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Article

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

by
Luis Johnson Kangale
1,2,
Didier Raoult
2,3,4 and
Fournier Pierre-Edouard
1,2,*
1
UMR VITROME, SSA, Aix-Marseille University, IRD, AP-HM, IHU-Méditerranée-Infection, 13385 Marseille, France
2
IHU-Méditerranée-Infection, 13385 Marseille, France
3
Department of Epidemiology of Parasitic Diseases, Aix Marseille University, IRD, AP-HM, MEPHI, 13385 Marseille, France
4
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2021, 12(2), 268-287; https://doi.org/10.3390/microbiolres12020019
Submission received: 29 January 2021 / Revised: 19 March 2021 / Accepted: 22 March 2021 / Published: 1 April 2021
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Abstract

:
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.

1. 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.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.3. 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.

2.4. 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.

2.5. Genomic Comparison

Clusters of Orthologous Groups (COG) [32] functional category comparison was carried out in Blastp. Degrees of genomic similarity were evaluated using the GGDC (http://ggdc.dsmz.de/ggdc.php, GGDC Genome-to-Genome Distance Calculator 2.1) [18] and Ortho-ANI (https://www.ezbiocloud.net/tools/orthoani, OrthoANI Tool Version 0.93.1) [19] software.

2.6. Phenotypic Characteristics of Strain Marseille-Q2390

Culturing of strain Marseille-Q2390 was attempted at various growth temperatures (4, 19, 28, 30, 37, and 45 °C) in 5% sheep blood-enriched Columbia agar (bioMérieux) under aerobic and anaerobic atmospheres (using GasPak™ EZ generators (Becton-Dickinson, Maryland, MD, USA)). A sporulation assay was undertaken by thermal shock. Bacteria were exposed to a temperature of 80 °C for 30 min. Then, bacterial growth was monitored for four days. The bacterial growth was also tested in various salinity (0, 20, 40, 50, 60, 80, and 100 g/L) and pH (5, 5.5, 6, 6.5, 7.5, 8.5, 9, and 10) conditions. Gram staining and motility from fresh colonies were observed using a DM1000 photonic microscope (Leica Microsystems, Nanterre, France) with a 40× objective lens and 10× ocular lens. Bacterial structure was evaluated by scanning electron microscope (Hitachi SUV5000) (Hitachi High-Technologies Corporation, Science & Medical Systems Business Group, Tokyo, Japan). Catalase and oxidase activities were investigated using BBL DrySlide, in accordance with the manufacturer’s instructions (Becton Dickinson, Le Pont de Claix, France). The biochemical characteristics were identified using API strips (API ZYM [33,34,35], API 20NE [36,37], API 20E [38,39], and API 50CH [40,41,42,43], bioMérieux).

2.7. 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].

2.8. 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).

3. Results and Discussions

3.1. 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.

3.2. 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-T17T (99.36%) [47], Pedobacter roseus strain CL-GP80T [48], P. soli strain 15–51T [49], P. borealis strain G-1T [50], P. alluvionis strain NWER-II11T [50], Pedobacter miscanthi strain RS10T [51], P. ginsenosidimutans strain THG-45T [52], P. suwonensis strain 15–52T [53], P. jejuensis strain THG-DR3T [54], P. kyonggii strain K-4-11-1T [55], P. nototheniae strain 36B243T [56], P. psychrotolerant strain V5RDT [55], P. zeae strain 22T [57], P. agri PB92T [58], P. terrae strain DS-57T [59], P. rhizosphaerae strain 01–96T [49], P. jeongneungensis strain BH45T [60], P. vanadiisoli strain XNV015T [61], P. humicola strain R135T [62], P. lithocola strain CCM 8691T [63], P. sandarakinus strain DS-27T [64], P. jamesrossensis strain CCM 8689T [63], P. petrophilus strain CCM 8687T [63], P. ginsengiterrae strain DCY49T [65], P. heparinus strain DSM 2366T [21], P. changchengzhani strain E01020T [66], P. seoulensis strain THG-G12T [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 15–51T, and P. kyungheensis strain THG-T17T 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.

3.3. 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 Table S2. We can note that the proteins [A], [Y], [Z], [W], and [X], 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.

3.4. 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 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 15–51T and P. borealis G1T, as previously described [49,50]. Strain Marseille-Q2390 differed from all other compared species of P. soli in the use of α-mannosidase, l-arabinose, D-xylose, D-galactose, D-fructose, L-rhamnose, methyl-αD-mannopyranoside, methyl-αD-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, D-saccharose, D-trehalose, D-melezitose, D-raffinose, starch, glycogen, gentiobiose, and D-turanose.

3.5. Antibiotic Susceptibility

Strain Marseille-Q2390 was susceptible to ceftriaxone, imipenem, ciprofloxacin, amikacin, gentamicin, streptomycin, doxycyclin, rifampicin, fosfomycin, and tigecycline, but it was resistant to benzylpenicillin, amoxicillin, ampicillin, daptomycin, metronidazole, and vancomycin (Table 6). Accordingly, strain Marseille-Q2390 was found to be resistant to antibiotics of the β-lactamin family.

3.6. Cellular Fatty Acids Analysis

The fatty acids were 13-methyl-tetradecanoic acid (54.5%), 9-hexadecenoic acid (11.1%), 11-hexadecenoic acid (8.6%), 3-hydroxy-15-methyl-hexadecenoic acid (5.5%), 15-methyl-hexadecenoic acid (3.5%), 3-hydroxy-13-methyl-tetradecanoic acid (3.5%), and 3-methyl-butanoic acid (3.0%). Minor amounts of other fatty acids included hexadecanoic acid (1.1%), 8-methyl-decanoic acid (1.7%), 14-methyl-hexadecenoic acid (1.1%), 9-methyl-decanoic acid (1.8%), and 8-pentadecenoic acid (1.1%) (Table 7). Comparing the fatty acid profile of strain Marseille-Q2390 with P. soli 15–51T, Marseille-Q2390 can be seen to differ from P. soli 15–51T due to the presence of 11:0 anteiso, 5:0 iso, and 15:1ω7. 13-methyl-tetradecanoic acid (15:0 iso) was found in all strains; thus, 15:0 iso could be used as a Pedobacter signature marker.

