Streptococcus xiaochunlingii sp. nov. E24 Isolated From the Oropharynx of Healthy Chinese Children

A Gram-positive, α-hemolytic, catalase-negative, facultative anaerobic and non-motile coccus was isolated form throat swabs taken from the oropharynx of healthy children. The genome was shown to be 1.950,659 bp long and contained 42.03 mol% G + C bases with 1,942 protein-coding and 53 RNA coding genes. The results of 16S rRNA gene sequencing strongly suggested that the strain is a member of the Streptococcus genus, with 98.04, 98.11, and 97.34% similarities to Streptococcus australis ATCC 700641T, Streptococcus rubneri LMG 27207T and Streptococcus parasanguinis ATCC 15912T, respectively. A sodA gene comparison exhibited a sequence identity of 92.6% with the closest strain Streptococcus australis ATCC 700641T. In silico DNA-DNA hybridization showed a highest DNA similarity value of 52% with Streptococcus australis ATCC 700641T. Comparing 18 biochemical traits, the similarities of the Streptococcus strain E24 were 72% with Streptococcus rubneri LMG 27207T, 78% with Streptococcus australis ATCC 700641T and 44% with Streptococcus parasanguinis ATCC 15912T. We suggest that based on the genotypic and phenotypic results that the strain E24 is a novel species of the Streptococcus genus and propose the name Streptococcus xiaochunlingii sp. nov. E24.


INTRODUCTION
The microbiota of the upper respiratory tract has been denoted as gatekeepers to respiratory health, since it prevent pathogens from overgrowing and disseminating toward the lungs (Man et al., 2017;Tsang et al., 2019). In the first week after birth, the upper respiratory microbiota of infants develop from a mixed toward a Streptococcus viridans-predominated bacterial community, followed by niche differentiation to Staphylococcus aureus and other bacteriae frequently leading to a composition of Moraxella spp., Dolosigranulum spp., and Streptococcus spp. (Bosch et al., 2016). In a previous study, we isolated Streptococcus strain E24 as one of 4 antagonistic strains which had bacteriostatic effects on Klebsiella pneumoniae, Proteus vulgaris, Enterobacter cloacae, and Acinetobacter Baumannii (Li et al., 2019).
In recent decades, due to improved phenotypic and molecular identification methods and with the development of the new taxonogenomics concept (Fournier et al., 2015), the genus Streptococcus has been significantly expanded with the addition of many newly discovered species (Huch et al., 2013;Ricaboni et al., 2017); more than 110 species are now recognized within the genus Streptococcus 1 . With 16S rRNA gene sequence analyses revealed that Streptococcus species were clustered into six groups comprising S. anginosus, S. mitis, S. mutans, S. bovis, S. pyogenes, and S. salivarius (Kawamura et al., 1995). Here we present the results of taxonogenomics concept analyses of a novel strain termed Streptococcus strain E24.

Collection of Bacterial Samples
Bacterial colonies from throat swabs of healthy children were cultivated aerobically for 24 h at 37 • C on agar plates enriched with sheep blood (5%) (Becton Dickinson, Heidelberg, Germany). During streptococci isolation a single, circular, unpigmented and α-hemolytic colony of 0.5-1.0 mm diameter emerged, which was purified by repeated streaking and finally stored at −80 • C. The study was conducted in accordance with the "Declaration of Helsinki" guidelines and approved by the Ethics Committee of Shenyang Medical College (approval number: No. 2015052902). Written informed consent was obtained from the legal representatives of the participating children.

