Sandaracinobacteroides hominis gen. nov., sp. nov., isolated from human skin

Strain SZY PN-1 T, representing a novel Gram-negative, aerobic, non-motile, rod-shaped and yellow-pigmented bacterium, was isolated from a skin sample of a healthy Chinese male. Growth occurred at pH 6.0–8.0 (optimum, pH 7.0) and 10–37 ℃ (optimum, 30 ℃) with 0–1.0% (w/v) NaCl in R2A agar. Comparative analysis of the 16S rRNA gene sequences revealed that strain SZY PN-1 T shared high similarities with two invalid-published species, “Sandaracinobacter sibiricus” RB16-17 (97.1%) and “Sandaracinobacter neustonicus” JCM 30734 (96.6%), respectively. Phylogenetic analysis of 16S rRNA gene sequences together with protein-concatemer tree showed that SZY PN-1 T formed a separate branch within the family Sphingosinicellaceae. The DNA G + C content of the strain SZY PN-1 T was 65.0% (genome). The polar lipid profile included phosphatidylethanolamine, phosphatidylglycerol, two sphingoglycolipids, diphosphatidylglycerol, five unidentified glycolipids, and seven unidentified lipids. The predominant fatty acids (> 10.0%) were identified as C18:1ω7c and/or C18:1ω6c, C17:1ω6c, C16:1ω7c and/or C16:1ω6c. The major respiratory quinone was Q-10. Based on the phenotypic and genotypic features, a novel genus and species, Sandaracinobacteroides hominis gen. nov., sp. nov. is proposed, with type strain SZY PN-1 T (= KCTC 82150 T = NBRC 114675 T).

Ping-Hua Qu and Hai-Min Luo have contributed equally to this work.

Isolation and cultivation
Skin samples of the antecubital fossa for culture were obtained from healthy people by culture swabs (MRC, China) during an investigation for the diversity of skin microbiota in 2019 at Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, PR China. The sampled cells were resuspended with a tube containing 2 mL of sterile saline solution, and then cultivated on BCYEα agar, R2A agar, and their modified medium at 28-32 °C under aerobic conditions. Single colonies were obtained by streaking onto fresh media several times.
A circular and yellow opaque colony, designed as SZY PN-1 T , was selected for taxonomic analysis due to a failure identification by matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) and low similarities of the 16S rRNA gene sequences compared with other species. The isolate was maintained as cells suspension in glycerol (30%, w/v) at − 80 °C.

Sequencing and phylogenetic analysis of 16S rRNA gene
For phylogenetic characterization, DNA extraction, primers, as well as PCR amplification of the 16S rRNA gene were described previously (Li et al. 2007). The amplicon was purified using a PCR purification kit (Sangon Biotech, China). Then, the purified PCR product was cloned into Escherichia coli DH5α chemically competent cells using pMD™ 19-T vector and sequenced on a Sanger platform as described by Giovannoni (Giovannoni et al. 1991). The cloned 16S rRNA gene sequence of SZY PN-1 T was compared with other sequences on the EzBioCloud server (http:// www. ezbio cloud. net) (Yoon et al. 2017) and the sequences of related species used for analysis were retrieved. The SZY PN-1 T strain sequence was aligned to those of related type strains using Clustal W (Larkin et al. 2007). Gaps at the 5' and 3' ends of the alignment were manually removed. Phylogenetic tree reconstructions were performed based on the neighbor-joining (NJ) (Saitou et al. 1987), maximumlikelihood (ML) (Felsenstein et al. 1981), and maximumparsimony (MP) (Fitch et al. 1971) algorithms using MEGA X (Kumar et al. 2018). The evolutionary distance and topology of the phylogenetic trees were evaluated by Tamura-Nei model (Nei and Kumar 2000) and the bootstrap analysis based on 1000 replicates (Felsenstein 1985). Rhodospirillum rubrum ATCC 11170 T was used as an outgroup.

