Bacteroides rhinocerotis sp. nov., isolated from the fresh feces of rhinoceros in Beijing Zoo

A Gram-negative strain, anaerobic, non-motile, non-spore-forming, rod-shaped bacterial strain named as NGMCC 1.200684 T was isolated from the fresh feces of rhinoceros in Beijing Zoo. Based on 16S rRNA gene sequences, phylogenetic analysis indicated that strain NGMCC 1.200684 T belonged to the genus Bacteroides and was most strongly related to the type strain of Bacteroides uniformis ATCC 8492 T (96.88%). The G + C content of the genomic DNA was determined to be 46.62%. Between strains NGMCC 1.200684 T and B. uniformis ATCC 8492 T, the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) were 93.89 and 67.60%, respectively. Strain NGMCC 1.200684 T can produce acid from fermentation of several substrates, including glucose, mannitol, lactose, saccharose, maltose, salicin, xylose, cellobiose, mannose, raffinose, sorbitol, trehalose, D˗galactose, and maltotriose. The major cellular fatty acids (> 10%) were identified as anteiso˗C15:0, iso˗C15:0, iso˗C14:0, and iso˗C17:0 3˗OH. The polar lipid profiles of strain NGMCC 1.200684 T were determined to contain diphosphatidyl glycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids, and two unknown amino-phospholipids. Based on phenotypic, phylogenetic, and chemotaxonomic characteristics, a novel species of the genus Bacteroides, Bacteroides rhinocerotis sp. nov. is proposed. The type strain is NGMCC 1.200684 T (= CGMCC 1.18013 T = JCM 35702 T).


Introduction
Bacteroides are anaerobic and mostly found in the gastrointestinal tract of animals and humans, besides Firmicutes, as well as control the gut microflora of mammals (Smith et al. 2006;Ley et al. 2008; Thomas et al. 2011). In addition to human fecal samples (Kim et al. 2022;Sun et al. 2022), a large number of novel Bacteroides species isolated from animals have been described recently. These include those found in the cecum of wild-derived house mice (Fokt et al. 2022;Clavel et al. 2010), the gut of a subterranean termite (Reticulitermes speratus) (Sakamoto and Ohkuma 2013), caecum of chicken (Irisawa et al. 2016;Saputra et al. 2015), a methanogenic reactor treating waste from cattle farms (Nishiyama et al. 2009;Ueki et al. 2008Ueki et al. , 2011, and chinchilla feces (Kitahara et al. 2011). Bacteroides spp. play diverse functions role as gut commensals, inducing both health-promoting and disease-promoting effects (e.g., Bacteroides fragilis) (Wexler 2007;Wang et al. 2021;Tan et al. 2019). In addition, Bacteroides species have an excellent ability to utilize the nutrients at hand. Bacteroid fermentation of carbohydrates produces a pool of volatile fatty acids, which are then reabsorbed through the large intestine and used as an energy source by the host, meeting a substantial amount of the host's daily energy needs (Hooper et al. 2002). As well, Bacteroides species have a tremendous capacity to use a wide range of dietary polysaccharides. Many dietary plant polysaccharides that are normally indigestible can be broken down by Bacteroides (e.g., amylose, amylopectin, and pullulan). Other organisms in the intestine do not have a series of sugar-utilizing enzymes owned by Bacteroides. But they can benefit from the presence of Bacteroides using sugars (generated by the glycosylhydrolases) (Sonnenburg et al. 2004). The wild animal intestinal microbiome, in particular, was recognized as an undisclosed environment with great bacterial diversity, and each animal can develop its own microbiota signature (Endo et al. 2010;Tsuchida and Ushida 2015). Nevertheless, there hasn't been much research done on rhinoceros' fresh feces. Only three new species have been isolated from rhinoceros' feces in the last 20 years that do not belong to the genus Bacteroides (Chen et al. 2017;Li et al. 2015Li et al. , 2016 was considered to belong to a potential novel species within the genus Bacteriodes. In this paper, we describe its taxonomic position from a polyphasic perspective.

