to PBS or M104 controls. Again, isolates CNCM I-6213, CNCM I-6214, CNCM I-6215, and CNCM I-6216 exhibited effects comparable to those observed with \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 (Figures S4 and S5).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we described eleven new isolates of the genus \u003cem\u003eAdlercreutzia\u003c/em\u003e from healthy European volunteers, nine of which were consistently assigned to \u003cem\u003eA. rubneri\u003c/em\u003e based on both ANI-based and phylogenetic analyses. However, taxonomic annotation proved more challenging for the remaining strains. This complexity is largely driven by the atypical distribution of genomic identity within the genus. Whereas many bacterial groups exhibit a distinct gap at 95% ANI, the \u003cem\u003eAdlercreutzia\u003c/em\u003e genomes analyzed here showed high density spanning this threshold.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In particular, strain CNCM I-6207 showed inconsistent placement depending on the method used, clustering with \u003cem\u003eA. rubneri\u003c/em\u003e according to ANI and GTDB-Tk, while appearing more closely related to \u003cem\u003eA. equolifaciens\u003c/em\u003e in the core-genome phylogeny. Such discordances likely reflect methodological limitations, including the effects of recombination and the influence of accessory genome content on phylogenetic inference.\u003c/p\u003e\n\u003cp\u003eSimilarly, strains CNCM I-6215 and CNCM I-6216, although positioned within the broader \u003cem\u003eA. rubneri\u003c/em\u003e lineage, formed long and well-supported branches that were clearly distinct from established \u003cem\u003eA. rubneri\u003c/em\u003e reference genomes. Their divergent placement, together with the absence of clear genomic thresholds supporting a confident species-level assignment, suggests that these strains represent divergent lineages and potentially undescribed taxa. In the absence of additional discriminating phenotypic, genomic, or ecological markers, we therefore chose to adopt a conservative taxonomic approach. Accordingly, CNCM I-6207, CNCM I-6215, and CNCM I-6216 were designated as \u003cem\u003eAdlercreutzia\u003c/em\u003e sp. strains, and the corresponding genomes were submitted to GenBank under this designation, pending further taxonomic resolution.\u003c/p\u003e\n\u003cp\u003eOur comparative genomic analyses also revealed a high degree of relatedness between the type strains of \u003cem\u003eA. rubneri\u003c/em\u003e and \u003cem\u003eA. hattorii\u003c/em\u003e. The pairwise ANI value between these genomes reached 96.72%, exceeding the commonly accepted 95-96% threshold for bacterial species delineation. This observation was further supported by full-length 16S rRNA gene sequence alignments, which showed a nucleotide identity of 98.4%. Notably, \u003cem\u003eA. rubneri\u003c/em\u003e and \u003cem\u003eA. hattorii\u003c/em\u003e were described only a few months apart, in September 2021 and December 2021, respectively. According to nomenclatural conventions, when two names are proposed for the same species, the earliest published name takes precedence. On this basis, and supported by robust genomic evidence, we propose that \u003cem\u003eA. hattorii\u003c/em\u003e should be considered a later heterotypic synonym of \u003cem\u003eA. rubneri\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe physiological and biochemical characteristics of the newly isolated strains were consistent with those reported for type strains of \u003cem\u003eA. equolifaciens\u003c/em\u003e, \u003cem\u003eA. hattorii\u003c/em\u003e, and \u003cem\u003eA. rubneri\u003c/em\u003e. The isolates were asaccharolytic, as they did not utilize any of the sugars tested in the API20A panel. Consistent with previous observations for \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450, growth was enhanced by supplementation of the culture medium with L-arginine. L-arginine is a key intestinal metabolite that serves as a substrate for both the gut microbial community and host intestinal cells, and its disrupted metabolism has been linked to inflammatory diseases and microbiota depletion. These findings suggest that L-arginine supplementation could be considered in formulations intended to support these bacteria\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe development of microbiome-based therapies for disease treatment is a complex and challenging process. Regulatory agencies, such as the FDA and EMA, require comprehensive documentation of quality, safety, and efficacy before approval. Consequently, the development of Live Biotherapeutic Products (LBPs) and Next Generation Probiotics (NGPs) must be thoroughly documented to meet these regulatory standards. A critical component of safety assessment is strain characterization, which includes evaluating antibiotic resistance through both genotypic and phenotypic approaches\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e. Antimicrobial resistance represents a significant public health concern, as gut microbiota can carry resistance genes that may be horizontally transferred to other bacteria via mobile genetic elements, such as plasmids. To address this, we determined the minimum inhibitory concentration (MIC) for a selected panel of antibiotics, chosen based on the characteristics of the strains and the target population\u003csup\u003e44\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e45\u003c/sup\u003e. In parallel, genomic analyses of our isolates identified tetracycline resistance genes (tetW or tetO), most of which were functionally validated through \u003cem\u003ein vitro\u003c/em\u003e assays. Importantly, no mobile genetic elements were detected in the genomes of our isolates, suggesting a low risk of horizontal gene transfer.