Corynebacterium dentalis sp. nov., a new bacterium isolated from dental plaque of a woman with periodontitis

Strain Marseille-P4122T is a new species from the order Corynebacteriales that was isolated from the dental plaque of a woman with periodontitis. It is a facultative anaerobic Gram-positive rod-shaped bacterium. Strain Marseille-P4122T exhibited a 98.19% sequence identity with Corynebacterium suicordis strain P81/02, the phylogenetically closely related species with standing in nomenclature. The draft genome size of strain Marseille-P4122T is 2.49 Mb with 60.1% G + C content. We propose that strain Marseille-P4122T (=CSURP4122) is the type strain of the new species Corynebacterium dentalis sp. nov.


Introduction
Corynebacterium genus belonging to family Corynebacteriaceae was first described in 1896 by Lehmann and Neumann [1]. It consists of Gram-positive rods and non-spore-forming bacteria with a high DNA G + C content [2]. Several species of this genus are implicated in human and animal diseases whereas others are members of normal flora on skin and mucous membranes [3][4][5]. Corynebacterium diphtheriae is the major pathogen in humans and causes diphtheria worldwide [6]. It is a large genus that regroups currently 132 species with 11 subspecies validly described with standing in nomenclature [7].
It is important to understand the implications of bacterial diversity in normal physiological functions and for disease [8].
Culturomics is a concept that develops different culture conditions to enlarge our knowledge of the human microbiota through the discovery of previously uncultured bacteria [9][10][11][12]. Once a bacterium is isolated, we use a taxono-genomics approach including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phylogenetic analysis, main phenotypic description and genome sequencing, to describe it [13,14].

Isolation and growth conditions
In 2015, we isolated from the dental plaque sample of a woman with periodontitis an unidentified bacterial strain. A screening was performed using MALDI-TOF MS on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously described [15]. The spectra obtained ( Fig. 1) were imported into MALDI BIOTYPER 3.0 software (Bruker Daltonics) and analysed against the main spectra of the bacteria included in two databases (Bruker and the constantly updated MEPHI databases). The study was validated by the ethics committee of the Institut Fédératif de Recherche IFR48 under number 2016- 2 New Microbes and New Infections, Volume 33 Number C, ---2020 010. Strain Marseille-P4122 T was first isolated in aerobic conditions after incubation in a culture bottle (bioMérieux, Marcy l'Etoile, France) supplemented with 5 mL sheep blood at 37°C.

Phenotypic characteristics
After the isolation step, the strain Marseille-P4122 T was cultured to obtain pure and isolated colonies on blood agar. The colonies were white and transparent. Bacterial cells were Gram-positive. The sporulation test (10 min at 80°C) was negative. Different growth temperatures (20, 28, 32, 37, 45 and 56°C), pH (5, 6, 7, 7.5, 8 and 8.5), NaCl content (5, 10 and 15 g/L) and atmospheres (aerobic, anaerobic and microaerophilic (CampyGEN; Oxoid, Basingstoke, UK)) were tested on 5% sheep-blood-enriched Columbia Agar. Strain Marseille-P4122 T is a very-easy-to-cultivate bacterium and grows in all these conditions except at 56°C. API ZYM and API Coryne tests (bioMérieux) were performed to determine specific phenotypic features for strain Marseille-P4122. The results are shown in Table 1. Using API 50CH strips (bioMérieux) the carbohydrate metabolism of strain Marseille-P4122 was evaluated according to the manufacturer's instructions (Table 2). Strain Marseille-P4122 T has enzymatic activities such as esterase (C4), esteraselipase (C8), lipase (C14), acid phosphatase, naphthol-AS-BIphosphohydrolase, α-glucosidase, β-glucosidase and urease, whereas only D-fructose and D-trehalose were positive for carbohydrate metabolism. All the other reactions tested were negative. Strain Marseille-P4122 T showed catalase-negative and oxidase-negative activities. A comparative study of the biochemical characteristics of this strain with other closely related Corynebacterium species is presented in Table 3. For scanning electron microscopy, a colony was collected from agar and immersed into a 2.5% glutaraldehyde fixative solution. The slide was gently washed in water, air-dried and examined with a TM4000 microscope. The cells appeared rod-shaped with a

CH
Erythritol -  (Fig. 2). Antimicrobial susceptibility testing was performed using the Etest strips (bioMérieux) method and the data obtained are summarized in Table 4. The major fatty acids found for this strain were hexadecanoic acid (44%) and 9-octadecenoic acid (36%). Very few other structures were described. No branched fatty acids were detected (Table 5).

