Chryseobacterium schmidteae sp. nov. a novel bacterial species isolated from planarian Schmidtea mediterranea

Marseille-P9602T is a Chryseobacterium-like strain that we isolated from planarian Schmidtea mediterranea and characterized by taxono-genomic approach. We found that Marseille-P9602T strain exhibits a 16S rRNA gene sequence similarity of 98.76% with Chryseobacterium scophthalmum LMG 13028T strain, the closest phylogenetic neighbor. Marseille-P9602T strain was observed to be a yellowish-pigmented, Gram-negative, rod-shaped bacterium, growing in aerobic conditions and belonging to the Flavobacteriaceae family. The major fatty acids detected are 13-methyl-tetradecanoic acid (57%), 15-methylhexadecenoic acid (18%) and 12-methyl-tetradecanoic acid (8%). Marseille-P9602 strain size was found from genome assembly to be of 4,271,905 bp, with a 35.5% G + C content. The highest values obtained for Ortho-ANI and dDDH were 91.67% and 44.60%, respectively. Thus, hereby we unravel that Marseille-P9602 strain is sufficiently different from other closed related species and can be classified as a novel bacterial species, for which we propose the name of Chryseobacterium schmidteae sp. nov. Type strain is Marseille-P9602T (= CSUR P9602T = CECT 30295T).

Phylogenetic analysis, and genomic comparison. Phylogenetic relationships were inferred from comparison of 16S rRNA gene sequences using MEGAX (version10.1) software 33,34 . Sequences were aligned using MUSCLE algorithm setup with default parameters, and numbers at the nodes were percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree. Only bootstrap values ≥ 50% were retained. For the Phylogenetic tree based on the core genes, we generated a core-gene alignment using Roary 3.13.0 35 with 70% identity. We obtained an alignment of 1535 core genes from which we inferred a phylogenetic tree using FastTree 2.1.10 36 . Degrees of genomic similarity were evaluated using the GGDC 37 (http:// ggdc. dsmz. de/ ggdc. php#) and Orthologous Average Nucleotide Identity 38 (https:// www. ezbio cloud. net/ tools/ ortho ani, OrthoANI Tool version 0.93.1) softwares. Comparison COG functional categories were carried out using Blast P (E-value 10-3, coverage 0.7 and identity percent 30%) against clusters of orthologous groups (COG) database.
Antibiotic susceptibility of Marseille-P9602 strain. Bacterial susceptibility to benzylpenicillin, amoxicillin, ampicillin, ceftriaxone, imipenem, ciprofloxacin, amikacin, gentamicin, streptomycin, daptomycin, doxycycline, metronidazole, rifampicin, fosfomycin, vancomycin and tigecycline was assessed using E-tests and a 0.5 McFarland concentration of Marseille-P9602 and LMG 13028 T strains. 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 51 .
Analysis of cellular fatty acids of strain Marseille-P9602. Cellular fatty acid methyl ester (FAME) analysis was performed by GC/MS for both Marseille-P9602 and LMG 13028 T strain. Fatty acid methyl esters were prepared as described by Sasser 52 and GC/MS analysis was realized as previously described 53 . Briefly, Marseille-P9602 and LMG 13028 T strains were inoculated in 5% sheep blood-enriched Columbia agar and incubated at 28 °C. 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).

