Abstract
A facultative anaerobic, catalase-negative, lactic acid-producing, non-endospore-forming, Gram-positive coccoid bacterium, strain MT/JULY 2010T, was isolated from the stomach of a Helicobacter pylori-positive Japanese female patient with pre-neoplastic gastric mucosa. This bacterium can form clusters, chains or pairs or occurs singly in liquid media. The growth ranges for temperature, pH and NaCl concentration were 20–45 °C, pH 5.0–7.0 and 1–4%, respectively. Cells exhibited an unusual tumbling motion when observed under the microscope using a hanging drop assay. Observation under an electron microscope showed some unidentified structures and numerous fimbriae-like appendages. The combined results of 16S rRNA gene sequence analysis and the overall genome-related index [average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH)] classified the bacterium as a novel species of the genus Streptococcus. It forms a monophyletic cluster with Streptococcus parasanguinis ATCC 15912T in the phylogenetic tree with 94.4% ANI. Furthermore, the conventional DNA–DNA hybridization assay and dDDH of the two strains identified their relatedness as 62.5% and 58.7% (both below the 70% threshold), respectively. The DNA G+C content, determined by nuclease P1-digested genomic DNA, was 42.6 mol%, while the digital G+C content calculated in silico was 41.8 mol%. Based on the phenotypic and genotypic results, strain MT/JULY 2010T represents a novel species within the genus. The name Streptococcus mobilis sp. nov. is proposed. The type strain is MT/JULY 2010T (=ATCC BAA-2258T=NBRC 107862T=NCBI 948105T=V10/022878T).
Keywords: digital DNA–DNA hybridization (dDDH), novel taxon, pre-neoplastic human stomach, Streptococcus, whole-genome sequence (WGS)
Introduction
The Austrian surgeon Albert Theodor Billroth first described Streptococcus in 1874 among patients with erysipelas and wound infections [1]. Louis Pasteur isolated the bacterium in 1879 from a female patient with puerperal fever [1]. The taxonomic name of the genus Streptococcus was published in 1884 with the publication of Streptococcus pyogenes by Rosenbach [2]. This genus currently comprises 145 species (https://lpsn.dsmz.de/ accessed on 22 August 2025). Streptococcus is spherical or ovoid, measuring less than 2 µm in diameter, and it forms chains or pairs in liquid media. They are facultative anaerobes, Gram-positive, catalase-negative, non-endospore-forming and non-motile and produce lactic acid via glucose fermentation [2]. The Mitis group of Streptococcus is part of the human microbiota in the oral cavity, upper respiratory, gastrointestinal and genitourinary tracts [2]. They are mesophilic and α-haemolytic. Identifying the species within the Mitis group, which comprises about 20 species, is challenging due to the close inter-species sequence similarities found in phylogenetic analyses based on 16S rRNA gene sequences [3]. The current taxonomic evaluation of species definition in bacteriology recommends the combined use of phenotypic and genomic data [4,5]. The latter includes 16S rRNA gene sequence analysis, DNA–DNA hybridization values, multilocus sequence analysis or whole-genome sequence (WGS) analysis to determine the species level in the Mitis group of the genus Streptococcus [3].
It was July 2010 when the strain MT/JULY 2010T (=ATCC BAA-2258T=NBRC 107862T=NCBI 948105T=V10/022878T) was isolated from multiple gastric biopsy specimens and aspirates of a Helicobacter pylori-positive 63-year-old Japanese woman with gastric erosion, active chronic gastritis, atrophy, intestinal metaplasia and atypia. The intra-lesional, intracellular, intranuclear and intravascular presence of these unidentified coccoid bacteria in various H. pylori-positive and -negative esophago-gastro-duodenopathies has been demonstrated using electron microscopy. This includes the classic gastric carcinogenic cascades: gastritis, atrophy, intestinal metaplasia, dysplasia and adenocarcinoma of the stomach. These human pathologies and microbiological data related to the unidentified bacteria have been presented at various international meetings [6].
In this report, the phenotypic and genomic characteristics of the unidentified coccoid bacterium, isolated from a gastric biopsy, strain MT/JULY 2010T, were investigated in comparison to the closely related Streptococcus parasanguinis ATCC 15912T, as they form an independent monophyletic cluster within the Mitis group. Based on phylogenetic tree analyses derived from the 16S rRNA gene sequence (~1,500 bp), the overall genome-related index (OGRI), along with amino acid compositions in the cell wall, including peptidoglycans, as well as morphological, physiological and biochemical characteristics, this strain is classified as a novel species within the genus Streptococcus, and a novel taxon is proposed for the strain MT/JULY 2010T (ATCC BAA 2258T): Streptococcus mobilis sp. nov.
