Pedobacter ghigonii sp. nov., Isolated from the Microbiota of the Planarian Schmidtea mediterranea

The planarian S. mediterranea is a platyhelminth with worldwide distribution that can regenerate any part of its body after amputation and has the capacity to eliminate a large spectrum of human bacterial pathogens. Surprisingly, the microbiota of S. mediterranea remains poorly investigated. Using the culturomics strategy to study the bacterial component of planarians, we isolated a new bacterial strain, Marseille-Q2390, which we characterized with the taxono-genomic approach that associates phenotypic assays and genome sequencing and analysis. Strain Marseille-Q2390 exhibited a 16S rRNA sequence similarity of 99.36% with Pedobacter kyungheensis strain THG-T17T, the closest phylogenetic neighbor. It is a white-pigmented, Gram-negative, and rod-shaped bacterium. It grows in aerobic conditions and belongs to the family Sphingobacteriaceae. The genome of strain Marseille-Q2390 is 5,919,359 bp-long, with a G + C content of 40.3%. By comparing its genome with other closely related strains, the highest Orthologous Average Nucleotide Identity (Ortho-ANI) and digital DNA-DNA hybridization (dDDH) values were 85.71% and 30.50%, respectively, which were found with Pedobacter soli strain 15-51T. We conclude that strain Marseille-Q2390T is sufficiently different from other nearby species to be classified within a new species for which we propose the name Pedobacter ghigonii sp. nov.


Introduction
The platyhelminth Schmidtea mediterranea is an invertebrate living in freshwater such as ponds, lakes, and rivers. It is used as a model of regeneration because of its unique capacity to regenerate after amputation [1]. In addition, planarians have been shown to be among the models useful for the investigation of the host-pathogen relationship in the context of human pathogens [2][3][4]. The microbiota profile of S. mediterranea remains poorly investigated [5,6]. Using a microbial culturomics approach [7], we investigated the S. mediterranea microbiota. Culturomics is a strategy in which diversified culture conditions are used to isolate a maximum of bacterial species [8,9]. Through this methodology, we isolated a bacterium [10], Marseille-Q2390, from S. mediterranea that could not be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [11,12]. The used the taxono-genomics strategy, which combines phenotypic assays and genome sequencing, to characterize this bacterium [13,14]. Regarding genotypic criteria, this was first based on the 16S rRNA gene [15], but the conventional low divergence between two 16S rRNA genes from two organisms resulted in a slight and limited bacterial description [16,17]. However, the use of the genome gave access to complete genetic information and made it possible to evaluate the degrees of genomic similarity using tools such as the Genome-to-Genome Distance Calculator (GGDC) [18] and Orthologous Average Nucleotide Identity (Ortho-ANI) [19]. This genus Pedobacter [20] belongs to the family Sphingobacteriaceae [21] and has mostly been isolated from the environment [21,22] and in animals [23]. The main characteristics of this genus are that it is rod-shaped, aerobic, Gram-negative, and does not involve the formation of endospores; it is catalase-, oxidase-, and phosphatase-positive; the major fatty acids are C15:0 iso, C17:0 iso 3-OH, C15:0 iso 2-OH and C16:1 ω7c; and the species are phylogenetically closely related at the 16S rRNA gene level (>95%), except Pedobacter saltans [20]. Here we describe the bacterium Marseille-Q2390, which exhibited enough genetic and phenotypic differences from closely related species to be classified in a new species for which the name Pedobacter ghigonii sp. nov. is proposed.

Culture of Schmidtea mediterranea
We used the S. mediterranea asexual clonal line ClW4 [23], which had been preserved in our laboratory for the previous ten years by cutting animals from tree fragments each month. The S. mediterranea were fed once per week with homogenized calf liver. Planarians were grown in filtered tap water at 19 • C. The water used was obtained by a device consisting of two 0.2 µm filters, one containing charcoal and ceramics (Fairey Industrial Ceramics Limited, Sulffolk, England), and the second being a simple 0.22 µm pore membrane (Thermo Scientific Nalgene Filtration Products, Mexico City, Mexico). Filtered water was checked for sterility prior to being used for S. mediterranea, using 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l'étoile, France) at different volumes (25,50,75, and 100 µL) and incubated at various temperatures (5,10,19,28,37, and 45 • C) for four days.

Isolation and Identification of the Bacteria from Schmidtea mediterranea
Before being used for experimentation, S. mediterranea worms were starved for two weeks, washed in filter-sterilized water, and then one ground worm was inoculated in Buffered Charcoal Yeast Extract (BCYE) (Oxoid Deutschland GmbH, Wesel, Germany), Luria Bertani (LB), and 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l'étoile, France). All inoculated media were incubated at 19, 28, and 37 • C. Each individual bacterial colony was harvested and identified by MALDI-TOF-MS (Microflex spectrometer; Bruker Daltonics, Bremen, Germany). The MALDI Biotyper RTC software was used to interpret the results according to the obtained score values: a colony was judged to be likely to be identified at the species level if it gained a score ≥ 2.0; probably identified at the genus level if it gained a score between 1.99 and 1.7; and not identified if it gained a score < 1.7.

