Noncontiguous finished genome sequence and description of Raoultibacter massiliensis gen. nov., sp. nov. and Raoultibacter timonensis sp. nov, two new bacterial species isolated from the human gut

Abstract As part of the culturomics project aiming at describing the human microbiota, we report in this study the description of the new bacterial genus Raoultibacter gen. nov. that includes two new species, that is, R. massiliensis sp. nov. and R. timonensis sp. nov. The R. massiliensis type strain Marseille‐P2849T was isolated from the fecal specimen of a healthy 19‐year‐old Saudi Bedouin, while R. timonensis type strain Marseille‐P3277T was isolated from the feces of an 11‐year‐old pygmy female living in Congo. Strain Marseille‐P2849T exhibited 91.4% 16S rRNA sequence similarity with Gordonibacter urolithinfaciens, its phylogenetic closest neighbor with standing in nomenclature. As well, strain Marseille‐P3277T exhibited 97.96% 16S rRNA similarity with strain Marseille‐P2849T. Both strains were Gram‐positive, motile, nonspore‐forming rod and form transparent microcolonies on blood agar in both anaerobic and microaerophilic atmospheres. The genome sizes of strain Marseille‐P2849T and strain Marseille‐P3277T were 3,657,161 bp and 4,000,215 bp, respectively. Using a taxono‐genomic approach combining the phenotypic, biochemical, and genomic characteristics, we propose the genus Raoultibacter gen. nov., which contains strains Marseille‐P2849T (= CSUR P2849T, = DSM 103407T) and Marseille‐P3277T (=CCUG 70680T, =CSUR P3277T) as type strains of the species R. massiliensis sp. nov., and R. timonensis sp. nov., respectively.


| INTRODUC TI ON
The human microbiota is a highly diverse consortium of microbes colonizing different regions of the human body. The role of the microbiota took an important interest in the scientific and medical communities as it was demonstrated to be involved in human health (Alegre, Mannon, & Mannon, 2014;Glenwright et al., 2017;Honda & Littman, 2016;Round & Mazmanian, 2009). For instance, a dysbiosis of the microbiota has been proven to be implicated in a growing number of pathologies and its modulation can have beneficial impacts on the host (Smits, Bouter, de Vos, Borody, & Nieuwdorp, 2013;Zak-Gołąb, Olszanecka-Glinianowicz, Kocełak, & Chudek, 2014). Over the past decade, great advances have been achieved by the development of next-generation DNA sequencing technologies, which led to a considerable progress in the study of different ecosystems including the intestinal microbiota (Margulies et al., 2005). However, many drawbacks appeared when using these molecular methods, such as the inability to distinguish between dead or living bacteria and the depth bias that neglects a minority but important bacterial species (Greub, 2012).
Consequently, a new approach "culturomics" was developed in our laboratory in order to exhaustively explore the microbial ecosystems and to increase the chance of isolating previously uncultured bacteria (Lagier et al., 2012(Lagier et al., , 2015. Culturomics relies on the multiplication of culture conditions (including the variation of temperature, media, atmosphere…) and is coupled by a rapid bacterial identification method, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The latter proved its efficiency in describing the human gut microbiota by reporting a significant number of previously uncultured and novel bacterial species . Nevertheless, we are still far from understanding the human microbiome since only around 2,776 human bacterial species have been isolated, knowing that up to 1,012 bacteria are estimated to be present in only 1 g of stool (Bilen et al., 2018;Hugon et al., 2015). In the present work, the two studied organisms, strains Marseille-P2849 T and Marseille-P3277 T , were isolated from the stool samples of a 19-year-old healthy Saudi Bedouin and an 11-year-old Congolese pygmy female, respectively. These bacteria were not identified using MALDI-TOF-MS. The sequencing and phylogenetic analysis of their 16S rRNA genes classified them as members of a new genus within the family Eggerthellaceae (Gupta, Chen, Adeolu, & Chai, 2013). This family contains the type genus Eggerthella and the genera Adlercreutzia, Asaccharobacter, Cryptobacterium, Denitrobacterium, Enterorhabdus, Gordonibacter, Paraeggerthella, Enteroscipio, Rubneribacter, and Slackia (Gupta et al., 2013). Among its members, Eggerthella lenta is commonly detected in humans and has been associated with bacteremia in patients with intraabdominal or gastrointestinal tract F I G U R E 1 Gel view comparing Raoultibacter massiliensis gen. nov., sp. nov. strain Marseille-P2849 T and strain Raoultibacter timonensis gen. nov., sp. nov. strain Marseille-P3277 T with other closely related species present in our matrix-assisted laser desorption/ionization timeof-flight mass spectrometry spectrum database. The gel view displays the raw spectra of loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a gray scale scheme code. The color bar and the right y-axis indicate the relation between the color of the peak and its intensity, in arbitrary units. Displayed species are indicated on the left pathologies, bacteremia complicated by spondylodiscitis, psoas abscess, and meningitis (Gardiner et al., 2015;Gardiner, Korman, & Junckerstorff, 2014;Wong, Aoki, & Rubinstein, 2014). We herein describe the new genus Raoultibacter gen. nov. within the family Eggerthellaceae using the taxono-genomic approach (Fournier & Drancourt, 2015). Strain Marseille-P2849 T (= CSUR P2849, = DSM 103407) is the type strain of the new species Raoultibacter massiliensis sp. nov and Marseille-P3277 T is the type strain of the species Raoultibacter timonensis sp. nov (=CCUG 70680, =CSUR P3277).