Methods
Isolation and maintenance
The strain MT/JULY 2010T was isolated at Okada Medical Clinic, Wickham Terrace, Brisbane, Queensland, Australia (latitude 27° 27′ 51.0″ S, longitude 153° 01′ 34.6″ E) in July 2010 from gastric biopsy specimens and aspirates of a H. pylori-positive 63-year-old Japanese woman with gastric erosion, active chronic gastritis, atrophy, intestinal metaplasia and atypia. The strain has been deposited in various culture collections and is maintained as ATCC BAA-2258T=NBRC 107862T=NCBI 948105T=V10/022878T.
Analysis of DNA base composition and DNA-DNA hybridization
Determining the DNA G+C content of the strain MT/JULY 2010T and conducting DNA–DNA hybridization analysis between the strains MT/JULY 2010T and S. parasanguinis ATCC 15912T were performed on cells grown in trypticase soy yeast extract medium (DAM medium No 92) at 37 °C for 72 h under anaerobic conditions. Genomic DNA from the two strains was extracted using the method of Marmur [7], with minor modifications from Saito and Miura [8]. The G+C content of the genomic DNA was determined by the method of Katayama-Fujimura et al. [9]. Briefly, genomic DNA was digested into nucleotides by nuclease P1 using a DNA-GC kit (Seikagaku Kogyo, Tokyo, Japan). The DNA G+C content was determined by HPLC (LC-10; Shimadzu, Kyoto, Japan) with an RP Aqueous column (Nomura Chemical Co. Ltd., Seto, Japan) and a UV-Vis spectrophotometric detector (SPD-10AV; Shimadzu) at 270 nm. The DNA–DNA hybridization study was performed using the fluorometric DNA–DNA hybridization method on a microdilution plate [10] at 41 °C in the presence of 50% formamide to determine the level of DNA–DNA relatedness. Hybridization was carried out five times, with the highest and lowest values excluded, and the mean of the remaining three experiments used for calculating DNA G+C content, to ensure reliable and valid results.
Phenotypic characterization
The studies were conducted at Osaka Metropolitan University (Osaka, Japan) or TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan). The cells were cultured in trypticase soy agar (BBL Becton Dickinson, USA) at 37 °C for 48 h under aerobic conditions unless the media and conditions were specified. Colony morphology, growth range of temperature (5–50 °C), pH (1.0–7.0) and NaCl concentration (1.0–6.5%) and biochemical characteristics including tests for catalase, oxidase and acid production from glucose were analysed. Favour G staining kit (Nissui Pharmaceutical, Tokyo, Japan) for Gram stain, API 20 STREP (bioMérieux, Marcy-L’Etoile, France) and API ZYM (bioMérieux) for biochemical analyses were used. As additional tests for examining biochemical characteristics of the strain MT/JULY 2010T, urea slope (Oxoid, Basingstoke, UK) for urease, TSI agar slant (Oxoid) or Kligler’s agar slant (bioMérieux) with lead acetate strip (Sigma-Aldrich, St. Louis, USA) for H2S production and DNase agar (bioMérieux) were used. For these biochemical tests, incubation was performed at 37 °C for over 72 h under anaerobic conditions. Motility was assessed using a needle inoculation method with semisolid thioglycolate media (BBL Becton Dickinson) with strain MT/JULY 2010T and control strains Escherichia coli ATCC 8739T and S. parasanguinis ATCC 15912T, respectively. A motility assay was also conducted using the hanging drop method with India ink. The strain MT/JULY 2010T and a clinical isolate of Streptococcus mitis used as the control were employed for the hanging drop assay. Motility was recorded using the CAMEDIA C-7070 (Olympus, Hachioji, Japan) with the MD Lens (MA151/30/65, Meiji Techno, Miyoshi, Japan) on the Olympus BX60 upright compound microscope. Transmission electron microscopy (TEM) and negative staining were performed at the Hanaichi Electron Microscopy Institute (Okazaki, Aichi, Japan), following established protocols. Colonies collected after 24 h of incubation at 37 °C under anaerobic conditions on 5% defibrinated horse blood Colombia agar (BBL Becton Dickinson) were used for electron microscopy. Amino acid compositions in the cell wall, including peptidoglycan, were measured using the ACQUITY UPLC H-Class system (Waters Corporation, Milford, USA), with a BEH C18, 2.1 (I.D.) × 150 mm, 1.7 µm column (Waters Corporation), a PDA eλ Detector and MassLynx V4.2 software. Cellular fatty acid (CFA) analyses were performed with the Sherlock Microbial Identification System (ver 6.0) (MIDI, USA), and the data were calculated by CLIN6.
