Alkaliphilus flagellatus sp. nov., Butyricicoccus intestinisimiae sp. nov., Clostridium mobile sp. nov., Clostridium simiarum sp. nov., Dysosmobacter acutus sp. nov., Paenibacillus brevis sp. nov., Peptoniphilus ovalis sp. nov. and Tissierella simiarum sp. nov., isolated from monkey faeces.

Non-human primates harbour diverse microbiomes in their guts. As a part of the China Microbiome Initiatives, we cultivated and characterized the gut microbiome of cynomolgus monkeys (Macaca fascicularis). In this report, we communicate the characterization and taxonomy of eight bacterial strains that were obtained from faecal samples of captive cynomolgus monkeys. The results revealed that they represented eight novel bacterial species. The proposed names of the eight novel species are Alkaliphilus flagellatus (type strain MSJ-5T=CGMCC 1.45007T=KCTC 15974T), Butyricicoccus intestinisimiae MSJd-7T (MSJd-7T=CGMCC 1.45013T=KCTC 25112T), Clostridium mobile (MSJ-11T=CGMCC 1.45009T=KCTC 25065T), Clostridium simiarum (MSJ-4T=CGMCC 1.45006T=KCTC 15975T), Dysosmobacter acutus (MSJ-2T=CGMCC 1.32896T=KCTC 15976T), Paenibacillus brevis MSJ-6T (MSJ-6T=CGMCC 1.45008T=KCTC 15973T), Peptoniphilus ovalis (MSJ-1T=CGMCC 1.31770T=KCTC 15977T) and Tissierella simiarum (MSJ-40T=CGMCC 1.45012T=KCTC 25071T).

Introduction

Gastrointestinal tracts accommodate diverse microbes, and those microbes together in a host gastrointestinal tract are called gut microbiomes (GMs) [1]. Many efforts have been made to characterize the microbial diversities of human [2-6] and animal GMs [7, 8], by culture-dependent and/or -independent methods [9-11]. Non-human primates (NHPs) are the most biologically relevant animal models for human studies [12]. The compositions and dynamics of NHP GMs were evaluated, and members of the genera , , , , and were cultivated and reported from the gastrointestinal tract of NHPs [13-17]. Based on analyses of major and large-scale investigations of human GMs [2-6], there are 5000–6000 bacterial species associated with humans. An exploration of cultivated human gut bacterial species diversity revealed that about 1500 bacterial species are recorded with valid and correct names, and that more gut bacterial species have been cultivated but not characterized or nominated [2]. Those cultivated but unnamed bacterial species have remained as ‘uncultivated’ in databases such as silva [18] and the Genome Taxonomy Database [19], and they are repeatedly claimed as ‘novel bacteria’ in later studies. Bacterial cultivation, characterization with polyphasic methods, and deposits in culture collections are essential to nominate a bacterial name following the rules of International Code of Nomenclature of Prokaryotes. Considering the large numbers (usually more than thousands) of bacterial isolates from one microbiome study, it is a challenge to characterize and nominate all bacterial isolates from microbiome studies.

Members of the family [20], such as and , are frequently detected in guts and they play important roles in host health. species, a group of 153 validly published and correct specific names (https://lpsn.dsmz.de/genus/clostridium) and represented by the type species [21], are Gram-positive, obligately anaerobic rods and form oval or spherical endospores. The genus is also a member of family [22] and contains seven validly published and correct specific names (https://lpsn.dsmz.de/genus/alkaliphilus). species are strictly anaerobic and their cells are usually straight to slightly curved rods. The genus of the family [23] is one of the largest genera of prokaryotes and members are widely distributed in natural environments and animal and human GMs. At the time of writing, 270 species names are validly published (https://lpsn.dsmz.de/genus/paenibacillus). species are usually straight to slightly curved rods and motile with flagella. The type species is and was isolated from decomposing plant materials and humus-rich soils. The genus is a member of the family [24]. species are non-spore-forming, Gram-positive, obligately anaerobic cocci, and the type species is . At the time of writing, 20 species of the genus have been validly published (https://lpsn.dsmz.de/genus/peptoniphilus). The genera and are members of the family and were proposed by Le Roy et al. [25] and Eeckhaut et al. [26], respectively. So far, only the type species is described (https://lpsn.dsmz.de/genus/dysosmobacter). is an obligately anaerobic, non-spore-forming and non-motile rod. The genus has four validly and correctly named species (https://lpsn.dsmz.de/genus/butyricicoccus), and they were isolated from human or animal faeces. Cells of the genus are anaerobic, non-motile and coccoid; the type species is . The genus belongs to the family [27]. species are obligately anaerobic, Gram-negative, non-spore forming rods. At the time of writing, five specific names have been validly published (https://lpsn.dsmz.de/genus/tissierella), and the type species is .

The China Microbiome Initiatives (CMI) integrated multiple studies of human and animal GMs and environmental microbiomes [28]. As a part of the CMI, we cultivated and characterized the GM of cynomolgus monkeys (Macaca fascicularis). In this report, we present the characterization and taxonomy of eight bacterial strains that were obtained from faecal samples of captive cynomolgus monkeys. The eight bacterial strains are affiliated with five families and were identified as new members of the genera , , , Alkaliphilus, Paenibacillus, and .

Methods

Sample collection and treatment

All faecal samples were from cynomolgus monkeys (M. fascicularis) at the experimental animal centre of the Institute of Neuroscience, Chinese Academy of Sciences, Suzhou, PR China. Fresh faecal samples were collected and maintained in airtight bags on dry ice, and were delivered immediately to the laboratory. The samples were diluted with sterile PBS and filtered through a 40 µm cell strainer and were treated with 70 % ethanol or heated at 85 °C for 30 min, as described in the literatures [29, 30].