4. 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-Q2390T. 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.

4.1. Protologue

Pedobacter ghigonii (ghi.go.ni’i/N.L. masc. Gen. from Ghigo, family name of Eric Ghigo a French researcher who works on planarians) is a bacterium belonging to the family Sphingobacteriaceae within the phylum Bacteroidetes. Type strain Marseille-Q2390T was isolated, on 5% sheep blood-enriched Columbia agar after 2 days at 28 °C in aerobic atmosphere at pH 7, from the microbiota of the planarian Schmidtea mediterranea. Colonies were small, circular, smooth, white, and convex. Cells were Gram-negative, rod-shaped, motile, and non-spore-forming bacilli showing positive catalase and oxidase activities. The fatty acids were 3-methyl-Butanoic acid, 13-methyl-tetradecanoic acid, 15-methyl-Hexadecenoic acid, 3-hydroxy-13-methyl-Tetradecanoic acid, 3-hydroxy-15-methyl-Hexadecenoic acid, 9-Hexadecenoic acid, and 11-Hexadecenoic acid. Positive activities were detected for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. Lipase (C14), α-chymotrypsin, and β-glucuronidase were the negative activities. Glucose, mannose, esculin ferric citrate, and maltose were assimilated, but not glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl-βD-xylopyranoside, D-galactose, D-fructose, L-sorbose, L-rhamnose, inositol, D-mannitol, D-sorbitol, methyl-αD-mannopyranoside, methyl-αD-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, D-saccharose, inulin, D-melezitose, D-raffinose, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium, or 5-ketogluconate. Positives reactions were observed for L-tryptophan, natrium pyruvate, N-acetyl-glucosamine, capric acid, malic acid, and trisodium citrate. No reaction was observed for L-lysin, L-ormithin, trinatrium citrate, natrium thiosulfate, indole production, gelatin, potassium nitrate, L-arginine, urea, adipic acid, or phenylacetic acid. The genome of strain Marseille-Q2390T is 5,919,359 bp-long with a G+C content of 40.3%. The 16S rRNA gene and genome sequences were deposited in GenBank under accession numbers LR797942 and CAESCM000000000.1, respectively. Type strain Marseille-Q2390T was deposited in the Collection de Souche de l’Unité des Rickettsies (CSUR).

4.2. Nucleotide Sequence Accession Numbers

The 16S rRNA gene sequence (BioProject: PRJEB37821) was deposited in GenBank under accession numbers LR797942.1. BioProject PRJNA224116, BioSample SAMEA6828267, and Assembly GCF_903166585.1 were deposited in GenBank under accession number NZ_CAESCM010000000, and consist of sequences CAESCM010000001–CAESCM010000041. The raw data from Illumina MiSeq paired-end sequencing (BioProject: PRJEB37821, Experiment: ERX4110733 and BioSample: SAMEA6830360 (ERS4557981)) were deposited in the sequence read archive (SRA) under run accession numbers ERR4143460.

4.3. Deposit in Culture Collections

Strain Marseille-Q2390T 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.

Author Contributions

L.J.K. conceived and realized the experiments, analyzed the data, prepared figures, and drafted the manuscript. D.R. and F.P.-E. designed the experiments, conceived the experiments, analyzed the data, and drafted the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The study was funded by the Méditerranée-Infection foundation, the National Research Agency under the program “Investissements d’avenir”, reference ANR-10-IAHU-03, and by Région Provence Alpes Côte d’Azur and European funding FEDER IHUBIOTK.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The 16S rRNA gene sequence (BioProject: PRJEB37821) was deposited in GenBank under accession numbers LR797942.1. BioProject PRJNA224116, BioSample SAMEA6828267, and Assembly GCF_903166585.1 were deposited in GenBank under accession number NZ_CAESCM010000000, and consist of sequences CAESCM010000001–CAESCM010000041. The raw data from Illumina MiSeq paired-end sequencing (BioProject: PRJEB37821, Experiment: ERX4110733 and BioSample: SAMEA6830360 (ERS4557981)) were deposited in the sequence read archive (SRA) under run accession numbers ERR4143460.

Acknowledgments

L.J.K. is a fellow of the Méditerranée-Infection foundation. The study was funded by the Méditerranée-Infection foundation, the National Research Agency under the program “Investissements d’avenir”, reference ANR-10-IAHU-03, and by Région Provence Alpes Côte d’Azur and European funding FEDER IHUBIOTK. We also thank Aurelia Caputo for submitting the 16S rRNA and genomic sequences to GenBank.

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.