DNA Extraction, Sequencing, Assembly, and Annotation
The genomic DNA of the strain Marseille-Q2390 was extracted using an EZ1 BioRobot and the EZ1 DNA tissue kit (Cat No./ID: 953034, Qiagen, Hilden, Germany). Genomic material was quantified using a Qubit assay (Life Technologies, Carlsbad, CA, USA) at 0.2 ng/µL, and then prepared and sequenced using the Mate-Pair strategy with a Miseq sequencer (Illumina, San Diego, CA, USA) [24] using the Nextera XT DNA sample prep kit (Illumina). The Miseq run were checked to evaluate quality using FastQC 0.11.8 [25] then trimmed using Trimmomatic 0.36.6 [26], with default parameters. Sequencing reads were assembled using Spades 3.12 [27], the "conservative" option was used to reduce the number of mismatches and short indels, and default parameters were applied. Genomic annotation of the strain Marseille-Q2390 was made using the Prokaryotic genome annotation 1.14.5 (Prokka) [28] with default parameters.

Phylogenetic Analysis
The taxonomic assignment was obtained by a BLASTn search in the nr database. A 98.65% sequence similarity threshold was used to delineate a new putative species by comparison with the phylogenetically closest species found in the nomenclature [29]. Phylogenetic relationships were inferred from the comparison of 16S rRNA sequences using the MEGAX 10.1 software [30,31], using the Maximum Likelihood Phylogenetic model. The sequences were aligned using the MUSCLE algorithm default parameters. Numbers at the nodes were percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree (bootstrap values ≥50% were retained). The scale bar indicated a 1% sequence divergence.

Antibiotic Susceptibility
After 48 h of growth, the colonies of the strain Marseille-Q2390 were suspended in saline to match the McFarland 0.5 turbidity standard. Columbia agar enriched with 5% sheep blood (bioMérieux) was inoculated with a suspension of the bacterial isolate. E-test strips (bioMérieux) were put on the surface of the 5% sheep blood-enriched Columbia agar, and the agar was incubated in an aerobic atmosphere at 28 • C for 48 h. The susceptibility of the strain Marseille-Q2390 was assessed for the benzylpenicillin, amoxicillin, ampicillin, ceftriaxone, imipenem, ciprofloxacin, amikacin, gentamicin, streptomycin, daptomycin, doxycycline, metronidazole, rifampicin, fosfomycin, vancomycin, and tigecycline. MICs were read at the point of intersection between the developed elliptical zone of inhibition and the test strip. Interpretation of the MICs was carried out according to NCCLS recommendations for bacterial isolates grown aerobically [44].

Analysis of Cellular Fatty Acids of the Strain Marseille-Q2390
Cellular fatty acid methyl ester (FAME) analysis was performed by GC/MS. Two samples were prepared with 120 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as described by Sasser [45] and GC/MS analysis was carried out as previously described [46]. 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). A spectral database search was performed using MS Search 2.0, operated with the Standard Reference Database 1A (NIST, Gaithersburg, MA, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).

MALDI-TOF-MS
MALDI-TOF-MS analysis showed that the spectrum of the strain Marseille-Q2390 corresponds to the spectrum of Pedobacter soli with a score of 1.8. This spectrum similarity score of 1.8 does not allow the classification of the strain Marseille-Q2390 as Pedobacter soli, because this value is less than 2. However, it was probably a strain belonging to the genus of Pedobacter at an earlier time since this score was between 1.7 and 1.99.

Phylogenetic Analysis
The gene 16S rRNA sequence from strain Marseille-Q2390 was 1519 bp. A sequence similarity calculation using the BLASTn search in the nr database indicated that the closest relatives of the strain Marseille-Q2390 were Pedobacter kyungheensis strain THG-T17 T (99.36%) [47], Pedobacter roseus strain CL-GP80 T [48] [21], P. changchengzhani strain E01020 T [66], P. seoulensis strain THG-G12 T [67], and P. schmidteae strain EGT [23], for which the similarity values and accesssion numbers are presented in Table 1. Although the species name Pedobacter wanjuense strain PL247-sym is not taxonomically correct, it is important to point out that there is a closely related 16S rRNA sequence in the genebank repository (KP277503.1) [68]. The 16S rRNA-based phylogenetic tree showed that strain Marseille-Q2390, P. soli strain 15-51 T , and P. kyungheensis strain THG-T17 T formed a monophyletic group with a high bootstrap value (54%), which was supported by both tree-making analyses ( Figure 1). Strain Marseille-Q2390 is a member of the family Sphingobacteriaceae [21] within the phylum Bacteroidetes [69], from the class of Sphingobacteriia [70] and the order of Sphingobacteriales [71], and of the genre Pedobacter [20] (Table 2). This result confirmed the data from the MALDI-TOF-MS analysis, showing that it is of the genus Pedobacter. Without this, the use of one gene (16S rRNA) would not have been sufficient to confirm such a result, so it would have been necessary to use the complete genome.

