TAXONOMIC DESCRIPTION Feng et al., Int J Syst Evol Microbiol 2018;68:3546–3550 DOI 10.1099/ijsem.0.003028 Eubacterium maltosivorans sp. nov., a novel human intestinal acetogenic and butyrogenic bacterium with a versatile metabolism Yuan Feng,1 Alfons J. M. Stams,1,2 Irene Sanchez-Andrea 1 and Willem M. de Vos1,3,* Abstract A novel anaerobic, non-spore-forming bacterium was isolated from a faecal sample of a healthy adult. The isolate, T designated strain YI , was cultured in a basal liquid medium under a gas phase of H2/CO2 supplemented with yeast extract (0.1 g lÀ1). Cells of strain YIT were short rods (0.4–0.7Â2.0–2.5 µm), appearing singly or in pairs, and stained Gram-positive. Catalase activity and gelatin hydrolysis were positive while oxidase activity, indole formation, urease activity and aesculin hydrolysis were negative. Growth was observed within a temperature range of 20–45 C (optimum, 35–37 C), and a pH range of 5.0–8.0 (optimum pH 7.0–7.5). Doubling time was 2.3 h when grown with glucose at pH 7.2 and 37 C. Besides acetogenic growth, the isolate was able to ferment a large range of monomeric sugars with acetate and butyrate as the main end products. Strain YIT did not show respiratory growth with sulfate, sulfite, thiosulfate or nitrate as electron acceptors. The T major cellular fatty acids of the isolate were C16 : 0 and C18 : 0. The genomic DNA G+C content was 47.8 mol%. Strain YI is affiliated to the genus Eubacterium, sharing highest levels of 16S rRNA gene similarity with Eubacterium limosum ATCC 8486T (97.3 %), Eubacterium callanderi DSM 3662T (97.5 %), Eubacterium aggregans DSM 12183T (94.4 %) and Eubacterium barkeri DSM 1223T (94.8 %). Considering its physiological and phylogenetic characteristics, strain YIT represents a novel species within the genus Eubacterium, for which the name Eubacterium maltosivorans sp. nov. is proposed. The type strain is YIT (=DSM 105863T=JCM 32297T). À1 The human intestinal tract is colonized by billions of com- supplemented with 5 mM Na2SO3, yeast extract (0.1 g l ; mensal micro-organisms that represent over a thousand BD BBL) and H2/CO2 (80 : 20, v/v, 1.7 atm) in the gas phase. species contributing to either health or disease. Among Throughout the enrichment, sulfite was not reduced but H2 others, intestinal microbes convert undigested carbohy- and CO2 were consumed, and acetate was produced. Subse- drates mainly into short-chain fatty acids, such as butyrate, quently, a pure culture was obtained by a combination of propionate and acetate [1–3]. As colonic fermentations are serial dilution and plating on solidified media, with 1 % generally anaerobic, redox balancing often involves the pro- noble agar (Sigma-Aldrich), under 1.7 atm of H2/CO2 duction of hydrogen, which can also be consumed by (80 : 20, v/v). methanogens, sulfur compounds respirers or homoaceto- Cell morphology, motility, Gram-staining and spore for- genic bacteria [4]. mation were studied by phase-contrast microscopy using a In the course of a study to enrich sulfidogenic bacteria from Zeiss AXIO Scope A1. Gram-staining was performed the human gut (approved by CCMO Netherlands, project according to standard procedures [7]. Survival due to ID: NL2907008109), we isolated a novel acetogenic bacterial spore formation was checked by placing the cultivation strain (YIT) sharing 97.3 % 16S rRNA gene similarity with bottle in an 80 C water bath for 20 min. Oxidase and cata- Eubacterium limosum ATCC 8486T [5]. Strain YIT was lase activities were tested as described by Florentino et al. enriched under anaerobic conditions in a basal liquid [8]. Indole and urease formation, as well as gelatin and medium prepared according to Stams et al. [6] aesculin hydrolysis were examined in duplicate by the API Author affiliations: 1Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands; 2IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal; 3RPU Immunobiology, Department of Bacteriology and Immunology, Faculty of Medicine, University of Helsinki, 00014 Helsinki, Finland. *Correspondence: Willem M. de Vos, [email protected] Keywords: acetogens; butyrate; maltose; hydrogen; human gut microbiota. Abbreviations: ANI, average nucleotide identity; DDH, DNA–DNA hybridization. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Eubacterium maltosivorans strain YIT is MH400075. The GenBank accession number of the assembled draft genome is GCA_002441855.1 One supplementary figure and one supplementary table are available with the online version of this article. 