References

  1. Elliott, S.A.; Alvarado, A.S. The history and enduring contributions of planarians to the study of animal regeneration. Wiley Interdiscip. Rev. Dev. Biol. 2012, 2, 301–326. [Google Scholar] [CrossRef]
  2. Abnave, P.; Mottola, G.; Gimenez, G.; Boucherit, N.; Trouplin, V.; Torre, C.; Conti, F.; Ben Amara, A.; Lepolard, C.; Djian, B.; et al. Screening in Planarians Identifies MORN2 as a Key Component in LC3-Associated Phagocytosis and Resistance to Bacterial Infection. Cell Host Microbe 2014, 16, 338–350. [Google Scholar] [CrossRef] [Green Version]
  3. Maciel, E.I.; Jiang, C.; Barghouth, P.G.; Nobile, C.J.; Oviedo, N.J. The planarian Schmidtea mediterranea is a new model to study host-pathogen interactions during fungal infections. Dev. Comp. Immunol. 2019, 93, 18–27. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Torre, C.; Ghigo, E. La planaire: Un ver immortel pour élucider la réponse immunitaire de l’homme. Méd. Sci. 2015, 31, 20–22. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Arnold, C.P.; Merryman, M.S.; Harris-Arnold, A.; A McKinney, S.; Seidel, C.W.; Loethen, S.; Proctor, K.N.; Guo, L.; Alvarado, A.S. Pathogenic shifts in endogenous microbiota impede tissue regeneration via distinct activation of TAK1/MKK/p38. eLife 2016, 5, e16793. [Google Scholar] [CrossRef] [PubMed]
  6. Lee, F.J.; Williams, K.B.; Levin, M.; Wolfe, B.E. The Bacterial Metabolite Indole Inhibits Regeneration of the Planarian Flatworm Dugesia japonica. iScience 2018, 10, 135–148. [Google Scholar] [CrossRef] [Green Version]
  7. Seng, P.; Abat, C.; Rolain, J.M.; Colson, P.; Lagier, J.-C.; Gouriet, F.; Fournier, P.E.; Drancourt, M.; La Scola, B.; Raoult, D. Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. J. Clin. Microbiol. 2013, 51, 2182–2194. [Google Scholar] [CrossRef] [Green Version]
  8. Lagier, J.-C.; Armougom, F.; Million, M.; Hugon, P.; Pagnier, I.; Robert, C.; Bittar, F.; Fournous, G.; Gimenez, G.; Maraninchi, M.; et al. Microbial culturomics: Paradigm shift in the human gut microbiome study. Clin. Microbiol. Infect. 2012, 18, 1185–1193. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Lagier, J.-C.; Hugon, P.; Khelaifia, S.; Fournier, P.-E.; La Scola, B.; Raoult, D. The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota. Clin. Microbiol. Rev. 2015, 28, 237–264. [Google Scholar] [CrossRef] [Green Version]
  10. Woese, C.R.; Kandler, O.; Wheelis, M.L. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 1990, 87, 4576–4579. [Google Scholar] [CrossRef] [Green Version]
  11. La Scola, B.; Raoult, D. Direct Identification of Bacteria in Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry. PLoS ONE 2009, 4, e8041. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Seng, P.; Drancourt, M.; Gouriet, F.; La Scola, B.; Fournier, P.; Rolain, J.M.; Raoult, D. Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. Clin. Infect. Dis. 2009, 49, 543–551. [Google Scholar] [CrossRef]
  13. Ramasamy, D.; Mishra, A.K.; Lagier, J.-C.; Padhmanabhan, R.; Rossi, M.; Sentausa, E.; Raoult, D.; Fournier, P.-E. A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. Int. J. Syst. Evol. Microbiol. 2014, 64, 384–391. [Google Scholar] [CrossRef]
  14. Fournier, P.-E.; Lagier, J.-C.; Dubourg, G.; Raoult, D. From culturomics to taxonomogenomics: A need to change the taxonomy of prokaryotes in clinical microbiology. Anaerobe 2015, 36, 73–78. [Google Scholar] [CrossRef] [PubMed]
  15. Drancourt, M.; Berger, P.; Raoult, D. Systematic 16S rRNA Gene Sequencing of Atypical Clinical Isolates Identified 27 New Bacterial Species Associated with Humans. J. Clin. Microbiol. 2004, 42, 2197–2202. [Google Scholar] [CrossRef] [Green Version]
  16. Pei, A.Y.; Oberdorf, W.E.; Nossa, C.W.; Agarwal, A.; Chokshi, P.; Gerz, E.A.; Jin, Z.; Lee, P.; Yang, L.; Poles, M.; et al. Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes. Appl. Environ. Microbiol. 2010, 76, 3886–3897. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Ochman, H.; Elwyn, S.; Moran, N.A. Calibrating bacterial evolution. Proc. Natl. Acad. Sci. USA 1999, 96, 12638–12643. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Auch, A.F.; Von Jan, M.; Klenk, H.-P.; Göker, M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genom. Sci. 2010, 2, 117–134. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Lee, I.; Kim, Y.O.; Park, S.-C.; Chun, J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 2016, 66, 1100–1103. [Google Scholar] [CrossRef] [PubMed]
  20. Margesin, R.; Shivaji, S. Pedobacter. In Bergey’s Manual of Systematics of Archaea and Bacteria; American Cancer Society: Atlanta, GA, USA, 2015; pp. 1–17. [Google Scholar]
  21. Steyn, P.L.; Segers, P.; Vancanneyt, M.; Sandra, P.; Kersters, K.; Joubert, J.J. Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. Proposal of the family Sphingobacteriaceae fam. nov. Int. J. Syst. Bacteriol. 1998, 48, 165–177. [Google Scholar] [CrossRef] [PubMed]
