DOI: 10.15468/u9swmc
title: Blautia argi sp. nov., a new anaerobic bacterium isolated from dog faeces

description: This dataset contains the digitized treatments in Plazi based on the original journal article Paek, Jayoung, Shin, Yeseul, Kook, Joong-Ki, Chang, Young-Hyo (2019): Blautia argi sp. nov., a new anaerobic bacterium isolated from dog faeces. International Journal of Systematic and Evolutionary Microbiology 69 (1): 33-38, DOI: 10.1099/ijsem.0.002981, URL: http://dx.doi.org/10.1099/ijsem.0.002981AbstractTwo isolates of a Gram-positive, non-motile, coccoid or oval-shaped anaerobic bacterium, designated strains N6H1-15 T and YH1_16, were isolated from faecal samples obtained from a mature dog. Analysis of 16S rRNA gene sequences indicated that the isolates belonged to the Blautia coccoides rRNA gene group (cluster XIVa) and were closely related to Blautia hansenii KCTC 5951 T, Blautia stercoris KCTC 5981 T, Blautia producta producta KCTC 3695 T and B. coccoides DSM 15327 T, with 96.7, 94.4, 94.2 and 93.9 % sequence similarity, respectively. The two isolates contained m -diaminopimelic acid within their peptidoglycans. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and the major fatty acids were C 16: 0 (18.5 %), C 16: 0 (18.0 %) and C 18: 1 cis 9 (16.2 %). The predominant metabolic end products of glucose fermentation were acetic and lactic acids, and the G+C content was 44.2 mol%. Thus, the polyphasic data suggest that the two new isolates represent a new species, proposed as Blautia argi sp. nov. The type strain is N6H1-15 T (= KCTC 15426= JCM 31394).The composition, diversity and function of the microbiome in the human and animal intestine have been demonstrated using molecular biological approaches such as metagenomics [1, 2]. Although the composition of the microbiome is variable, the Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria and Fusobacteria groups comprise approximately 90 % of intestinal bacteria [1, 3]. The intestinal genus Blautia was first proposed by Liu et al. in 2008 and includes a number of Gram-positive, non-motile, coccoid or oval-shaped obligate anaerobes that produce acetate, ethanol, lactate, and succinate as the end products of glucose fermentation [4]. The genus Blautia currently comprises 13 species with validly published names [4 – 6]. In addition, two species belonging to the genus Clostridium, nine species belonging to the genus Ruminococcus, and a number of species within the Firmicutes [4, 7] have been reclassified as Blautia species. During a previous investigation into the diversity of the intestinal microbiota in various animals, two novel strains, designated N6H1- 15 T and YH1_16, were isolated from a faecal sample taken from a mature pet dog (Argi lupus familiaris). In the present study, a polyphasic taxonomic approach was used to evaluate the taxonomic position of these isolates, leading to their identification as a new species of Blautia.Samples (1 g) of mature dog faeces were obtained and placed immediately in sterile tubes under an anaerobic atmosphere generated using the GasPak EZ anaerobe pouch system (Becton Dickinson). Samples were stored at ‒70 Ǫ C. When required, the samples were homogenized and serially diluted (up to a 10 ‒8) in sterile saline solution [0.85 % (w/v) NaCl]. Volumes were spread-plated onto Reinforced Clostridial Medium (RCM; Difco) supplemented with 5 % sheep blood and incubated anaerobically at 37 Ǫ C for 48 h using a Bactron anaerobic system (Sheldon) with a gas phase of N 2: H 2: CO 2 (88: 7: 5 %, v/v) [8]. Blautia hansenii KCTC 5951 T, Blautia stercoris KCTC 5981 T, Blautia producta KCTC 3695 T and Blautia coccoides DSM 15327 T were used as reference strains for phenotypic characterization and chemotaxonomic analyses. Isolates and reference strains were routinely cultured on RCM agar with 5 % sheep blood at 37 Ǫ C and preserved in 10 % (w/v) skim milk (BD) or 20 % (v/v) glycerol at ‒70 Ǫ C [8].Near-complete 16S rRNA gene sequences of the isolates were determined according to the method of Chang et al. [9]. Preliminary sequence comparisons were conducted using the 16S rRNA gene sequences of prokaryotic type strains with validly published names retrieved from the EzTaxon-e program (www.ezbiocloud.net/) and GenBank databases [10]. The sequences were manually aligned with