Description of Sandaracinobacter Hominis 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 people. Growth of SZY PN-1 T optimally occurred at pH 7.0, at 30 ºC and tolerate up to 1.0 % (w/v) NaCl. According to the absorption spectrum, carotenoid was present in the cells. Comparative analysis of the 16S rRNA gene revealed that strain SZY PN-1 T shared high similarities with Sandaracinobacter sibiricus RB16-17 T (97.1 %) and Sandaracinobacter neustonicus JCM 30734 T (96.6 %), respectively. Phylogenetic analysis of 16S rRNA gene sequences together with protein-concatamer tree showed that SZY PN-1 T formed a separate branch within the genus Sandaracinobacter. The DNA G+C content of the strain SZY PN-1 T was 65.0 % (genome). The polar lipid prole included phosphatidylethanolamine, phosphatidylglycerol, two sphingoglycolipids, diphosphatidylglycerol, ve unidentied glycolipids and seven unidentied lipids. The predominant fatty acids (> 10.0 %) were identied as C 18:1 ω7c and/or C 18:1 ω6c, C 17:1 ω6c, C 16:1 ω7c and/or C 16:1 ω6c. The major respiratory quinone was ubiquinone Q-10. Based on the phenotypic and genotypic features, we proposed Sandaracinobacter hominis sp. nov. with type strain SZY PN-1 T (= KCTC 82150 T = NBRC 114675 T ). esculin ferric citrate, negative reactions for glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, L-xylose, d-adonitol, methyl β-d-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, N-acetylglucosamine, arbutin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-sucrose, d-trehalose, inulin, d-melezitose, d-ranose, starch, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate are observed. The major cellular fatty acids are Summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c), C 17:1 ω6c and summed feature 3 16:1 naphthol-AS-BI-phosphohydrolase and β-galactosidase, negative for Voges-Proskauer reaction, H 2 S production, β-glucuronidase, β-glucosidase, N-Acetyl-β-glucosaminidase, α-fucosidase, nitrate, nitrate reduction, indole production, acid production from glucose, arginine dihydrolase, urease activity, assimilation of caprate, malate, citrate, phenylacetate, glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, l-xylose, d-adonitol, methyl β-d-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, d-cellobiose, d-lactose, d-melibiose, d-sucrose, d-trehalose, inulin, d-melezitose, d-ranose, starch,


Introduction
The genus Sandaracinobacter, which belongs to the family Sphingosinicellaceae within the order Sphingomonadales, comprises two species with validly published names: Sandaracinobacter sibiricus RB16-17 T (Yurkov et al. 1997) and Sandaracinobacter neustonicus JCM 30734 T (Lee et al. 2020). Members of this genus are Gram-negative, long rods, aerobic bacteria, forming yellow to yellow-orange colonies for carotenoid pigments. Up to date, the members of the ecology of genus Sandaracinobacter and related Sphingosinicellaceae spp. were reported from environmental samples including varied lakes (Yurkov and Gorlenko. 1990; Gich et al. 2006;Cai et al. 2018;Phurbu et al. 2020), seawater (Lee et al. 2020), soil (Jia et al. 2015), and so on. In this study, we described a member of genus Sandaracinobacter, designated SZY PN-1 T , which was isolated from a skin sample of a healthy human.

Isolation and cultivation
Cutaneous samples of the antecubital fossa for culture were obtained from healthy people during an investigation for the diversity of skin microbiota in 2019, in the Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, PR China. Culture swabs (Miraclean, China) were used to collect samples from the antecubital fossa from each volunteer. And then the swabs were placed into a tube containing 2 mL of sterile saline solution, respectively. The fresh sample was immediately brought into the laboratory and inoculated onto BCYEα agar, R2A agar and their modi ed medium at 28-32 °C under aerobic conditions. Single colonies were obtained by streaking onto fresh medium several times.
Among those single colonies, a circular and yellow opaque colony (strain designed as SZY PN-1 T ) was obtained. Subsequently, cell lysate was used as template DNA for universal bacterial 16S rRNA gene PCR followed by direct Sanger sequencing to obtain partial nucleotide sequences, which resulted in a lower similarity comparing with other species. The strain was subjected to taxonomic analysis based on phenotypic, physiological and phylogenetic studies and maintained as cells suspension in glycerol (30 %, w/v) at -80 °C.