Culture media, bacterial isolation and cultivation

The following media were used for bacterial cultivation: FAB (Fastidious anaerobe broth) medium (LA4550, Solarbio) and YCFA (yeast extract, casein hydrolysate, fatty acids) medium [31], modified GAM (Gifu anaerobic medium) medium (mGAM) [2, 32], and modified R-medium named by Dione N et al [33]. The mGAM medium (per 1 l) contained 10 g casitone, 3 g soya peptone, 15 g proteose peptone, 13.5 g digested serum, 5 g yeast extract, 2 g beef extract powder, 1.2 g liver extract, 0.3 g soluble starch, 0.5 g l-cysteine, 0.5 g l-arginine, 0.3 g l-tryptophan, 2 g NaHCO3, 2.5 g KH2PO4, 3 g NaCl, 0.15 g CH2(SH)COONa, 2.46 g CH3COONa, 0.01 g haemin, 0.001 g resazurin, 0.3 g glucose, 0.3 g d-galactose, 0.3 g cellobiose, 0.3 g mannose, 0.3 g fructose, 0.3 g rhamnose, 0.3 g palatinose, 0.3 g inulin, 15 g agar, adjusted pH to 7.2, sterilized at 115 °C for 25 min. The modified R medium was prepared from two solutions that were prepared, sterilized and separated: solution A (per 900 ml) consisted of 6 g casein hydrolysate, 5 g peptone, 5 g yeast extract, 1 g glucose, 1 g inulin, 1 g d-fructose, 1 g cellobiose, 1.5 g NaCl, 0.1 g MgSO4. H2O, 5 ml haemin (0.1%, w/v), 1 ml resazurin (0.1%, w/v), 20 ml (2%, v/v) rumen fluid, 15 g agar, adjusted pH to 7.2, sterilized at 112 °C for 15 min. Solution B (per 100 ml) consisted of 0.4 g l-cysteine, 1 g ascorbic acid, 0.1 g glutathione, 2 g α-ketoglutarate, 0.45 g K2HPO4, 0.9 g KH2PO4, adjusted pH to 7.2, filtered using a 0.2 µm micro filter.

Sterilized agar plates were inoculated with dilutions of pretreated faecal samples and incubated at 37 °C under strictly anaerobic conditions with N2 (85 %), H2 (10 %) and CO2 (5 %) in an anaerobic chamber (Electrotek AW400SG workstation). Colonies appeared after cultivation for 2, 5 and 10 days were picked and re-streaked on agar plates of same media. Bacterial purity was evaluated by observation of morphology, 16S rRNA gene and genome sequencing.

Cell morphology observation and chemotaxonomic determinations

Cell morphology was determined by transmission electron microscopy (JEM-1400, jeol). The utilization of carbon sources was determined using the 96-well Biolog AN MicroPlate that contained 95 different carbon substrates [34]. Bacteria strains were cultured in liquid mGAM medium for 2 days, then cells were harvested. Cellular fatty acids were extracted and methylated according to the standard midi protocol (Sherlock Microbial Identification System, version 6.0). The identification was performed by GC (HP 6890 Series GC System; Agilent) [35]. Polar lipids were separated by two-dimensional thin-layer chromatography (TLC plates coated with silica gel, 1010 cm; Merck). Chromatography was performed using chloroform–methanol–water (65 : 25 : 4, by vol.) for the first dimension, followed by chloroform–methanol–acetic acid–water (80 : 12 : 15 : 4, by vol.) for the second dimension [36]. Total lipids were detected with 10 % ethanolic molybdatophosphoric acid (Sigma). Aminolipids were detected with 0.4 % solution of ninhydrin (Sigma) in butanol. Phospholipids were detected with Zinzadze reagent (molybdenum blue spray reagent, 1.3 %; Sigma) and glycolipids were detected with 0.5 % α-naphthol sulphuric acid reagent.

Fermentative production of short-chain fatty acids

Bacterial strains were cultivated for 72 h in mGAM broth at 37 °C under strictly anaerobic conditions. Short-chain fatty acids (SCFAs) were measured using GC-MS. Culture (1 ml) was extracted with 1 ml ethyl acetate. The supernatant liquid was prepared for GC-MS analysis, which was performed on a GCMS-QP2010 Ultra with an auto sampler (Shimadzu) and the DB-wax capillary column (30 m, 0.25 mm i.d., 0.25 µm film thickness, Agilent Technologies). The temperature of oven was programmed from 35 to 130 °C at 5 °C min−1 gradient, to 230 °C at 30 °C min−1 gradient, with 16 min hold. Injection of 2 µl sample was performed at 230 °C. The carrier gas, helium, flowed at 1.0 ml min−1. Electronic impact was recorded at 70 eV.

16S rRNA gene sequencing and phylogenetic analysis

Complete 16S rRNA gene sequences of isolates were obtained using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′- GGTTACCTTGTTACGACTT-3′) [37]. 16S rRNA gene sequences similarities were determined using the EzBioCloud server [38]. Multiple alignments of sequences were performed using the Clustal W [39]. The phylogenetic trees were reconstructed by the neighbour-joining method [40] according to Kimura’s two-parameter model [41] in mega X [42], by the maximum-likelihood method [43] based on the Tamura–Nei model, and the maximum-parsimony method [44] based on the subtree-pruning-regrafting search method. The statistical reliability of the trees was calculated by bootstrap analysis with 1000 replications [45].

