Description of Prevotella rectalis sp. nov., a new bacterium isolated from human rectum

Using a taxonogenomics method, we describe here a Gram-negative bacterium named Prevotella rectalis sp. nov., strain Marseille-P4334T (= CSUR P4334) isolated from the rectum. Strain Marseille-P4334T has a genome that measure 3.03 Mbp with 43.3 mol% G + C content.


Introduction
Studying the diversity of bacteria is fundamental to understanding their involvement in human diseases and normal physiological functions [1]. To unveil human microbial diversity, the culturomics approach, based on diversified culture conditions, was designed to isolate as yet uncultured species and to complement 16S rRNA metagenomics [2][3][4]. Furthermore, a new taxonomic strategy, named taxonogenomics, was developed to include the analysis of complete genome sequences in combination with phenotypic characteristics [5]. Herein, we report a detailed description of Prevotella rectalis sp. nov., for which the type strain Marseille-P4334 T was isolated from the human rectum.

Isolation and growth conditions
In 2017, we isolated an unidentified bacterium strain from a human rectum. The study was approved by the Institut Federatif de Recherche 48 (agreement number 09-022, Marseille, France), and the patient's consent was obtained. After three tests using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), the strain was not identified. Screening was carried out on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously described [6]. Spectra obtained from the strain (Fig. 1) were imported and analysed using BIOTYPER 3.0 software against the Bruker database that was permanently updated with the local MEPHI database (https://www.mediterranee-infection. com/urms-data-base).

Strain identification
The 16S rRNA gene was sequenced to classify this bacterium. Amplification was performed using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequenced using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and 3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously described [7]. The 16S rRNA nucleotide sequence was assembled and corrected using CODONCODE ALIGNER software (http://www.codoncode.com). Strain Marseille-P4334 T exhibited a 96.4% rRNA sequence identity with Prevotella corporis strain JCM 8529 (GenBank accession number: NR_113099.1), its phylogenetically closest species with standing in nomenclature (Fig. 2). Considering this value less than a threshold value established previously [8], we consequently proposed to classify strain Marseille-P4334 T as a new species within the genus Prevotella in the phylum Bacteroidetes.

Phenotypic characteristics
Colonies of strain Marseille-P4334 T were black and smooth with a mean diameter of 0.5-1 mm. Bacterial cells were Gramnegative bacilli ranging from 1 to 3 μm in length and 0.4 to 0.6 μm in width. Strain Marseille-P4334 T showed catalase-positive and oxidase-negative activities. Electron micrograph showing the shapes of this new bacterial strain ( Fig. 3) was performed with the Hitachi TM4000 instrument (Hitachi Group, Krefeld, Germany). A comparative study of the biochemical characteristics of strain Marseille-P4334 T with other closely related species in phylum Bacteroidetes is presented in Table 1. Results of biochemical tests performed using the API ZYM and 50 CH strips (bioMérieux) are shown in Table 2.

Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot with the EZ1 DNA tissue kit (Qiagen, Hilden, Germany) and then  New Microbes and New Infections, Volume 36 Number C, July 2020 sequenced on a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired End (Illumina), as previously described [9]. The assembly was performed using a pipeline containing several softwares (VELVET [10], SPADES [11] and SOAP DENOVO [12]), and trimmed data (MISEQ and TRIMMOMATIC [13] softwares) or untrimmed data (only MISEQ software). GAPCLOSER was used to reduce assembly gaps. Scaffolds <800 bp and scaffolds with a  depth value < 25% of the mean depth were removed. The best assembly was selected using different criteria (number of scaffolds, N50, number of N). Strain Marseille-P4334 T had a genome size of 3 039 397 bp with 43.3 mol% G + C content. The degree of genomic identity of this strain with closely related species was calculated using ORTHOANI software [14]. Results of this analysis are displayed in Fig. 4. For strain Marseille-P4334 T , ORTHOANI values among closely related species ranged from 68.57% between Prevotella corporis and Prevotella bergensis to 68.70% between Prevotella bergensis and Prevotella salivae. When P. rectalis was compared with these closely related species, values ranged from 69.91% with P. salivae to 76.38% with P. corporis.

Conclusion
On the basis of unique phenotypic features, including the MALDI-TOF spectrum, a 16S rRNA sequence divergence >1.3% and an ORTHOANI value > 95% with the phylogenetically closest species with standing in nomenclature, we formally proposed strain Marseille-P4334 T as the type strain of Prevotella rectalis sp. nov., as a new member within the phylum Bacteriodetes.

Description of Prevotella rectalis sp. nov
Prevotella rectalis (rec.ta'lis. L. gen. n. rectalis pertaining to rectum, the chamber that begins at the end of the large intestine from which this bacterium was isolated). It is a Gramnegative bacterium, non-motile and non-spore-forming. Strain Marseille-P4334 T is the type strain of Prevotella rectalis sp. nov., and shows catalase-positive and oxidase-negative activities. The strain grows strictly under anaerobic conditions at 37°C. The potential pathogenicity of the type strain Marseille-P4334 T (= CSUR P4334) is unknown. This strain has a genome size of 3.03 Mbp long with a 43.3 mol% G + C content. The 16S rRNA gene sequence and whole-genome shotgun sequence of strain Marseille-P4334 were deposited in GenBank under accession numbers LS488976 and UWTY00000000, respectively. It has been isolated from the human rectum.

Conflicts of interest
None to declare.