Enterococcus burkinafasonensis sp. nov. isolated from human gut microbiota

Strain Marseille-Q0835T is an aerobic, non-motile and non-spore-forming Gram-positive coccus isolated from the stools of a Burkinabe woman. In this report, we present its phenotypic description including MALDI-TOF mass spectrometry analysis and genome sequencing. Strain Marseille-Q0835T; 2.9768-Mb genome exhibited a 41.9 mol% G+C content and 2699 predicted genes. Considering phenotypic features and comparative genome studies, we propose the strain Marseille-Q0835T as the type strain of Enterococcus burkinafasonensis sp. nov., a new species within the family Enterococcaceae.


Introduction
Culturomics strategy is a high-throughput culturing method [1]. This strategy consists of the diversification of culture conditions and uses matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF/MS) for identification, to study the human microbiota [1][2][3]. Culturomics has played a fundamental role in resolving the gaps in 16S rRNA genetargeted metagenomics [4]. It has been reported that culturomics has contributed up to 66.2% towards updating the repertoire of isolated human bacterial and archaeal species [2]. Taxonogenomics is a concept used for the description of new species that includes phenotypic data, MALDI TOF/MS data and genome sequencing [5,6]. In this study, we report a human gut isolate representative of a novel Enterococcus species purposely named Enterococcus burkinafasonensis.

Isolation and growth conditions
In September 2018, a fresh stool sample was collected from an apparently healthy 28-year-old Burkinabe woman who was admitted for diagnosis check-up in the Regional Tuberculosis Control Centre, Bobo-Dioulasso, Burkina Faso. A stool sample was sent to the collaborative laboratory at IHU in Marseille, France for culturomics analysis, which isolated an unidentified bacterial strain from the stool. The study was validated by the Science and Health Research Ethics Committee of Bobo-Dioulasso, under number (N/Ref.002-2018-CEIRS). The bacterium here referred to as strain Marseille-Q0835 was isolated on Columbia sheep blood agar after a 24-hour incubation under aerobic atmosphere at 37°C and pH 7.5. Purified colonies could not be identified by MALDI-TOF MS. The screening was performed on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany), as previously described [7]. The obtained

16S rRNA gene sequencing
The 16S rRNA gene was sequenced in an attempt to classify this bacterium. Amplification was performed using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequencing using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously described [8]. The 16S rRNA gene nucleotide sequences were assembled and corrected using CODONCODE ALIGNER software (http://www. codoncode.com). BLASTn research was conducted using nucleotide databases for cross-species comparison (https:// blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_ TYPE=BlastSearch&LINK_LOC=blasthome). The search was limited to records that include sequences from type material, and exclude uncultured/environmental sample sequences. The result showed that strain Marseille-Q0835 exhibited a 97.80% sequence identity with Enterococcus gallinarum strain LMG 13129 (GenBank accession number NR_104559.2), the phylogenetically closest species with standing in nomenclature (Fig. 2). We consequently classify strain Marseille-Q0835 as representative of a new species within the genus Enterococcus, family Enterococcaceae, phylum Firmicutes.

Phenotypic characteristics
Colonies were smooth, white with entire edges with a mean diameter of 1 mm. The bacterial cells were Gram-positive cocci, non-motile, non-spore forming with a mean diameter of 0.7 μm (Fig. 3). Enterococcus sp. Marseille-Q0835 T showed negative catalase and oxidase activities. API 50CH and API ZYM tests (bioMérieux, La Balme les Grottes, France) were performed at 37°C under aerobic conditions and the results are summarized in Table 1. Table 2 compares the characteristics of Enterococcus sp. nov. strain Marseille-Q0835 T with other bacterial species (Table 2).

Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France) with the EZ1 DNA tissue kit and then sequenced on MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera XT Paired end (Illumina), as previously described [9]. The assembly was performed with a pipeline incorporating different softwares (VELVET [10], SPADES [11] and SOAP DENOVO [12]) on trimmed data (TRIMMOMATIC [13]) or raw data. GAPCLOSER was used to reduce assembly gaps. Scaffolds <800 bp and scaffolds with a depth value < 25% of the mean depth were removed [14]. The best assembly was selected by using different criteria (17 scaffolds, 19 contigs). The genome of strain Marseille-Q0835 T is 2.9768 Mb long with a 41.9 mol% G+C content and contains 2699 predicted genes.
The strain Marseille-Q0835 T genome is 2.9768 Mb long, with a G-C content of 41.9%.

Nucleotide sequence accession number
The 16S rRNA gene and genome sequences were deposited in GenBank under accession number LR742708 and NZ_CACSLH000000000, respectively.