\u003c/p\u003e\n\u003cp\u003eOrally administered microbial agents face numerous challenges in achieving clinical efficacy as they pass through the gastrointestinal tract. Their viability and cellular integrity can be hampered by the highly acidic conditions of the stomach and subsequent exposure to bile salts in the small intestine\u003csup\u003e46\u003c/sup\u003e. Furthermore, as most gut commensals, they are often highly sensitive or intolerant to oxygen\u003csup\u003e47\u003c/sup\u003e. To address these challenges, we evaluated the tolerance of our isolates to oxygen, bile salts, and pH, with the goal of optimizing production and administration methods to ensure that the bacteria reach their target site in the gut and exert their beneficial effects. Under our experimental conditions, the isolates exhibited relative tolerance to oxygen, which may facilitate production and encapsulation strategies. However, they were unable to grow under either acidic or basic conditions, indicating limited survival in the stomach. Encapsulation thus represents a promising delivery method, with the choice of encapsulation materials being critical for protecting the therapeutic agent. Extensive research will be required to identify processes that ensure host safety while allowing reliable and scalable production\u0026nbsp;\u003csup\u003e48\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e49\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e50\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding bile salt tolerance, some isolates were sensitive even at low concentrations (2%), highlighting the need for specific delivery strategies, as discussed above for pH and oxygen. In contrast, other isolates were able to grow at higher bile concentrations, accompanied by morphological changes suggestive of bile-induced biofilm formation. Biofilm formation is a known bacterial strategy to survive the bactericidal effects of bile during gastrointestinal transit\u003csup\u003e51\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e52\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e53\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e54\u003c/sup\u003e, although it has been primarily observed in pathogenic species. Further investigation using electron microscopy could clarify the morphological changes observed, as bile salts are known to directly affect the bacterial cell surface\u003csup\u003e55\u003c/sup\u003e. The bile resistance observed in several isolates may also be attributed to bile salt hydrolase (BSH) activity. BSH enzymes, which are widespread in the human gut microbiota, contribute to bacterial survival and persistence in the gastrointestinal tract by deconjugating bile salts\u003csup\u003e56\u003c/sup\u003e. Beyond microbial survival, BSH enzymes play a critical role in host metabolism as bile salt deconjugation has been linked to cholesterol reduction and decreased weight gain\u003csup\u003e57\u003c/sup\u003e. Bile acids, synthesized in the liver, are released into the duodenum and reabsorbed in the colon through enterohepatic circulation, influencing metabolic pathways via nuclear receptors such as the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5\u003csup\u003e58\u003c/sup\u003e. Activation of these receptors by bile acids may modulates lipid, glucose, and cholesterol metabolism\u003csup\u003e59\u003c/sup\u003e, making them promising targets for drug development in metabolic diseases, including MASLD. Investigating whether our isolates possess BSH activity and the capacity to influence these pathways is therefore of significant interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e assays demonstrated that both the pellet and supernatant of our isolates exert strain-dependent inhibitory effects on the NF-\u0026kappa;B pathway in intestinal epithelial cells and hepatocytes, indicating potential anti-inflammatory properties. These results are consistent with our previous findings using the type strain \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450\u003csup\u003e11\u003c/sup\u003e. Notably, none of the isolates carry genes involved in equol production from daidzein, indicating that this function is not required for these anti-inflammatory effects. However, the strains may metabolize other polyphenols, such as resveratrol, as previously reported for non-equol-producing strains, including \u003cem\u003eA. rubneri\u003c/em\u003e \u003cem\u003eResAG\u003c/em\u003e\u003cem\u003e-91\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e. This raises the possibility that the anti-inflammatory activity of these isolates could be enhanced through polyphenol supplementation.\u003c/p\u003e\n\u003cp\u003eTo further investigate the mechanism of action on the NF-\u0026kappa;B pathway, we examined the implication of Toll-Like-Receptor-2 (TLR2) using HEK293 reporter cell lines. TLR2 is a host receptor expressed on epithelial cell membrane and activated by cell-wall components of Gram positive bacteria. TLR2 interacts with the myeloid differentiation factor 88 (MyD88), an adaptator protein which leads to the activation of NF-\u0026kappa;B and to the induction of pro-inflammatory cytokines\u003csup\u003e60\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e61\u003c/sup\u003e. In this study, we used the control cell line HEK Null1, which do not express TLR2, to determine whether the observed inhibition of the NF-\u0026kappa;B pathway was dependent of TLR2. We found that both supernatant and bacterial cell-wall components (pellet) had a mild effect on control HEK cells, whereas they strongly activated TLR2-dependent NF-\u0026kappa;B pathway, although variability exist between strains under basal conditions. We did not find evidence that the anti-inflammatory properties were specific to the TLR2 pathway in inflammatory conditions as an inhibition of the inflammatory pathway was observed similarly in both reporter cell lines (HEK Null1 and HEK TLR2). Therefore, the mechanism of action by which our isolates exert their beneficial effects needs to be further clarified. Notably, it would be interesting to investigate whether the anti-inflammatory properties of the strains are linked to our observations with bile acids. Indeed, receptors such as FXR and TGR5 are known to inhibit NF-\u0026kappa;B activity, thus lowering inflammation\u003csup\u003e62\u003c/sup\u003e. However, in the \u003cem\u003ein vitro\u003c/em\u003e set-up no bile salts were added. If we hypothesize that our strains possess BSH activity, they may be able to deconjugate bile salts. This, in turn, could activate FXR or TGR5 receptors, leading to the repression of NF-\u0026kappa;B activity and providing further anti-inflammatory properties.