Description of Gemella massiliensis sp. nov., a new bacterium isolated from the human gut.

Thanks to its ability to isolate previously uncultured bacterial species, culturomics has dynamized the study of the human microbiota. A new bacterial species, Gemella massiliensis Marseille-P3249 T , was isolated from a sputum sample of a healthy French man. Strain Marseille-P3249 T is a facultative anaerobe, catalase negative, Gram positive, coccus and unable to sporulate. The major fatty acids were C 16:0 (34%), C 18:1n9 (28%), C 18:0 (15%) and C 18:2n6 (13%). Its 16S rRNA sequence exhibits a 98.3% sequence similarity with Gemella bergeri strain 617-93, its phylogenetically closest species with standing in nomenclature. Its digital DNA-DNA hybridization (dDDH) and OrthoANI values with G. bergeri of only 59.7 ± 5.6% and 94.8%, respectively. These values are lower than the thresholds for species delineation (>70% and >95%, respectively). This strain grows optimally at 37°C and its genome is 1.80 Mbp long with a 30.5 mol% G+C content. Based on these results, we propose the creation of the new species Gemella massilienis sp. nov., strain Marseille-P3249 T (= CSUR P3249 = DSMZ 103940).


Introduction
The human microbiota has been strongly correlated to health and diseases (1) with numerous projects being launched to study its residing bacterial population (2,3). Indeed, the metagenomics approach led to the production of a signi cant amount of data which helped the scienti c community to better understand the residing population of different microbiota (4). However, several drawbacks can be faced when adopting this approach, such as the presence of unclassi ed genomic sequences, depth bias and inability to have biological material for further manipulation and studies (5). Thus, culturomics was developed in order to re-introduce the culture approach in the human microbiota description using a more sophisticated methodology (6). The latter relies on culturing samples with 18 different conditions assessing their bacterial content by direct seeding on solid media (7). Isolated colonies are e ciently identi ed using MALDI-TOF MS or 16S rRNA gene sequencing whenever MALDI-TOF MS fails (8,9).
Using this approach, a signi cant number of new bacterial species was isolated with some being correlated to previously detected operational taxonomic units (OTUs) (7,10,11). Recently, in our institute research was focused on the respiratory microbiota in order to assess its role in health or disease development (12). Accordingly, and as part of the project aiming to decipher the human microbiota, culturomics was applied to human sputum samples with the aim of pro ling its bacterial content. Using this process, a new bacterial species, Gemella massiliensis strain Marseille-P3249 T , was isolated. Herein, we report the taxonogenomic description of this new species that was isolated from the sputum of a healthy French man (13).

Material And Methods
Growth conditions A bacterial strain was isolated from a sputum sample from a healthy Frenchman by culturomics in order to explore the human microbiome. The study was approved by the ethics committee of the Institut Federatif de Recherche IFR48 under the number 09-022 and then the patient the patient gave his formal agreement by signing the informed consent. Thus optimal growth conditions of strain Marseille-P3249 were evaluated using various culture conditions. Culture assays were done at 28, 37, 45 and 55°C under anaerobic (GENbag anaer, bioMérieux), microaerophilic (GENbag Microaer, bioMérieux) and aerobic conditions. Tolerance to acidity and halotolerance were evaluated independently with growth assays at pH 6, 6.5, 7 and 8.5 and by using 0, 5, 10, 50, 75 and 100 g/L NaCl concentrations, respectively.

Morphological, Biochemical And Antibiotic Susceptibility Analysis
The main biochemical features of strain Marseille-P3249 were tested using API strips (ZYM, 50CH and 20A (bioMérieux, France)). Motility and Gram stain were checked using a DM1000 photonic microscope (Leica Microsystems, Nanterre, France). Additionally, sporulation was evaluated after exposing a bacterial suspension to a 20 minutes heat shock at 80°C. Cell morphology images were obtained using a scanning electron (SEM) microscope (TM4000 Plus, Hitachi High-Technologies Corp., Tokyo, Japan). Cellular fatty acid methyl ester (FAME) analyses were performed with GC/MS with 10 mg of bacterial biomass per tube. GC/MS and FAME analyses were performed as previously reported (14).

DNA Extraction And Genome Sequencing
A total of 82.1 ng/µL of genomic DNA (gDNA) were extracted from strain Marseille-P3249 as previously described (14). gDNA was sequenced using the MiSeq technology (Illumina Inc, San Diego, CA, USA) with the Mate-pair strategy and were run and barcoded with 11 additional projects using the Nextera Mate-Pair sample prep kit (Illumina) as formerly described (14). The DNA fragment size ranged from 1.5 kb up to 11kb with an optimal size of 6.29 kb. No size selection was done and 177.24 ng of tagmented fragments were circularized. The circularized DNAs were sheared mechanically to smaller fragments with an optimal size at 1393 bp on the Covaris device S2 in T6 tubes (Covaris, Woburn, MA, USA). Using a high sensitivity bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA), the library pro le was visualized with a nal concentration of 15.59 nmol/l. The latter were normalized at 2nM and pooled with other samples, and nally diluted to 15pM. Automated cluster generation and sequencing run were performed in a single 2x251-bp run. Total information of 9.5 Gb was obtained from a 1050 K/mm2 cluster density with a cluster passing quality control lters of 92.5 % (18,644,000 passing lter paired-reads). Within this run, the index representation for strain Marseille-P3249 T was determined to 4.67%. The 870,362 paired reads were trimmed, assembled, annotated and analyzed as previously described (14).

Phylogenetic Analysis
For phylogenetic analyses, 16S rRNA gene sequences of closely related species were recovered from the 16S RNA database of "The All-Species Living Tree" Project of Silva (LTPs121) (15). Muscle was used for sequence alignment and phylogenetic inferences were generated using the approximately-maximumlikelihood method within the FastTree software (16,17).

GMA
Description of Gemella massiliensis sp. nov.
We propose strain Marseille-P3249 is the type strain of the new species Gemella massiliensis sp. nov. (mas.il.i.en'sis, L. gen. fem., adj., massiliensis, pertaining to Massilia, the Latin name of the city of Marseille, where this bacterium was discovered). Strain Marseille-P3249 is a facultative anaerobic bacterium but grows optimally at 37°C under aerobic conditions. Using a 50 CH strip, this strain exhibits positive reactions for D-fructose, amygdaline and L-sorbose. Positive reactions are also observed for esterase (C4), esterase lipase (C8), leucine arylamidase, phosphatase acid and naphtol phosphohydrolase using an API strip. In addition, using an API 20A (bioMérieux), positive reactions are observed for esculin ferric citrate only.