Metabacillus schmidteae sp. nov., Cultivated from Planarian Schmidtea mediterranea Microbiota

: Taxonogenomics combines phenotypic assays and genomic analysis as a means of char-acterizing novel strains. We used this strategy to study Marseille-P9898 T strain, an aerobic, motile, Gram-negative, spore-forming, and rod-shaped bacterium isolated from planarian Schmidtea mediterranea . Marseille-P9898 T is catalase-positive and oxidase-negative. The major fatty acids detected are 12-methyl-tetradecanoic acid, 13-methyl-tetradecanoic acid, and hexadecanoic acid. Marseille-P9898 T strain shared more than 98% sequence similarity with the Metabacillus niabensis strain 4T19 T (98.99%), Metabacillus halosaccharovorans strain E33 T (98.75%), Metabacillus malikii strain NCCP-662 T (98.19%), and Metabacillus litoralis strain SW-211 T (97.15%). Marseille-P9898 strain belongs to Metabacillus genus. Genomic analysis revealed the highest similarities with Ortho-ANI and dDDH, 85.76% with Metabacillus halosaccharovorans, and 34.20% with Bacillus acidicola , respectively. These results show that the Marseille-P9898 T strain is a novel bacterial species from Metabacillus genus, for which we propose the name of Metabacillus schmidteae sp. nov. (Type strain Marseille-P9898 T = CSUR P9898 T = DSM 111480 T ). lipase leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α -chymotrypsin, acid phosphatase, α -galactosidase, β -galactosidase, β -glucosidase, and N-acetyl- β -glucosaminidase; no assimilate to α -mannosidase, α -fucosidase, erythritol, D-arabinose, D-ribose, L-xylose, D-adonitol, L-sorbose, L-rhamnose, Dulcitol, Inositol, D-sorbitol, methyl- α D-mannopyranoside, arbutin, glycogen, xylitol, D-turanose, D-tagatose, D-fucose, L-fucose, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, L-arginin, L-lysin, L-ormithin, trinatriumcitrat, natriumthiosulfat, urea, indole production, gelatin, potassium nitrate, capric acid, adipic acid, and phenylacetic acid. T strain sensitive to amikacin, fosfomycin, benzylpenicillin, ciproﬂoxacin, amoxicillin, ceftriaxone, daptomycin, doxycycline, rifampicin, vancomycin, and ampicillin. Fatty acids 12-methyl-tetradecanoic acid (62.6%), 13-methyl-tetradecanoic acid acid acid acid T type


Introduction
The birth of genomics, followed by the development of Next Generation Sequencing (NGS) methods, has allowed the characterization, classification, and nomenclature of many prokaryotic species using a taxonogenomics strategy combining phenotypic assays and genome sequencing [1][2][3]. Phenotypic assays are based on the analysis of morphological, physiological, chemical, and biochemical features of an organism [4]. Genotypic characterization, i.e., the analysis of the genetic material, is essential for species description and sheds light on the evolutionary relationships between various lineages. For the genotypic characterization, first, the gene 16S rRNA sequences are used to determine sequence similarity and for phylogenetic analysis [5]. Then Digital DNA-DNA hybridization values (dDDH) that evaluate the degree of genetic similarity between two genomes have been used for bacterial species demarcation by providing a constant numerical threshold (dDDH value > 70%) for species boundary [6].
Planarian Schmidtea mediterranea is an invertebrate living in freshwater and an excellent organism model to investigate the human host-pathogen relationship [7][8][9]. To understand the role of planarian S. mediterranea microbiota in the immune response, we studied its composition and isolated by culturomics [10], a bacterial strain (Marseille-P9898).
In this study, we have characterized the taxonogenomic properties of this strain, designated Marseille-P9898 (=CSUR P9898 T = DSM 111480 T ), which is a novel bacterial species named Metabacillus schmidteae sp. nov.

Planarian Schmidtea Mediterranea Culture
S. mediterranea flatworms (asexual clonal line ClW4) were kept in tap water filtered at 19 • C. Filtered water was obtained by filtration through a filter containing charcoal and ceramics (Fairey Industrial Ceramics limited, Suffolk, England), and a 0.2 µm membrane (Thermo Scientific Nalgene filtration Products, Mexico City, Mexico). The sterility of filtered water was analyzed before any utilization. Microbiological analysis of filtered water was performed by inoculation of filtered water (25,50,75, and 100 µL) in 5 % sheep bloodenriched Columbia agar plate (bioMérieux, Marcy l'étoile, France) and by incubation at various temperatures (5, 10, 19, 28, 37 and 45 • C). After four days, the absence of bacterial colonies was assessed.

