Megasphaera vaginalis sp. nov. and Anaerococcus vaginimassiliensis sp. nov., new bacteria isolated from vagina of French woman with bacterial vaginosis

Using the culturomics method, two strains were isolated, identified and characterized following the taxonogenomics concept. Megasphaera vaginalis sp. nov. strain Marseille-P4512 (= CSURP4512) and Anaerococcus vaginimassiliensis sp. nov. strain Marseille-P4857 (= CSURP4857) were isolated from the vagina of a French woman. The phylogenic tree, phenotypic criteria and genomic analysis described here clearly show that these two bacteria are different from previously known bacterial species with standing in nomenclature and new members of Firmicutes phylum.


Introduction
Healthy vaginal microbiota is a complex dynamic ecosystem, mainly dominated by Lactobacillus spp. and classified into five community state types (CST) depending on the following majority species: CST I (Lactobacillus crispatus), CST II (Lactobacillus gasseri), CST III (Lactobacillus iners) and CST V (Lactobacillus jensenii) [1,2]. These beneficial bacteria are the first line of defence against vaginal pathogens through competition and production of inhibitory compounds [3,4]. Bacterial vaginosis is a common infection due to an imbalance of the vaginal flora with an increase in CST IV, which is represented by anaerobic pathogenic bacteria, such as Atopobium sp., Gardnerella sp. and Sneathia sp.
The development of culturomics, combined with taxonogenomic analysis, has enabled the description of many previously unknown bacterial species [5,6]. Thanks to this strategy, our laboratory has characterized several new bacteria isolated from the vagina [7][8][9].
We report here the description of two new designated species, Megasphaera vaginalis sp. nov. strain Marseille-P4857 and Anaerococcus vaginimassiliensis sp. nov. strain Marseille-P4512, belonging to the Firmicutes phylum.
French woman with bacterial vaginosis. These were strains Marseille-P4857 and Marseille-P4512. The patient provided informed consent, and the study was authorized by the ethics committee of the Institut Federatif de Recherche IFR48 under the number 09-022. The vaginal swabs were directly seeded in Petri dishes containing 5% sheep blood agar (BioMérieux, Marcy l'Étoile, France) and incubated under anaerobic condition (Thermo Scientific, Dardilly, France) at 37°C after 3 days. Identification was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) as previously reported [19]. The spectra generated were analysed by Biotyper 3.0 software, which is regularly incremented with the local URMS database (https://www.mediterranee-infection. com/urms-data-base). Misidentification with MALDI-TOF MS led to amplification of the 16S rRNA gene using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and then sequencing using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and 3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously reported [20]. All 16S rRNA nucleotide sequences were assembled and edited using CodonCode Aligner software (http://www.codoncode.com). Once a consensus sequence is obtained, it is submitted to the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/ nucleotide/) and a comparative analysis of nucleotides by BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM= blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) is performed. Hence, the sequences phylogenetically closest to the only typical species are recovered to build the phylogenetic trees.

Genome characteristics
Genomic DNA extraction was performed with the EZ1 biorobot using the EZ1 DNA tissue kit (Qiagen, Hilden, Germany), and sequencing was performed on the MiSeq instrument (Illumina Inc., San Diego, CA, USA) using the Nextera Mate Pair and Nextera XT Paired End (Illumina) sample preparation kit, as previously described [20]. The genomic assembly was carried out using the three following softwares: Velvet [23], Spades [24] and Soap Denovo [25]. MiSeq and Trimmomatic [26] softwares were used for trimmed or untrimmed sequences. To reduce assembly gaps, GapCloser software [27] was used. Best assembly was determined using different criteria, such as the number of scaffolds, N50 or number of N. Scaffolds were deleted when their nucleotide number was <800 bp and their depth value < 25% of the mean depths. Genome annotation of these two species was performed as described elsewhere [28].
In addition, the Genome-to-Genome Distance Calculator web server available online (http://ggdc.dsmz.de) made it possible to assess the similarity between the genomes being compared and to replace the DNA-DNA hybridization (DDH) with a digital DDH (dDDH) [29]. Average nucleotide identity analysis was also evaluated using the OAT software [30].

Results
Strain identification and phylogenetic analysis Attempts to identify the strains cultivated on blood agar by mass spectrometry failed, indicating that these isolates were not known from the MALDI-TOF database. Therefore, their generated spectra were added to the local database. 16S rDNA-based similarity analysis of strain Marseille-P4857 and strain Marseille-P4512 against GenBank exhibited highest nucleotide sequence similarities of 95.12% with Megasphaera micronuciformis strain AIP 412.00 (accession number NR_025230.1) and 96.78% with Anaerococcus tetradius strain CCUG 46590 (accession number NR_041941.1), being respectively the two phylogenetically closest species. As these similarity values were below the 98.65% threshold recommended for the delimitation of new bacterial species [29,31], strain Marseille-P4857 and strain Marseille-P4512 were considered potentially new species within the phylum Firmicutes. The phylogenetic trees of Megasphaera spp. (Fig. 1a) and Anaerococcus spp. (Fig. 1b) show their positions concerning their respective closely related species with a validly published name. In addition, the shape of each bacterium (shown in Fig. 2) was obtained from the Hitachi TM4000 instrument.

Genomic analysis
The size of the genomes of strains Marseille-P4857 and Marseille-P4512 were 2 206 375 and 1 836 452 bp with 50.2 and 33.1 mol% G + C content, respectively. The genomic assembly was carried out into 17 contigs for Marseille-P4857 and into one scaffold for Marseille-P4512. Indeed, 2137 and 1826 were assigned as predicted genes for Marseille-P4857 and Marseille-P4512, respectively. In addition, 2032 and 1722 protein-coding genes and 56 and 61 RNA genes were found from the respective genomes of Marseille-P4857 and Marseille-P4512. The comparison of the genomes of M. vaginalis and A. vaginimassiliensis in terms of size and G + C content, as well as the number of genes compared with their phylogenetically closest species is presented in Table 3. Using dDDH analysis, values ranged from 17.7% between M. massiliensis and Megasphaera paucivorans to 27.0% between M. micronuciformis and Megasphaera stantonii. At the end of the dDDH analysis of Anaerococcus species used in this study, we obtained values ranging from 20.2% between A. prevotii ACS-065-V-Col13 and Anaerococcus mediterraneensis strain Marseille-P2765 to 33.6% between A. vaginalis ATCC 51170 and A. mediterraneensis strain Marseille-P2765. These values are lower than the 70% threshold used for the delineation of prokaryotic species, confirming that these three strains represent new species. The dDDH values obtained from genome analysis of the species studied here are shown in Table 4.