  22. Viana, A.T.; Caetano, T.; Covas, C.; Santos, T.; Mendo, S. Environmental superbugs: The case study of Pedobacter spp. Environ. Pollut. 2018, 241, 1048–1055. [Google Scholar] [CrossRef]
  23. Kangale, L.J.; Raoult, D.; Ghigo, E.; Fournier, P.-E. Pedobacter schmidteae sp. nov., a new bacterium isolated from the microbiota of the planarian Schmidtea mediterranea. Sci. Rep. 2020, 10, 1–12. [Google Scholar] [CrossRef] [Green Version]
  24. Ravi, R.K.; Walton, K.; Khosroheidari, M. MiSeq: A Next Generation Sequencing Platform for Genomic Analysis. Methods Mol. Biol. 2018, 1706, 223–232. [Google Scholar] [CrossRef] [PubMed]
  25. Wingett, S.W.; Andrews, S. FastQ Screen: A tool for multi-genome mapping and quality control. F1000Research 2018, 7, 1338. [Google Scholar] [CrossRef] [PubMed]
  26. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [Green Version]
  28. Seemann, T. Prokka: Rapid Prokaryotic Genome Annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef]
  29. Meier-Kolthoff, J.P.; Göker, M.; Spröer, C.; Klenk, H.-P. When should a DDH experiment be mandatory in microbial taxonomy? Arch. Microbiol. 2013, 195, 413–418. [Google Scholar] [CrossRef]
  30. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  31. Tamura, K.; Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 1993, 10, 512–526. [Google Scholar] [CrossRef] [PubMed]
  32. Tatusov, R.L.; Galperin, M.Y.; Natale, D.A.; Koonin, E.V. The COG database: A tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000, 28, 33–36. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Unaogu, I.C.; Gugnani, H.C.; Boiron, P. The enzymatic profile of some pathogenic aerobic actinomycetes as determined by api-zym method. J. Med. Mycol. 1999, 9, 235. [Google Scholar]
  34. Gruner, E.; Von Graevenitz, A.; Altwegg, M. The API ZYM system: A tabulated review from 1977 to date. J. Microbiol. Methods 1992, 16, 101–118. [Google Scholar] [CrossRef]
  35. Humble, M.W.; King, A.; Phillips, I. API ZYM: A simple rapid system for the detection of bacterial enzymes. J. Clin. Pathol. 1977, 30, 275–277. [Google Scholar] [CrossRef] [Green Version]
  36. Søgaard, P.; Gahrn-Hansen, B.; Zhou, H.P.; Frederiksen, W.; Hui-Ping, Z. An Investigation of Three Commercial Methods for Rapid Identification of Non-Enteric Gram-Negative Rods. Acta Pathol. Microbiol. Scand. Ser. B Microbiol. 2009, 94, 357–363. [Google Scholar] [CrossRef]
  37. Bilkey, M.K.; Bremner, D.A.; Cameron, G.L.; Garner, J.G. Comparison of five commercial methods for the identification of non- fermentative and oxidase positive fermentative gram negative Bacilli. N. Z. J. Med. Lab. Technol. 1988, 42, 8–12. [Google Scholar]
  38. Swanson, E.C.; Collins, M.T. Use of the API 20E system to identify veterinary Enterobacteriaceae. J. Clin. Microbiol. 1980, 12, 10–14. [Google Scholar] [CrossRef] [Green Version]
  39. Smith, P.B.; Tomfohrde, K.M.; Rhoden, D.L.; Balows, A. API System: A multitube micromethod for identification of Enterobacteriaceae. Appl. Microbiol. 1972, 24, 449–452. [Google Scholar] [CrossRef]
  40. Véron, M.; Le Minor, L. Nutrition and taxonomy of “enterobacteriaceae” and related bacteria. III. Nutritional characters and differentiation of the taxonomic groups (author’s transl). Ann. Microbiol. 1975, 126, 125–147. [Google Scholar]
  41. Bergey, D.H.; Krieg, N.R.; Holt, J.G. Bergey’s Manual of Systematic Bacteriology; Williams and Wilkins: Baltimore, MD, USA, 1984. [Google Scholar]
  42. Rogosa, M.; Sharpe, M.E. An approach to the classification of the Lactobacilli. J. Appl. Bacteriol. 1960, 22, 329–340. [Google Scholar]
  43. Sharpe, M.E.; Hill, L.R.; LaPage, S.P. Pathogenic Lactobacilli. J. Med. Microbiol. 1973, 6, 281–286. [Google Scholar] [CrossRef] [Green Version]
  44. Jorgensen, J.H.; Turnidge, J.D. Susceptibility Test Methods: Dilution and Disk Diffusion Methods. 15.6 Pack. Shipp. Infect. Subst. 2015, 1253–1273. [Google Scholar] [CrossRef]
  45. Sasser, M. Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids; MIDI Technical note #101: Newark, DE, USA, 1990; revised 2001. [Google Scholar]
  46. Dione, N.; Sankar, S.; Lagier, J.-C.; Khelaifia, S.; Michele, C.; Armstrong, N.; Richez, M.; Abrahão, J.; Raoult, D.; Fournier, P.-E. Genome sequence and description of Anaerosalibacter massiliensis sp. nov. New Microb. New Infect. 2016, 10, 66–76. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Yang, J.-E.; Shin, J.-Y.; Park, S.-Y.; Mavlonov, G.T.; Yi, E.-J.; Lee, E.-H.; Lee, J.M.; Yi, T.-H. Pedobacter kyungheensis sp. nov., with ginsenoside converting activity. J. Gen. Appl. Microbiol. 2012, 58, 309–316. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Hwang, C.Y.; Choi, D.H.; Cho, B.C. Pedobacter roseus sp. nov., isolated from a hypertrophic pond, and emended description of the genus Pedobacter. Int. J. Syst. Evol. Microbiol. 2006, 56, 1831–1836. [Google Scholar] [CrossRef]