Isolation and growth conditions
The strain NGMCC 1.200684 T was isolated from rhinoceros' feces. The feces samples were collected, immediately placed in anaerobic PBS solution containing 1% cysteine, and transferred into an anaerobic glove box (Shanghai Longyue Co., Ltd) that was 90% N 2 , 5% H 2 , and 5% CO 2 . Pipetting was used to disperse the suspended feces, which were then filtered through 70 μm and 40 µm cell sieves. Following that, the filtrate was serially diluted up to 10 ˗7 , and 100 μl of each of the last four dilutions was respectively plated on modified Gifu anaerobic broth (mGAM; HB8518, Hopebio) agar plates and YCFA agar plates. As described above, plates were incubated for 3 days at 37 ℃ in an anaerobic glove box. Strain NGMCC 1.200684 T was isolated from an mGAM agar plate of 10 ˗4 series diluted fecal samples, which were heat-treated. Single colonies were picked and grown on modified GAM agar plates. This procedure was repeated until pure cultures were obtained and stored at − 80 °C in mGAM broth supplemented with 20% glycerol (w/v). Reference strains Bacteroides fluxus DSM 22534 T , Bacteroides rodentium DSM 26882 T , and B. uniformis DSM 6597 T were obtained from DSMZ, and maintained under the same conditions.

Morphological, physiological, and biochemical characterization
For the purposes of phenotypic, chemotaxonomic, and phylogenetic characterization, the strain NGMCC 1.200684 T was grown on mGAM agar or in liquid medium at 37 °C and anaerobic cultivation 3 days, unless otherwise stated. Gram staining was carried out using a Gram staining kit (G1060, Solarbio), and optical microscopy (CX-31, Olympus) was used to evaluate the results. Cellular morphology and the presence of spores were examined by scanning electron microscopy (Merlin compact, ZEISS). Growth was examined in environments that were aerobic, anaerobic, and microaerophilic, which were produced using a bio-incubator, AnaeroPackTM˗Anaero, and MicroAeroTM˗MicroAero (Mitsubishi Gas Chemical Co, Inc.). Cell motility was performed depending on the development of turbidity in an anaerobic tube containing mGAM semisolid medium (Tittsler and Sandholzer 1936). The activities of catalase and oxidase were investigated with 3% (v/v) hydrogen peroxide solution and oxidase test strips (M153, LAND BRIDGE), respectively. According to the manufacturer's instructions, physiological and biochemical tests were conducted using the VITEK 2 ANC card of anaerobic bacteria identification test (bioMérieux), API ZYM Kit (bioMérieux), and API 20A systems (bioMérieux). Other phenotypic traits, including temperature, pH for growth, and salt tolerance, were evaluated using the methods previously described (Sun et al. 2022;Yu et al. 2019). Cellular polar lipids were extracted with chloroform-methanol filtration and identified by twodimensional TLC (Minnikin and Abdolrahimzadeh 1974). The processes of saponification, methylation, extraction, and measurement of cellular fatty acids followed those previously reported (Sakamoto et al. 2002). Using the Microbial Identification System (MIDI) (Sasser 1990), the fatty acid composition of strain NGMCC 1.200684 T was examined as per Sasser's (1990) instructions.

Phylogenetic and genome sequencing analyses
The 16S rRNA gene of strain NGMCC 1.200684 T was amplified using universal primers: 27F (5ʹ˗AGA GTT TGA TCC TGG CTC A˗3ʹ), 1492R (5ʹ˗GGT TAC CTT GTT ACG ACT T˗3ʹ). PCR products were sequenced using a BigDye ™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 3730 XL Genetic Analyzer (Applied Biosystems). The closest recognized relatives of the novel isolates were identified and downloaded by comparing the 16S rRNA gene sequence (1412 bp) of strain NGMCC 1.200684 T to those available in the EzBioCloud database (www. ezbio cloud. net). The isolate sequences were aligned with 16S rRNA gene sequences obtained from EzBioCloud using the multiple sequence alignment program Clustal_X software (version 2.0) (Thompson et al. 1997). The trimmed alignment was converted to mega format for phylogenetic analyses. Phylogenetic consensus trees were constructed using the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods with MEGA_X (Kumar et al. 2018;Felsenstein 1981) and evaluated using 1000 bootstrap replicates (Saitou and Nei 1987;Kluge and Farris 1969). Evolutionary distance was obtained by the two-parameter method of Kimura (Kimura 1980). The genomic DNA from pure cultures of strain NGMCC 1.200684 T was extracted using the TIANamp Bacteria DNA Kit (DP302, Tiangen) following the manufacturer's instructions. The Illumina PE150 platform was used to sequence the genome. Using the algorithm outlined by Yoon et al. (Yoon et al. 2017), the OrthoANI determined the average nucleotide identity (ANI) values using EzBioCloud (www. ezbio cloud. net/ tools/ ani). Version 3.0 of the Genome-to-Genome Distance Calculator (GGDC) (http:// ggdc. dsmz. de/ ggdc. php) was used to determine the digital DNA˗DNA hybridization (dDDH) values (Auch et al. 2010;Meier-Kolthoff et al. 2013). The phylo-genomic tree is constructed using a concatenated alignment of 120 conserved bacterial single-copy genes with GTDB-Tk v. 1.5.1 (Parks et al. 2018;Chaumeil et al. 2020).

