Anaerostipes Faecalis Sp. Nov. Isolated from Swine Faeces

A novel, strictly anaerobic, gram-negative, segmented lamentous bacterium (SFB), strain AGMB03513 T , was isolated from the faeces of a 5-month-old pig. Comparative analysis of 16S rRNA gene sequences indicated that strain AGMB03513 T forms a lineage within the genus Anaerostipes and is most closely related to Anaerostipes butyraticus DSM 22094 T (= KCTC 15125 T , 95.8%), Anaerostipes hadrus DSM 3319 T (= KCTC 15606 T , 95.5%), Anaerostipes caccae DSM 14662 T (= KCTC 15019 T , 94.0%), and Anaerostipes rhamnosivorans DSM 26241 T (= KCTC 15316 T , 93.4%). Phylogenetic analysis based on the 16S rRNA gene and whole genome sequencing analysis revealed that its closest relatives are members of the family Lachnospiraceae and that the closest related is Anaerostipes butyraticus. Strain AGMB03513 T grows at temperatures of between 30 and 45°C within a pH range of 7.0 to 9.0, and in medium containing up to 1.5% NaCl. Cells were found to utilize d-glucose, d-mannitol, d-lactose, d-saccharose, d-maltose, d-xylose, l-arabinose, d-mannose, and d-sorbitol, and acetate was identied as the major end product of metabolism. The DNA G+C content of the strain is 37.0 mol%. Average nucleotide identity (ANI) values obtained in comparisons of strain AGMB03513 T with reference strains of species in the genus Anaerostipes were between 71.0% and 75.7%, which are below the ANI criterion for interspecies identity. The major components of cellular fatty acids were C 12:0 , C 16:0 , and C 18:0 . On the basis of phenotypic, phylogenetic, biochemical, chemotaxonomic, and genomic characteristics, we consider it reasonable to assign novel species status to strain AGMB03513 T , for which we propose the name Anaerostipes faecalis sp. nov. The type strain AGMB03513 T (=KCTC 25020 T =NBRC 114896 T ). 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.


Introduction
In the past few decades, antibiotics have been used indiscriminately to prevent diseases and promote livestock growth. However, this indiscriminate usage has given rise to the increasing emergence of antibiotic-resistant bacteria, which affects not only livestock but also humans (Gong et al., 2014). In recent years, antibiotic alternatives such as prebiotics and probiotics have been used with increasing frequency (Mingmongkolchai and Panbangred, 2018). Identifying viable alternatives to antibiotics is of particular importance in the pork industry, in which premature mortality accounts for approximately 15% of the stock. In this regard, the use of probiotics and prebiotics as antibiotic alternatives can improve health and limit disease by nurturing healthy intestinal microbiota through the promotion of optimal initial microbial colonization (Nowland et al., 2019). In suckling piglets, the bacterial genera Bacteroides, Oscillibacter, Escherichia, Shigella, Lactobacillus, and unclassi ed Ruminococcaceae are the primary components of the intestinal microbiota. In contrast, after weaning, species in the genera Acetivibrio, Dialister, Oribacterium, Succinivibrio, and Prevotella become increasingly prominent (Mach et al., 2015). Analysis of the intestinal contents of piglets, has revealed that the dominant genus Lactobacillus plays an important role in disease prevention by reducing gut populations of pathogenic bacteria (Beasley, 2004). Furthermore, a higher abundance of Prevotella has been found to be associated with increased body weight, which is correlated with the production of luminal secretory IgA by Prevotella within the gut (Mach et al., 2015). However, our current understanding of the role of the gut microbiota in promoting livestock health is notably limited, and therefore, there is a pressing need for further studies on the characterization of intestinal microorganisms, the effects of these microorganisms on host health, and microbial community manipulation (Nowland et al., 2019).
The genus Anaerostipes, within the phylum Firmicutes, was initially proposed by Schwiertz et al. (2002), who classi ed Anaerostipes caccae within this new genus and assigned it to the family Lachnospiraceae. The members of Anaerostipes are non-motile, rod-shaped, gram-variable obligate anaerobes (Schwiertz et al., 2002). At the time of writing, the genus Anaerostipes comprised four species with validly published names (www.bacterio.net/anaerostipes.html) (Euzéby, 1997). In this study, we isolated a novel strain, AGMB03513 T , from swine faeces, which was characterized through phenotypic, biochemical, phylogenetic, and chemotaxonomic analyses, and on the basis of our ndings, we propose that this strain represents a novel species within the genus Anaerostipes.