Culture Conditions from Schmidtea mediterranea and Strain Isolation
Worms were starved for 2 weeks, washed in filter-sterilized water. One worm was smashed in a tube with a pestle in 500 µL of PBS, then 100 µL of the crushed mixture was inoculated in 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l'étoile, France) and incubated at 28 • C. Individual bacterial colonies were harvested and identified by MALDI-TOF-MS (Microflex spectrometer; Bruker Daltonics, Bremen, Germany) [22]. The MALDI Biotyper RTC software was used to interpret results according to obtained score values. The database used was the MaldiBiotyperDBUpdate_V9. 8468 MSP from the Bruker Daltonics. A bacterial colony is likely identified at the species level for a score ≥ 2.0; probably identified for a score between 1.99 and 1.7, but not identified for a score < 1.7. Profile spectrum of the whole cell was obtained after analysis as previously described [22].

DNA Extraction, Sequencing, Assembly, and Annotation
Genomic DNA extraction (gDNA) from the Marseille-P9898 strain was performed using an EZ1 BioRobot and EZ1 DNA tissue kit (Cat No./ID: 953034, Qiagen, Hilden, Germany). Then gDNA was quantified by a Qubit assay (Life Technologies, Carlsbad, CA, USA). Here, we used two technologies for Marseille-P9898 strain sequencing. First, gDNA was normalized at 0.2 ng/µL, then prepared and sequenced using the Mate-Pair strategy with a Miseq sequencer (Illumina, San Diego, CA, USA), as previously described [23]. Second, gDNA was normalized at 1.5 ng/µl and sequenced using nanopore strategy with a Minion sequencer (Oxford Nanopore MinION™) [24,25]. The reads of Miseq and MinION run were examined using FastQC 0.11.8 to evaluate quality [26]. Two sequencing reads were assembled using Spades [27] genome assembler software for regular and single-cell projects (Galaxy 3.12.0+galaxy1). The "conservative" option was used to reduce mismatches number and short indels. Default parameters were applied for each software. Genomic annotation was obtained using Prokka [28] software (Galaxy version 1.14.5+galaxy1).

Phylogenetic Analysis
For the taxonomic assignment, we used the nr database (Standard databases) for the BLASTn search. A sequence similarity threshold of 98.65% by comparison with the phylogenetically closest species with standing in nomenclature was used to delineate a putative novel species [29]. Phylogenetic relationships were inferred from the comparison of 16S rRNA sequences using MEGAX 10.1 software [30,31]. The method for estimating phylogenetic trees was maximum likelihood. Sequences were aligned using the MUSCLE algorithm with default parameters. Numbers shown at the nodes are percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree. Only bootstrap values ≥ 50% were retained. Scale bare indicates a 0.01% sequence divergence.

Chemotaxonomic Analysis
Cellular fatty acid methyl ester (FAME) analysis of Marseille-P9898 strain and Metabacillus niabensis strain 4T19 T was performed by GC/MS. Fatty acid methyl esters were prepared as published by Sasser [46] and GC/MS analysis was realized as previously described [47]. Briefly, fatty acid methyl esters were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500-SQ 8 S, Perkin Elmer, Courtaboeuf, France). Spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).

Phenotypic and Biochemical Characteristics
The Marseille-P9898 strain was isolated on 5% Columbia agar enriched with sheep blood (bioMérieux) after 2 days of growth at 28 • C in an aerobic atmosphere. This strain grows at a temperature ranging from 19 to 50 • C, and under pH from 7.5 to 10 (alkaline). Marseille-P9898 strain can grow at NaCl concentration lower than 10 g/L (Table 1). After 4 days of incubation at 28 • C on blood-enriched Columbia agar, Marseille-P9898 strain colonies were white, round, and smooth, with a 3-5 mm diameter and a convex shape (Supplementary data Figure S1). Electron microscopy revealed that Marseille-P9898 strain cells are rod-shaped and have a 2.70 µm mean length, a 0.42 µm mean width, and a polar flagellum (Figure 1). Bacterial cells are Gram-negative ( Figure 2), motile, and spore-forming bacilli (Supplementary data Figure S2). The endospore formation occurs in the terminal position. Marseille-P9898 strain is catalase positive and oxidase negative. Bacterial metabolism was assessed using API 50CHB/E, API 20NE, API Zym, and API 20E strips ( Table 2). Marseille-P9898 strain differs from Metab. halosaccharovorans, Metab. niabensis, Metab. malikii and Metab. litoralis compared to α-glucosidase, L-rhamnose, methyl-αD-glucopyranoside, glycogen, potassium gluconate, natriumpyruvat, glucose, trisodium citrate, because Marseille-P9898 strain needs these substrates to grow. Interestingly, identification by MALDI-TOF-MS of Marseille-P9898 strain showed a score of 1.77, matching with Metabacillus niabensis (spectrum in supplementary data Figure S3). Because this value was less than 2, the Marseille-P9898 strain cannot be identified as Metabacillus niabensis. This score only indicates that the Marseille-P9898 strain belongs to the Metabacillus genus. To define the species type, we performed a genomic approach.

