Miniphocibacter massiliensis gen. nov., sp. nov., a new species isolated from the human gut and its taxono‐genomics description

Abstract With the aim of describing the human microbiota by the means of culture methods, culturomics was developed in order to target previously un‐isolated bacterial species and describe it via the taxono‐genomics approach. While performing a descriptive study of the human gut microbiota of the pygmy people, strain Marseille‐P4678T has been isolated from a stool sample of a healthy 39‐year‐old pygmy male. Cells of this strain were Gram‐positive cocci, spore‐forming, non‐motile, catalase‐positive and oxidase‐negative, and grow optimally at 37°C under anaerobic conditions. Its 16S rRNA gene sequence exhibited 89.69% of sequence similarity with Parvimonas micra strain 3119BT (NR 036934.1), its phylogenetically closest species with standing in nomenclature. The genome of strain Marseille‐P4678T is 2,083,161 long with 28.26 mol% of G+C content. Based on its phenotypic, biochemical, genotypic and proteomic profile, this bacterium was classified as a new bacterial genus and species Miniphocibacter massiliensis gen. nov., sp. nov. with the type strain Marseille‐P4678T.


| INTRODUC TI ON
Ever since it has been proved to play a role in the human health and diseases, the human gut microbiota took more attention and descriptive studies have been exhaustively done toward unveiling its microbial content (Clemente, Ursell, Parfrey, & Knight, 2012). For example, the gut microbiota composition has been shown to play a role in malnutrition, Crohn's disease, inflammatory bowel diseases, etc. and thus became a therapeutic target in several cases (Million et al., 2016;Rigottier-Gois, 2013;Tidjani Alou et al., 2017;Vétizou et al., 2015;Würdemann et al., 2009). Yet, with the expansion of culture-independent techniques, scientists' thought that the human microbiota could be more efficiently described with no need of re-adapting culture approaches. Indeed, with metagenomics, loads of data have been reported out of which some were significant and others were not since several drawbacks can be faced throughout the process such as the depth bias, presence of operational taxonomic units (OTUs) inability to identify certain pathogenic species or distinguish dead from living bacteria, and thus rendering a part of the human gut population neglected, not identified or described (Greub, 2012). Thus, culturomics was developed, based on sophisticated culture methods, targeting previously uncultured bacterial species . The latter was able to report a significant number of nonhuman and new bacterial species along with correlating its sequences to different OTUs . New bacterial species are described with the means of taxono-genomics approach, which relies on characterizing the understudied organism at the phenotypic, biochemical, proteomic and genomic level in order to confirm the novelty of the understudied organism whenever full genomes are available for comparison at wider range (Fournier & Drancourt, 2015;Lagier et al., 2016). Knowing that 1g of human stool might contain up to 1012 bacterial cells and that only around 2,000 species were isolated by culture, motivate us to purse our efforts in describing the human gut microbiota by culturomics. (Hugon et al., 2015;Raoult & Henrissat, 2014). Herein, we describe a new bacterial species, Miniphocibacter massiliensis gen.nov, sp. nov. strain Marseille-P4678 T using the taxono-genomics approach. This bacterium was isolated from the stool samples of a healthy pygmy male.

| Strain Marseille-P4678 T isolation and identification
Shipment of the stool samples was done using a special medium C-Top Ae-Ana (Culture Top, Marseille, France) and stored at our laboratory at -80°C for further analysis. To assess bacterial content using culturomics, the stool sample was diluted with phosphate buffer saline and incubated in an anaerobic culture bottles (BD BACTEC ® , Plus Anaerobic/F Media, Le Pont de Claix, France) supplemented with 5% (V/V) sheep blood and 5% (V/V) sterile-filtered cow rumen at 37°C. Subculturing assays were done on 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l'Etoile, France) for bacterial colonies isolation. Isolated colonies were identified using MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry; Microflex Lt [Bruker Daltonics, Bremen, Germany]) as previously described Seng et al., 2010). Whenever MALDI-TOF MS fails to identify the tested organism, 16S rRNA gene sequencing was performed as formerly done for further phylogenetic analysis (Drancourt, Berger, & Raoult, 2004). CodonCode Aligner tool (http://www. codoncode.com) served for sequence optimization and alignment.