Materials And Methods
Isolation of the bacterial strain and culture conditions Strain AGMB03513 T was isolated from faeces of swine raised in the National Institute of Animal Science (Wanju, Republic of Korea). The collected samples were immediately maintained under anaerobic conditions and subsequently transported to the laboratory. Isolation was performed using an anaerobic chamber (Coy Laboratory Products, Michigan, USA) containing an atmosphere of 86% nitrogen, 7% hydrogen, and 7% carbon dioxide. The sample was serially diluted in a saline solution [0.85% (w/v) NaCl] and spread on tryptic soy agar containing 5% (v/v) sheep blood (TSAB). After incubation for 72 h at 37°C, single colonies were isolated. Identi cation was performed based on 16S rRNA gene sequencing, and taxonomic analysis was performed based on phylogenetic, phenotypic, biochemical, chemotaxonomic, and genomic analyses. The isolate was stored at -80°C in 10% (w/v) skim milk. For the purpose of comparative studies, we used strains of the four existing members of the genus, namely, Anaerostipes butyraticus KCTC 15125 T , Anaerostipes hadrus KCTC 15606 T , Anaerostipes caccae KCTC 15019 T , and Anaerostipes rhamnosivorans KCTC15316 T as reference species.
16S rRNA gene sequencing and phylogenetic analysis Genomic DNA was extracted from strain AGMB03513 T grown on Reinforced clostridial medium (RCM) (MB Cell, Republic of Korea; 10.0 g peptone, 5.0 g NaCl, 10.0 g beef extract, 3.0 g yeast extract, 5.0 g dextrose, 1.0 g starch, 0.5 g l-cysteine hydrochloride monohydrate, and 3.0 g sodium acetate per litre) supplemented with agar (15.0 g per litre) using the phenol:chloroform:isoamyl alcohol method (Wilson et al., 1990). The extracted genomic DNA was used to amplify the near complete sequence of the 16S rRNA gene by using the universal primer pair 785F (5′-GGATTAGATACCCTGGTA-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′). The ampli ed 16S rRNA gene was sequenced commercially by Macrogen Inc. (Republic of Korea). The complete 16S rRNA sequence was assembled by comparison with the whole-genome sequence and identi ed using the EZBioCloud (Yoon et al., 2017) and GenBank/EMBL/DDBJ (http://www.ncbi.nlm.nih.gov/blast) databases. Respective sequences of the 16s rRNA of the isolate and related strains were aligned using CLUSTAL W (Thompson et al., 1997), and phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA) 7.0.26 software (Kumar et al., 2016). Evolutionary distances were calculated using Kimura's two-parameter model (Kimura, 1980). Phylogenetic trees based on sequences of the 16S rRNA gene were reconstructed according to the neighbour-joining (NJ) (Saitou and Nei, 1987), maximum-likelihood (ML) (Fitch, 1971), and maximum-parsimony (MP) (Felsenstein, 1981) algorithms with bootstrap analysis (1000 replications).

Phenotypic and biochemical analyses
For the purposes of phenotypic and biochemical analyses, the AGMB03513 T isolate was grown on RCM agar for 24-48 h. Cell morphology was observed using an Eclipse 80i phase-contrast microscope (Nikon), a SUPRA 55VP scanning electron microscope (Carl Zeiss, Germany), and a Tecnai 10 transmission electron microscopy (FEI, USA). Gram staining was performed using a Gram stain kit (Difco) according to the manufacturer's instructions. KOH tests were based on determinations of the formation of viscous and mucoid strings within 15 s (Suslow, 1982). To determine the optimal growth conditions, cells were incubated at a temperature of 37°C, over a temperature range of 10 to 50°C (at 5°C intervals), and at eight different pH values (4, 5, 6, 7, 8, 9, 10, and 11). Salt tolerance was determined by growing cells in media with NaCl concentrations ranging from 0.5% to 4.0% (at 0.5% intervals). Growth was measured using a DU 700 UV-visible spectrophotometer (Beckman Coulter, CA, USA). Catalase activity was con rmed based on bubble formation using a catalase reagent (bioMérieux, #55561), and oxidase activity was veri ed based on the production of a purple colouration by using an oxidase reagent (bioMérieux, #55635). Spore formation was assessed using the Schaeffer-Fulton method with malachite green (Schaeffer and Fulton, 1933), whereas for characterization of biochemical properties, cells were analysed using API 20A, Rapid ID 32A, and ZYM strips (bioMérieux, Marcy-l'Étoile, France).