16S rRNA sodA Gene Analyses
The nucleic acids of the strain were extracted for gene amplification and cloning of the 16S rRNA gene was accomplished by using a commercial DNA Purification Kit (Wizard Genomic DNA Purification Kit, Promega, Madison, WI, United States) and the 16S rRNA universal primers 27F/1492R (5 -AGAGTTTGATCMTGGCTCAG-3 and 5 -GGYTACCTTGTTACGACTT-3 , respectively). The sequence obtained by sequencing was aligned using Basic Local Alignment Search Tool (BLAST) of NCBI. We collected 16S rRNA gene sequences of all known Streptococcus species from the NCBI database (Pontigo et al., 2015). Phylogenetic trees were constructed with two different algorithms, namely neighborjoining (Saitou and Nei, 1987) and maximum-likelihood (Guindon and Gascuel, 2003) by close-neighbor-interchange (CNI) (search level = 2, random additions = 100) using MEGA version 7.0 (Kumar et al., 2016) and the reliability of the nodes was estimated by bootstrap analysis (1000 replications) (Felsenstein, 1985). Additionally, we analyzed the sodA gene since it has been proposed for phylogenetic characterization especially for differentiating streptococci (Poyart et al., 1998) with the primers forward: 5 -CCITAYICITAYGAYGCIYTIGARCC-3 and reverse: 5 -ARRTARTAIGCRTGYTCCCAIACRTC-3 leading to a 430 bp fragment.
Cell sizes were calculated from images captured on a scanning electron microscope. Bacteria were fixed in glutaraldehyde phosphate (2.5%) buffer overnight at 4 • C. They were then washed twice with phosphate buffer (PBS), fixed for 30 min in a 1% osmium tetroxide solution (SPI supplies Inc., West Chester, PA, United States) and then washed 3 times with PBS before being dehydrated with 50, 70, 80, 90, 95 (twice), and 100% ethanol (three times), respectively (5 min per ethanol concentration). After natural drying, each sample was subjected to ion sputtering and then the gold was sprayed onto the surface and then viewed on a Hitachi 3400N scanning electron microscope (Hitachi High Technologies, Japan).
Sporulation was tested with Streptococcus strain E24 solutions (concentration: 1 × 10 6 cfu/mL) divided into two groups, from which one group was heated to 80-90 • C for 20 min and the other served as control. Then 50 µl solutions of each group were applied on nutrient agar plates containing 5% sheep blood. The strains were incubated under normal conditions at 37 • C overnight to observe the bacterial colony formation. No colony growth was observed on the plate of the heat treatment group on the next day.

Analysis of Fatty Acids in Bacteria
Fatty acid methyl esters (FAMEs) were analyzed using a gas chromatography/mass spectrometry [see (Dione et al., 2016) and (Sasser, 1990)]. FAMEs were prepared from a sample consisting of 40 mg of bacterial biomass, separated using an MS column (Elite 5) and examined with a mass spectrometry (Clarus 500-SQ 8 S, PerkinElmer, France) followed by appropriate searches of spectral databases.

Extraction of DNA and Sequencing of the Genome
Genomic DNA was extracted using the SDS methodology (Lim et al., 2016). Agarose gel electrophoresis (HE-120, Tanon Science & Technology Inc., Shanghai, China) was used to detect the DNA, which was analyzed and quantified with a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, United States). 1 µg samples were used as preparations of DNA and sequence libraries were constructed using a NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, MA, United States), with appropriate index codes. Samples were sonicated to sizes of about 350 bp and fragments endpolished, A-tailed and ligated for subsequent amplification by PCR. The PCR products were purified and libraries evaluated to determine the distribution of sizes using real-time PCR (Agilent 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, United States). Beijing Novogene Bioinformatics Technology Co., Ltd sequenced the genome (Illumina NovaSeq PE150, Illumina Inc., San Diego, United States).

Genome Sequencing Assembly and Annotation
After preprocessing, appropriate data were gathered using SOAP (version 2.04) (Li et al., 2008(Li et al., , 2010, SPAdes (Bankevich et al., 2012) and ABySS (Simpson et al., 2009) assembly software, and finally integrated using CISA software. The preliminary assembly results were optimized and holes filled with GapCloser (version 1.12) to obtain the final assembly result. Fragments below 500 bp were filtered out. Encoded genes were predicted using GeneMarkS software. The protein-coding sequences produced with BLAST were annotated by comparison with the NR, GO, KEGG, COG, PFAM, TCDB, and Swiss-Prot protein databases.

Genome Comparison
Average nucleotide identity (ANI) was used to assess genomic similarity and to define species with values of 95%. The ANI analysis was performed with OAT (Yoon et al., 2017) and DNA-DNA hybridization (DDH) was used to assess the genomic similarity and to define species with 70% hybridization values (Tindall et al., 2010). The Genome-to-Genome Distance Calculator (GGDC) (Meier-Kolthoff et al., 2013) was employed to conduct a digital DNA-DNA hybridization (dDDH) analysis. The adjacent to the Streptococcus strain E24 located strains were selected based on BLAST alignment and the phylogenetic tree constructed from 16S rRNA gene results data.