| Ethical requirements and sample collection
Strain Marseille-P2849 T was isolated in April 2016 from the stool sample of a 19-year-old healthy Bedouin male living in Saudi Arabia. As for strain Marseille-P3277 T , it was isolated in June 2016 from the stool specimen of an 11-year-old healthy Pygmy female living in Congo. The fecal specimens were preserved at 4°C and sent to Marseille, where they were stored at −80°C in 2015. The donors gave a signed informed consent, and the study was validated by the ethics committee of the Institut Federatif de Recherche 48 under number 09-022.

| Isolation of the strains
Stool samples were diluted with phosphate-buffered saline (Life Technologies, Carlsbad, CA, USA) and multiple culture conditions were applied as previously described (Jean-Christophe Lagier et al., 2016).
Bacterial growth assessment was done by directly culturing samples from the blood culture bottles on Columbia blood agar (Biomerieux, France). Strain Marseille-P2849 T was isolated after stool sample's incubation in an anaerobic blood culture bottle (Becton-Dickinson, BACTEC Plus anaerobic/F Media, Le pont de Claix, France) supplemented with 5 ml filtered rumen for 7 days at 37°C. Similarly, strain Marseille-P3277 T was isolated after 2 days of stool sample incubation in an anaerobic blood culture bottle supplemented with 5 ml sterile F I G U R E 2 Phylogenetic tree highlighting the position of Raoultibacter massiliensis strain gen. nov., sp. nov. strain Marseille-P2849 T and Raoultibacter timonensis gen. nov., sp. nov. strain Marseille-P3277 T relative to other closely related species. Strains and their GenBank accession numbers of 16S rRNA gene are indicated in brackets. Sequences were aligned using ClustalW, with default parameters and phylogenetic inferences obtained using the neighbor-joining method with 500 bootstrap replicates, within MEGA7 software. The scale bar represents a 2% nucleotide sequence divergence sheep blood and 5 ml filtered rumen at 37°C. Colonies were purified by selecting independent colonies directly from the plate and subculturing it.

| Strain identification by MALDI-TOF-MS and 16S rRNA gene sequencing
Identification of bacterial colonies was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis as previously described (Seng et al., 2010).
Each 16S rRNA sequence was compared with the nr database of the National Center for Biotechnology Information using the BLAST software (https://blast.ncbi.nlm.nih.gov). Compared to its phylogenetically closest species with standing in nomenclature, a 95% similarity threshold was used to define a new genus and a 98.65% similarity threshold was used to define a new species (Kim, Oh, Park, & Chun, 2014). The mass spectrum and 16S rRNA sequence of the newly isolated species were submitted in the URMITE (https://www. mediterranee-infection.com/article.php?laref=256&amp;titre=urmsdatabase) and EMBL-EBI databases, respectively.

| Phylogenetic tree
For phylogenetic analysis, sequences of the phylogenetically closest species were obtained after performing a BLASTn search within the 16S rRNA database of "The All-Species Living Tree" Project of Silva (The SILVA and "All-species Living Tree Project (LTP)" taxonomic frameworks, 2013). Alignment was performed using CLUSTALW (Thompson, Higgins, & Gibson, 1994) and MEGA software (Kumar, Tamura, & Nei, 1994) was used for phylogenetic inferences generation using the maximum likelihood method.