representatives of closely related genera using the bacterial 16S rRNA secondary structure model [11]. Only regions available for all sequences (conserved and variable regions) that showed unambiguous alignment were used to reconstruct the phylogenetic trees. The alignment and neighbourjoining tree [12] analyses were performed using the PHYLIP [13] and jPHYDIT programs [14]. Three algorithms were employed to reconstruct phylogenetic trees: neighbourjoining [15], maximum-likelihood [16] and maximumparsimony [17]. The reliability of the resulting trees was evaluated using bootstrap analyses [18] based on 1000 resamplings. Atopobium minutum ATCC 32267 T was used as an outgroup.Author affiliations: 1 ABS Research Support Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea; 2 Korean Collection for Oral Microbiology and Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju 501-759, Republic of Korea. *Correspondence: Young-Hyo Chang, yhchang@kribb.re.krKeywords: genome; Blautia argi; anaerobic; phylogenetic; new species; dog.Abbreviations: AL, aminolipid; ANI, average nucleotide identity; DPG, diphosphatidylglycerol; DAP, m -diaminopimelic acid; GGD, genome-to-genome distance; GL, glycolipid; PG, phosphatidylglycerol; PL, phospholipid; RCM, reinforced clostridial medium.The GenBank/ EMBL / DDBJ accession numbers for the 16S rRNA gene sequences of strain N6H1-15 T are KX025138. The NCBI accession number for the whole-genome sequence of strain N6H1-15 T is CP030280.Two supplementary figures and two supplementary tables are available with the online version of this article.The genomic DNA of strain N6H1-15 T was prepared as described by Tamaoka et al. [19]. The DNA concentration was determined by the Epoch Microplate Spectrophotometer (BioTek Instruments) at wavelengths of 260 and 280 nm [20]. The genomic DNA was sequenced using the PacBio RSII platform by Macrogen. A 20 kb DNA library was reconstructed according to the manufacture’ s protocol and sequenced using single-molecule real-time sequencing with P6 DNA polymerase and C4 chemistry. A total of 713 317 789 bp (328×coverage) was read with 126 570 subreads. The de novo assembly was performed by RS HGAP Assembly 3.0 [21] with the default option. Genome annotation was conducted by the NCBI Prokaryotic Genome Annotation Pipeline [22] through the NCBI 1 Genome submission portal (GenomeSubmit at http://ncbi.nlm.nih.gov). The GeneBank accession number of the genome of strain N6H1-15 T is CP030280.The genome average nucleotide identity (ANI) analysis was conducted using the calculator provided by ChunLab (www. ezbiocloud.net/tools/ani). The genome-to-genome distance (GGD) analysis was performed using the GGD calculator (GGDC) provided by the German Collection of Microorganisms and Cell Cultures (http://ggdc.dsmz.de). Whole genome sequences and their GenBank accession numbers of the strains for ANI and GGD analyses were downloaded from the GenBank database (www.ncbi.nlm.nih.gov/ genome) and are listed in Tables S1 and S 2 (available in the online version of this article).Cell morphology and flagellation were observed under a phase-contrast microscope (E600, Nikon) and a transmission electron microscope (CM20, Philips), respectively [8]. Gliding motility was determined on 0.4 % (w/v) semi-solid RCM medium containing 5 % bovine blood serum [9]. Gram staining of isolates was performed using a bioḾerieux Gram stain kit according to the manufacturer’ s instructions. Endospores of cells previously incubated at 37 Ǫ C for 5 days on RCM agar plates containing 5 % sheep blood were observed by phase-contrast microscopy after staining with 5 % malachite green and safranin [8]. The temperature, pH, and salinity ranges were determined in RCM broth containing 5 % bovine blood serum. The temperature range supporting growth was evaluated in 5 Ǫ C increments between 10 and 60 Ǫ C. The pH range for growth was assessed in 1 pH unit increments within the range of 3.0 to 10.0, and the salinity range allowing growth was tested in 1 % (w/v) increments in NaCl concentrations ranging from 0 to 6 % [9]. Additional phenotypic and biochemical characterization was performed using API ZYM, 32A and API 50 CH test strips with 50CHB/E medium according to the manufacturer’ s instructions.Cells were incubated at 37 Ǫ C for 2 days in PYG broth [1 % (w/v) bactopeptone, 1 % (w/v) glucose, and 0.05 % (w/v) yeast extract] supplemented with 5 % bovine serum to identify the products of anaerobic fermentation. The culture broth was filtered through a 0.22 µm pore size filter, mixed with an equal volume of 10 % (v/v) phosphoric acid, and analysed using a GC system (Agilent Technologies) equipped with a flame-ionization detector and a HP-INNO-Wax column (10 m ×250 µm×0.25 µm; Agilent Technologies). Helium was used as the carrier gas at a flow rate of 1 ml min ‒1. The oven temperature was programmed to increase from 50 to 170 Ǫ C at a rate of 10 Ǫ C min ‒1. The injector and detector temperatures were set to 250 Ǫ C [8, 9].For cellular fatty acid analysis, the isolates were cultured at 37 Ǫ C for 2 days on RCM blood agar (pH 7.0; Difco) and then harvested from the fourth quadrant of quadrantstreaked plates. The fatty acids were saponified, methylated and extracted according to the standard protocol in the MIDI/Hewlett Packard Microbial Identification System, analysed by GC (6890N and 7683 autosampler; Agilent Technologies), and identified using the BHIBLA library (version 4.0) of the Microbial Identification System [23].The structure of the peptidoglycan was examined as described previously [8] with a slight modification in which two-dimensional TLC on cellulose plates was used instead of paper chromatography. Polar lipids were extracted and separated from 100 mg of freeze-dried cellular material using a two-stage protocol, as previously described by Tindall [24]. TLC plates were stained with 5 % molybdophosphoric acid to detect all lipids. The DNA G+C content was determined by reversed-phase HPLC, as described by Mesbah et al. [25].Cells of the isolates were Gram-positive and coccoid or oval shaped. They lacked flagella and endospores, and were nonmotile (Fig. S1); all characteristics have been previously reported for members of the genus Blautia [26]. The optimal growth temperature was 37 Ǫ C, the optimal NaCl concentration for growth was 1 % and the optimal pH for growth was pH 7.0. The isolates and reference strains showed similar profiles for biochemical tests: they were positive for b- galactosidase, a- galactosidase, L- arabinose, galactose, glucose, fructose, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, melibiose, sucrose and gentiobiose, but negative for alkaline phosphatase and a- fucosidase, glycerol, erythritol, xylose, adonitol, sorbose, dulcitol, mannitol sorbitol, mannose, raffinose, inulin, glycogen, D- frocose and L-arabitol. Detailed phenotypic characteristics of strains N6H1-15 T and YH1_16 are presented in Table 1 and are described below.Phylogenetic analysis revealed that the isolates belonged to the Blautia coccoides rRNA gene group (cluster XIVa) whose members are closely related to B. hansenii KCTC 5951 T, B. stercoris KCTC 5981 T, B. producta KCTC 3695 T and B. coccoides DSM 15327 T. In the phylogenetic tree, the isolates formed a cluster with the type strains of Blautia species, joining B. hansenii KCTC 5951 T with a bootstrap value of 97 % (Fig. 1). Isolates N6H1-15 T and YH1_16 had closely related 16S rRNA gene sequence similarity values to: B. hansenii KCTC 5951 T (96.7 %), followed by B. stercoris KCTC 5981 T (94.4 %), B. producta KCTC 3695 T (94.2 %) and B. coccoides DSM 15327 T (93.9 %). The isolates showed less than 93 % similarity to other species belonging to the genus Blautia within the family Lachnospiraceae.The complete genome size of strain N6H1-15 T was 3 297 975 bp, longer than those of the other Blautia species, B. hansenii KCTC 5951 T (3 053 221 bp). The ANI value between strain N6H1-15 T and type strains of related strains ranged from 65.84 to 77.09 % when analysed by the ANI calculator of the ChunLab or the Kostas Laboratory, respectively (Table S1). The GGD values between strain N6H1- 15 T and related strain B. hansenii KCTC 5951 T were&gt;24.5 %, which is the threshold for bacterial species discrimination (Table S2) [27, 28]. These results indicate that strain N6H1-15 T represents a novel Blautia species.Since the isolates and their closest relative, B. hansenii KCTC 5951 T, exhibited less than 97 % 16S rRNA gene sequence similarity, ANI and GGD were performed to more accurately differentiate these closely related strains. Isolates N6H1-15 T and YH1_16 were closely related to each other (approx. 