Genome sequencing and assembly
The complete genome sequencing was performed by hybrid assembly using short reads from the HiSeq 2500 (Illumina Inc., San Diego, California, USA) and long reads from MinION (Oxford Nanopore, Oxford, UK). DNA libraries for Illumina sequencing were prepared using Nextera DNA Flex (Illumina) and then sequenced on a HiSeq 2500 platform in 251 bp paired-end mode. For MinION libraries, 1.5–2.5 µg of DNA was sheared using g-TUBE (Covaris Inc., Woburn, Massachusetts, USA) to obtain 8,000 bp fragments. One microgram of the sheared DNA was converted to each library using the Ligation Sequencing Kit (SQK-LSK108) (Oxford Nanopore). The read data was assembled using the Flye assembler ver 2.7. Assembled contigs were polished using BWA ver 0.7.17 and Pilon ver 1.23 with HiSeq sequence reads. Raw reads can be found from DDBJ under BioProject accession number PRJDB19917 (PSUB024865), and the complete annotated genome has been deposited in DDBJ under accession number AP041823. The genome sequence data, presented as a single complete contig, was submitted to the dFAST annotation server to determine the genomic features. The assembled genome was subjected to average nucleotide identity (ANI) calculations using the ANI calculator from Kostas lab, available at https://enveomics.scigap.org/ or pyani (ANIm method) based on MUMmer alignment with the closest type strain genomes. The genome sequence data were also uploaded to the Type (Strain) Genome Server (TYGS), available at https://tygs.dsmz.de, for a whole genome-based taxonomic analysis [11] with the closest type strain genomes and Streptococcus strains belonging to mitis group. The analysis also utilized recently introduced methodological updates and features [12]. Information on nomenclature, synonymy and associated taxonomic literature was provided by TYGS’s sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at https://lpsn.dsmz.de) [12]. The results were provided by the TYGS on 21 August 2025.
16S rRNA gene sequencing
The 16S rRNA gene sequences were obtained independently through PCR amplification and Sanger sequencing. The 16S rRNA gene was amplified using flanking primers designed in this study. The PCR amplicons were purified by gel cutting using the Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA). The amplicons were sequenced using the primers shown in Table S1 (available in the online Supplementary Material). The amplicon/primer premix was sent to Eurofin Genomics Co. Ltd. (Tokyo, Japan) and sequenced using the Sanger sequencing protocol with the ABI PRISM® 3130 Genetic Analyzer System (Applied Biosystems, USA). The 16S rRNA gene contigs were assembled with the Seqman program, and the phylogenetic tree based on the sequence analysis was constructed using the Megalign program from Lasergene’s DNAstar software package. The 16S rRNA gene sequences are available at DDBJ under the accession numbers LC877506–LC877509.
Results
Identification of the novel species
Preliminary analysis with the partial 16S rRNA gene sequence analysis showed that the strain MT/JULY 2010T was closely related to S. parasanguinis ATCC 15912T, based on nucleotide BLAST searches or gene sequence homology. Conventional DNA–DNA hybridization studies revealed that the DNA–DNA relatedness value between the strain MT/JULY 2010T and S. parasanguinis ATCC 15912T was 62.5%, which is below the 70% threshold. The DNA G+C content of the strain MT/JULY 2010T was 42.6 mol%, as determined by HPLC analysis of the nucleoside P1-digested genomic DNA. These results suggest that the strain MT/JULY 2010T could be a novel species in the genus Streptococcus.
Phenotypic characterization