Genome sequencing and analysis

Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega) and the genomic DNA library was sequenced on an Illumina Hiseq X-ten platform. All good-quality paired reads were assembled using SPAdes software (version 3.9.0) [46]. The average nucleotide identity (ANI) values with closely related and available genomes were calculated using OAT software at www.ezbiocloud.net/sw/oat along with teh UPGMA dendrogram (unweighted pair group method with arithmetic mean) [47]. The genomic distances, digital DNA–DNA hybridization (dDDH), were calculated by using the Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/) [48]. Genome analysis using the Check M indicated that the genomes of eight strains were not contaminated [49].

Culture preservation

Bacterial strains were cultured in liquid medium for 2 days. We stored our own cultures (1 ml) in the lab by addition of an equal volume of 65 % (v/v) glycerol (1 ml), and was storing at −80 °C for long-term preservation. All type strains assigned by this study were deposited at China General Microbiological Culture Collection Center (CGMCC) and the Korean Collection for Type Culture (KCTC), and strain numbers are included in the species descriptions.

Results and discussion

Source and isolation of the bacteria

Strains MSJ-1T, MSJ-2T, MSJ-4T, MSJ-5T, MSJ-6T, MSJd-7T, MSJ-11T and MSJ-40T were isolated from faeces samples of Macaca fascicularis. Strain MSJ-1T was obtained from the sample after enrichment, strains MSJ-4T and MSJ-5T were obtained from samples after heat treatment (85 °C for 30 min), and strains MSJ-2T, MSJ-6T, MSJd-7T, MSJ-11T and MSJ-40T were obtained from samples after 70 % ethanol treatment for 30 min. Strains MSJ-1T, MSJ-2T, MSJ-4T and MSJ-11T were successfully cultivated first with FAB, strain MSJ-5T first from YCFA, strain MSJd-7T first from modified mGAM, and MSJ-40T first from modified R media. However, we later demonstrated that they all grew with mGAM medium.

Bacterial growth and cell morphology

Strains MSJ-1T, MSJ-2T, MSJ-4T, MSJ-5T, MSJ-6T, MSJd-7T, MSJ-11T and MSJ-40T were strictly anaerobic bacteria. They grew on mGAM agar at 37 °C and formed visible colonies after 1–10 days. Colonies of MSJ-2T, MSJ-4T, MSJd-7T and MSJ-40T were white, colonies of MSJ-5, MSJ-6 and MSJ-11 were grey, and colonies of MSJ-2 were tiny and translucent. No pigments were observed. Additional features are provided in the species description. Cellular morphology was examined with transmission electron microscopy and are shown in Fig. 1. Cells of strains MSJ-1T and MSJd-7T were spherical-shaped. Cells of strains MSJ-2T, MSJ-4T, MSJ-5T, MSJ-6T, MSJ-11T and MSJ-40T were rod-shaped. Flagella were observed for strains MSJ-4T, MSJ-5T, MSJ-6T, MSJ-11T and MSJ-40T, but not for MSJ-1T and MSJd-7T. Additional features of those strains are detailed in the species description.

Fig. 1.

Cellular morphology (transmission electron microscopy) of eight bacteria isolated from M. fascicularis. The names of each bacterium and the size of bars are showed in each picture.

Assimilation of carbon sources and fermentative production of SCFAs

The assimilation of 95 carbon sources were tested with Biolog AN MicroPlates and the results are recorded in Fig. 2(a). The eight bacteria showed different carbon source spectra, and in total 75 out of the 95 carbon sources were metabolized. We found that mono- and di-saccharides were preferred by the strains, which are extensively found in guts [50]. The eight bacteria all assimilated five carbon sources, i.e. d-fructose, l-fucose, d-galacturonic acid, palatinose and pyruvic acid.

Fig. 2.

Assimilation of the carbon sources on Biolog AN MicroPlates (a, purple indicates positive and white indicates negative) and production of short-chain fatty acids in mGAM medium (b). The mixed standards contain six short-chain fatty acids. 1, Acetic acid; 2, propanoic acid; 3, isobutyric acid; 4, butyric acid; 5, isovaleric acid; 6, valeric acid.

Many gut microbes produce SCFAs that are related to host health [50, 51]. We determined the production of SCFAs in an mGAM broth that contained glucose, d-galactose, cellobiose, mannose, fructose, rhamnose, palatinose and inulin. The results showed that each strain produced unique profiles of SCFAs (Fig. 2b). Butyric acid was produced by MSJ-1T, MSJ-4T, MSJ-5T, MSJ-6T, MSJ-11T and MSJ-40T. Propionic acid was produced MSJ-4T, MSJ-11T, MSJ-6T and MSJ-40T. Acetic acid was produced by MSJ-1T, MSJ-2T, MSJ-4T, MSJ-5T, MSJ-6T, MSJ-11T and MSJ-40T. In addition to the above SCFAs, strains MSJ-6T and MSJ-11T produced also branched SCFAs of isobutyric acid and/or isovaleric acid. Strain MSJd-7T did not produce the six SCFAs detected in this study.

Cellular fatty acid and polar lipid profiling

The chemotaxonomic cellular fatty acid and polar lipid profiles for the eight bacteria were determined and are summarized. As shown in Tables 1 and 2, the eight bacteria had different cellular fatty acid profiles, but they all had C14 : 0, C16 : 0, C18 : 0 and anteiso C15:0. Taking 10 % as cutoff value for predominant cellular fatty acids, MSJ-1T had C16 : 0 (19.9 %), MSJ-2T had C16 : 0 (20.1 %), MSJ-4T had C14 : 0 (15.2 %) and C16 : 0 (24.5 %), MSJ-5T had C16 : 0 (10.6 %) and iso-C13 : 0 (11.3 %)/anteiso-C17 : 0 (14.5 %)/anteiso-C15 : 0 (15.7 %)/iso-C16 : 0 (16.8 %), MSJ-6T had C16 : 0 (20.6 %)/C18 : 0 (10.5 %) and iso-C16 : 0 (17.1 %)/anteiso-C15 : 0 (25.6 %), MSJd-7T had C14 : 0 (10.9 %)/C16 : 0 (26.8 %)/C18 : 0 (22.0 %) and iso-C17:1 ω5c (12.8 %), MSJ-11T had C14 : 0 (19.8 %)/C16 : 0 (37.4 %)/C18 : 0 (11.7 %), and MSJ-40T had mainly iso-C15 : 0 (62.2 %). Polar lipid profiling showed that all eight bacteria had diphosphatidylglycerol and phosphatidylglycerol, but were different from each other in terms of the presence or not of phosphatidylethanolamine, phosphatidylmethylethanolamine, unknown phospholipids, unknown lipids and unknown glycolipids, as detailed in Tables 1 and 2, and Fig. S1 (available in the online version of this article).