  49. Kwon, S.-W.; Son, J.-A.; Kim, S.-J.; Kim, Y.-S.; Park, I.-C.; Bok, J.-I.; Weon, H.-Y. Pedobacter rhizosphaerae sp. nov. and Pedobacter soli sp. nov., isolated from rhizosphere soil of Chinese cabbage (Brassica campestris). Int. J. Syst. Evol. Microbiol. 2011, 61, 2874–2879. [Google Scholar] [CrossRef]
  50. Gordon, N.S.; Valenzuela, A.; Adams, S.M.; Ramsey, P.W.; Pollock, J.L.; Holben, W.E.; Gannon, J.E. Pedobacter nyackensis sp. nov., Pedobacter alluvionis sp. nov. and Pedobacter borealis sp. nov., isolated from Montana flood-plain sediment and forest soil. Int. J. Syst. Evol. Microbiol. 2009, 59, 1720–1726. [Google Scholar] [CrossRef]
  51. Chen, Z.; Tian, W.; Sun, F.; Chen, Y.; Han, H.; Yao, L.; Zhang, Z. Pedobacter miscanthi sp. nov., isolated from Miscanthus sinensis. Int. J. Syst. Evol. Microbiol. 2019, 69, 3344–3349. [Google Scholar] [CrossRef] [PubMed]
  52. Yang, J.-E.; Son, H.-M.; Lee, J.M.; Shin, H.-S.; Park, S.-Y.; Lee, D.-G.; Kook, M.; Yi, T.-H. Pedobacter ginsenosidimutans sp. nov., with ginsenoside-converting activity. Int. J. Syst. Evol. Microbiol. 2013, 63, 4396–4401. [Google Scholar] [CrossRef] [Green Version]
  53. Kwon, S.-W.; Kim, B.-Y.; Lee, K.-H.; Jang, K.-Y.; Seok, S.-J.; Kwon, J.-S.; Kim, W.-G.; Weon, H.-Y. Pedobacter suwonensis sp. nov., isolated from the rhizosphere of Chinese cabbage (Brassica campestris). Int. J. Syst. Evol. Microbiol. 2007, 57, 480–484. [Google Scholar] [CrossRef]
  54. Kook, M.; Park, Y.; Yi, T.-H. Pedobacter jejuensis sp. nov., isolated from soil of a pine grove, and emended description of the genus Pedobacter. Int. J. Syst. Evol. Microbiol. 2014, 64, 1789–1794. [Google Scholar] [CrossRef] [PubMed]
  55. Chaudhary, D.K.; Lee, S.D.; Kim, J. Pedobacter kyonggii sp. nov., a psychrotolerant bacterium isolated from forest soil. Int. J. Syst. Evol. Microbiol. 2017, 67, 5120–5127. [Google Scholar] [CrossRef]
  56. Kämpfer, P.; Irgang, R.; Fernández-Negrete, G.; Busse, H.-J.; Poblete-Morales, M.; Fuentes-Messina, D.; Glaeser, S.P.; Avendaño-Herrera, R. Proposal of Pedobacter nototheniae sp. nov., isolated from the spleen of a black rock cod (Notothenia coriiceps, Richardson 1844) from the Chilean Antarctica. Antonie Leeuwenhoek 2019, 112, 1465–1475. [Google Scholar] [CrossRef] [PubMed]
  57. Gao, J.-L.; Sun, P.; Mao, X.-J.; Du, Y.-L.; Liu, B.-Y.; Sun, J.-G. Pedobacter zeae sp. nov., an endophytic bacterium isolated from maize root. Int. J. Syst. Evol. Microbiol. 2017, 67, 231–236. [Google Scholar] [CrossRef]
  58. Roh, S.W.; Quan, Z.-X.; Nam, Y.-D.; Chang, H.-W.; Kim, K.-H.; Kim, M.-K.; Im, W.-T.; Jin, L.; Kim, S.-H.; Lee, S.-T.; et al. Pedobacter agri sp. nov., from soil. Int. J. Syst. Evol. Microbiol. 2008, 58, 1640–1643. [Google Scholar] [CrossRef] [Green Version]
  59. Yoon, J.-H.; Kang, S.-J.; Oh, T.-K. Pedobacter terrae sp. nov., isolated from soil. Int. J. Syst. Evol. Microbiol. 2007, 57, 2462–2466. [Google Scholar] [CrossRef]
  60. Jung, J.; Park, W. Pedobacter jeongneungensis sp. nov., isolated from forest soil. J. Microbiol. 2012, 50, 660–664. [Google Scholar] [CrossRef]
  61. Wang, Z.; Tan, Y.; Xu, D.; Wang, G.; Yuan, J.; Zheng, S. Pedobacter vanadiisoli sp. nov., isolated from soil of a vanadium mine. Int. J. Syst. Evol. Microbiol. 2016, 66, 5112–5117. [Google Scholar] [CrossRef]
  62. Dahal, R.H.; Kim, J. Pedobacter humicola sp. nov., a member of the genus Pedobacter isolated from soil. Int. J. Syst. Evol. Microbiol. 2016, 66, 2205–2211. [Google Scholar] [CrossRef] [PubMed]
  63. Švec, P.; Králová, S.; Busse, H.-J.; Kleinhagauer, T.; Pantůček, R.; Mašlaňová, I.; Cnockaert, M.; Vandamme, P.; Staňková, E.; Gelbíčová, T.; et al. Pedobacter jamesrossensis sp. nov., Pedobacter lithocola sp. nov., Pedobacter mendelii sp. nov. and Pedobacter petrophilus sp. nov., isolated from the Antarctic environment. Int. J. Syst. Evol. Microbiol. 2017, 67, 1499–1507. [Google Scholar] [CrossRef]
  64. Yoon, J.-H.; Lee, M.-H.; Kang, S.-J.; Park, S.-Y.; Oh, T.-K. Pedobacter sandarakinus sp. nov., isolated from soil. Int. J. Syst. Evol. Microbiol. 2006, 56, 1273–1277. [Google Scholar] [CrossRef] [Green Version]
  65. Hoang, V.-A.; Kim, Y.-J.; Nguyen, N.-L.; Min, J.-W.; Yang, D.-C. Pedobacter ginsengiterrae sp. nov., isolated from soil of a ginseng field. Int. J. Syst. Evol. Microbiol. 2013, 63, 1273–1279. [Google Scholar] [CrossRef]
  66. He, R.-H.; Liu, Z.-W.; Yu, Y.; Li, H.-R.; Du, Z.-J. Pedobacter changchengzhani sp. nov., isolated from soil of Antarctica. Antonie Leeuwenhoek 2019, 112, 1747–1754. [Google Scholar] [CrossRef]
  67. Ngo, H.T.T.; Son, H.-M.; Park, S.-Y.; Kim, K.-Y.; Yi, T.-H. Pedobacter seoulensis sp. nov., isolated from soil of a bamboo field. Antonie Leeuwenhoek 2014, 105, 961–970. [Google Scholar] [CrossRef]
  68. Corsaro, D.; Wylezich, C.; Walochnik, J.; Venditti, D.; Michel, R. Molecular identification of bacterial endosymbionts of Sappinia strains. Parasitol. Res. 2016, 116, 549–558. [Google Scholar] [CrossRef]
