Vitreoscilla massiliensis sp. nov., Isolated From the Stool of an Amazonian Patient

Strain SN6T is a non-motile and non-spore-forming gram-negative bacterium which was isolated from the stool sample of an Amazonian patient. The optimum growth was observed at 37 °C, pH 7, and 0–5 g/l of NaCl. Based on the 16S rRNA gene sequence similarity, the strain SN6T exhibited 97.5% identity with Vitreoscilla stercoraria strain ATCC_15218 (L06174), the phylogenetically closest species with standing in nomenclature. The predominant fatty acid was hexadecenoic acid (31%). The genomic DNA G + C content of the strain SN6T was 49.4 mol %. After analysis of taxonogenomic data, phenotypic and biochemical characteristics, we concluded that strain SN6T represents a new species of the genus Vitreoscilla for which the name Vitreoscilla massiliensis sp.nov is proposed. The type strain is SN6T (=CSUR P2036 = LN870312 = DSM 100958).


Introduction
This strain was isolated from the stool specimen of an obese Amazonian patient as part of the culturomics study [1] to search for microaerophilic bacteria from human gut. The genus Vitreoscilla was first described by Pringsheim in 1951, after having proposed the family Vitreoscillaceae in 1949. In 1986, Strohl et al. proposed three new species with validated names (Vitreoscilla stercoraria, Vitreoscilla beggiatoides and Vitreoscilla filiformis) of this genus [2]. In 2013, through the use of new-generation sequencing tools, the genus Vitreoscilla was placed in the Neisseriaceae family on the basis of its branching in the 16S rRNA gene tree [3]. Within its clade, members of the genus were the only ones capable of evolving in different habitats.
Since the use of the culturomic concept, the repertoire of bacteria isolated from the human digestive microbiota [4] has expanded considerably. The characterization of these new species is based on a ribosomal RNA sequencing coupled with a taxonogenomics description, a strategy combining a comparison of genomic analysis and phenotypic characteristics, including the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) spectrum. In the present study, we used this approach to facilitate the identification and the description of this novel species named Vitreoscilla massiliensis sp.nov.

Materials and Methods
Vitreoscilla massiliensis SN6 T was isolated by cultivation on 5% sheep blood agar under microaerophilic conditions at 37 °C for 48 h and the strain could not be identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
(MALDI-TOF MS). The bacterial spectrum obtained was incremented in our database and its comparison with those of BioTyper database spectra and our own collection did not allow for its identification. Sequencing of 16S rRNA gene of the strain SN6 T showed a nucleotide sequence similarity of 97.5% with V. stercoraria strain (ATCC 15218) and V. stercoraria strain Göttingen 1488-6 (NR_025894.1), the phylogenetically closest species with standing in nomenclature (Fig. 1).

Optimal Growth
Growth at various temperatures (28 °C, 37 °C, 42 °C, 45 °C) in different atmospheres (aerobic, microaerophilic using CampyGen from Thermo Scientific and anaerobic using AnaeroGenTM from bioMérieux) was tested by culture on Columbia agar (bioMérieux) after 48 h of incubation. The salinity acceptance limit of SN6 T strain was investigated by culture on a home-made culture medium consisting of a Columbia agar culture medium (Sigma-Aldrich, Saint-Quentin Fallavier, France) modified by adding (per liter) 5 g MgCl2 6H2O, 5 g MgSO4 7H2O, 2 g KCl, 1 g CaCl2 2H2O; 0.5 g NaBr, 0.5 g NaHCO3, and 2 g glucose with various NaCl concentrations 0, 5, 10, 25, 50, and 75 g/L. The pH range (6; 6.5; 7; 8.5) for growth was also determined and pH was adjusted by addition of HCl or NaOH.

Biochemical and Chemotaxonomic Analysis
The abilities of the strain SN6 T to use various substrates as sole carbon sources were evaluated using the API 20NE and API 50CH (bioMérieux) and the presence of some enzyme activities using APIZYM following the manufacturer's instructions. All tests were performed in duplicate. Susceptibility to antimicrobial agents was determined by the disk (i2a, Montpellier, France) diffusion method [5] on Mueller-Hinton agar in a Petri dish (BioMerieux) after 48 h of incubation at 37 °C under aerobic conditions. The interpretation of inhibition diameters to the manual measurement using a ruler was done using a Sirscan system© (i2a, Montpellier, France) according to the criteria proposed by the Comité de l'Antibiogramme of the French Society for Microbiology [6]. Cellular fatty acid methyl ester (FAME) analysis was performed by GC/MS. Two samples were prepared with approximately 65 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as previously described by Sasser [7]. Sequences were aligned using CLUSTALW, and phylogenetic inferences are obtained with kimura two-parameter models using the maximum-likelihood method within the MEGA software. Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 1,000 times to generate majority consensus tree. Scale bar indicates 1% nucleotide sequence divergence. The scale bar represents 500 nm GC/MS analyses were carried out as previously described by Dione et al. [8]. 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).