| Optimal growth conditions
Several growth attempts were done with various culture conditions in order to determine the optimal growth of strain Marseille-P4678 T .

| Biochemical, antibiotic resistance, and phenotypic characteristics of strain Marseille-P4678 T
To biochemically describe strain Marseille-P4678 T , API strips (ZYM, 20A and 50CH; bioMérieux) were used according to manufacturer's protocol. Sporulation ability was tested by performing a culture assay of a previously heat-shocked bacterial suspension (80°C; 20 min).
Gram staining and motility were evaluated using a DM1000 photonic microscope (Leica Microsystems, Nanterre, France) at 100 × oil immersion lens, and cell morphology was determined using an electron microscope as formerly done (Bilen et al., 2018).
GC/MS and cellular fatty acid analysis of strain Marseille-P4678 T were carried out using 20 mg (dry weight) of bacterial biomass per tube as previously done . SQ8s mass spectrometer (Perkin Elmer, Courtaboeuf, France) was used for short-chain fatty acids measurements along with Clarus 500 chromatography (Zhao, Nyman, & Jönsson, 2006). Isobutyric, propionic, butyric, isovaleric, caproic, valeric, enanthic, and isocaproic (Sigma-Aldrich; Lyon, France) were used. Calibrations were done using acidified water (pH 2-3 with HCl 37%), and SCFA were examined using three samples and three controls. Centrifugation of the culture medium was done for 5 min at 16,000 × g in order to get rid of the bacteria and its debris. Collected supernatant's pH was fixed between two and three and spiked with 2-ethylbutyric acid as the internal stan- caproic, and enanthic acid), and 74 m/z (propanoic and isocaproic acid). All data were collected and processed using Turbomass 6.1 (Perkin Elmer, Courtaboeuf, France). The peak areas of the associated SIR chromatograms were used for quadratic internal calibration calculations. All coefficients of determination were more than 0.999. Back-calculated standards and calculated quality controls (0.5 and 5 mm) represented accuracy with less than 15% deviation.
Blank results of the control were subtracted from while analyzing samples.

| DNA extraction and genome sequencing
Strain Marseille-P4678 T genomic DNA (gDNA) was extracted by first performing acid glass beads wash (G4649-500 g Sigma) using a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5 m/s) for 90s. Two hours later, a lysozyme incubation at 37°C was done prior to DNA extraction on the EZ1 biorobot (Qiagen) with EZ1 DNA tissues kit. 50 μl gDNA of 15.8 ng/ μl concentration was eluted. Concentration was measured using a Qubit assay with the high sensitivity kit (Life Technologies, Carlsbad, CA, USA). MiSeq technology (Illumina Inc, San Diego, CA, USA) was used with the mate-pair strategy for gDNA sequencing along with 11 other projects and barcoded with the with the Nextera Mate Pair sample prep kit (Illumina). 1.5 μg of gDNA was used for library preparation according to Nextera mate-pair Illumina guidelines. Tagmentation was done using with a mate-pair junction adapter and simultaneously fragmented. Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) was used for fragments profile validation with a DNA 7500 labchip. Fragments' DNA sizes ranged between 1.5 and 11 kb with an optimal size at 5.933 kb. Additionally, circularization (600 ng of tagmented fragments) was performed with no size selection.
The circularized DNA was mechanically sheared to small fragments with optima on a bimodal curve at 475 and 1,207 bp on the Covaris device S2 in T6 tubes (Covaris, Woburn, MA, USA). The library final concentration was measured and visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA) as 27.85 nmol/l. The latter was normalized at 2 nM and pooled. Denaturation was done, and a dilution step to 19 pM was performed prior to pool loading. A single 2 × 251-bp run was performed with an automated sequencing and cluster generation method. Total information of 4.6 Gb was obtained from a 471 K/ mm 2 cluster density with a cluster passing quality control filters of 98.7% (9,100,000 passing filter paired reads). Within this run, the index representation for strain Marseille-P4678 T was determined to 20.97%. The 1,908,234 paired reads were trimmed and assembled as previously done . Extra-genomic feature was obtained using Rast tool (Aziz et al., 2008;Brettin et al., 2015;Overbeek et al., 2014). PHAST was used for phage detection (Zhou, Liang, Lynch, Dennis, & Wishart, 2011), RNAmmer for F I G U R E 1 Phylogenetic tree representing the position of strain Marseille-P4678 T relative to other closely related species rRNA (Lagesen et al., 2007), and Artemis was used for genome circular representation (Kumar, Tamura, & Nei, 1994). dDDH (DNA-DNA hybridization) between the genomes was obtained using the online GGDC tool (http://ggdc.dsmz.de/ggdc.php#).