  69. Krieg, N.R.; Ludwig, W.; Euzéby, J.P.; Whitman, W.B. Bacteroidetes phyl. nov. In Bergey’s Manual of Systematics of Archaea and Bacteria; Wiley: Hoboken, NJ, USA, 2015; pp. 1–2. [Google Scholar]
  70. Kämpfer, P. Sphingobacteriia class. nov. In Bergey’s Manual of Systematics of Archaea and Bacteria; Wiley: Hoboken, NJ, USA, 2015; p. 1. [Google Scholar]
  71. Kämpfer, P. Sphingobacteriales ord. nov. In Bergey’s Manual of Systematics of Archaea and Bacteria; Wiley: Hoboken, NJ, USA, 2015; pp. 1–11. [Google Scholar] [CrossRef]
Microbiolres 12 00019 g001 550
Figure 1. Phylogenetic tree based on 16S rRNA sequence comparison highlighting the position of strain Marseille-Q2390 relative to other closely related species.
Figure 1. Phylogenetic tree based on 16S rRNA sequence comparison highlighting the position of strain Marseille-Q2390 relative to other closely related species.
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Figure 2. Heatmap generated with Orthologous Average Nucleotide Identity (Ortho-ANI)values calculated using the OAT software, comparing Pedobacter ghigonii and other closely related species with standing in the nomenclature. The color code indicates the closest species with green and the farthest with red. The strain Marseille-Q2390 is mainly grouped with the genus Pedobacter soli and, with less similarity, Pedobacter kyungheensis [19].
Figure 2. Heatmap generated with Orthologous Average Nucleotide Identity (Ortho-ANI)values calculated using the OAT software, comparing Pedobacter ghigonii and other closely related species with standing in the nomenclature. The color code indicates the closest species with green and the farthest with red. The strain Marseille-Q2390 is mainly grouped with the genus Pedobacter soli and, with less similarity, Pedobacter kyungheensis [19].
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Figure 3. Comparison of gene functional categories of the strain Marseille-Q2390 and other Pedobacter species.
Figure 3. Comparison of gene functional categories of the strain Marseille-Q2390 and other Pedobacter species.
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Figure 4. Gram staining of the strain Marseille-Q2390 at 100× magnification.
Figure 4. Gram staining of the strain Marseille-Q2390 at 100× magnification.
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Figure 5. Transmission electron microscopy of the strain Marseille-Q2390. Bacterium was rod-shaped and without flagellum. Scale bar = 5 µm.
Figure 5. Transmission electron microscopy of the strain Marseille-Q2390. Bacterium was rod-shaped and without flagellum. Scale bar = 5 µm.
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Table
Table 1. Taxonomic assignment obtained by a BLASTn search of 16S rRNA genes of the strain Marseille-Q2390.
Table 1. Taxonomic assignment obtained by a BLASTn search of 16S rRNA genes of the strain Marseille-Q2390.
Marseille-Q2390
Names CoverIdentification PercentageAccession
Pedobacter kyungheensis strain THG-T17T82%99.36%NR_132668.1
Pedobacter roseus strain CL-GP80T89%98.68%NR_043555.1
Pedobacter soli strain 15-51T 97%98.59%NR_115008.1
Pedobacter borealis strain G-1T92%98.23%NR_044381.1
Pedobacter alluvionis strain NWER-II11T92%98.21%NR_044382.1
Pedobacter miscanthi strain RS10T95%97.99%NR_164958.1
Pedobacter ginsenosidimutans strain THG-45T95%97.93%NR_108685.1
Pedobacter suwonensis strain 15-52T94%97.78%NR_043543.1
Pedobacter jejuensis strain THG-DR3T93%97.68%NR_133810.1
Pedobacter kyonggii strain K-4-11-1T95%97.65%NR_159165.1
Pedobacter nototheniae strain 36B243T91%97.62%NR_164976.1
Pedobacter psychrotolerant strain V5RDT89%97.57%NR_152669.1
Pedobacter zeae strain 22T 97%97.57%NR_156064.1
Pedobacter agri PB92T95%97.45%NR_044339.1
Pedobacter terrae strain DS-57T97%97.43%NR_044005.1
Pedobacter rhizosphaerae strain 01-96T97%97.31%NR_122096.1
Pedobacter jeongneungensis strain BH45T95%97.30%NR_132685.1
Pedobacter vanadiisoli strain XNV015T95%97.30%NR_153693.1
Pedobacter humicola strain R135T95%97.03%NR_149278.1
Pedobacter lithocola strain CCM 8691T97%96.90%NR_156883.1
Pedobacter sandarakinus strain DS-27T97%96.82%NR_043665.1
Pedobacter jamesrossensis strain CCM 8689T97%96.68%NR_156882.1
Pedobacter petrophilus strain CCM 8687T97%96.61%NR_156885.1
Pedobacter ginsengiterrae strain DCY49T91%96.55%NR_109023.1
Pedobacter heparinus strain DSM 2366T99%96.49%NR_074519.1
Pedobacter changchengzhani strain E01020T99%96.09%NR_164993.1
Pedobacter seoulensis strain THG-G12T92%96.08%NR_145561.1
Pedobacter schmidteae EGT 100%96.12%LS453293.1
Table
Table 2. Classification and general features of the strain Marseille-Q2390.
Table 2. Classification and general features of the strain Marseille-Q2390.
PropertyTerm
Current classificationDomain: Bacteria [10]
Phylum: Bacteroidetes [69]