Phylogenetic Analysis
The gene 16S rRNA sequence from the Marseille-P9898 strain had a size of 1548 bp. Using phylogenetic comparison with the GenBank database, we found that Marseille-P9898 strain had similarity at the level of an 16S rRNA gene sequence with Metabacillus niabensis strain 4T19 T [45] [68] (98.07%). The most closely related species to the Marseille-P9898 strain was the Metab. niabensis strain 4T19 T , Metab. halosaccharovorans strain E33 T , Metab. malikii strain NCCP-662 T and Metab. litoralis strain SW-211 T . A phylogenetic tree based on the maximum-likelihood algorithm revealed that the Marseille-P9898 strain is shared in a cluster of members of the Metabacillus genus (Figure 3), belonging from Bacillaceae family and Firmicutes phylum. On the phylogenetic tree, it was clear that the Marseille-P9898 strain presented a distinct taxon separated from other species of genus Bacillus. We selected nine closely related species to make a comparison genomic (Table 3  and Supplementary Data Table S1).
dDDH hybridization values were obtained using GGDC software and were reported in Table 3. Marseille-P9898 strain shows values ranged from 22.30% with Metab. niabensis to 34.20% with B. acidicola. These values were below the 70% threshold recognized for delineation of distinct species [32]. Ortho-ANI values ranged from 67.64% with Mesob. subterraneus to 85.76 % with Metab. halosaccharovorans, which was lower than the 95% threshold used to discriminate bacterial species [33] (Figure 4 and Table 4). These results were sufficient to separate the Marseille-P9898 strain from Metab. halosaccharovorans strain E33 T , Metab. niabensis strain 4T19 T , Metab. malikii strain NCCP-662 T , Metab. litoralis strain SW-211 T , and other most closely related species of genus Metabacillus. The distribution of genes into COG categories was similar among all compared genomes ( Figure 5 and Table 5). Taken together, these results confirmed that the Marseille-P9898 strain belongs to a separate Bacillus species.
dDDH hybridization values were obtained using GGDC software and were reported in Table 3. Marseille-P9898 strain shows values ranged from 22.30% with Metab. niabensis to 34.20% with B. acidicola. These values were below the 70% threshold recognized for delineation of distinct species [32]. Ortho-ANI values ranged from 67.64% with Mesob. subterraneus to 85.76 % with Metab. halosaccharovorans, which was lower than the 95% threshold used to discriminate bacterial species [33] (Figure 4 and Table 4). These results were sufficient to separate the Marseille-P9898 strain from Metab. halosaccharovorans strain E33 T , Metab. niabensis strain 4T19 T , Metab. malikii strain NCCP-662 T , Metab. litoralis strain SW-211 T , and other most closely related species of genus Metabacillus. The distribution of genes into COG categories was similar among all compared genomes ( Figure 5 and Table 5). Taken together, these results confirmed that the Marseille-P9898 strain belongs to a separate Bacillus species.