Genome sequences analysis
Whole-genome sequencing was performed for strain SZY PN-1 T using 100 bp paired-end sequencing method with the Illumina Hiseq 2000 platform. The raw data were filtered, and high-quality paired-end reads were assembled using the soapdenovo version 2.04 (Yarza et al. 2014). The completeness and contamination of the assembled genome sequence were evaluated using Checkm (Abbas et al. 2014). For phylogenomic tree reconstruction, marker genes were extracted from 18 genomes available for the family Sphingosinicellaceae using AMPHORA2 (Parks et al. 2015). Sequences of the amino acid were aligned separately using MUSCLE (Wu and Scott 2012) and were checked to remove the poorly aligned regions via Gblocks (Castresana et al. 2000). Then, cleaned alignments were concatenated using perl script (https:// github. com/ nylan der/ catfa sta2p hyml). The protein-concatemer tree was generated using the RAxML method by applying the default parameter (Edgar 2004) and visualized using the online Tree of Life program version 4.2 (https:// itol. embl. de) (Stamatakis 2014).
Activities of oxidase, catalase and urease, Voges-Proskauer (VP), gelatin liquefaction, H 2 S production, nitrate reduction, hydrolysis of starch, casein and aesculin, Tweens 20, 40, 60 and 80 were investigated according to the conventional procedures as previously described (Aslanzadeh 2006;Hansen and Sørheim 1991;Smibert and Krieg 1994). Other biochemical activities of strain SZY PN-1 T were performed using API 20NE, API ZYM, and API 50CH kits (bioMérieux) according to the manufacturer's instructions. All tests were performed in duplicate, and Polymorphobacter fuscus CGMCC 1.12714 T was used as control.

Chemotaxonomic analysis
The fatty acid profiles, polar lipids, and respiratory quinones of strain SZY PN-1 T were analyzed in this study. The cellular fatty acid profiles were determined for SZY PN-1 T and reference strains grown on R2A plates incubated at 30 °C for 72 h. Cellular fatty acid methyl ester profiles were prepared and analyzed according to the standard protocol of the Microbial Identification System (Sherlock version 6.2; MIDI database: TSBA6) using a gas chromatograph (7890B, Agilent). Polar lipids of strain SZY PN-1 T were extracted, separated by two-dimensional thin-layer chromatography on Silica gel 60 plates (Merck; Germany) and further analyzed according to the methods as previously described (Minnikin et al. 1979;Collins and Jones 1980). Respiratory quinones were extracted, purified, and analyzed using high-performance liquid chromatography (HPLC) (Kroppenstedt et al. 1982) following the process of Collins et al. (1977). The Bacteriochlorophyll α (BChl α) and carotenoid pigment analysis were performed using the middle-late logarithmic phase as described by Saga et al. (2005). Then, the cells were washed with NaClsaturated and the pigments were extracted with acetone/ methanol (7:2, v/v). The absorption spectrum of the cell extractive at 200−900 nm was analyzed by using Gen5™ (Biotek).

16S rRNA gene sequence and phylogenetic characterization
Comparison of the 16S rRNA gene sequence of SZY PN-1 T (1409 bp) with those of other species showed that the most similar sequence was that of strain "Sandaracinobacter sibiricus" RB16-17 (97.1% similarity), followed by strain "Sandaracinobacter neustonicus" JCM 30734 (96.6% similarity; Lee et al. 2020) and other type strains with less than 94.2% similarity within the family Sphingosinicellaceae, which were lower than the threshold (98.65%) for bacterial species demarcation (Kim et al. 2014). The maximum-likelihood tree based on 16S rRNA gene sequences demonstrated that strain SZY PN-1 T formed a monophyletic clade and clustered closer to the genus "Sandaracinobacter" (Fig. 1). A similar result was obtained when using the neighbor-joining and maximum-parsimony algorithms ( Fig. S1-2, available in the online version of this article).