Bacterial colonies of strain MT/JULY 2010T and S. parasanguinis ATCC 15912T, when cultured at 37 °C for 48 h on soybean casein digested agar, appeared creamy, buttery, smooth, round and convex, measuring about 1 mm in diameter. Colonies on 5% horse blood agar were α-haemolytic. The strains were endospore negative and the sizes of the bacterial cells of strain MT/JULY 2010T and S. parasanguinis ATCC 15912T under the microscope BX50F (Olympus, Japan) were 0.8–1.0×1.0–1.2 and 0.7–0.8×0.9–1.0 µm, respectively. The common characteristics of these two strains were that they were Gram-positive (Fig. 1) and -negative for catalase and oxidase. Furthermore, they produced H2S gas and DNase and occurred singly, in pairs, in short chains or in clusters. Both strains were observed to grow within the temperature range of 20–45 °C, pH 5.0–7.0 and NaCl concentration 1–4% (data not shown). No differences in chemical reactions were seen between these two strains with API ZYM and acid production from glucose. The biochemical and physiological characteristics that differentiate the strain MT/JULY 2010T from S. parasanguinis ATCC 15912T are shown in Table 1. The strain MT/JULY 2010T demonstrated positive reactions for hippurate hydrolysis, as well as the production of pyrrolidonyl arylamidase, while these characteristics were negative for S. parasanguinis ATCC 15912T. In contrast, S. parasanguinis ATCC 15912T was positive for the production of arginine hydrolase, β-glucuronidase and acid from trehalose. The strain MT/JULY 2010T was resistant to azithromycin, whereas S. parasanguinis ATCC 15912T was susceptible. Both strains were susceptible to vancomycin. The amino acid compositions of the cell walls in the two strains were glutamine, glycine, alanine and lysine (Table 1). The ratios of these amino acids (glutamine:glycine:alanine:lysine) relative to glutamine were 1 : 1.42 : 2.94 : 1.42 for S. parasanguinis ATCC 15912T and 1 : 0.63 : 4.83 : 1.43 for the strain MT/JULY 2010T, respectively. Furthermore, Table 2 shows the fatty acid compositions of strains MT/JULY 2010T and S. parasanguinis ATCC 15912T. The data from the strain MT/JULY 2010T matched Streptococcus bovis-GC subgroup A (currently classified as Streptococcus gallolyticus) [13], with a similarity index (SI) of 0.120, whereas no matches were found for S. parasanguinis ATCC 15912T. CFAs C17 : 0, C19 : 0 iso, C17 : 1 ω6c, C17 : 1 ω9c and C15 : 0 2OH were found in the strain MT/JULY 2010T but were absent in S. parasanguinis ATCC 15912T. In contrast, CFAs C9 : 0, C12 : 0 anteiso, C13 : 0, C16 : 0 anteiso, C20 : 0 iso, C17 : 1 anteiso A, C14 : 0 iso 3OH, and the summed feature (un 18.846/C19 : 1 ω6c, C19 : 1 ω6c/0.846 /C19 cyclo ω10c/C19 ω6) were identified in S. parasanguinis ATCC 15912T but not in the strain MT/JULY 2010T. The predominant saturated CFAs were C14 : 0 and C16 : 0, while the predominant unsaturated CFAs were C16 : 1 ω9c and the summed feature (C18 : 1 ω7c or C18 : 1 ω6c) for both strains. The summed feature C16 : 1 ω7c or C16 : 1 ω6c was a major fatty acid of the strain MT/JULY 2010T, but not of S. parasanguinis ATCC 15912T.
Fig. 1. Gram staining of (a) S. mobilis sp. nov., (b) S. parasanguinis, (c) Staphylococcus aureus and (d) E. coli.
Table 1. Characteristics differentiating the strain MT/JULY 2010T and S. parasanguinis ATCC 15912T.
| Characteristic | Strain MT/JULY 2010T | S. parasanguinis ATCC 15912T |
|---|---|---|
| Hydrolysis of | ||
| Hippurate | + | – |
| Production of | ||
| Arginine hydrolase | – | + |
| β-Glucuronidase | – | + |
| Pyrrolidonyl arylamidase | + | – |
| Acid production from | ||
| Trehalose | – | + |
| Antibiotic sensitivity | ||
| Azithromycin | R | S |
| Vancomycin | S | S |
| Cell wall amino acid composition (relative to glutamine) | ||
| Glutamine | 1.0 | 1.0 |
| Glycine | 0.63 | 1.42 |
| Alanine | 4.83 | 2.94 |
| Lysine | 1.43 | 1.42 |
The data presented in the table are based on the results of multiple biochemical tests, including API 20 STREP, antibiotic sensitivity and amino acid compositions of the cell wall, including peptidoglycan, in these strains. Both strains tested positive for the hydrolysis of aesculin and the activities of alkaline phosphatase, esterase (C4), α-galactosidase, β-galactosidase, α-glucosidase, naphthol-AS-BI-phosphohydrolase, leucine aminopeptidase (arylamidase), valine arylamidase, acid phosphatase, α-chymotrypsin and acid production from glucose, lactose and raffinose. Both strains were negative for acetoin production (Voges–Proskauer); the activities of esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase; and acid production from l-arabinose, inulin, mannitol, d-ribose, sorbitol, starch and glycogen.
Table 2. CFA compositions (%) of the strain MT/JULY 2010T and the strain S. parasanguinis ATCC 15912T.