Phylogenetic Analysis
The newly determined 16S rRNA (Figures 1A,B)

Phenotypic Characteristics
Morphological features were determined using cells cultured aerobically at 37 • C on enriched nutrition agar (vide supra) for 24 h, when the bacteria became punctate, gray and formed α-hemolytic colonies having edges that were undulated. Gram staining was performed using the classical Gram stain procedure (Austrian, 1960) and light microscopy images showed Grampositive cocci (Figure 2A). Cells had an average diameter of about 5 µm as viewed by an electron microscopy ( Figure 2B). Salt tolerance was determined by the growth of the strains in the presence of 2.5 g/L. The isolate was found to be unable to grow at or below 22 • C but could grow at up to 42 • C, with 37 • C being the optimal temperature. The cells were facultative anaerobe, and the pH for optimal growth was 7 (Table 1 and  Supplementary Table 1).

Antibiotic Susceptibility
Antibiotic susceptibility tests of Streptococcus strain E24 revealed susceptibility to vancomycin, cefepime, Rina thiazole amine, penicillin, chloramphenicol and clindamycin, but resistance to ceftriaxone, ampicillin, and cefotaxime.

Genome Properties
A draft of the genome of Streptococcus strain E24 revealed that it was comprised of 1,950,659 bp with a 42.03% G + C content (Figure 3). It is comprised of five scaffolds (five contigs). Among the 1,994 genes predicted, 1,942 encoded proteins and 52 RNAs (3 genes were 5S rRNA, 1 gene 16S rRNA, 1 gene 23S rRNA and 47 genes tRNA). Of these, 1,525 genes (78.52%) were specified with putative functions according to COG or NR BLAST analysis. Twentytwo genes were ORFs (1.13%) and the remaining genes were responsible for hypothetical proteins. Table 4 shows the COG functional categories of the sorted genes. The sequence of the genome was recorded in the NCBI database (accession no. VFSG00000000).

Comparisons of Genomes
The genome sequence length of Streptococcus strain E24 (1,950 Mb) was less than S. pseudopneumoniae ATCC BAA-960 T Frontiers in Microbiology | www.frontiersin.org  Hemolysis

DISCUSSION
The phenotypic analysis obtained from API strips ( Table 2) revealed, that the Streptococcus strain E24 was the only one which hydrolyzed hippurate and there were essential differences between the nine analyzed streptococci, which has been described also for other Streptococcus strains in which the probability that a strain was correctly identified by nine phenotypic tests was 90-100% (Raemy et al., 2013). The similarities of the 16S rRNA gene sequences from Streptococcus strain E24 with the closest related streptococci were 98.04, 98.11, and 97.34% for Streptococcus australis ATCC 700641 T , Streptococcus rubneri LMG 27207 T and Streptococcus parasanguinis ATCC 15912 T , whereas the sodA gene comparison exhibited a sequence identity of 92.6% with the closest strain Streptococcus australis ATCC 700641 T . These similarities are in line with another newly identified Streptococcus strain exhibiting 98.7 and 92.6% similarities of 16S rRNA and rpoB gene sequences between the Streptococcus timonensis sp. nov. and the Streptococcus infantis strain JCM 10157 T (Ricaboni et al., 2017). There were also essential differences of G + C contents and genome sequence lengths between Streptococcus strain E24 and eight other streptococci, but also within the 8 streptococci species, which has been described also for other Streptococcus strains (Thompson et al., 2013) and attributed to frequent occurrence of horizontal gene transfers (Bellanger et al., 2009;Harvey et al., 2011;Zhang et al., 2011). Most obviously, the dDDH analysis revealed 24 to 52% similarities between Streptococcus strain E24 and 11 comparison strains and the similarities between the 12 different streptococci strains was between 23.5 and 57.8%, which is less than the proposed limit of 70% similarity for discrimination of Streptococcus species (Auch et al., 2010;Thompson et al., 2013).
In summary, based on the genotypic, phylogenetic and phenotypic results, it is concluded that the Streptococcus strain E24 is a novel species of the genus Streptococcus, which is distinct from its closest phylogenetic neighbors Streptococcus australis ATCC 700641 T , Streptococcus rubneri LMG 27207 T and Streptococcus parasanguinis ATCC 15912 T and for which the name Streptococcus xiaochunlingii sp. nov.E24 is proposed.

DESCRIPTION OF Streptococcus xiaochunlingii sp. nov. E24
Streptococcus xiaochunlingii (xiao'chun' lingi.i. N.L. gen masc. named after Professor Xiao Chunling, who is the leader of the laboratory in which the strain has been isolated).
Streptococcus strain E24 is a non-motile, non-spore-forming, facultative anaerobic and Gram-positive coccus, isolated from

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession numbers can be found below: https://www.ncbi. nlm.nih.gov/, VFSG00000000; https://www.ncbi.nlm.nih.gov/, MN592637.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Shenyang Medical College (approval number: No. 2015052902). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.