Phylogenetic analysis
Approximately 1459 bases of the 16S rRNA gene were sequenced, and comparative analysis of the sequence indicated that strain AGMB03513T is closely related to species in the genus Anaerostipes. AGMB03513T showed sequence similarities of between 93.3% and 95.8% with the reference bacteria, with highest similarity to A. butyraticus 35-7T (KCTC 15125; 95.8%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain AGMB03513T is a species within the family Lachnospiraceae (Fig. 1).
Phenotypic and biochemical characteristics
Cells of strain AGMB03513T were found to be strictly anaerobic, gram-negative, non-motile, and formed spores. The strain failed to grow on RCM agar incubated in air or in an atmosphere containing 5% CO2, whereas under anaerobic conditions, cells grew in several long chains of connected rods, referred to as segmented filamentous bacteria (SFB) (Figs. S1 and S2). Colonies grown on RCM agar were circular, convex, white, opaque, and shiny, and grew at temperatures of between 35 and 45°C (optimum at 37°C). In RCM broth, cells were found to grow at pH values ranging from 7 to 9 (optimum pH 7) and NaCl concentrations up to 1.5%. The isolate was observed to utilize carbon sources, such as d-glucose, d-mannitol, d-lactose, d-saccharose, d-mannose, d-sorbitol, and d-raffinose, and to a limited extent, l-leucine. As final products of fermentation, strain AGMB03513T produces acetate and small amounts of propionate and butyrate, the latter of which is the final fermentation product of the reference strain of A. caccae used in the present study (Table 1). However, none of the four reference strains were found to produce acetate as the final fermentation product. Furthermore, strain AGMB03513T showed no evidence of either catalase or oxidase activity.
Chemotaxonomic and genomic characteristics
The major cellular fatty acids (>10%) of strain AGMB03513T were C12:0 (20.8%), C16:0 (16.8%), and C18:0 (11.9%). Comparatively, the major fatty acids of the four reference strains in this study are as follows: A. caccae DSM 14662T: C12:0 (29.7%), C18:0 DMA (12.5%), and C18:0 ALDE (19.5%); A. butyraticus DSM 22094T: C12:0 (32.0%), C18:0 (12.1%), and C18:0 ALDE (12.2%); A. rhamnosivorans DSM 26241T: C12:0 (32.1%), C16:0 (9.4%), and C18:0 ALDE (12.6%); and A. hadrus DSM 3319T: C11:0 DMA (13.2%), C12:0 (24.5%), C18:0 DMA (15.4%), and C18:0 ALDE (22.7%). Details of the cellular fatty acid profiles of strain AGMB03513T and the reference strains are shown in Table 2. Strain AGMB03513T was found to contain the following polar lipids: three glycophosphoaminolipids, four glycolipids, four unidentified lipids, one glycophosphoaminolipid, one glycoaminolipid, one phospholipid, and an aminolipid (Fig. S4).
The genome of strain AGMB03513T is 2,544,126 bp in length and contains 2,492 coding sequences, and genes encoding 10 rRNA and 59 tRNAs. The ANI and AAI values obtained based on comparisons between strain AGMB03513T (JABRXE000000000) and strains of the four congeneric species A. butyraticus JCM 17466 (BLYI00000000), A. caccae NCIMB 13811T (CP036345), A. hadrus ATCC 29173T (AMEY00000000), and A. rhamnosivorans 1y-2T (CP040058) were 75.3%, 71.0%, 75.5%, and 71.2% and 73.2%, 66.6%, 72.5%, and 66.7%, respectively, and the respective dDDH values were 19.5%, 20.2%, 20.1%, and 21.4%. These values were found to be notably lower than the threshold values of ANI and AAI (95%–96 %) and dDDH (70 %) for differentiating bacterial species.