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General features of genome and genomic DNA G+C contents

The eight bacteria had different genome sizes, MSJ-1T was the smallest (2.1 Mbp) and MSJ-6 T was the largest genome (5.2 Mbp). Genome sequencing data and some basic features of coding density and G+C contents are listed in Table 3. The genomic DNA G+C molar contents of MSJ-1T, MSJ-2T, MSJ-4T, MSJ-5T, MSJ-6T, MSJd-7T, MSJ-11T and MSJ-40T were 30.65, 58.27, 30.46, 31.71, 49.3, 50.29, 30.38, 44.49 and 30.39 mol%, respectively.

Table 3.

Genome features of the eight bacterial strains from M. fascicularis

Genome features	MSJ-1T	MSJ-2T	MSJ-4T	MSJ-5T	MSJ-6T	MSJd-7T	MSJ-11T	MSJ-40T
Genome Size (bp)	2 102 036	3 161 374	3 811 517	3 614 516	5 239 947	2 711 934	4 014 245	4 088 863
G+C content (mol%)	30.65	58.27	30.46	31.71	49.3	50.29	30.38	30.39
Completeness (%)	98.6	94.63	99.19	97.87	98.66	99.33	100	99.13
Contamination (%)	0.7	0.67	0.93	0.24	0	0.67	0.57	1.98
Number of contigs	14	2	70	51	64	27	18	53
N50 of contigs (bp)	300 523	3 156 307	406 816	534 169	198 601	357 991	1 137 742	199 075
Gene number	2037	3296	3532	3591	4800	3023	4017	4177

The eight bacterial strains represent novel taxa

Based on the 16S rRNA gene and genomic data, we further investigated the phylogenetic and phylogenomic relationships of the eight bacteria to their closely related and currently validly nominated bacterial taxa (Figs 3, S2 and S3). ANI scores based on whole genomes were used to generated UPGMA dendrogram trees (Fig. 4). Combining the results from DNA molecule analysis and the phenotypic characterization, we concluded that MSJ-1T, MSJ-2T, MSJ-4T, MSJ-5T, MSJ-6T, MSJd-7T, MSJ-11T and MSJ-40T represented novel species of the currently known genera (for details, see the following sections).

Fig. 3.

Phylogenetic trees of the eight bacteria strains based on 16S rRNA gene sequences and using the neighbour-joining algorithm showing the relationships between the eight novel bacteria and their closely related micro-organisms. (a) The phylogenetic tree of strains MSJ-5T, MSJ-4T, MSJ-11T, closely related species in the genera and , and the type species of the other genera in the family . (b) The phylogenetic tree of strains MSJ-2T, MSJd-7T, closely related species in the genera and , and the type species of the other genera in the family . (c) The phylogenetic tree of strain MSJ-6T, closely related species in the genus , and the type species of the other genera in the family Paenibacilaceae. (d) The phylogenetic tree of strain MSJ-40T, closely related species in genus , and the type species of the other genera in the family . (e) The phylogenetic tree of strain MSJ-1T, closely related species in the genus , and the type species of the the other genera in the family . GenBank accession numbers are given in parentheses. Bootstrap percentages (>50 %) based on 1000 replicates are shown at the nodes. Phylogenetic trees based on the maximum-likelihood method (Fig. S2) and the maximum-parsimony method (Fig. S3) are available as supplementary materials with the online version. DSM 4136 (X90515) was used as an outgroup. Bar, 0.05 substitutions per nucleotide position.

Fig. 4.

UPGMA phylogenetic trees and ANI heat maps based on whole genomes. Each of the eight UPGMA phylogenetic trees and the ANI heat maps displays the connections between a novel bacterial taxon and its closely related neighbours, the new names proposed in this study are showed in red. GenBank accession numbers of the genomes are shown in parentheses.

Strain MSJ-1T

The phylogenetic trees revealed that strain MSJ-1T clustered within the previously described genus clade, supported by a 100 % bootstrap value (Fig. 3d, S2d and S3d). Strain MSJ-1T was closely related to WAL10418T (96.82 % identity), DSM 100661T (95.93 %) and DSM 10020T (95.64 %) [24, 52, 53]. In the phylogenomic tree, strain MSJ-1T also formed a separate branch located in the genus clade (Fig. 4). The ANI and dDDH values between strain MSJ-1T and its closest neighbour DSM 100661T (GCA 900106515.1) were 78.79 and 20.50 %, respectively (Fig. 4). At the time of writing, the genus contains 20 species with validly published names [54]. Cells of members are non-spore-forming, obligately anaerobic and coccus-shaped. In addition to its unique 16S rRNA gene and genome sequences, MSJ-1T contains the predominant fatty acid of C16 : 0, which is consistent with most species of the genus (Table 1), although the fatty acids of other members in the genus are more diverse [55]. Based on phenotypic, chemotaxonomic and phylogenetic results, as well as phylogenomic and genome data, we suggest that strain MSJ-1T represents a novel species affiliated with the genus and the name Peptoniphilus ovalis sp. nov. is proposed.