| Fatty acid | Strain MT/JULY 2010T | S. parasanguinis ATCC 15912T |
|---|---|---|
| Saturated | ||
| C9 : 0 | nd | 0.2 |
| C12 : 0 | 0.8 | 1.0 |
| C12 : 0 anteiso | nd | 0.1 |
| C13 : 0 | nd | 0.1 |
| C13 : 0 anteiso | 0.1 | 0.2 |
| C14 : 0 | 13.6 | 13.6 |
| C16 : 0 | 23.7 | 15.9 |
| C16 : 0 anteiso | nd | 0.4 |
| C17 : 0 | 0.1 | nd |
| C18 : 0 | 2.3 | 1.5 |
| C19 : 0 iso | 0.5 | nd |
| C20 : 0 | 0.4 | 0.3 |
| C20 : 0iso | nd | 0.6 |
| Unsaturated | ||
| C15.1 iso F | 0.4 | 1.0 |
| C16 : 1 ω5c | 0.8 | 0.7 |
| C16 : 1 ω9c | 12.7 | 15.0 |
| C17 : 1 anteiso A | nd | 0.4 |
| C17 : 1 iso w5c | 1.0 | 3.0 |
| C17 : 1 ω6c | 0.1 | nd |
| C17 : 1 ω8c | 0.2 | 0.3 |
| C17 : 1 ω9c | 0.2 | nd |
| C19 : 1 iso I | 1.3 | 3.8 |
| C18 : 1 ω5c | 0.5 | 0.1 |
| C18 : 1 ω9c | 6.5 | 6.1 |
| C20 : 1 ω7c | 0.6 | 0.8 |
| C20 : 1 ω9c | 5.8 | 4.5 |
| Hydroxy | ||
| C13 : 0 2OH | 0.9 | 2.2 |
| C14 : 0 2OH | 0.4 | 0.6 |
| C14 : 0 iso 3OH | nd | 0.4 |
| C15 : 0 2OH | 0.8 | nd |
| C15 : 0 3OH | 0.6 | 0.6 |
| C16 : 0 3OH | 0.5 | 0.8 |
| Methylated | ||
| C19 : 0 10-methyl | 0.7 | 0.5 |
| Summed features | ||
| C12 : 0 aldehyde? (unknown 10.928) or C14 : 0 3OH and 16 : 1 iso I | 0.3 | 0.5 |
| C16 : 1 ω7c and C16 : 1 ω6c | 12.7 | 6.2 |
| C17 : 1 iso ω9c or C16 : 0 10-methyl | 0.4 | 0.7 |
| C18 : 1 ω7c or C18 : 1 ω6c | 18.5 | 17.6 |
| C18 : 2 ω6, 9c and C18 : 0 ante | 0.5 | 0.4 |
| un 18.846/C19 : 1 ω6c, C19 : 1 ω6c/0.846/C19cy or C19 : 0 cyclo ω10c/C19 ω6 | nd | 0.1 |
nd, not detected.
The motility tests with semisolid media were negative for the strain MT/JULY 2010T and S. parasanguinis ATCC 15912T. Interestingly, cells of strain MT/JULY 2010T exhibited an unusual tumbling motion through the hanging drop method, whereas such motion was not observed in the control strain (S. mitis). Ultrastructural studies conducted by electron microscopy on strain MT/JULY 2010T identified the cell sizes as 0.2–0.75 µm (average 0.3 to 0.5 µm) in diameter, with the presence of unidentified structures, peritrichous fimbriae-like appendages with terminal discs in large numbers, pili, multiple pedicles and blebs present on the surface (Fig. 2).
Fig. 2. Electron microscopic images of the strain MT/JULY 2010T. (a) The negative staining of the strain revealed the presence of a capsule (C), as well as pili and fimbriae with terminal bulbs (Tb). Additionally, there was an unidentified structure (U). Surrounding the cells, multiple small round structures were observed, which correspond to the terminal bulbs of the fimbriae and pili. (b) TEM revealed numerous fimbriae (F) and unidentified structure (U). Bar scale: 100 nm.
Genomic characterization
The pairwise similarity analysis of the 16S rRNA gene sequence using nucleotide blast searches for gene sequence homology revealed that strain MT/JULY 2010T was closely related to S. parasanguinis ATCC 15912T, with 98.6% similarity. The complete genome of strain MT/JULY 2010T was sequenced and annotated with dFAST (DDBJ Fast Annotation and Submission Tool). Its total length was 2,111,493 bp and L50 was 1. The number of coding sequences was 1,980, while the numbers of rRNA and tRNA were 12 and 60, respectively. The in silico DNA G+C content was 41.8 mol%. In accordance with Riesco and Trujillo [14], the genomic data of the strain MT/JULY 2010T were subjected to computational OGRI. Two-way ANI analysis using the calculator from Kostas lab showed that strain MT/JULY 2010T had an ANI of 94.41% with S. parasanguinis ATCC 15912T (Table 3). In addition, ANIs of 95%, 94.80 and 94.55% were found with the recently reported novel species Streptococcus caecimuris DSM 110150T, Streptococcus taoyuanensis ST2 and Streptococcus dentalis S1, respectively [15,17]. Furthermore, genome-based taxonomic analysis was performed by uploading the genome sequence to the TYGS. The digital DNA–DNA hybridization (dDDH) values using formula d4 were 58.7% for S. parasanguinis ATCC 15912T and 57.8% for S. dentalis S1. Higher dDDH values were observed with the recently reported novel species S. caecimuris DSM 110150T (60.8%) and S. taoyuanensis ST2 (60.1%) [15,16]. The results show that strain MT/JULY 2010T does not belong to any species found in the TYGS database and possibly is a novel species. The phylogenetic tree, inferred from GBDP distances calculated from the 16S rRNA gene and genome sequences, is shown in Figs3 4, respectively.