| Strain Marseille-P4678 T identification
The mass spectrum of strain Marseille-P4678 T was missing in the current MALDI-TOF MS Bruker database. Thus, its identification with the means of this technique was not possible. Consequently, 16S rRNA gene sequencing analysis showed that the understudied organism has more than 5% sequence divergence with Parvimonas micra strain 3119B T (NR 036934.1), its phylogenetically closest species with standing in nomenclature ( Figure 1). Thus, according to Kim et al. (2014), we propose the isolation of a new bacterial genus. To analyze at the proteomic level strain Marseille-P4678 T , a gel view comparing the available mass spectra of the phylogenetically closest species with standing in nomenclature was done (Figure 2b). The gel view shows that strain Marseille-P4678 T has a unique peaks profile that does not match completely with any of the implemented organisms. This stands by the fact that this strain represents a new bacterial genus. The left y-axis indicates the running spectrum number acquired from successive spectra loading. The intensity of the peaks is indicated with the different gray scale, and the y-axis indicates the relation between the peak color and its intensity of the latter are non-saccharolytic and rely on peptone for growth (Ezaki et al., 2001;Ueki et al., 2009). Recently, many protein anaerobic degraders were reported such as Clostridium thiosulfatireducens (Hernández-Eugenio et al., 2002), Clostridium tunisiense (Thabet et al., 2004), and Proteiniphilum acetatigenes (Chen & Dong, 2005). These bacteria were characterized by the use of proteinaceous compounds. When comparing strain Marseille-P4678 T to its phylogenetically close species with standing in nomenclature, we determine that most of these species are also proteinaceous compounds users. For example, Anaerosphaera aminiphila, isolated from a methanogenic reactor, has been reported to be able to degrade several types of amino acids, similarly to P. micra, Finegoldia, and Peptostreptococcus species, which are also member of the Gram-positive anaerobic cocci family (Ueki et al., 2009). This highlights the potential use of strain Marseille-P4678 T as protein, peptide, and amino acid degraders in the ecosystem.

| CON CLUS ION
Culturomics has proved that culture is an essential method that should be adapted in complementarity with metagenomics when describing the human microbiota and especially the gut. and 37°C under microaerophilic and anaerobic conditions but optimally under anaerobic conditions at 37°C. It tolerates a pH range between 6 and 8.5 and NaCl concentration more than 100 g/L.
Using The type strain is Marseille-P4678 T and was isolated from the stool sample of a healthy 39-year-old pygmy male from Congo.

CO N FLI C T O F I NTE R E S T
None to be declared.

AUTH O R CO NTR I B UTI O N S
MB isolated, described, and wrote the manuscript. MF helped in the taxono-genomics description. TN helped in the genomic sequencing.
MR helped in genome sequencing. ZD helped in writing and critical analysis of the manuscript. PF helped in writing and critical analysis of the manuscript. DR designed the project, and helped in writing, reviewing, and critical analysis. FC designed the study, analyzed the data, and wrote the manuscript.

E TH I C S S TATEM ENT
An approval under the number 09-022 was obtained from the Ethic Committee of the Institut Fédératif de Recherche 48 along with a signed consent from the sample's donor. The donor was a healthy 39-year-old pygmy male, and collection was done according to Nagoya protocol from Republic of Congo.

DATA ACCE SS I B I LIT Y
Genome sequences of strain Marseille-P4678 T were deposited in EMBL-EBI and can be accessed with the following accession numbers: LT934441 and OCTQ00000000.
The type strain is Marseille-P4678 T and was deposited in two international strain collection institutes with the following accession numbers: CSUR P4678 T = CCUG 71375 T .
Strain Marseille-P4678 T typical mass spectrum can be ac-