Discussion
Planarian S mediterranea is an organism model to investigate the regeneration [72] and host-pathogen relationship [7][8][9]. To understand the implication of microbiota in the S. mediterranea antimicrobial response, we investigated its composition. While, recently, we have already published the identification of Pedobacter schmidteae sp. nov., as a novel bacterium isolated from this microbiota [73], here, we report the identification of a second novel bacteria species, which belongs to Metabacillus genus called Metabacillus schmidteae sp. nov.
Description of novel bacterial species requires several approaches, the most recurrent one being the taxonogenomic approach [1][2][3]. Using this approach, we describe the main phenotypic and genotypic features of the Marseille-P9898 strain. The use of MALDI-TOF-MS showed a protein profile, but it did not allow the discrimination between the Marseille-P9898 strain and Metabacillus niabensis. 16S rRNA gene sequence presented a value of more than 98.65% [29], a cut-off to demarcate new species. We know that the conventional low divergence between two 16S rRNA genes from two organisms results in a slight and limited bacterial description [74,75]. Nonetheless, gene 16S rRNA sequence gives us the information concerning genomic sequence analysis by tools such as Genometo-Genome Distance Calculator (GGDC) [32] and Orthologous Average Nucleotide Identity (Ortho-ANI). Genomics allows the evaluation of the degrees of genomic similarity among species [33]. Taken together, genomic data showed that Marseille-P9898 is a novel bacterial species of genus Metabacillus called Metabacillus schmidteae sp. nov.
Until now, Metabacillus have been isolated from diverse environments including soil, hyper-saline aquatic, and marine coastal region [11]. We revealed that Metabacillus schmidteae is also hosted by aquatic animals such as planarians, precisely in their microbiota. We have observed that Metabacillus schmidteae is a Gram-negative bacteria (fresh and older cultures) and it is the first Gram-negative one from Metabacillus genus, which is usually Gram-positive or Gram-variable stained [11]. It possesses a polar motile flagellum, and it is strictly aerobic as other species of the genus Metabacillus. Metabacillus schmidteae can make spores under harsh conditions as reported for other species, except for Metabacillus weihaiensis [11]. In addition, biochemical analysis shows that Metabacillus (Metabacillus schmidteae Marseille-P9898 T , Metabacillus niabensis 4T19 T , Metabacillus halosaccharovorans E33 T, and Metabacillus litoralis SW-211 T ) use as substrates for their growth, D-galactose, D-glucose, D-fructose, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, Dtrehalose, and D-raffinose. Chemotaxonomic analysis indicates that Metabacillus (Metabacillus schmidteae Marseille-P9898 T , Metabacillus niabensis 4T19 T , Metabacillus halosaccharovorans E33 T, and Metabacillus litoralis SW-211 T ) produce membrane proteins such 15:0 iso (13-methyl-tetradecanoic acid), 15:0 anteiso (12-methyl-tetradecanoic acid), 17:0 anteiso (15methyl-Hexadecanoic acid). The data of chemotaxonomic and biochemical analysis could be used as a protein or biochemical marker to identify the genus metabacillus. Metabacillus schmidteae is now the second bacteria newly identified in planarian microbiota [73]. Its contribution in regeneration, as well as the antimicrobial capacity of the planarian, remains to be investigated.

Conclusions
Using taxonogenomic analysis, we described and characterized the Marseille-P9898 strain, hereby for the first time isolated from planarian S. schmidteae microbiota. Marseille-P9898 is phylogenetically related to Metabacillus genus. Chemotaxonomic and biochemical analysis and genomics comparison allowed considering Marseille-P9898 as a novel species of Metabacillus genus. For this strain Marseille-P9898, we propose the name of Metabacillus schmidteae sp. nov. (Marseille-P9898 T = CSUR P9898 T = DSM 111480 T ).

Nucleotide Sequence Accession Number
The 16S rRNA gene sequence and genome sequence were deposited in GenBank under accession numbers LR797940 and CAESCH000000000.1, respectively. Raw data of Illumina MiSeq paired-end and MinION sequencing were deposited in EMBL-EBI under run accession ERR4143811 and ERR4143810; and experiment Accession ERX4111084 and ERX4111083, respectively.

Deposit in Culture Collections
Marseille-P9898 T strain was deposited in the CSUR and DSMZ strain collections under numbers CSUR P9898 and DSM 111480, respectively.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/microbiolres12020021/s1, Figure S1: Colonies color Colonies color of Metabacillus schmidteae, Figure S2: Electron Micrograph of Metabacillus schmidteae Spore, Figure S3: MALDI-TOF spectrum of Metabacillus schmidteae, Table S1: GenBank accession numbers of the type strains. Data Availability Statement: The 16S rRNA gene sequence and genome sequence were deposited in GenBank under accession numbers LR797940 and CAESCH000000000.1, respectively. Raw data of Illumina MiSeq paired-end and MinION sequencing were deposited in EMBL-EBI under run accession ERR4143811 and ERR4143810; and experiment Accession ERX4111084 and ERX4111083, respectively.

Conflicts of Interest:
The authors have no conflicts of interest to declare. The funding sources had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.