Phylogenetic and genomic analyses
The 16S rRNA gene sequences of strain NGMCC 1.200684 T (1412 bp) were determined. The 16S rRNA gene sequences of strain NGMCC 1.200684 T and related type species of the genus Bacteroides were aligned, and a phylogenetic tree was constructed using Parabacteroides distasonis ATCC 8503 T as an outgroup (Fig. 1). According to the findings of phylogenetic analyses based on 16S rRNA gene sequences using the NJ, ML, and MP techniques, strain NGMCC 1.200684 T and the closely related species formed a separate branch within the genus Bacteroides. The phylogenetic analysis and EzBioCloud database searches indicated that the type strains of B. uniformis ATCC 8492 T , B. rodentium JCM 16496 T , and B. fluxus YIT 12057 T had similar sequences to NGMCC 1.200684 T , with approximate similarity values of 96.88%, 95.56%, and 93.45%, respectively. A phylo-genomic tree based on whole genomes was reconstructed (Fig. 2). The result showed that NGMCC 1.200684 T was clustered with the type strains of B. uniformis ATCC 8492 T in the same clade, and they have a high bootstrap value (98%). The average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) results showed that B. uniformis ATCC 8492 T was the closest strain, with values of 93.89% and 67.60%, which were lower than the classification limits of 95% and 70% of international standards (Wayne 1988) ( Table 2). We concluded that strain NGMCC 1.200684 T represented a novel species within the genus Bacteroides.
The TIANamp Bacteria DNA Kit was used to extract genomic DNA from cells cultured in the mGAM broth (DP302, Tiangen). The genome was sequenced by the Illumina PE150 platform. The size of the strain NGMCC 1.200684 T genome was 4.88 Mb. 76 high-quality scaffolds were produced from 1,126 Mb of clean readings after de novo assembly. The isolate's DNA G + C content was 46.62%, which was within the range (40-48%) previously described for the genus Bacteroides (Shah 1992). In the draft genome, the genome carried 62 ncRNA genes, including    Fig. S2). Further genome mining revealed strain NGMCC 1.200684 T genome sequence encodes the starch utilization system (Sus), which is made up of susABCDEFG genes and can degrade various oligosaccharides into monosaccharides or disaccharides by periplasmic glycan-degrading enzymes like susA and susB. The sus system was also found in Kim et al.'s study (Kim et al. 2022) (Supplementary Table S2).

Taxonomic conclusion
On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic studies, we propose that strain NGMCC 1.200684 T be classified as representing a novel species of the genus Bacteroides, for which the name Bacteroides rhinocerotis sp. nov. is proposed.
The type strain, NGMCC 1.200684 T (= CGMCC 1.18013 T = JCM 35702 T ), was isolated from rhinoceros' feces. The DDBJ/ENA/GenBank accession numbers for the 16S rRNA gene and genome sequences of the type strain are OP931997 and JAPDHT000000000, respectively.
Author contributions XL and LS carried out the data analysis, wrote, and revised the manuscript. XL, PLS, LG, and WXS performed the experiments. ZGX and ML participated in the data analysis. LS and CQ supervised the project. All authors reviewed and approved the final manuscript.

Conflict of interest
The authors declare that there is no conflict of interest.
Ethics approval and consent to participate In this study, the collection and the analysis of animal feces did not involve animal ethics.