Strain identification
The 16S rRNA gene was sequenced to classify this bacterium. Amplification was carried out using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequencing using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and 3500xL Genetic Analyzer capillary3500xL sequencer (Thermofisher, Saint-Aubin, France), as previously described [16]. The 16S rRNA nucleotide Human Human Pig Human Human    sequences were assembled and corrected using CODONCODE ALIGNER software (http://www.codoncode.com). Strain Marseille-P4122 T exhibited a 98.19% sequence identity with Corynebacterium suicordis strain P81/02 (GenBank accession number NR042151.1), the phylogenetically closest species with standing in nomenclature (Fig. 3a). The rpoB gene that encodes the β subunit of bacterial RNA polymerase was targeted to discriminate the Corynebacterium species [17]. Corynebacterium dentalis strain Marseille-P4122 T was close to strains Corynebacterium auriscanis and Corynebacterium resistens (Fig. 3b). Considering these phylogenetic criteria, we consequently classify this strain as a member of a new species within the genus Corynebacterium, family Corynebacteriaceae, phylum Actinobacteria.

Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France) with the EZ1 DNA tissue kit and then sequenced on the MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired end (Illumina), as previously described [18]. The assembly was performed with a pipeline incorporating different softwares (VELVET [19], SPADES [20] and SOAP DENOVO [21]), and trimmed data (MISEQ and TRIMMOMATIC [22] 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). The genome of Corynebacterium dentalis strain Marseille-P4122 T is 2 303 041 bp long with a 60.1% G + C content. The degree of genomic similarity of strain Marseille-P4122 T with closely related species was estimated using the ORTHOANI software [23]. Values among closely related species (Fig. 4)   New Microbes and New Infections, Volume 33 Number C, ---2020

Conclusion
Based on the results from unique phenotypic characteristics, including API galleries tests, MALDI-TOF spectrum, and phylogenetic and genomic analysis such as 16S rRNA sequence similarity <98.7% and ORTHOANI value < 95% with the phylogenetically closest species with standing in nomenclature, we formally proposed strain Marseille-P4122 T as the type strain of Corynebacterium dentalis sp. nov.
Description of Corynebacterium dentalis sp. nov.
Corynebacterium dentalis (den.ta'lis. N.L. masc. adj. dentalis referring to the teeth surrounded by dental plaque from which this strain was isolated). The strain grows easily in varied conditions. Optimum growth of colonies was obtained at 37°C on 5% sheep-blood-enriched Columbia Agar in <24 hours. They appear white and transparent. Corynebacterium dentalis is a Gram-positive rod-shaped bacterium with a mean length of 1 μm and a mean diameter of 0.5 μm. Strain Marseille-P4122 T produced esterase, lipase, acid phosphatase, naphthol-AS-BIphosphohydrolase, α-glucosidase, β-glucosidase, urease, Dfructose and D-trehalose. But no activity was observed with trypsin, β-galactosidase, α-glucosidase, glycerol, D-arabinose, Dribose, D-xylose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-lactose, D-saccharose, glycogen, D-fucose and D-arabitol. Strain Marseille-P4122 T is catalase-negative. It is susceptible to rifampicin, ciprofloxacin, amoxicillin, penicillin G, doxycycline and vancomycin, but resistant to erythromycin. The genome size of Corynebacterium dentalis strain Marseille-P4122 T is about 4.04 Mb with 60.1 mol% G + C content. The GenBank Accession number for the 16S rRNA gene sequence of strain Marseille-P4122 T is LT897837 and for the whole-genome shotgun project is OCTS00000000.This strain was isolated from the dental plaque of a woman with periodontitis.

Nucleotide sequence accession number
The 16S rRNA gene and genome sequences were deposited in GenBank under accession numbers LT897837 and OCTS00000000, respectively.