Class: Sphingobacteriia [70]
Order: Sphingobacteriales [71]
Family: Sphingobacteriaceae [21]
Genus name: Pedobacter [20]
Species name: ghigonii
Specific epithet: Pedobacter ghigonii
Type strain: Marseille-Q2390
Species statussp. nov.
Gram stainNegative
Cell shapeRod-shaped
MotilityMotile
SporulationNon-spore-forming
Temperature range for growth4–30
Temperature optimum28
pH range for growth5.5–10
pH optimum7.5
pH categoryNeutro-alkalophilic
Lowest NaCl concentration for growth0
Highest NaCl concentration for growth20 g/L
Salinity optimum9 g/L
O2 conditions for strain testingAerobiosis
Catalase Positive
OxydasePositive
HabitatGut microbiota of Schmidtea mediterranea
Biotic relationshipSymbiotic
Table
Table 3. Main genomic characteristics of the strain Marseille-Q2390 and other closely related Pedobacter species.
Table 3. Main genomic characteristics of the strain Marseille-Q2390 and other closely related Pedobacter species.
NameContigsSize (bp)CDSsGC%tRNAsrRNAsRefseq
P. soli386,006,4204,92340.5496NZ_FMZH00000000.1
P. ghigonii415,921,5344,87040.3497CAESCM000000000.1
P. kyungheensis676,358,6425,27040.5526NZ_JSYN00000000.1
P. borealis2165,544,9174,61038.4503NZ_JAUG00000000.1
P. zeae155,444,8024,56740.3493NZ_JACIEF000000000.1
P. alluvionis206,037,6455,00638.4464NZ_RCCK00000000.1
P. kyonggii736,186,1835,10738.8508NZ_SIXF00000000.1
P. ginsenosidimutans866,517,5535,30138.7525NZ_LMZQ00000000.1
P. suwonensis405,803.8314,73839.5473NZ_FOJM00000000.1
P. terrae635,755,1014,.78338.8463NZ_FNCH00000000.1
Table
Table 4. Digital DNA-DNA hybridization (dDDH) values obtained through a comparison of all studied genomes using the Genome-to-Genome Distance Calculator (GGDC), formula 2 (DDH estimates based on identities/HSP length).
Table 4. Digital DNA-DNA hybridization (dDDH) values obtained through a comparison of all studied genomes using the Genome-to-Genome Distance Calculator (GGDC), formula 2 (DDH estimates based on identities/HSP length).
Digital DNA-DNA Hybridization123456789
P. borealis23.90
P. ginsenosidimutans23.3045.70
P. zeae24.5026.1025.70
P. kyonggii23.6045.8044.4025.50
P. ghigonii30.5024.5023.8024.0023.80
P. alluvionis23.5031.7031.1026.3031.1023.90
P. soli56.4023.7023.5024.4023.5030.5023.40
P. suwonensis24.2026.7026.3025.2026.0023.6027.5023.90
P. terrae23.6028.4027.6025.3027.5023.6030.3023.7031.70
Taxa: 1, P. kyungheensis; 2, P. borealis 3, P. ginsenosidimutans; 4, P. zeae; 5, P. kyonggii; 6, P. ghigonii; 7, P. alluvionis; 8, P. soli; 9, P. suwonensis; 10, P. terrae. The strain Marseille-Q2390 had a higher percentage of hybridization with Pedobacter soli and P. kyungheensis compared to other species, but these hybridization values were below the threshold of 70% recognized for the delimitation of bacterial species [18].
Table
Table 5. Physiological characteristics of strain Marseille-Q2390 and phylogenetically related species of the genus Pedobacter.
Table 5. Physiological characteristics of strain Marseille-Q2390 and phylogenetically related species of the genus Pedobacter.
Characteristics12345678910
Gram-staining----------
Sporulation----------
Growth temperature range (°C)4–304–354–304–404–354–301–370–3215–304–30
Aerobic growth++++++++++
Source PlanarianRhizosphereSoilSoilSoilSoilRhizosphereSoilMaize rootFloodplain
Colony colorWhitePinkish yellowReddish pinkPinkOrangePinkPinkish yellowLight salmonPinkish yellowReddish pink
Catalase++++++++++
Oxidase++++++++++
Enzyme activity (API ZYM):
Alkaline phosphatase++++++++++
Esterase (C4)+++-++++++
Esterase lipase (C8)++++++++++
Lipase (C14)----------
Leucine arylamidase++++++++++
Valine arylamidase++++++++++
Cystine arylamidase+++----+++
Trypsin+++--+++++
α-chymotrypsin-----+-NA--
Acid phosphatase++++++++++
Naphtol-AS-BI-P.hydrolase+++++++-++
α-galactosidase++++-+-+++
β-galactosidase+++++++-++
β-glucuronidase--+-----+-
α-glucosidase+++-++++++
β-glucosidase++v+++++++
N-acetyl-β-glucosaminidase++++++++++
α-mannosidase-+-+-+-++-
α-fucosidase++++-+++++
Assimilation of (API 50 CH/B):
Glycerol---NA------
Erythritol---NA------
D-Arabinose---NA- -NA++
L-Arabinose-+++++++++
D-Ribose--------++
D-Xylose-++NA+ +NA+-
L-Xylose---NA------
D-Adonitol---NA-----+
Methyl-βD-Xylopyranoside---NA+NANANA-+
D-Galactose-++ +NA+-++
D-Glucose++++++++++
D-Fructose-++NA +NA+NA++
D-Mannose++++++++++
L-Sorbose---NA ---NA-+
L-Rhamnose-+++-+++-+
Dulcitol---- ---NA--
Inositol----------
D-Mannitol----------
D-Sorbitol----------
Methyl-αD-Mannopyranoside-++NA+NA+-++
Methyl-αD-Glucopyranoside-++NA+NA+NA++
N-Acetylglucosamine-+++++++++
Amygdalin-++NA+NA+-++
Arbutin-++NA+NA+NA++
Esculin ferric citrate+++NA++++++
Salicin-+++++++++
D-Cellobiose-++++NA+NA++
D-Maltose++++++++++
D-Lactose-+++++++++
D-Melibiose-+++++++++
D-Saccharose-+++++++++
D-Trehalose-++NA+NA+NA++
Inulin---NA+NA-NA-+
D-Melezitose-+-NA-NA+NA--
D-Raffinose-++NA+NA+NA++
Starch-++-+++NA-+
Glycogen-+-+--+-+-
Xylitol---NA-NA-NA--
Gentiobiose-++NA+NA+NA-+