Genome sequence and phylogenetic characterization
The genomic size of the strain SZY PN-1 T was 3.53 Mbp and the DNA G + C content was 65.0%. The protein-concatemer tree based on 29 marker genes indicated that the novel strain SZY PN-1 T clustered within the genus "Sandaracinobacter", forming a clade with the strain "S. neustonicus" JCM 30734 (Fig. 2). The threshold limits (95.0-96.0% ANI, 95% AAI and 70% dDDH) for delineation of bacterial species were considered as recommended (Chun et al. 2018;Thompson et al. 2013). The results confirmed that strain SZY PN-1 T represented a novel genomic species within the family Sphingosinicellaceae, with ANI values ≤ 85.0%, AAI values ≤ 76.0%, and dDDH values ≤ 21.1%. The detailed characteristics of the genomes of the strain SZY PN-1 T and other type strains within the family Sphingosinicellaceae are listed in Table S1.

Morphological, physiological, and biochemical characterization
Strain SZY PN-1 T showed good growth on R2A agar and BCYEα agar; weak growth on Columbia blood agar, MH agar, TSA, and LB agar; but not on Haemophilus chocolate 2 agar, chocolate agar with PolyViteX (PVX agar, Bio-caring, China), CHAB agar and MacConkey agar (Biocaring, China). After incubation on R2A at 30 ℃ for 72 h, the colonies were 1-2 mm in diameter, circular, convex, a little hard and yellow colored. Strains were able to grow at 1 3 10-37 ℃ (optimum, 30 ℃), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of up to 1.0% (w/v) NaCl with optimum at non-additional NaCl on R2A. Cell of strain SZY PN-1 T was observed to be Gram-negative, aerobic, nonspore-forming and non-motile. The strain was enhanced by the presence of 5% CO 2 . Transmission electron microscopy image showed that the strain was a rod, 0.71-0.97 µm long and 0.53-0.63 µm wide without flagella, as shown in Fig. S3.

Taxonomic conclusion
Based on the phenotypic and genotypic features, strain SZY PN-1 T was observed to be a novel member of the family Sphingosinicellaceae. Although sharing the highest 16S rRNA gene sequence similarities, closely phylogenetic distance, similar physiological, cellular fatty acid, and polar lipid characteristics with the genus "Sandaracinobacter", strain SZY PN-1 T should be classified as a novel species of a new genus in the family Sphingosinicellaceae. The major issue is that the genus "Sandaracinobacter" is now considered as an illegitimate name, for the type strain of the type species is not available from any public collections. Therefore, we proposed Sandaracinobacteroides hominis gen. nov., sp. nov., which is resembling the genus "Sandaracinobacter".
The type strain SZY PN-1 T (KCTC 82150 T = NBRC 114675 T ) was isolated from a skin sample of a Chinese male. The GenBank accession number for the 16S rRNA gene sequence of the strain SZY PN-1 T is MW135304. The GenBank/EMBL/DDBJ/PIR accession number for the Whole Genome Shotgun projects of the strain SZY PN-1 T is JADCUC000000000.
Author contributions YL and CC designed the research and project outline. PHQ, HML, JHF, SL, LD and YZM performed isolation, deposition, and identification. PHQ, HML and JHF analyzed the data. PHQ, HML and WJL drafted the manuscript. All authors read and approved the final manuscript.

Declarations
Conflict of interest All the authors have declared that there are no conflicts of interest.
Ethical approval All experiments involving human subjects were carried out according to the institutional review board protocols approved by the Medical Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine in China (BE2019-165). Informed consent was obtained from all subjects. Taxa: 1, SZY PN-1 T ; 2, "S. neustonicus" JCM 30734 (Lee et al. 2020); 3, Polymorphobacter fuscus CGMCC 1.12714 T . All data are from this study unless otherwise indicated. Values are the percentage of total fatty acids. Symbols: -, not detected; tr, trace (< 0.8%) Summed features represent fatty acids that cannot be separated using gas-liquid chromatography with the Sherlock microbial identification (MIDI) system. Summed feature 3 contained C 16:1 ω7c and/or C 16:1 ω6c; Summed feature 8 contained C 18:1 ω7c and/or C 18:1 ω6c Fatty acids (%) 1 2 3 Saturated straight chain C 12:0 5.