003028 ã 2018 IUMS Downloaded from www.microbiologyresearch.org by IP: 128.214.144.1493546 On: Thu, 15 Nov 2018 07:39:36 Feng et al., Int J Syst Evol Microbiol 2018;68:3546–3550 Table 1. Selected physiological and biochemical characteristics that differentiate strain YIT from its closest described relatives The major fatty acids are shown in bold. +, Positive; À, negative; W, weak (after 5 days less than 3 mM of substrates were consumed). Both strains stained Gram-positive, and were non-motile, non-spore-formers. Both strains were able to use H2/CO2, CO, glucose, fructose, ribose, lactate, pyru- vate, mannitol, erythritol, vanillate, cysteine and betaine, and showed weak growth on soluble starch, but were not able to use lactose, arabinose, cel- lobiose, galactose, rhamnose, melibiose, succinate, glycine, serine, glycerol or ethanol. DMA denotes dimethylacetal. Characteristic E. limosum ATCC 8486T* Strain YIT† Growth temperature 25À45 20–45 (optimum, C) (30À37) (35À37) pH range 5.0–8.0 5.0–8.0 (optimum) (7.0–7.2) (7–7.5) DNA G+C content (mol%) 47.2 47.8 Genome size (Mbp) 4.37 4.58 Sugars D-xylose w† À Maltose À† + Mannose À† + Sucrose À† + Raffinose À + Acids Formate W W Vanillate +† + Alcohol Methanol + W Cellular fatty acids (%)† Saturated straight-chain 12 : 0 0.3 0.2 14 : 0 16.8 6.3 14 : 0 DMA 1.1 0.1 16 : 0 38.6 44.5 16 : 0 aldehyde 3.8 3.0 16 : 0 DMA 9.6 9.1 18 : 0 4.6 10.5 18 : 0 aldehyde 0.6 1.5 18 : 0 DMA 1.0 3.3 20 : 0 0.3 1.1 Unsaturated straight-chain 16 : 1!5c 0.2 0.7 16 : 1!7c 1.7 1.2 16 : 1!7c DMA 0.8 0.2 18 : 1!7c 8.1 7.7 18 : 1!7c DMA 1.3 0.9 Saturated branched-chain 15 : 0 0.3 0.2 15 : 0 anteiso 0.2 À 15 : 0 iso 0.2 À 17 : 0 cyclopropane 1.4 0.8 17 : 0 cyclo DMA 0.2 0.1 19 cycloprop-11,12 6.1 7.2 19 cyclo 11,12 DMA 1.2 1.7 *Data for E. limosum ATCC 8486T were from Genthner et al. [23] (except where otherwise indicated). †Data from this study. 20A test (bioMerieux) according to the manufacturer’s To determine the temperature range and optimum, strain instructions. YIT was grown in the basal medium supplemented with Downloaded from www.microbiologyresearch.org by IP: 128.214.144.1493547 On: Thu, 15 Nov 2018 07:39:36 Feng et al., Int J Syst Evol Microbiol 2018;68:3546–3550 À1 20 mM glucose under 1.7 atm of N2/CO2 (80 : 20, v/v) gas yeast extract l ) for 2 days. Cells were harvested and analy- phase without yeast extract and incubated for up to 6 weeks sis was performed at the Identification Service of the Deut- within the temperature range from 15 to 60 C (at 5 C sche Sammlung von Mikroorganismen und Zellkulturen intervals, 37 C was tested as well) at pH 7.2. The optimum (DSMZ). pH was tested in the same medium at 37 C but bicarbonate To obtain genomic DNA, strain YIT was grown in the afore- and N /CO were omitted. Different buffer systems were 2 2 mentioned basal medium containing 20 mM glucose under employed to give different pH ranges and the gas phase con- 1.7 atm of N2/CO2 (80 : 20, v/v) for 48 h at 37 C. Biomass was tained only N . For pH higher than 7.0, 20 mM Tris was 2 harvested by centrifugation at 13 000 g for 5 min at 4 C. used; for pH 6.0–7.0, 20 mM PIPES was added; and for pH Genomic DNA was isolated by using a MasterPure device 6–4, 20 mM citrate/phosphate buffer was used. Hence, (Epicentre) and purified via a Wizard Genomic DNA Purifi- growth over the pH range from 4.0 to 8.5 was tested at 0.5 cation Kit (Promega) following the manufacturers’ instruc- unit intervals, with incubation at 37 C for up to 6 weeks. tions. Illumina MiSeq sequencing was performed at GATC Both temperature and pH tests were run in triplicate. Biotech and assembled using the Edena v3.130110 and IDBA- To explore the physiological properties of strain YIT, a vari- UD v1.1.1 assemblers and merged [11, 12]. The assembled ety of substrates including sugars, organic acids, amino draft genome had a size of 4.5 Mbp and the sequence has been acids and sugar alcohols (see Table 1) were added to the deposited at GenBank under accession number basal medium to a final concentration of 20 mM without GCA_002441855.1. The 16S rRNA gene sequence (1512 bp) yeast extract, unless mentioned otherwise. Cultures were was obtained from the draft genome and deposited at Gen- incubated under 1.7 atm of N2/CO2 (80 : 20, v/v) at 37 C Bank under accession number MG015881.