Table 1.

Cellular fatty acids and polar acid compositions of strains MSJ-1T, MSJ-4T, MSJ-11T, MSJ-5T, MSJ-40T and closely related type strains of the families , and

Strains: 1, MSJ-1T; 2, CCUG 9988T [55]; 3, WAL 10408T [55]; 4, MSJ-4T; 5, MSJ-11T; 6, DSM 100320T [20]; 7, DSM 4474T [20]; 8, MSJ-5T; 9, CGMCC 1.5124T [63]; 10, DSM 21761T [63]; 11, MSJ-40T; 12, DSM 18095T [27]. Major fatty acid components are indicated with bold text. –, Not detected; tr, trace amount (<1.0 %).

Strains	1	2	3	4	5	6	7	8	9	10	11	12
Fatty acids (>10 % of total fatty acids):
C16 : 0	19.9	14.4	24.0	24.5	37.4	17.7	19.4	10.6	5.0	8.5	7.2	20.0
C18 : 0	6.8	–	–	3.8	11.7	13.1	3.1	7.7	1.3	4.4	5.1	1.5
C14 : 0	4.9	5.4	2.9	15.2	19.8	10.8	43.4	5.4	3.2	4.9	9.0	8.0
C18:1 ω9c	4.2	20.2	22.6	1.1	2.22	1.3	–	–	4.0	4.2	–	–
anteiso-C15 : 0	4.0	–	–	4.1	2.1	–	–	15.7	1.4	3.3	2.3	–
anteiso-C17 : 0	3.1	1.6	–	0.9	1.0	1.3	1.5	14.5	–	–	0.8	–
iso-C16 : 0	2.1	–	–	0.9	–	–	–	16.8	–	–	–	tr
iso-C15 : 0	1.2	2.6	–	9.8	1.1	–	–	9.6	41.4	27.9	62.2	44.5
iso-C13 : 0	–	–	–	9.3	–	–	–	11.3	9.7	10.5	4.5	–
C18:2 ω6,9c /ante-C18 : 0	3.6	22.0	21.1	0.6	–	–	–	–	–	–	–	–
Polar lipids*	DPG, PG, PE, PL1, PL2, PL3, L	–	–	DPG, PG, PE, PL1, PL2, PL3, L	DPG, PG, PE, PL1, PL2, PL3, L, APL	PG, PE, PL	–	DPG, PG, PE, PME, PL, L	–	–	DPG, PG, GL1, GL2, GL3, PL1, PL2	–

*APL, aminophospholipid; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PME, phosphatidylmethylethanolamine; PL, unknown phospholipids; L, unknown lipid; GL, unknown glycolipid.

Description of Peptoniphilus ovalis sp. nov.

Peptoniphilus ovalis sp. nov. (o.va’lis. L. masc. adj. ovalis, pertaining to an egg, egg-shaped).

Cells are non-mobile cocci with diameters of approximately 0.6–0.8 µm, and no flagellum. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0. Produces butyric acid and acetic acid from fermentation. After 2 days of cultivation on mGAM agar plate, colonies are 1–2 mm in diameter, white, circular, entire, opaque and smooth. Cells metabolize dextrin, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, α-d-glucose, glucose-6-phosphate, lactulose, d-mannose, melibiose, 3-methyl-d-glucose, palatinose, l-rhamnose, glyoxylic acid, α-hydroxybutyric, β-hydroxybutyric, α-ketobutyric acid, α-ketovaleric acid, d- and l-lactic acid, d-lactic acid methyl ester, d-malic acid, pyruvic acid, pyruvic acid methyl ester, urocanic acid, l-alanyl-l-histidine, l-glutamic acid, l-glutamine, l-serine, 2′-deoxy adenosine, inosine, thymidine, uridine, thymidine-5′-monophosphate and uridine-5′-monophosphate. The predominant cellular fatty acid is C16 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids and an unknown lipid. Genome size is 2 102 036 bp and the G+C content is 30.65 mol%.

The type strain is MSJ-1T (=CGMCC 1.31770T=KCTC 15977T) and was isolated from faecal samples of M. fascicularis.

Strain MSJ-2T

MSJ-2T was closely related to DSM 106889T (95.78 % 16S rRNA gene identity) [25], DSM 18026T (95.15 %) and JCM 18333T (94.72 %) [56, 57]. The phylogenetic tree indicated that MSJ-2T formed a heterogeneous cluster with members of and (Figs 3b, S2b and S3b). The OrthoANI tree clearly separated MSJ-2T and from and (Fig. 4). Furthermore, the G+C molar content of strain MSJ-2T (58.27 mol%) was closer to that of (58.9 mol%) than to (53.2 %) and (55.0%). Thus, MSJ-2T was more likely a member of genus . ANI and dDDH values between strain MSJ-2T and the closest cultivated neighbour DSM 106889T (GCA 005121165.1) were 74.09 and 21.20 %, respectively (Fig. 4), suggesting they represented different species within the genus . Cells of strain MSJ-2T were long rods, with no flagella. The predominant fatty acid of MSJ-2T was C16 : 0 (20.1 %), which distinguished this organism from DSM 106889T (C16 : 0<1 %) [25] (Table 2). Based on the chemotaxonomic, phylogenetic and genomic results described above, we conclude that strain MSJ-2T represents a novel species affiliated to the genus and the name Dysosmobacter acutus sp. nov. is proposed.

Table 2.