| Morphologic observation and growth conditions
Following Gram staining, bacterial cells were observed using a Leica DM 2500 photonic microscope (Leica Microsystems, Nanterre, France) with a 100X oil immersion lens. A wet mount was performed to determine motility of both bacteria and a Leica DM 1000 photonic F I G U R E 3 Gram staining of (a) Raoultibacter massiliensis gen. nov., sp. nov. strain Marseille-P2849 T and (b) Raoultibacter timonensis gen. nov., sp. nov strain Marseille-P3277 T . Transmission electron microscopy images of R. massiliensis gen. nov., sp. nov. strain Marseille-P2849 T (c) and R. timonensis gen. nov., sp. nov strain Marseille-P3277 T (d) using a Tecnai G20 transmission electron microscope (FEI Company). The scale bar represents 200 nm microscope (Leica Microsystems) at a 1,000× total magnification. A Tecnai G20 (FEI company, Limeil-Brevannes, France) electron microscope was used for bacterial cell imaging at an operating voltage of 60 kV, as previously described (Elsawi et al., 2017).
GENbag is commercially available, disposable sachet containing different chemical compounds (activated charcoal, sodium ascorbate, and others) used in the production of an anaerobic environment free of elemental oxygen gas (O 2 ) or microaerophilic environment with 5% of elemental oxygen gas. Different growth temperatures (25, 28, 37, 45, 55°C), pH values (6-8.5), and NaCl concentrations (5-100 g/L) were also tested.  For strain Marseille-P2849 T , total information of 4.5 Gb was obtained from a 477 K/mm 2 cluster density with a cluster passing quality control filters of 94.8% (8,444,000 passing filter paired reads).

| Biochemical analysis, fatty acid methyl ester analysis, and antibiotic susceptibility testing
Within this run, the index representation for strain Marseille-P2849 T was determined to be of 8.34%. For strain Marseille-P3277 T , total information of 6.3 Gb was obtained from a 673 K/mm 2 cluster density with a cluster passing quality control filters of 95.4% (12,453,000 clusters). Within this run, the index representation for this strain was determined to be of 7.29%. The 769,472 and 907,611 paired reads of strains Marseille-P2849 T and Marseille-P3277 T , respectively, were trimmed, assembled, annotated, and analyzed using the same pipeline adapted in our previous studies (Elsawi et al., 2017).

| Strain identification by MALDI-TOF-MS and 16S rRNA sequencing
Matrix-assisted laser desorption/ionization-TOF-MS failed to identify strains Marseille-P2849 T and P3277 T at the genus and species levels (score <1.7). The spectra of strain Marseille-P2849 T and Marseille-P3277 T were added to our URMS database (Supporting Information Figure S1). A gel view comparing the available mass spectrum of the new isolated species to the mass spectrum of its phylogenetically close species was done (Figure 1). Mass spectrum of each organism was unique and did not match any other spectrum, confirming the novelty of both studied strains.   Note. Values represent the percentage of total identified fatty acid methyl esters only (aldehydes, dimethyl acetals and unidentified "summed features" described previously were not included). Data of the close species were taken as reported by Selma et al. (2014).  Notes. The total is based on either the size of the genome in base pairs or the total number of protein-coding genes in the annotated genome.

| Genomic properties
The draft genome of strain Marseille-P2849 T was 3,657,161-bp long with a G+C content of 59.02 mol% (Table 4; Figure 4a). It was composed of nine scaffolds (35 contigs). Of the 3,073 predicted genes, 3,025 were protein-coding genes and 48 were RNAs (one complete rRNA operon and 45 tRNA genes). A total of 2,365 proteins (76.86%) were assigned to COGs and 253 genes were identified as ORFans The genome statistics are presented in Table 4, and the distribution of genes into COGs functional categories is summarized in Table 5.

The distribution of functional classes of predicted genes of strains
Marseille-P2849 T and Marseille-P3277 T according to the COGs of proteins is summarized in Figure 5.

| D ISCUSS I ON
Culturomics is a high-throughput culture approach that enabled the isolation of approximately 1,057 bacterial species including 247 new species from the human gut in our laboratory (Jean-Christophe Lagier et al., 2016). Along with the development of culturomics, a new polyphasic approach, taxonogenomics, was developed in order to describe novel bacterial species using their biochemical, proteomic, and genomic properties (Fournier & Drancourt, 2015). This approach has the advantage of exhibiting a higher inter and intralaboratory reproducibility when compared

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. Note. The number of proteins per genome is indicated in bold. The strains of the species included in the genomic analysis were given in Table 6 28.

E TH I C S S TATEM ENT
The donors gave a signed informed consent, and the study was validated by the ethics committee of the Institut Federatif de Recherche 48 under number 09-022.

DATA ACCE SS I B I LIT Y
The 16S