Table 3. ANI and dDDH values between the strain MT/JULY 2010T and closely related type strains.
| Reference genome | ANI | In silico DNA–DNA hybridization | ||
|---|---|---|---|---|
| % | Fragments | dDDH (d4, in %) | C.I. (d4, in %) | |
| Streptococcus taoyuanensis ST2 | 94.80 | 7,389 | 60.1 | 57.2–62.8 |
| S. caecimuris DSM 110150 | 95.00 | 7,175 | 60.8 | 58.0–63.6 |
| S. parasanguinis ATCC 15912 | 94.40 | 7,440 | 58.7 | 55.9–61.5 |
| S. dentalis S1 | 94.55 | 7,070 | 57.8 | 55.0–60.6 |
| S. lactarius CCUG 66490 | 87.78 | 5,286 | 35.1 | 32.6–37.6 |
| S. australis NCTC13166 | 86.53 | 2,649 | 27.2 | 24.8–29.7 |
| S. xiaochunlingii E24 | 86.42 | 2,363 | 26.2 | 23.8–28.7 |
| S. rubneri DSM 26920 | 86.21 | 2,536 | 26.5 | 24.1–29.0 |
| S. koreensis KCOM 2890 | 86.03 | 2,620 | 26.5 | 24.2–29.0 |
| S. ilei I-G2 | 85.92 | 2,667 | 26.6 | 24.3–29.1 |
| S. humanilactis IMAU99125 | 82.69 | 886 | 25.1 | 22.8–27.6 |
| S. vulneris DM3B3 | 82.02 | 907 | 24.4 | 22.1–26.9 |
| S. himalayensis HTS2 | 80.82 | 430 | 22.3 | 20.0–24.7 |
ANI was calculated using the ANI calculator at the Enveomics Gateway from Kostas Lab at Georgia Institute of Technology (https://enveomics.scigap.org/), based on reciprocal best-hit alignments (two-way ANI). dDDH values and confidence intervals (C.I.) were calculated using the recommended settings of the GGDC 4.0. formula d4: sum of all identities found in high-scoring segment pairs (HSPs) divided by overall HSP length. dDDH values >70% are considered as same species).
Fig. 3. Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from 16S rRNA gene sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 67.3%. The tree was rooted at the midpoint. 16S rRNA gene sequence from Lactococcus lactis subsp. lactis, a member of the family Streptococcaceae but outside the genus Streptococcus, was included as a reference taxon.
Fig. 4. Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 90.3%. The tree was rooted with Lactococcus lactis subsp. lactis, a member of the family Streptococcaceae but outside the genus Streptococcus as outgroup.
Discussion
The strain MT/JULY 2010T was a mesophilic, α-haemolytic, Gram-positive, coccoid bacterium. Its morphological features resembled those of Streptococcus species under a microscope, except for the unusual tumbling motion. This observation was surprising, as Streptococcus species are classically considered non-motile. Few historical reports have described motility-like behaviour in certain streptococci. However, subsequent analyses often revealed that these bacteria were misidentified and later reclassified into different genera [18,19]. This tumbling motion in strain MT/JULY 2010T is characterized by flipping, rotating, vibrating and non-swimming motility. Under certain conditions, the motions observed in this strain could be mistaken for Brownian motion. The non-swimming motility of the strain MT/JULY 2010T resulted in a negative result with the conventional motility test using semisolid media. However, these motions could be observed with the hanging drop assay. There were differences in the biochemical characteristics, antibiotic susceptibility, CFAs and amino acid components of the cell walls, including peptidoglycans, between the strain MT/JULY 2010T and S. parasanguinis ATCC 15912T. CFA’s study of the strain MT/JULY 2010T matched with S. gallolyticus with a low SI. S. gallolyticus is a Gram-positive bacterium that causes bacteraemia, endocarditis and gastrointestinal diseases [20]. The amino acid compositions of bacterial cell walls in these two strains were consistent with those of Gram-positive bacteria [21]. The levels of glutamic acid, glycine and lysine, relative to alanine, were lower in the strain MT/JULY 2010T compared to those in S. parasanguinis ATCC 15912T. The ratio of alanine to glycine was 2 : 1 for S. parasanguinis ATCC 15912T and 7.7 : 1 for the strain MT/JULY 2010T. Recent studies lead to the description of several new species closely related to strain MT/JULY2010ᵀ, including S. caecimuris [15], S. taoyuanensis [16] and S. dentalis [17]. To evaluate the distinctiveness of strain MT/JULY2010ᵀ, a comparative analysis was performed based on the available phenotypic and biochemical characteristics, as well as CFA compositions reported for these species (Tables S2 and S3). Limited phenotypic and biochemical information is available for S. caecimuris DSM 110150ᵀ, and therefore, a comprehensive comparative analysis could not be performed. Although the overall phenotypic profiles exhibit similar characteristics, differences were observed in several biochemical reactions and major fatty acid components. In particular, MT/JULY2010ᵀ showed distinct differences in major fatty acids compared to S. taoyuanensis [16] and S. dentalis [17], whereas CFA information for S. caecimuris is not available. A clear difference was observed in motility and NaCl tolerance between MT/JULY2010ᵀ and other species. However, the phenotypic features (Tables1 2, S2 and S3), including biochemical characteristics and CFA compositions, are not sufficient for conclusive species delineation. The integration of phenotypic data with genotypic analysis such as ANI, dDDH (Table 3) and phylogenomic analyses (Figs3 4) provides more reliable evidence for species-level identification. The results from both genetic and phenotypic comparisons in this study confirm that strain MT/JULY2010ᵀ represents a distinct taxon.
