Taxono-genomics and description of Gordonibacter massiliensis sp. nov., a new bacterium isolated from stool of healthy patient

Using the taxono-genomics concept, we describe here a strictly anaerobic Gram-positive bacillus. This strain was isolated from the stool sample of a 50-year-old healthy Bedouin woman. The 16S rRNA gene sequence analysis and the whole-genome sequencing showed that this isolate belonged to the genus Gordonibacter in the family Eggerthellaceae. Based on these criteria, we propose the creation of Gordonibacter massiliensis sp. nov., strain Marseille-P2775T (= CSUR P2775).


Introduction
Members of the genus Gordonibacter are Gram-positive bacteria belonging to the recent family Eggerthellaceae [1]. These species are part of the human gut microbiota and have the capacity to metabolize polyphenols from diet into bioavailable metabolites known as urolithin [2,3]. During the last decades, culturomics studies have brought insight into the human microbiota, which has led to the discovery of previously uncultured bacteria [4,5]. Culturomics, including different culture conditions, is completed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification and sequencing of the 16S rRNA gene, in order to explore the microbial diversity of the human gut [6,7]. This new bacterial species was described using a combination of genotypic and phenotypic characteristics according to the previously reported taxono-genomics approach [8,9].
Herein, we give details of the isolation and taxono-genomics characters of strain Marseille-P2775 T , which is the type strain of Gordonibacter massiliensis sp. nov.

Isolation and growth conditions
The strain was isolated in 2016 from the stool sample of a 50year-old healthy Bedouin woman living in the Jazan region of Saudi Arabia. This study was performed in Franceafter approval from the ethics committee of the King Abdulaziz University (Saudi Arabia) and the local ethics committee of the IFR48 (Marseille, France) under numbers 014-CEGMR-2-ETH-P and 09-022, respectively. Isolation and growth conditions of strain were performed as previously described [10]. The initial growth of strain Marseille-P2775 was obtained after 2 days of incubation in a Colombia agar supplemented with 5% sheep's blood (COS, bioMérieux, Marcy l'Étoile, France) under strict anaerobic conditions at 37°C. Identification of this bacterial strain was attempted using MALDI-TOF mass spectrometry. The screening was performed on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously reported [11]. The spectra obtained were saved into MALDI BIOTYPER 3.0 software (Bruker Daltonics) and analysed against the main

Phenotypic characteristics
Growing on Columbia blood agar, colonies of strain Marseille-P2775 appeared beige with a mean diameter of 1 mm. Bacterial cells were Gram-positive, strictly anaerobic short-rod bacilli with a mean length of 1.2 μm and 0.5 μm in diameter (Fig. 3). Strain Marseille-P2775 was motile, non-haemolytic and nonspore-forming. It presented catalase-positive and oxidasenegative activities. Carbohydrate metabolism and enzymatic characteristics of the strain Marseille-P2775 were tested under strict anaerobic conditions at 37°C using API 50 CH and ZYM, respectively (Table 1). A comparative study of the differential characteristics of this strain with other closely related species is shown in Table 2. Cellular fatty acid methyl ester (FAME) analysis was performed as previously described [15,16]. The major fatty acids were 9-octadecenoic acid (41%) and hexadecanoic acid (24%). Several branched structures were also described with lower abundances (Table 3).

Genome sequencing
The DNA genomic extraction was performed using the EZ1 biorobot and the EZ1 DNA tissue kit. Genomic DNA (gDNA) was quantified by a Qubit assay. The sequencing was performed using MiSeq technology (Illumina, San Diego, CA, USA) with the Paired-End (Illumina). The assembly was performed with SPADES software [17]. The reads with low quality were trimmed using TRIMMOMATIC software [18]. GAPCLOSER software [19] was used to reduce the assembly gap. Scaffolds < 800 bp and scaffolds with a depth value < 25% of the mean depth were removed. The total length of the G. massiliensis genome is 3.9 megabases encompassing 1 contig with a G + C content of 65.1 mol%. The predicted genes analysis reported 3248 genes. The degree of genomic similarity of strain Marseille-P2775 with closely related species was estimated using the ORTHOANI software [20].  (Fig. 4).
Gordonibacter massiliensis (mas.si.li.en'sis. L. fem. adj., from massiliensis of Massilia, the Latin name of Marseille where the strain was first cultivated) is a Gram-positive, motile, non-sporeforming and obligate anaerobic coccobacillus. Bacterial cells had   a mean diameter of 0.5 μm. Colonies appear beige on blood agar after 48 h of incubation at 37°C in an anaerobic environment. Major cellular fatty acids were 9-octadecenoic acid (41%) and hexadecanoic acid (24%). Catalase is positive but oxidase is negative. Utilization of L-arabinose and D-glucose distinguishes strain Marseille-P2775 from among the closest bacterial species. Also mannose, raffinose, fucose and β-glucosidase are not produced. The strain Marseille-P2775 is isolated from the stool sample of healthy woman living in Saudi Arabia. The G + C content of the genomic DNA is 65.1 mol%.

Conclusion
Based on phenotypic, genomic and phylogenetic analyses, we formally propose the creation of Gordonibacter massiliensis sp. nov., represented here by the strain Marseille-2775. This strain was isolated from a stool sample of a 50-year-old healthy Bedouin woman living in the Jazan region of Saudi Arabia.

Nucleotide sequence accession numbers
The 16S rRNA and genome sequences were deposited in GenBank under accession numbers LT558845 and LT827128, respectively.

Deposit in culture collections
Strain Marseille-P2775 T was deposited in the Collection de Souches de l'Unité des Rickettsies under the following number: CSUR P2775.

Conflicts of interest
None to declare.

Funding sources
This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the programme Investissements d'avenir, reference ANR-10-IAHU-03, the Région Provence Alpes Côte d'Azur and European funding FEDER PRIMI.