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWithin the Actinomycetota phylum, \u003cem\u003eBifidobacteria\u003c/em\u003e are the most extensively studied in relation to human health, but interest in \u003cem\u003eCoriobacteria\u003c/em\u003e, particularly the genus \u003cem\u003eAdlercreutzia\u003c/em\u003e, is rapidly increasing. In this study, we isolated and characterized eleven new non-equol-producing \u003cem\u003eAdlercreutzia\u003c/em\u003e strains from the stool of healthy French volunteers. The anti-inflammatory properties previously reported for \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450 were also observed in these isolates. Thorough \u003cem\u003ein vitro\u003c/em\u003e assays revealed specific functional and physiological traits, providing important insights into their mechanisms of action and informing their potential clinical and industrial applications.\u003c/p\u003e\n\u003cp\u003eFurther studies are needed to ensure the safety of these strains. Additionally, validating their beneficial effects in more complex models, such as gut organoids or gut-on-chip systems, will allow a more accurate assessment of their therapeutic potential and generate data required for regulatory approval.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll sequencing data generated in this study have been submitted to the EMBL-EBI\u0026rsquo;s European Nucleotide Archive (ENA) and are available under the BioProject accession number PRJEB97395. All genome assemblies have also been submitted under the same BioProject and are accessible via NCBI GenBank.\u003c/p\u003e\n\u003cp\u003eEthics declaration\u003c/p\u003e\n\u003cp\u003eStool samples from which the strains were isolated were anonymously collected from healthy volunteers. In accordance with French regulations, this collection was declared to the French Ministry of Higher Education, Research and Innovation (no. DC-2015-2513) and received approval from the Ethical committee (Comit\u0026eacute; de Protection des Personnes CPP EST I) in March 2016. All participants received written information and provided informed consent prior to participation.\u003c/p\u003e\n\u003cp\u003eAuthors contributions\u003c/p\u003e\n\u003cp\u003eC.C. and F.P.O. wrote the manuscript. C.C. designed the study, performed the experiments, and analyzed the data. M.B. performed microbial characterization and conducted the \u003cem\u003ein vitro\u003c/em\u003e assays. A.L. contributed to figure preparation, data interpretation, and manuscript drafting and revision. B.Q. performed genome sequencing. E.M. and N.L. contributed to data interpretation and manuscript writing and editing. F.P.O. performed the bioinformatics genome analyses. P.Y.M. and H.M.B. supervised the project, secured funding, and critically revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by Novobiome. Additional support was provided by the MetaGenoPolis grant (ANR-11-DPBS-0001), which funded part of the infrastructure used in this project. We thank the Bioaster team (Samuel Bellais and Vincent Thomas) for strain isolation.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eP.Y.M. is co-founder and CEO of Novobiome. H.M.B. is co-founder and scientific advisor of Novobiome. C.C., M.B., and A.L. are employees of Novobiome. 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Pathog.\u003c/em\u003e \u003cstrong\u003e2007\u003c/strong\u003e, \u003cem\u003e43\u003c/em\u003e (2), 78\u0026ndash;87. https://doi.org/10.1016/j.micpath.2007.04.002.\u003c/li\u003e\n\u003cli\u003eRuiz, L.; S\u0026aacute;nchez, B.; Ruas-Madiedo, P.; De Los Reyes-Gavil\u0026aacute;n, C. G.; Margolles, A. Cell Envelope Changes in Bifidobacterium Animalis Ssp. Lactis as a Response to Bile. \u003cem\u003eFEMS Microbiol. Lett.\u003c/em\u003e \u003cstrong\u003e2007\u003c/strong\u003e, \u003cem\u003e274\u003c/em\u003e (2), 316\u0026ndash;322. https://doi.org/10.1111/j.1574-6968.2007.00854.x.\u003c/li\u003e\n\u003cli\u003eBourgin, M.; Kriaa, A.; Mkaouar, H.; Mariaule, V.; Jablaoui, A.; Maguin, E.; Rhimi, M. Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e9\u003c/em\u003e (6), 1122. https://doi.org/10.3390/microorganisms9061122.\u003c/li\u003e\n\u003cli\u003eLong, S. 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Syndr.\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e26\u003c/em\u003e (2), 86\u0026ndash;96. https://doi.org/10.7570/jomes.2017.26.2.86.\u003c/li\u003e\n\u003cli\u003eSipka, S.; Bruckner, G. The Immunomodulatory Role of Bile Acids. \u003cem\u003eInt. Arch. Allergy Immunol.\u003c/em\u003e\u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e165\u003c/em\u003e (1), 1\u0026ndash;8. https://doi.org/10.1159/000366100.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"508\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrains characteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMorphology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003eCoccobacilli ; single or chains\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth conditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003eAnaerobic ; 37\u0026deg;C ; pH 7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram stain\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpore formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCatalase\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOxidase\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDNase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49.4094%;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePhysiological characteristics of the newly isolated strains and \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 