Although strain MT/JULY 2010T was isolated from gastric mucosal biopsy specimens, it has low acid resistance with an optimum growth range at pH 5.0–7.0. While the gastric lumen is highly acidic, the mucus layer has a relatively higher pH, which can support the colonization and growth of strain MT/JULY 2010T. Furthermore, in pre-neoplastic mucosa, hypochlorhydria (low stomach acid) is common due to the loss of parietal cells [22,23]. H. pylori-mediated chronic gastritis also contributes to hypochlorhydria [24,25], and therefore, less acid-tolerant strains like strain MT/JULY 2010T could grow in such environments. Additionally, the consistent observation of the coexistence of H. pylori and intracellular cocci bacteria suggests that urease produced by H. pylori might create a suitable environment. Further investigation is needed to uncover the mechanisms that support survival of strain MT/JULY 2010T in the gastric environment.
The phylogenetic trees constructed from the 16S rRNA gene sequence analysis demonstrated that the strain MT/JULY 2010T consistently clustered with S. parasanguinis ATCC 15912T, which is in the Mitis group [26]. Identifying a novel species in the Mitis group of the genus Streptococcus is challenging due to overlapping phenotypic features and close gene sequence relationships, including ANI unreliability [3]. The 16S rRNA sequence similarities among the type strains within the Mitis group of the genus Streptococcus are very high despite differences in the whole genome [3]. The similarity value of the 16S rRNA gene sequence for the strain MT/JULY 2010T and S. parasanguinis ATCC 15912T necessitated further testing of genomic DNA–DNA relatedness between them because the homology value of the 16S rRNA gene in these two species was very close to the threshold value (98.6%) used to discriminate between them [27]. The conventional DNA–DNA hybridization study demonstrated that the DNA–DNA relatedness value between the strain MT/JULY 2010T and S. parasanguinis ATCC 15912T was 62.5%, which did not meet the 70% recommended guideline for species delineation by Wayne et al. [28] and supported the heterogeneity of these two strains. WGS analysis further confirmed the heterogeneity of these two strains, revealing dDDH values of 58.7% and ANI of 94.4%. Further analysis of the genome of strain MT/JULY 2010T identified the presence of a pleiotropic regulator of exopolysaccharide synthesis in biofilm formation, lipopolysaccharide choline phosphotransferase LicD1, Rrf2 family transcriptional regulator and flagellar hook-length control protein FliK. Although the entire fliK gene sequence was detected, no other known flagella-related genes were identified. The genes associated with heat shock proteins (for instance, grpE, groES and groEL) and toxins (for instance, toxin higB and exfoliative toxin A) were also identified. Nevertheless, this novel bacterium was isolated from the gastric biopsy specimens of a 63-year-old H. pylori-positive Japanese woman with gastric erosion, active chronic gastritis, atrophy, intestinal metaplasia and atypia. Furthermore, motility such as unusual tumbling motion may be an important factor contributing to bacterial invasiveness and subsequent pathogenicity. The presence of these genetic traits and phenotypic features suggests that the strain MT/JULY 2010T could be pathogenic and of significant clinical relevance in gastritis.
In conclusion, the strain MT/JULY 2010T, isolated from gastric aspirates and gastric mucosal biopsy specimens of a H. pylori-positive Japanese female, represents a novel species in the genus Streptococcus based on phenotypic and genotypic characteristics. The name Streptococcus mobilis sp. nov. is proposed for the strain MT/JULY 2010T. Further investigations are warranted to determine the nature of this new bacterium, including its potential association with human pathology.
Description of Streptococcus mobilis sp. nov.
Streptococcus mobilis (moʹbi.lis. L. masc. adj. mobilis, movable, moving, named for its tumbling motion) sp. nov.