Genome Sequencing and Assembly
DNA of strain SN6 T was extracted on the EZ1 biorobot (Qiagen) with a EZ1 DNA tissues kit after pretreatment by a lysozyme incubation at 37 °C, as previously described [9]. Genomic DNA (gDNA) was quantified by a Qubit assay with the high-sensitivity kit (Life technologies, Carlsbad, CA, USA) and sequenced on the MiSeq Technology (Illumi-naInc, San Diego, CA, USA) with the mate pair strategy, as previously described [9]. The gDNA was barcoded in order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit (Illumina). The assembly of the genome was carried out with the help of a pipeline that allowed the creation of an assembly with different softwares (Velvet [10], Spades [11] and Soap Denovo [12], on trimmed (MiSeq and Trimmomatic [13] softwares) or untrimmed data (only MiSeq software). For each of the six assemblies performed, GapCloser [12] was used to reduce gaps. Then, contamination with Phage Phix was identified (blastn against Phage Phix174 DNA sequence) and eliminated. Finally, scaffolds whose size was less than 800 bp were removed and scaffolds whose depth value was lower than 25% of the mean depth were removed (identified as possible contaminants). The best assembly was selected using different criteria (number of scaffolds, N50 and number of N). Spades gave the best assembly of this strain, with a depth coverage of 98.

Results and Discussion
Based on the sequence similarity threshold values of the 16S rRNA gene that delineate a new species according to the recommendations of Stackebrandt and Ebers [23], the strain SN6 T can, therefore, be classified as a new species of the genus Vitreoscilla and was accordingly named V. massiliensis SN6 T [24].

Genome Properties
The genome is 3,716,289 bp long with 49.4% GC content (Fig. 2, Table 2). It is composed of 10 scaffolds (composed of 13 contigs). Of the 3 716 predicted genes, 3 627 were protein-coding genes and 89 were RNAs (5 genes are 5S rRNA, 5 genes are 16S rRNA, 5 genes are 23S rRNA and 74 genes are TRNA genes). A total of 2,263 genes (62.3%) were assigned with putative function (by cogs or by NR blast). 475 genes were identified as ORFans (13.1%). The 1 3 remaining genes were described as hypothetical proteins (744 genes ≥ 20.5%). A summary of the distribution of V. massiliensis genes into the different COGs categories is presented in Table 3.

Genome Comparison
The draft genome sequence and the G + C content of V. massiliensis (3.71 MB and 49.4%) is larger than that of Na not available a Data from Mayfield et al. [25] b, c Data from Strohl et al. [2] d Data from Vaz-Moreira et al. [26] e Data from Chang et al. [27] f Data from Chern et al. [28] g Data from Kwong et al. [ ). Also, the gene content of V. massiliensis is larger than that of (3,627and 2,440 respectively). The distribution of genes into COG categories was similar in all 7 compared genomes (Fig. 3). All genomes were compared with V. massiliensis using Orthologous average nucleotide identity. The OrthoANI analysis showed that identity nucleotide value is 76.4% with V. stercoraria which is lower than 95% (Fig. 4). Likewise, we obtained similar results for the analysis of the digital DNA-DNA hybridization (dDDH) with 31.60% between V. massiliensis and V. stercoraria (Table 4).

Conclusion
Based on the phenotypic characteristics, and phylogenetic and genomic analyses of strain SN6 T , we suggest the creation of a new species within the Vitreoscilla genus, for which the name V. massiliensis sp. nov., is proposed.
T h e t y p e s t r a i n i s S N 6 T ( = C S U R P2036 = LN870312 = DSM 100958) and was isolated from the stool specimen of an obese Amazonian patient.
Acknowledgements The authors thank the Xegen Company (http:// www. xegen. fr/) for automating the genomic annotation process. They also thank Aurelia Caputo for submitting the genomic sequences to GenBank.
This work has benefited from the French State support, managed by the 'Agence Nationale pour la Recherche', including the "Programme d'Investissement d'avenir" under the reference Méditerranée Infection Fig. 2 Graphical circular map of the genome of Vitreoscilla massiliensis strain SN6 T from outside to the center: genes on the forward strand colored by COG categories (only genes assigned to COG), genes on the reverse strand colored by COG categories (only gene assigned to COG), RNA genes (tRNAs green, rRNAs red), GC content, and GC skew (Color figure online)