Strain AGMB03513T was also found to contain meso-diaminopimelic acid (DAP) in the cell wall (Fig. S3), which is synthesized from l-aspartate, and l-aspartate via tetrahydrodipicolinate (THDPA) as an intermediate product (Rodionov et al., 2003, Xu et al., 2019). Within cells, l-aspartate is converted to THDPA via activity of the lysC, asd, dapA, and dapB gene products (Rodionov et al., 2003), and there are several pathways whereby THDPA is converted to DAP (Xu et al., 2019), among which is the succinylase pathway containing the enzyme encoded by dapDH. In addition, the new isolate expresses the dapL and dapF genes that play roles in the meso-DAP/L-lysine biosynthetic pathway.
DNA G+C content
The G + C content of strain AGMB03513T genomic DNA was found to be 37.0 mol%, which compared with the values of 45.5−46.0 mol%, 44.0 mol%, 44.5 mol%, and 37.0 mol% obtained for the reference strains of A. caccae, A. butyraticus, A. rhamnosivorans, and A. hadrus, respectively (Allen-Vercoe et al., 2012) (Table 1).
Taxonomic conclusions
Phylogenetic tree analysis based on 16S rRNA gene sequences revealed that strain AGMB03513T is grouped in the family Lachnospiraceae and closely related to species in the genus Anaerostipes. The strain AGMB03513T showed 93.3%−95.5% identity to the four reference strains with respect to the 16S rRNA gene sequence and showed clear similarities as well as differences with respect to phenotypic, biochemical, chemotaxonomic, and genomic characteristics. On the basis of this evidence, we consider it reasonable to designate strain AGMB03513T as a novel species in the genus Anaerostipes, for which the name Anaerostipes faecalis sp. nov. is proposed.
Description of Anaerostipes faecalis sp. nov.
Anaerostipes faecalis sp. nov. (fae.ca’lis. L. fem. adj. faecalis derived from faeces).
Cells were long rod-shaped, gram-negative, non-motile, and non-spore forming obligate anaerobes. SEM images revealed a segmented filamentous bacterial morphology. Colonies cultured for 24–48 h on RCM agar were circular, convex, white, opaque, and shiny. Growth occurred at temperatures between 30 and 45°C (optimum 37°C) within a pH range from 7 to 9 (optimum pH 7). API 20A strip analysis indicated that cells produce acid from d-glucose, d-mannitol, d-lactose, d-saccharose, d-mannose, and d-sorbitol, whereas acid production from d-maltose, d-xylose, and l-arabinose was weakly positive, and no acid was produced from salicin, glycerol, d-cellobiose, d-melezitose, d-raffinose, d-rhamnose, or d-trehalose. Neither indole nor urease was detected. Additionally, esculin and gelatin hydrolysis were absent. API Rapid ID 32A strip analysis revealed positive reactions for alkaline phosphatase and the fermentation of d-mannose and d-raffinose, whereas negative reactions were detected for urease, arginine dihydrolase, α-galactosidase, β-galactosidase, β-galactosidase-6-phosphate, α-glucosidase, β-glucosidase, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, α-fucosidase, nitrate reduction, indole production, arginine arylamidase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Leucine arylamidase activity was, however, found to be weakly positive. API ZYM strip analysis indicated positive reactions for alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, whereas esterase (C4) and leucine arylamidase showed weakly positive activity. In contrast, negative reactions were observed for esterase lipase (C8), lipase (C14), valine arylamidase, crystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, β-glucosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. As end products of fermentation, cells produce acetate, propionate, and butyrate. meso-DAP was identified as the diagnostic cell-wall diamino acid. The cell polar lipid profile comprised three glycophosphoaminolipids, four glycolipids, four unidentified lipids, one glycophosphoaminolipid, one glycoaminolipid, one phospholipid, and an aminolipid, and the major cellular fatty acids (>10%) were C12:0, C16:0, and C18:0. The G + C content of genomic DNA was 37.0 mol%.
The type strain AGMB03513T (=KCTC 25020T= NBRC 114896T) was isolated from swine faeces. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AGMB03513T is MT534274, and the GenBank/EMBL/DDBJ accession number for the whole-genome sequence of strain AGMB03513T is JABRXE000000000.