Cellular fatty acids and polar acids compositions of strains MSJ-2T, MSJ-6T, MSJd-7T and closely related type strains of the families and Paenibacilaceae

Strain: 1, MSJ-2T; 2, DSM 106889T [25]; 3, DSM 18026T [25]; 4, MSJ-6T; 5, JCM 31620T [65]; 6, DSM 27603T [65]; 7, MSJd-7T; 8, ATCC TSD-102T [66]; 9, DSM 23266T [66]. Major fatty acid components are indicated with bold text. –, Not detected; tr, trace amount (<1.0 %).

Strains	1	2	3	4	5	6	7	8	9
Fatty acids (>10 % of total fatty acids):
C16 : 0	20.1	tr	14.3	20.6	29.7	7.3	26.8	10.2	–
C18 : 0	8.7	tr	1.7	10.5	2.4	1.7	22.0	2.3	1.5
C14 : 0	7.7	2.4	11.5	5.2	3.4	1.5	10.9	15.7	6.4
C18:1 ω9c	4.5	tr	–	–	–	14.7	2.51	14.7	–
anteiso-C15 : 0	8.5	15.2	3.0	25.6	16.7	53.4	4.6	–	–
iso-C16 : 0	–	tr	–	17.1	3.5	18.9	7.3	–	–
iso-C15 : 0	3.4	24.2	8.3	8.3	3.9	3.3	–	2.3	1.5
iso-C13 : 0	1.6	tr	11.8	–	–	–	–	1.6	13.8
iso- C17:1 ω5c	–	–	–	–	–	–	12.8	–	–
Polar lipids	DPG, PG, GL1, GL2, GL3, PL	–	–	DPG, PG, PE, GL1, GL2, L	DPG, PG, PE, PL, APL, L	DPG, PG, PE, APL	DPG, PG, PL1, PL2, PL3, GL1, GL2, GL3, L1, L2	–	–

*APL, aminophospholipid; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PL, unknown phospholipids; L, unknown lipid; GL, unknown glycolipid.

Description of Dysosmobacter acutus sp. nov.

Dysosmobacter acutus (a.cu’tus. L. masc. adj. acutus, sharp, pointed referring to atypical cell shape).

Cells are non-mobile long rods with sharp ends. No flagellum. The cell size is approximately 0.5–0.6×2.7–2.9 µm. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0. Colonies are <1 mm in diameter after 5 days of incubation at 37 °C on mGAM agar plates and flat, circular, entire, translucent and smooth. Fermentative production is isovaleric acid, isobutyric acid and acetic acid. Cells metabolize cellobiose, dextrin, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, α-d-glucose, glucose-6-phosphate, lactulose, d-mannose, melibiose, 3-methyl-d-glucose, palatinose, l-rhamnose, acetic acid, formic acid, glyoxylic acid, α-ketobutyric acid, propionic acid, pyruvic acid and pyruvic acid methyl ester. The predominant cellular fatty acid is C16 : 0. The major lipids are phosphatidylglycerol and two unknown glycolipids. Genome size is 3 161 374 bp and the G+C content is 58.27 mol%.

The type strain is MSJ-2T (=CGMCC 1.32896T=KCTC 15976T), which was isolated from a faecal sample of M. fascicularis.

Strains MSJ-4T and MSJ-11T

Based on the phylogenetic and phynogenomic trees (Figs 3 and 4, S2a, S3a), strains MSJ-4T and MSJ-11T formed a cluster that was well-separated from the valid members of the genus [21]. MSJ-4T was closely related to DSM 15099T (96.85 % 16S rRNA gene identity) and NCTC 9836T (96.78 %) [58, 59] MSJ-11T was closely related to ATCC 25776T (98.33 %) [60]. The ANI and dDDH values of strain MSJ-4T to its closest neighbour NCTC 9836T (GCA900461105.1) were 78.07 and 22.10 %, respectively. The ANI and dDDH values of strain MSJ-11T to its closest cultivated neighbour KCTC 15555 T (GCA 900626095.1) were 74.93 and 18.50 %, respectively. Our results revealed that the major fatty acids of strains MSJ-4T and MSJ-11T were C16 : 0 and C14 : 0, which is consistent with the majority of species within genus [61, 62] (Table 1). Strains MSJ-4T and MSJ-11T belonged to genus and could be differentiated from each other and from other species of genus . Therefore, we conclude that strain MSJ-4T represents a novel species and the name Clostridium simiarum sp. nov. is proposed, and that strain MSJ-11T also represents a novel species for which the name Clostridium mobile sp. nov. is proposed.

Description of Clostridium simiarum sp. nov.

Clostridium simiarum (si.mi.a’rum. L. gen. pl. n. simiarum, of monkeys).

Cells are fat rods with blunt ends, 0.5–0.9×1.4–2.0 µm, and have peritrichous flagella. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0. Produces white, flat, circular, entire, opaque, smooth colonies with a diameter of 2–3 mm after 2 days of incubation at 37 °C on mGAM agar plates. Cells produce butyric acid, propanoic acid and acetic acid, isovaleric acid and isobutyric acid during fermentation. Assimilates the following carbon sources: dextrin, d-fructose, l-fucose, d-galacturonic acid, palatinose, acetic acid, formic acid, pyruvic acid and pyruvic acid methyl ester. The predominant cellular fatty acids are C16 : 0 and C14 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids and an unidentified lipid. Genome size is 3 811 517 bp and the G+C content is 30.46 mol%.

The type strain is MSJ-4T (=CGMCC 1.45006T=KCTC 15975T) and was isolated from faecal samples of M. fascicularis.

Description of Clostridium mobile sp. nov.

Clostridium mobile (mo’bi.le. L. neut. adj. mobile, motile).