D-Turanose-++NA+NA+NA++
D-lyxose---NA-NA--+-
D-Tagatose---NA-NA--+-
D-Fucose-----NA-NA--
L-Fucose--------+-
D-Arabitol---NA -NA-NA--
L-Arabitol--- - ----
Potassium Gluconate----------
Potassium 2-ketoGluconate----------
Potassium 5-ketogluconate--+-------
API 20E
Natriumthiosulfat-NANANA -NANA-NA NA
L-tryptophan+NANA--NANA-NA NA
Indole production-NA--------
API 20NE
Potassium nitrate-NA--------
L-arginine-NA+------+
Urea-NA--------
Gelatin-++NA ++++++
Capric acid+NANA+-----NA
Adipic acid-NANA------NA
Malic acid+NANA--+----
Trisodium citrate+NANA-----+-
Phenylacetic acid-NANA------NA
Taxa: 1, Marseille-Q2390; 2, Pedobacter soli 15–51T [49]; 3, P. borealis G1T [50]; 4, P. kyungheensis strain THG-T17T [47]; 5, P. terrae strain DS-57T [59]; 6, P. ginsenosidimutans strain THG-45T [52]; 7, P. suwonensis strain 15–52T [53]; 8, P. kyonggii strain K-4-11-1T [55]; 9, Pedobacter zeae strain 22T [57]; 10, Pedobacter alluvionis strain NWER-II11T [50]. The data were completed using previously described [47,49,50,52,53,55,57,59] characteristics and those obtained in the present study. positive (+);negative (-); NA, data not available.
Table
Table 6. Antimicrobial susceptibility and MIC values of strain Marseille-Q2390.
Table 6. Antimicrobial susceptibility and MIC values of strain Marseille-Q2390.
Drug (Antibiotics)CC µg/mLP. ghigonii MIC µg/mL
Benzylpenicelin0.016–256>256
Amoxicillin0.016–256>256
Ampicillin0.016–256>256
Ceftriaxone0.016–256128
Imipenem0.002–320.047
Ciprofloxacin0.002–320.25
Amikacin 0.016–2561
Gentamicin0.64–10240.5
Streptomicin0.064–10246
Daptomycin0.016–256>256
Doxycyclin0.016–2561.5
Metronidazole0.016–256>256
Rifampicin0.002–3216
Fosfomycin0.064–1024192
Vancomycin0.016–256>256
Tigecyclin0.016–2564
CC: tested range of drug concentration in µg/mL, MIC: minimum inhibition of concentration in µg/mL.
Table
Table 7. Cellular fatty acid composition of strain Marseille-Q2390 and related species of the genus Pedobacter.
Table 7. Cellular fatty acid composition of strain Marseille-Q2390 and related species of the genus Pedobacter.
Fatty AcidsName12345678910
Straight-Chain Saturated
10:0 Decanoic acid tr--1.92------
14:0 Tetradecanoic acid trtr1.0---tr1.22.60.7
15:0 Pentadecanoic acid tr-2.0------1.1
16:0 Hexadecanoic acid 1.14.1-2.541.94.71.62.2tr0.7
14:0 2-OH2-hydroxy-tetradecanoic acid-trtr----trtr-
16:0 3-OH 3-hydroxy-hexadecanoic acid tr2.12.2-1.61.4tr1.4tr-
17:0 2-OH2-hydroxy-hexadecenoic acid-----1.7-tr--
Branched Saturated
11:0 anteiso 8-methyl-decanoic acid 1.1---------
13:0 anteiso 10-methyl-dodecanoic acid tr---------
17:1 anteiso 14-methyl-hexadecenoic acid 1.11.02.3-1.2--1.5tr-
15:0 anteiso -tr2.11.762.13.3trtrtr1.7
5:0 iso 3-methyl-butanoic acid 3.0---------
8:0 iso 6-methyl-octanoic acid tr---------
11:0 iso 9-methyl-decanoic acid 1.8tr------tr-
14:0 iso 12-methyl-tridecanoic acid tr---------
15:0 iso 13-methyl-tetradecanoic acid 54.529.625.424.828.330.135.427.037.036.6
17:1 iso 15-methyl-hexadecenoic acid 3.5trtr-1.2--trtr-
15:0 3-OH iso 3-hydroxy-13-methyl-tetradecanoic acid 3.52.03.34.413.5-2.42.63.0-
16:0 3-OH iso3-hydroxy-13-methyl-hexadecanoic acid-trtr-tr--trtr-
17:0 3-OH iso 3-hydroxy-15-methyl-hexadecenoic acid 5.512.314.620.120.418.2-12.87.913.7
Mono-Unsaturated
14:1ω5 9-tetradecenoic acid tr--1.33----tr-
15:1ω7 8-pentadecenoic acid 1.7------- -
16:1ω7 9-hexadecenoic acid 11.133.130.630.027.724.527.229.331.420.2
16:1ω5 11-hexadecenoic acid 8.61.42.7-1.7tr1.41.41.421.1
17:1ω99-hexadecanoic acid-6.96.66.516.13.47.44.74.53.4
18:1ω9 9-octadecenoic acid tr---------
18:2ω6 9,12-octadecadienoic acid tr---------
18:1ω511-octadecenoic acid-tr1.3-tr--trtr-
Taxa: 1, Marseille-Q2390; 2, Pedobacter soli 15–51T [49]; 3, P. borealis G1T [50]; 4, P. kyungheensis strain THG-T17T [47]; 5, P. terrae strain DS-57T [59]; 6, P. ginsenosidimutans strain THG-45T [52]; 7, P. suwonensis strain 15–52T [53]; 8, P. kyonggii strain K-4-11-1T [55]; 9, Pedobacter zeae strain 22T [57]; 10, Pedobacter alluvionis strain NWER-II11T [50]. tr, trace (<1%); -, not detected.
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Kangale, L.J.; Raoult, D.; Pierre-Edouard, F. Pedobacter ghigonii sp. nov., Isolated from the Microbiota of the Planarian Schmidtea mediterranea. Microbiol. Res. 2021, 12, 268-287. https://doi.org/10.3390/microbiolres12020019

AMA Style

Kangale LJ, Raoult D, Pierre-Edouard F. Pedobacter ghigonii sp. nov., Isolated from the Microbiota of the Planarian Schmidtea mediterranea. Microbiology Research. 2021; 12(2):268-287. https://doi.org/10.3390/microbiolres12020019

Chicago/Turabian Style

Kangale, Luis Johnson, Didier Raoult, and Fournier Pierre-Edouard. 2021. "Pedobacter ghigonii sp. nov., Isolated from the Microbiota of the Planarian Schmidtea mediterranea" Microbiology Research 12, no. 2: 268-287. https://doi.org/10.3390/microbiolres12020019

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