1945\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReactions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eIndole (IND)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eUrea (URE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eGlucose (GLU)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eMannitol (MAN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eLactose (LAC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eSaccharose (SAC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eMaltose (MAL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eSalicin (SAL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eXylose (XYL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eArabinose (ARA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eGelatin (GEL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eEsculin (ESC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eGlycerol (GLY)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eCellobiose (CEL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eMannose (MNE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eMelezitose (MLZ)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eRaffinose (RAF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eSorbitol (SOR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eRhamnose (RHA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68.8742%;\"\u003e\n \u003cp\u003eTrehalose (TRE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.1258%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Biochemical properties of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe newly isolated strains and \u003cem\u003eA. equolifaciens\u003c/em\u003e DSM 19450\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;margin-left: -0.25pt;border-collapse: collapse;width: 1014px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:92.35pt;border:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eBacterial strains\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" style=\"width: 667.9pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eAntibiotics MIC (\u0026micro;g/mL)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:84.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eMetronidazole\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:84.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eChloramphenicol\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eVancomycin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eClindamycin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eImipenem\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003ePiperacillin-tazobactam\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.2pt;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eTetracycline\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.35pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eAe DSM 19450\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,032\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,094\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,75\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,125-0,38\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.35pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003eCNCM I-6207\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e0,19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri Light\";color:black;'\u003e1,5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(252, 228, 214);padding: 0cm 3.5pt;height: 15.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;text-align:center;text-indent:0cm;font-size:11.0pt;font-family:\"Calibri Light\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Calibri 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anaerobes\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adlercreutzia species, inflammation, metabolic diseases, live biotherapeutic product, gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-8574898/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8574898/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Adlercreutzia, a prevalent genus in the human gut microbiota, gains growing interest due to the potential beneficial effects on human health, particularly in the context of metabolic and inflammatory diseases. Previous work showed the A. equolifaciens type strain exhibits anti-inflammatory properties, but whether this effect is strain-specific or linked to equol production remained unclear.\nIn this study, we isolated 11 novel Adlercreutzia strains from healthy European volunteers. Whole-genome sequencing classified most isolates closely related to A. rubneri or A. equolifaciens species. Phenotypic characterization revealed all isolates are obligate anaerobes, asaccharolytic, non-equol producers and exhibit strain-dependent tolerance to oxygen and bile salts, opening up possibilities for therapeutic options. Functional assays demonstrated that all strains exert significant anti-inflammatory effects in vitro by downregulating NF-κB activation in human intestinal epithelial and liver cells. These findings reveal potent anti-inflammatory activity independent of equol production, positioning these novel, well-characterized Adlercreutzia strains as promising candidates for microbiome-based therapies targeting metabolic diseases.","manuscriptTitle":"Comparative genomics and functional characterization of 11 newly isolated non-equol-producing Adlercreutzia strains with anti-inflammatory properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 05:57:55","doi":"10.21203/rs.3.rs-8574898/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b349bc6b-4301-496b-b09c-397cf80c55c5","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64095892,"name":"Biological sciences/Biotechnology"},{"id":64095893,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-18T10:41:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 05:57:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8574898","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8574898","identity":"rs-8574898","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}