91.5 % DNA relatedness), indicating that these strains were members of the same species. The ANI and GGD values of the novel isolate N6H1-15 T and its most closely related reference strain, B. hansenii KCTC 5951 T, indicated a low genomic relatedness of 77.1 and 24.5 %, respectively. These results also suggest that the isolates are members of a species distinct from those represented by other reference strains.The relative G+C content values were calculated based on Escherichia coli KCTC 2441 T DNA (51.9 mol%) as the standard. The mean DNA G+C content of the isolates and of the reference strains B. hansenii, B. stercoris, B. producta and B. coccoides were 44.2, 43.8, 45.7, 49.8 and 46.6 mol%, respectively. These values were within the typical G+C content range of members of the genus Blautia, which is 37–50 mol% [26].Metabolic end products of fermentation resulting from growth of the isolates in PYG broth containing 5 % bovine serum included acetic acid (47.5 mM), lactic acid (15.6 mM) and formic acid (0.7 mM). Although some differences were observed between the isolates and the reference strains, the major end product, acetic acid, was consistent with that identified in other members of the genus Blautia [26, 29].The predominant cellular fatty acids (mean value&gt;10 % of total fatty acids) of the isolates were C 14: 0 (18.5 %), C 16: 0 (18.0 %) and C 18: 1 cis 9 (16.2 %) (Table 2). The fatty acid profiles of the isolates were therefore similar to those of other members of the genus Blautia. In contrast, the profile of minor fatty acids was different to those in other closely related species. For example, the two isolates and strain B. hansenii KCTC 5951 T differed from all other strains in terms of the production of C 16: 0 as a minor fatty acid component. This information supports the hypothesis that the isolates are distinct among species of the genus Blautia. The fatty acid profiles of the isolates and the other closely related species are shown in Table 2.Table 1. Differential characteristics of isolates and closely related speciesStrains: 1, isolates; 2, Blautia hansenii KCTC 5951 T; 3, Blautia stercoris KCTC 5981 T; 4, Blautia producta KCTC 3695 T and 5, Blautia coccoides DSM 15327 T. +, Positive;, negative;, no data; A, acetic acid; F, for-‒ ND mic acid; L, lactic acid. Capital letters represent an amount of product equal to or greater than 1 mg per 100 ml of cultures and small letters represent an amount of product less than 1 mg per 100 ml of cultures in end products.Characteristic 1 2 3 4 5 End products A, L A, L A (l, A (l, A, L (f) (f) f) f) (f) Enzyme activities: Esterase (C4) – – – – + Acid phosphatase – – – + – a- Glucosidase + – + + + b- Glucosidase + – + + + N -Acetyl-b- – + – – – glucosaminidase Arginine dihydrolase + – + – – a - Arabinosidase + – + + + Glutamic acid – + – + – decarboxylase Mannose fermentation – – – + + Raffinose fermentation – – – + + DNA G+C content (mol%) 44.2 43.8 45.7 49.8 46.6Peptidoglycan analysis revealed that the isolates and reference strains contained meso -diaminopimelic acid (m -DAP) as the diagnostic amino acid. The presence of the m -DAP– type peptidoglycan (A31; DSMZ) is typical for the members of this phylogenetic cluster [30]. The polar lipid profiles of the isolates revealed diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG) to be the major polar lipids, with phospholipids (PLs) and aminolipids (ALs) and glycolipid (GL)comprising the minor polar lipids (Fig. S2).In conclusion, the novel isolates N6H1-15 T and YH1_16 shared the characteristic peptidoglycan type (m -DAP), major polar lipids (DPG, PG), major metabolic end products (acetic and lactic acids), and the major fatty acids C 14: 0, C 16: 0, and C 18: 1 cis 9. Phylogenetic analysis (Fig. 1) also indicated that the novel isolates belong to the same clade within the Blautia coccoides rRNA group (cluster XIVa) [29]. The 16S rRNA gene sequence similarity between the isolates and closely related reference strains was &lt;96.7 %, i.e. below the recently defined threshold for delineation of bacterial species [31]. The data presented in this study indicate that the two isolates can be differentiated from other members of the genus Blautia and represent a novel species, for which the name Blautia argi sp. nov. is proposed.