Chemotaxonomic and genomic characteristics
Fermentation end products were characterized in cell-free supernatants derived from cells cultured for 2 days in RCM broth by high-performance liquid chromatography (Shimadzu, Kyoto, Japan) equipped with Aminex TM Organic Acid Columns (Bio-Rad, CA, USA). The diamino acid in cell walls was determined using a previously described method (Komagata and Suzuki, 1988). Fatty acid pro les were determined in cells grown on RCM agar at 37°C for 24 h. The cellular fatty acids were saponi ed, methylated, and extracted according to instructions of the Chemical Analysis System (MIDI, DE, USA), and subsequently identi ed using gas chromatography (GC-2010; Shimadzu) and Sherlock TM Chromatographic Analysis System software package (Anaerobe Database version 6.4). Diamino acids in cell wall peptidoglycans were analysed as described previously (Schleifer and Kandler, 1972). Polar lipids were extracted using a chloroform/methanol method and analysed by two-dimensional thin-layer chromatography following a previously described method (Kates, 1986). Brie y, after extracting polar lipids from 100 mg freeze-dried cells, silica gel 60 F 254 aluminium-backed thin layer plates (Merck) were dotted with samples and subjected to two-dimensional development, with a rst mobile phase solvent of chloroform/methanol/distilled water (65:25:4, v/v) followed by a second mobile phase solvent of chloroform/methanol/acetic acid/distilled water (40:7.5:6:2, v/v). Whole-genome sequencing using the NovaSeq 6000 system (Illumina, San Diego, CA, USA) was performed at Macrogen Inc. to determine the G + C content of genomic DNA and for genomic analysis. The average nucleotide identity (ANI) and the average amino acid identity (AAI) were calculated using ChunLab's online ANI calculator with the OrthoANI algorithm (Lee et al., 2016) and AAI calculator tools (http://enve-omics.ce.gatech.edu/aai/) (Rodriguez and Konstantinidis, 2016). Digital DNA-DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator (GGDC) version 2.1 (Meier-Kolthoff et al., 2013). Whole-genome analysis was performed using CLgenomics TM software (ChunLab, Republic of Korea) and the UniProt database (https://www.uniprot.org).

Phylogenetic analysis
Approximately 1459 bases of the 16S rRNA gene were sequenced, and comparative analysis of the sequence indicated that strain AGMB03513 T is closely related to species in the genus Anaerostipes.
AGMB03513 T showed sequence similarities of between 93.3% and 95.8% with the reference bacteria, with highest similarity to A. butyraticus 35-7 T (KCTC 15125; 95.8%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain AGMB03513 T is a species within the family Lachnospiraceae (Fig.  1).

Phenotypic and biochemical characteristics
Cells of strain AGMB03513 T 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% CO 2 , whereas under anaerobic conditions, cells grew in several long chains of connected rods, referred to as segmented lamentous 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, dmannitol, d-lactose, d-saccharose, d-mannose, d-sorbitol, and d-ra nose, and to a limited extent, l-leucine.
As nal products of fermentation, strain AGMB03513 T produces acetate and small amounts of propionate and butyrate, the latter of which is the nal 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 nal fermentation product. Furthermore, strain AGMB03513 T showed no evidence of either catalase or oxidase activity.
Strain AGMB03513 T 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 AGMB03513 T 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 AGMB03513 T is grouped in the family Lachnospiraceae and closely related to species in the genus Anaerostipes. The strain AGMB03513 T 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 AGMB03513 T as a novel species in the genus Anaerostipes, for which the name Anaerostipes faecalis sp. nov. is proposed.
Description of Anaerostipes faecalis sp. nov.
The type strain AGMB03513 T (=KCTC 25020 T = NBRC 114896 T ) was isolated from swine faeces. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AGMB03513 T is MT534274, and the GenBank/EMBL/DDBJ accession number for the whole-genome sequence of strain AGMB03513 T is JABRXE000000000. All presented data were obtained in the present study. Values are percentages of total cellular fatty acids.
TR, trace amount (< 1%); −, not detected. Only fatty acids accounting for more than 1% in at least one of the strains are shown. Major components (>10%) are highlighted in bold.

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