The type strain of this species is Gram-positive, facultatively anaerobic, non-endospore-forming cocci that occur singly, in pairs, in short chains or clustered masses in a liquid medium. Alfa-haemolysis is visible after 72 h of incubation at 37 °C on horse blood agar under anaerobic conditions. Colonies grow after 48 h under aerobic conditions at 37–45 °C on trypticase soy agar to 1 mm in diameter, exhibiting a creamy, buttery, smooth, round and convex shape. Cells measure 0.8–1.0×1.0–1.2 µm in size. Cell growth occurs at temperatures ranging from 20 to 45 °C, with a pH of 5–7, and in 1–4% (w/v) NaCl. The strain exhibits an unusual tumbling motion characterized by flipping, rotating and vibrating. Bacterial cells possess unidentified structures, multiple peritrichous fimbriae-like appendages and pili with terminal discs. Multiple pedicles, blebs and vesicles may be present on the cell membrane surface. Strain is negative for urease, catalase, oxidase, arginine hydrolase, β-glucuronidase, esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, β-glucosidase, N-acetyl-β-phosphohydrolase, α-mannosidase and α-fucosidase. The strain is positive for alkaline phosphatase, esterase (C4), α-galactosidase, β-galactosidase, α-glucosidase, leucine aminopeptidase (arylamidase), valine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and pyrrolidonyl arylamidase. Acid production from glucose, lactose and d-raffinose is positive, while acid production from l-arabinose, inulin, d-mannitol, d-ribose, d-sorbitol, d-trehalose, AmiDon starch and glycogen is negative. Acetoin production (Voges–Proskauer) is negative. The hydrolysis of aesculin and hippurate is positive. The strain produces H2S gas and DNase. Major fatty acids include C14 : 0, C16 : 0, C16 : 1 ω9c and the summed features (C16 : 1 ω7c or C16 : 1 ω6c) and (C18 : 1 ω7c or C18 : 1 ω6c). The following fatty acids are present in smaller amounts: C15 : 0 2OH, C17 : 0, C17 : 1 ω6c, C17 : 1 ω9c and C19 : 0 iso. The DNA G+C content of the type strain is 42.6 mol%.
The type strain MT/JULY 2010T (=ATCC BAA-2258T=NCBI 948105T=NBRC 107862T=V10/022878T) was isolated from gastric aspirates and gastric mucosal biopsy specimens of a H. pylori-positive 63-year-old Japanese woman with dyspeptic symptoms, who had pre-neoplastic mucosal changes in the stomach, in Brisbane, Queensland, Australia.
16S rRNA gene sequences of strain MT/JULY 2010T are available under GenBank accession LC877506–LC877509. Raw reads can be found from DDBJ under BioProject accession number PRJDB19917 (PSUB024865), and the complete annotated genome has been deposited in DDBJ under accession number AP041823.
Supplementary material
Acknowledgements
We thank TechnoSuruga Laboratory Co. Ltd and Hanaichi Electron Microscopy Institute. We are grateful to Em. Professor S. Kamiya at Kyorin University, Professor D. Graham at Baylor College of Medicine, Professor R. Peek Jr. at Vanderbilt University, Dr G. Adkins FRCP and Professor H. Miwa at Hyogo Medical College for their continuous encouragement and support.
Abbreviations
- ANI
average nucleotide identity
- CFA
cellular fatty acid
- dDDH
digital DNA–DNA hybridization
- OGRI
overall genome-related index
- SI
similarity index
- TEM
transmission electron microscopy
- TYGS
type (strain) genome server
- WGS
whole-genome sequence
Footnotes
Funding: This study was supported in part by a collaborative research grant from Sakura Corporation in Matsue, Shimane, Japan, as well as basic research grants from Osaka Prefecture University and Osaka Metropolitan University.
Ethical statement: This study was approved by the Okada Medical Research Ethics Committee as a number of 21010. All procedures were conducted in accordance with the ethical standards.
Accession No: 16S rRNA gene sequences of strain MT/JULY 2010T are available under GenBank accession LC877506–LC877509. Raw reads can be found from DDBJ under BioProject accession number PRJDB19917 (PSUB024865), and the complete annotated genome has been deposited in DDBJ under accession number AP041823.
Consent to publish: Written informed consent was obtained from the participant included in the study.
Contributor Information
Takayuki Okada, Email: drokada@ozemail.com.au.
Sharda Prasad Awasthi, Email: getsharda@omu.ac.jp.
Atsushi Hinenoya, Email: hinenoya@omu.ac.jp.
Noritoshi Hatanaka, Email: n.hatanaka@omu.ac.jp.
Shota Nakamura, Email: nshota@gen-info.osaka-u.ac.jp.
Daisuke Motooka, Email: daisukem@gen-info.osaka-u.ac.jp.
Shinji Yamasaki, Email: yshinji@omu.ac.jp.
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