Results and discussion
Phylogenetic analysis and genomic comparison. The gene 16S rRNA sequence from Marseille-P9602 strain was observed to be 1513 bp-long. A sequence similarity calculation using BLASTn search in the nr database indicated that the closest relatives of Marseille-P9602 strain are Chryseobacterium scophthalmum LMG 13028 T strain 1,39 , Chryseobacterium piscium LMG 23089 T strain 54 , C. balustinum NBRC 15053 T strain 1 , C. indoltheticum LMG 4025 T strain 55 , C. taihuense THMBM1 T strain 55 , C. ureilyticum F-Fue-04IIIaaaa T strain 7 , C. aquaticum 10-46 T strain 6 , C. lactis KC1864 T strain 8 , C. soldanellicola NBRC 100864 T strain 9 , C. formosense CC-H3-2 T strain 56 ,C. aureum 17S1E7 T strain 57 , C. hominis NF802 T strain 58 , C. timonianum G972 T strain 59 C. polytrichastri YG4-6 T strain 10 , C. echinoideorum CC-CZW010 T strain 60 , C. xinjiangense TSBY-67 T strain 61 , C. endophyticum CC-YTH209 T strain 62 , C. taiwanense BCRC 17412 T strain 63 , C. vrystaatense R-23566 T strain 64 , C. joostei LMG 18212 T strain 65 , C. geocarposphaerae 91A-561 T66 , and C. gleum NBRC 15054 T strain 2 , whose similarity values, coverage and accesssion strain numbers are shown in Table 1. Therefore, Marseille-P9602 strain belongs to Chryseobacterium genus 1 within the Flavobacteriaceae family 67 and the Bacteroidetes phylum 68 ( Table 2). The 16S rRNA-based phylogenetic tree showed that Marseille-P9602, C. scophthalmum LMG 13028 T , C. piscium LMG 23089 T and C. balustinum NBRC 15053 T strains form a monophyletic group (Fig. 1A). Core genome tree showed that strains are different from each other (Fig. 1B). The genomic sequence from Marseille-P9602 strain was assembled into 56 contigs for a total size of 4,276,845 bp (Cover, 56x; N 50 , 151,068; L 50 , 9) with a 33.5% G + C content. A total of 3881 predicted protein-coding genes were identified, along with 9 rRNAs, 67 tRNAs, 1 tmRNA and 1 repeat region; and this genome was compared with other closely related Chryseobacterium genomes (Table 3). Based on the Digital DNA-DNA hybridization values (dDDH) obtained using GGDC software, Marseille-P9602 strain values ranged from 21.40% with C. aureum and C. lactis to 44.60% with C. scophthalmum (Table 3). These values were below the 70% threshold recognized for the delimitation of bacterial species. Ortho-ANI values of Marseille-P9602 strain ranged from 76.65% C. aureum to 91.67% with C. scoph- www.nature.com/scientificreports/ thalmum, which is lower than the 95% threshold used to distinguish species (Table 3). These values of genomic comparison showed that Marseille-P9602 strain is probably a novel species in the Chryseobacterium genus. The distribution of genes in COG functional categories is presented in Fig. 2 and Table 4. Few differences were observed between these species. In addition, by comparison of the genomes of Marseille-P9602 strain and of the 11 closest species, we highlighted 100 specific and unique genes to the Marseille-P9602 strain (Supplementary data S1). Taken together, these results confirm that Marseille-P9602 strain belongs to a separate Chryseobacterium species.
Phenotypic analysis and biochemical characteristics. Marseille-P9602 strain was isolated on COS agar after 2 days at 28 °C in aerobic atmosphere at pH 7.5. We observed that Marseille-P9602 strain grows at temperatures ranging from 4 to 30 °C in aerobic atmosphere and at pH values ranging from 6.5 to 9 (Neutroalkalophilic bacterium). In contrast, LMG 13028 T strain grows at pH 6. Marseille-P9602 strain grows at salinity concentrations lower than 12 g of NaCl/l; however, in contrast, LMG 13028 T strain needs a NaCl concentration lower than 25 g/l. After 4 days culture on COS agar, Marseille-P9602 strain colonies were observed to be yellowish, small (0.4 mm median diameter), circular with a convex shape and smooth. Bacterial cells (Fig. 3) are Gramnegative (Fig. 3A), rod-shaped, non-spore-forming bacilli and non-motile, but without any flagellum. Their mean length and width are 3.15 µm and 0.66 µm, respectively (Fig. 3B). Marseille-P9602 strain was found to be oxidase positive and catalase negative. Bacterial metabolism was characterized using API 50CHB/E, API 20NE, API Zym and API 20E strips (Table 5). Marseille-P9602 strain differs from C. scophthalmum, C. indoltheticum, C. piscium, and C. balustinum regarding catalase, α-glucosidase, inositol and urea.

Conclusion
Based on the results obtained by the taxono-genomic approach, we confirm that Marseille-P9602 strain belongs to a novel species from Chryseobacterium genus. We propose the name of Chryseobacterium schmidteae Marseille-P9602 T strain. To date, this novel strain has never been identified in any other environment.