Cells are rods with size of approximately 0.4–0.7×2.9–9.9 µm, and have flagella at both ends. Strictly anaerobic, with growth at 37 °C and pH 7.0. Colonies are grey, convex, circular, entire, and opaque with a diameter of 2–3 mm after 2 days of incubation at 37 °C on mGAM agar plates. The SCFAs produced by anaerobic fermentation are butyric acid, propanoic acid and acetic acid. Cells metabolize cellobiose, dextrin, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, α-d-glucose, glucose-6-phosphate, d-mannose, melibiose, 3-methyl-d-glucose, palatinose, l-rhamnose, turanose, glyoxylic acid, α-ketobutyric acid, pyruvic acid and pyruvic acid methyl ester. The predominant cellular fatty acids are C16 : 0, C14 : 0 and C18 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified aminophospholipid, three unknown phospholipids and an unidentified lipid. Genome size is 4 014 245 bp and the G+C content is 30.38 mol%.

The type strain is MSJ-11T (=CGMCC 1.45009T=KCTC 25065T) and was isolated from faecal samples of M. fascicularis.

Strain MSJ-5T

Strain MSJ-5T was closely related to CGMCC 1.5124T (96.29 % 16S rRNA gene identity) and DSM 21761T (96.14 %) [63, 64] (Figs 3a, S2a and S3a). Phylogenetic and phylogenomic trees revealed that strain MSJ-5T was in the genus clade. The ANI and dDDH values of strain MSJ-5T to its closest neighbour DSM 21761T (GCA 000018325.1) were 75.27 and 21.20 %, respectively (Fig. 4). Strain MSJ-5T had rod-shaped and motile cells, which was consistent with the description of the genus , and had G+C content of 31.71 mol%, within the range of 28–36 mol% for the genus [63]. The predominant fatty acid compositions vary among species but iso-C15 : 0, iso-C13 : 0, C16 : 0 and C14 : 0 are the major components in most of species, as detected in strain MSJ-5T. Anteiso-C15 : 0 and anteiso-C17 : 0 were detected in strain MSJ-5T and distinguish this isolate from other species (Table 1). Based on the polyphasic analysis, strain MSJ-5T should be classified as representing a novel species of the genus for which the name Alkaliphilus flagellatus sp. nov. is proposed.

Description of Alkaliphilus flagellatus sp. nov.

Alkaliphilus flagellatus (fla.gel.la'tus. L. neut. n. flagellum, a whip; L. part. adj. flagellatus, flagellated).

Cells are rods, 0.7–1.0×2.2–4.2 µm, and have bundled flagella at both ends. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0, . Produces grey, low convex, circular, entire, opaque colonies with a diameter of 1–2 mm after 2 days of incubation at 37 °C on mGAM agar plates. Fermentative products are butyric acid and acetic acid. Cells metabolize dextrin, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, d-glucosaminic acid, α-d-glucose, glucose-6-phosphate, lactulose, d-mannose, melibiose, 3-methyl-d-glucose, palatinose, l-rhamnose, α-ketobutyric acid, α-ketovaleric acid, pyruvic acid, pyruvic acid methyl ester, l-alanyl-l-glutamine, l-glutamic acid, l-glutamine, glycyl-l-glutamine, l-methionine, l-serine, l-threonine, 2′-deoxy adenosine, inosine, thymidine and uridine. The predominant cellular fatty acids are iso-C16 : 0, anteiso-C15 : 0, anteiso-C17 : 0, iso-C13 : 0 and C16 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, an unknown phospholipid and an unknown lipid. Genome size is 3 614 516 bp and the G+C content is 31.71 mol%.

The type strain is MSJ-5T (=CGMCC 1.45007T=KCTC 15974T) and was isolated from faecal samples of M. fascicularis.

Strain MSJ-6T

Strain MSJ-6T was closely related to JCM 31620T [65], with 96.94 % 16S rRNA gene sequence identity. The phylogenetic and phylogenomic analysis showed that strain MSJ-6T was in the clade (Figs 3 and 4, S2c, S3c). The genome size of MSJ-6T was 5 239 947 bp. The ANI and dDDH values of strain MSJ-6T to its closely related neighbour DSM 23593T (GCA 008084145.1) were 72.99 and 19.50%, respectively. The unique 16S rRNA and genome sequence is one of the characteristics of MSJ-6T. The predominant cellular fatty acids of species are anteiso-C15 : 0, C16 : 0, iso-C16 : 0 and iso-C15 : 0. Strain MSJ-6T shared this profile, but the presence of C18 : 0 differentiates it from other species (Table 2). In addition to diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, which is shared by species, MSJ-6T had two unknown glycolipids (Fig. S1). In terms of phenotypic, chemotaxonomic, phylogenetic and genomic features, strain MSJ-6T should be classified as representing a novel species of the genus for which the name Paenibacillus brevis sp. nov. is proposed.

Description of Paenibacillus brevis sp. nov.

Paenibacillus brevis sp. nov. (bre’vis. L. masc. adj. brevis, short, denoting the formation of short rods).

Cells are ovoid to short rods with size of approximately 0.3–1.42×2.57–3.57 µm, have 1–2 flagella. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0. After 5 days of incubation at 37 °C on mGAM agar plates, colonies are 1–2 mm in diameter, grey, circular, entire and translucent. The SCFAs produced by anaerobic fermentation are acetic acid, valeric acid, propanoic acid, butyric acid and isobutyric acid. Cells metabolize amygdalin, cellobiose, α-cyclodextrin, β-cyclodextrin, dextrin, dulcitol, i-erythritol, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, d-glucosaminic acid, α-d-glucose, glucose-1-phosphate, glucose-6-phosphate, m-inositol, lactose, lactulose, maltose, maltotriose, d-mannitol, d-mannose, melezitose, melibiose, 3-methyl-d-glucose, methyl α-d-galactoside, methyl β-d-galactoside, palatinose, raffinose, l-rhamnose, salicin, d-sorbitol, stachyose, sucrose, trehalose, turanose, acetic acid, fumaric acid, glyoxylic acid, α-ketobutyric acid, α-ketovaleric acid, propionic acid, pyruvic acid, pyruvic acid methyl ester, urocanic acid, l-alanine, glycyl-l-methionine, l-methionine, l-phenylalanine, l-serine, l-valine and l-valine plus l-aspartic acid. The predominant cellular fatty acids are anteiso-C15 : 0, C16 : 0, iso-C16 : 0 and C18 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unknown glycolipids and an unknown lipid. Genome size is 5 239 947 bp and the G+C content is 49.3 mol%.