Genomic Comparison
The genome sequence from strain Marseille-Q2390 was assembled into 41 contigs for a total size of 5,921,534 bp (N50, 292.871; L50, 7; coverage, 20×) with a G + C content of 40.3%. A total of 4.870 predicted protein-coding genes were identified, along with 7 rRNA, 49 tRNAs, and 1tmRNA ( Table 3). The genome of strain Marseille-Q2390 was compared with those of P. kyungheensis, P. zeae, P. alluvionis, P. borealis, P. ginsenosidimutans, P. kyonggii, P. soli, P. suwonensis, P. terrae, and P. suwonensis. With regard to contigs, size, CDSs, GC%, tRNAs, and rRNAs, all strains were shown to have different characteristics. Digital DNA-DNA hybridization (dDDH) values obtained using the GGDC software for the strain Marseille-Q2390 ranged from 23.60% with P. suwonensis and P. terrae to 30.50% with Pedobacter soli and P. kyungheensis (Table 4 and Table S1). As strain Marseille-Q2390 was mostly clustered with Pedobacter soli and P. kyungheensis (30.50%), such values were lower than the 70% threshold recognized to delineate bacterial species [18]. Accordingly, Marseille-Q2390 is a new species of Pedobacter. Similarly, the Ortho-ANI values ( Figure 2 and Table S1) ranged from 79.48% with P. suwonensis to 85.71% with P. soli, which was lower than the 95% threshold used to discriminate species [19]. The strain Marseille-Q2390 was grouped with the genus Pedobacter soli with a lower identity percentage, and thus Marseille-Q2390 was found to be a new species of Pedobacter. The distribution of genes in COG functional categories is presented in Figure 3 and , which are typically involved in several functions in bacteria, was not produced in the genus Pedobacter. Moreover, the other proteins were produced in all the species studied, which means that the quality of the proteins was similar in all compared species; but the quantity produced was different from one species to another species. Thus, with the genomic data we could confirm that strain Marseille-Q2390 belongs to a separate Pedobacter species.

Phenotypic Characteristics of Strain Marseille-Q2390
Strain Marseille-Q2390 was isolated on Columbia agar after two days at 28 °C in an aerobic atmosphere at pH 7. Strain Marseille-Q2390 grew at temperatures ranging from 4 to 30 °C in an aerobic atmosphere and at pH values ranging from 6 to 10 (neutro-alkalophilic bacterium). It also grew at salinity concentrations lower than 9 g/L. After four days of culture on Columbia agar, colonies of strain Marseille-Q2390 were white, small (0.3 mm median diameter), circular with a convex shape, and smooth. Bacterial cells were Gramnegative (Figure 4), rod-shaped, non-spore-forming, and motile bacilli, but without any flagellum. Their mean length and width were 2.25 µm and 0.86 µm, respectively ( Figure  5). Strain Marseille-Q2390 exhibited positive oxidase and catalase activities. Positive and negative reactions obtained using API 50CHB/E, API 20NE, API Zym, and API 20E strips are show in Table 5. These data were compared to those of closely related species data, including P. soli 15-51 T and P. borealis G1 T , as previously described [49,50]. Strain Mar-

Phenotypic Characteristics of Strain Marseille-Q2390
Strain Marseille-Q2390 was isolated on Columbia agar after two days at 28 • C in an aerobic atmosphere at pH 7. Strain Marseille-Q2390 grew at temperatures ranging from 4 to 30 • C in an aerobic atmosphere and at pH values ranging from 6 to 10 (neutroalkalophilic bacterium). It also grew at salinity concentrations lower than 9 g/L. After four days of culture on Columbia agar, colonies of strain Marseille-Q2390 were white, small (0.3 mm median diameter), circular with a convex shape, and smooth. Bacterial cells were Gram-negative (Figure 4), rod-shaped, non-spore-forming, and motile bacilli, but without any flagellum. Their mean length and width were 2.25 µm and 0.86 µm, respectively ( Figure 5). Strain Marseille-Q2390 exhibited positive oxidase and catalase activities. Positive and negative reactions obtained using API 50CHB/E, API 20NE, API Zym, and API 20E strips are show in Table 5. These data were compared to those of closely related species data, including P. soli 15-51 T and P. borealis G1 T , as previously described [49,50].

Conclusions
On the basis of phenotypic and genomic data, we confirmed that strain Marseille-Q2390 belongs to a new species within the Pedobacter genus for which we propose the name Pedobacter ghigonii sp. nov. strain Marseille-Q2390 T . This strain is abundant among the Schmidtea mediterranea planarians but has not yet been identified in any other environment. Identification of the strain Marseille-Q2390 makes it possible to study its involvement in the gut microbiota of planarians.

Deposit in Culture Collections
Strain Marseille-Q2390 T was deposited in the CSUR strain collections under number Q2390.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/microbiolres12020019/s1, Table S1: dDDH and OrthoANI analysis of phylogenetically related species of the strain Marseille-Q2390, Table S2: Functional annotation of strain Marseille-Q2390 predicted gene according to the COGs database.