16S rRNA analysis and phylogeny
For phylogenetic characterization, The DNA extraction, primers used, as well as PCR ampli cation of the 16S rRNA were described previously (Li et al. 2007). The amplicon was puri ed by using a PCR puri cation kit (Sangon Biotech, China). Then, the puri ed PCR product was cloned into Escherichia coli DH5α chemically competent cells using PMD-19T vector and sequenced by 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.ezbiocloud. net) (Yoon et al. 2017) and the sequences of related species used for the analysis were retrieved. The SZY PN-1 T strain sequence was aligned to those of related type strains using clustal_w. 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), maximum-likelihood (ML) (Felsenstein et al. 1981) and maximum-parsimony (MP) (Fitch et al. 1971) algorithms by using the mega version 7.0 (Kumar et al. 2016). The evolutionary distance and topology of the phylogenetic trees were evaluated by Kimura's twoparameter model (Kimura 1980) and 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 100bp paired-end sequencing method with the Illumina Hiseq 2000 platform. The raw data was ltered, and high quality paired-end reads were assembled using the soap de novo version 2.04 (Yarza et al. 2014). The completeness and contamination of the assembled genome sequence were evaluated by 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 amino acid were aligned separately by using muscle (Wu and Scott 2012) and were checked to remove the poorly aligned regions via Gblocks (Castresana et al. 2010). Then, cleaned alignments were concatenated using perl script (https://github.com/ nylander/catfasta2phyml). The protein-concatamer 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).
The genomic relatedness of strains SZY PN-1 T to the Whole Genome Shotgun sequences of all species type strains in the Sandaracinobacter available in public databases was determined by several methods: average nucleotide identity (ANI) and average amino acid identity (AAI). The ANI with the blastn algorithm (ANIb) and using the MUMmer ultrarapid aligning tool (ANIm) were calculated by the online Ribocon GmbH-version 3.0.20 software (http://jspecies. ribohost.com/jspeciesws/) (Richter et al. 2015). The AAI values were calculated using the CompareM software of the online server (https://github.com/dparks1134/CompareM).
Activities of oxidase, catalase and urease, Voges-Proskauer (VP), gelatin liquefaction, H 2 S production and nitrate reduction, hydrolysis of starch, casein, aesculin and 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). 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 NaCl-saturated and the pigments were extracted with acetone/methanol (7:2, v/v). The absorption spectrum of the cell extractive at 200 900 nm was analysed by using Gen5™ (Biotek). 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.

Chemotaxonomy
The fatty acid pro le, polar lipids and respiratory quinones of strain SZY PN-1 T were analysed in this study. The cellular fatty acid pro les were determined for SZY PN-1 T and reference strains grown on R2A plates incubated at 30 °C for 72 h. Fatty acids were harvested, saponi ed, methylated and extracted according to standard protocols as described for the Sherlock Microbial Identi cation System (Sasser 1990). The analysis of cellular fatty acid was performed using a gas chromatograph (7890B, Agilent) with the MIDI Microbial Identi cation System using the TSBA6 method and the Microbial Identi cation Sherlock software package version 6.1 (MIDI 2008). Polar lipids of strain SZY PN-1 T were extracted, separated by two-dimensional thin-layer chromatography on silica gel G 60 plates (Merck; Germany) and further analysed according to the method described by Minnikin et al. (Minnikin et al. 1979) and Collins and Jones (Collins and Jones 1980). Respiratory quinones were extracted, puri ed and analysed by using high-performance liquid chromatography (HPLC) (Kroppenstedt et al. 1982) following the process of Collins et al. (1977).  (Fig. 1). A similar result was obtained when using the neighborjoining and maximum-parsimony algorithms ( Fig. S1-2, available in the online version of this article), which also revealed that strain SZY PN-1 T representing a novel species within the genus Sandaracinobacter.

Phenotypic characteristics
Strain SZY PN-1 T showed good growth on R2A 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 (Bio-caring, China). After incubation for 72 h at 30 ℃ on R2A agar media, the colonies were 1-2 mm in diameter, circular, convex, a little hard and yellow coloured. Strains were able to grow at temperatures ranging between 10 and 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 growth at nonadditional NaCl on R2A. Cell of strain SZY PN-1 T was observed to be Gram-negative, aerobic, non-sporeforming 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 agella, as shown in Fig. 3. The absorption spectrum of pigments extracted from the cells showed two peaks at 452 and 478 nm (Fig S3), indicating the presence of carotenoids. No peaks were detected above 600 nm, which showed that the strain SZY PN-1 T was absent in BChl α.

Taxonomic conclusion
Based on the low 16S rRNA gene sequence and other phenotypic characteristics, strain SZY PN-1 T could be distinguished from the related species within the genus Sandaracinobacter. The physiological and chemotaxonomic characteristics, including cellular fatty acid and polar lipid pro les, also indicated that strain SZY PN-1 T was similar to the species within the genus Sandaracinobacter. These results revealed that strain SZY PN-1 T should be classi ed as representing a novel species of the genus Sandaracinobacter, for which we propose the name Sandaracinobacter hominis sp. nov.