The type strain is MSJ-6T (=CGMCC 1.45008 T=KCTC 15973T) and was isolated from faecal samples of M. fascicularis.

Strain MSJd-7T

Strain MSJd-7T was closely related to ATCC TSD-102T [66], with 97.15 % 16S rRNA gene sequence identity. The genome size of MSJd-7T is 2 711 934 bp. The phylogenetic and phylogenomic analysis revealed that strain MSJd-7T was a member of the genus clade (Figs 3 and 4, S2b, S3b). The ANI and dDDH values of strain MSJd-7T to its closest neighbour ATCC TSD-102T (GCA 002157465.1) were 75.88 and 21.10%, respectively. species have diverse cellular fatty acid compositions, but the predominant components are C14 : 0, C18 : 0 and C16 : 0 in most species of the genus. The presence of iso-C17:1 ω5c is characteristic of MSJd-7T and distinguished it from other species (Table 2). According to the phenotypic, chemotaxonomic, phylogenetic and genomic data, strain MSJd-7T should be classified as representing a novel species of the genus for which the name Butyricicoccus intestinisimiae sp. nov. is proposed.

Description of Butyricicoccus intestinisimiae sp. nov.

Butyricicoccus intestinisimiae (in.tes.ti.ni. si’mi.ae. L. neut. n. intestinum, intestine; L. fem. n. simia, a monkey; N.L. gen. n. intestinisimiae, of the monkey intestine, where the type strain dwells).

Cells are cocci with a diameter of approximately 1.46–1.85 µm, and no flagellum. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0, respectively. Colonies are 2–3 mm in diameter after 2 days of incubation at 37 °C on mGAM agar plates, white, convex, circular, entire, opaque and smooth. Anaerobic and fermentative production of valeric acid. Cells metabolize i-erythritol, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, d-glucosaminic acid, α-d-glucose, glucose-6-phosphate, d-mannose, melibiose, 3-methyl-d-glucose, palatinose, l-rhamnose, d-malic acid, pyruvic acid, succinamic acid, succinic acid and succinic acid mono-methyl ester. The predominant cellular fatty acids are C16 : 0, C18 : 0, iso-C17 : 1 ω5c and C14 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, four unknown phospholipids, three unknown glycolipids and two unknown lipids. Genome size is 2 711 934 bp and the G+C content is 50.29 mol%.

The type strain is MSJd-7T (=CGMCC 1.45013=KCTC 25112) and was isolated from faecal samples of M. fascicularis.

Strain MSJ-40T

Strain MSJ-40T was closely related to DSM 23816T (94.2 % 16S rRNA gene sequence identity), DSM 18095T (94.13 %) and DSM 105185T (92.9 %) [27, 67, 68]. According to the phylogenetic and phylogenomic trees (Figs 3 and 4, S2e, S3e), MSJ-40T clustered with members of genus . Thus, strain MSJ-40T was likely a member of the genus . As previously reported for species [27], cells of strain MSJ-40T were rod-shaped. The ANI and dDDH values of strain MSJ-40T to its closest related neighbour DSM 105185T (GCA 009695605.1) were 74.61 and 22.40 % (Fig. 4). The predominant fatty acid of MSJ-40T was iso-C15 : 0, which is a characteristic of the genus [27, 68] (Table 1). At the time of writing, the genus has five described species with validly published names, and strain MSJ-40T is different from them according to phenotypic, chemotaxonomic, phylogenetic and genomic features. Thus, strain MSJ-40T should be classified as representing a novel species of the genus and the name Tissierella simiarum sp. nov. is proposed.

Description of Tissierella simiarum sp. nov.

Tissierella simiarum (si.mi.a'rum. L. gen. pl. n. simiarum, of monkeys).

Cells are rod-shaped, approximately 0.6–0.8×1.0–3.3 µm, and have flagella at both ends. Strictly anaerobic, heterotrophic growth at 37 °C and pH 7.0. Colonies are 3–5 mm in diameter after 2 days of incubation at 37 °C on mGAM agar plates, white, flat, circular, entire, opaque and smooth. The SCFAs produced by fermentation are isovaleric acid, butyric acid, isobutyric acid, propanoic acid and acetic acid. Cells metabolize cellobiose, dextrin, d-fructose, l-fucose, d-galactose, d-galacturonic acid, gentiobiose, α-d-glucose, glucose-6-phosphate, lactulose, d-mannose, melibiose, 3-methyl-d-glucose, palatinose, l-rhamnose, glyoxylic acid, α-ketobutyric acid, pyruvic acid and pyruvic acid methyl ester. The predominant cellular fatty acid is iso-C15 : 0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, three unknown glycolipids and two unknown phospholipids. Genome size is 4.09 Mb and the G+C content is 30.4 mol%.

The type strain is MSJ-40T (=CGMCC 1.45012T=KCTC 25071T) and was isolated from faecal samples of M. fascicularis.

The type strain is MSJ-40T (=CGMCC 1.45012T=KCTC 25071T) and was isolated from faecal samples of M. fascicularis.

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