Characterization of a novel Gram‐stain‐positive anaerobic coccus isolated from the female genital tract: Genome sequence and description of Murdochiella vaginalis sp. nov.

Abstract Strain Marseille‐P2341T, a nonmotile, nonspore‐forming, Gram‐stain‐positive anaerobic coccus, was isolated in the vaginal specimen of a patient with bacterial vaginosis using culturomics. Its growth occurred at temperatures ranging from 25 to 42°C, with pH between 6.5 and 8.5, and at NaCl concentrations lower than 5%. The major fatty acids were C18:1n9 (27.7%) and C16:0 (24.4%). Its genome is 1,671,491 bp long with 49.48 mol% of G+C content. It is composed of 1,501 genes: 1,446 were protein‐coding genes and 55 were RNAs. Strain Marseille‐P2341T shared 97.3% of 16S rRNA gene sequence similarity with Murdochiella asaccharolytica, the phylogenetically closest species. These results enabled the classification of strain Marseille‐P2341T as a new species of the genus Murdochiella for which we proposed the name Murdochiella vaginalis sp. nov. The type strain is strain Marseille‐P2341T (=DSM 102237, =CSUR P2341).

). An imbalance in this flora is observed in bacterial vaginosis.
The vaginal microflora diversity of a patient suffering from bacterial vaginosis was first described by Schröder in 1921(Pal et al., 2011. This dysbiosis is characterized by a progressive decrease or even a lack of normal Lactobacillus flora accompanied by an increased pH of the vaginal lumen and an abnormal proliferation of previously underrepresented bacteria and Gram-stain-negative anaerobic bacteria (Gardnerella vaginalis, Atopobium vaginae, Mobiluncus curtisii, etc.) (Pépin et al., 2011;Shipitsyna et al., 2013). The mechanism of bacterial vaginosis is unknown; its empirical treatment and relapse rate is estimated at 50% at 3 months (Bretelle et al., 2015). This disturbance is associated with some complications in pregnant women such as miscarriage, chorioamnionitis, and preterm birth (Bretelle et al., 2015;Svare, Schmidt, Hansen, & Lose, 2006).
Initially studied using conventional culture methods, the understanding of the human vaginal microbiota was enhanced through the use of molecular techniques involving sequencing and phylogenetic analysis of the 16S rRNA gene (Lamont et al., 2011). These molecular methods enabled the detection of fastidious and uncultured bacteria such as bacterial vaginosis-associated bacteria (BVAB): BVAB1 BVAB2, and BVAB3 (Fredricks, Fiedler, & Marrazzo, 2005). In order to identify all bacteria (uncultured and fastidious) present in the vagina and involved in this alteration, we studied normal vaginal flora and those from bacterial vaginosis using the concept of "microbial culturomics," based on the multiplication of culture conditions with variations in temperature, media, pH, and atmospheric conditions, and rapid bacterial identification using matrix-assisted laser-desorption/ ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) (Lagier et al., 2012. This microbial culturomics approach enabled us to isolate a new member of the Murdochiella genus that did not correspond to other species of this genus. This strain is designated as Marseille-P2341 T . The Murdochiella genus was created in 2010, to include strain recovered from a human abdominal wall abscess and in a sacral pilonidal cyst aspirate (Ulger-Toprak, Liu, Summanen, & Finegold, 2010). This genus has only one valid species: Murdochiella asaccharolytica.
The description of new bacterial species is based on phenotypic and genotypic characteristics but has some limitations (Chan, Halachev, Loman, Constantinidou, & Pallen, 2012;Vandamme et al., 1996). In this manuscript we use taxonogenomics, a new approach combining classic characteristics with the proteomic information obtained from MALDI-TOF MS and the description of the annotated whole genome (Fournier & Drancourt, 2015;Fournier, Lagier, Dubourg, & Raoult, 2015), to describe Murdochiella vaginalis sp. nov.

| Sample ethics and strain isolation
Using a Sigma Transwab (Medical Wire, Corsham, United Kingdom), the vaginal specimen of a 33-year-old French woman was collected F I G U R E 1 Phylogenetic tree highlighting the position of Murdochiella vaginalis strain Marseille-P2341 T relative to other close strains. GenBank accession numbers of each 16S rRNA are noted in parenthesis. Sequences were aligned using Muscle v3.8.31 with default parameters and phylogenetic inferences were obtained using the neighbor-joining method with 500 bootstrap replicates, within MEGA6 software. The scale bar represents a 2% nucleotide sequence divergence and transported to the La Timone hospital in Marseille (France).
At the time the sample was collected, she was not being treated with any antibiotics. The study was authorized by the local IFR48 ethics committee (Marseille, France) under agreement number 09-022 and the patient also signed written consent. After sampling, the specimen was preincubated in a blood culture bottle (BD Diagnostics, Le Pontde-Claix, France) enriched with 4 ml of rumen that was filter-sterilized through a 0.2 μm pore filter (Thermo Fisher Scientific, Villebon-sur-Yvette, France) and 3 ml of sheep's blood (bioMérieux, Marcy l'Etoile, France). After different preincubation periods (1, 3, 7, 10,15, 20, and 30 days), 50 μl of the supernatant was inoculated on Schaedler agar (BD Diagnostics) and then incubated for 7 days under anaerobic conditions at 37°C.

| Strain identification by MALDI-TOF MS and 16S rRNA gene sequencing
Isolated colonies were deposited in duplicate on a MTP 96 MALDI-TOF target plate (Bruker Daltonics, Leipzig, Germany) for identification with a microflex spectrometer (Bruker), as previously described (Seng et al., 2009). All obtained protein spectra were loaded into the MALDI Biotyper Software (Bruker Daltonics) and compared, as previously described (18), using the standard pattern-matching algorithm, which compared the acquired spectrum with those present T A B L E 1 Classification and general features of Murdochiella vaginalis Marseille-P2341 T The gel view displays the raw spectra of loaded spectrum files arranged with a pseudo-gel like appearance. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a gray scale scheme code. The right y-axis indicates the relation between the color of a peak and its intensity, in arbitrary units. Displayed species are indicated on the left in the library (the Bruker database and our constantly updated database). If the score was greater than 1.9, the bacterium was considered to be identified at the species level. If not, identification failed and to achieve identification for unidentified colonies, the 16S rRNA gene was sequenced using fD1-rP2 primers (Eurogentec, Angers, France) and the obtained sequence was matched against the NCBI database using the BLAST algorithm (Drancourt et al., 2000).

Properties Terms
As suggested, if the 16S rRNA gene sequence similarity value was lower than 95% or 98.7%, the strain was defined as a new genus or species, respectively (Kim, Oh, Park, & Chun, 2014;Stackebrandt & Ebers, 2006).

| Phylogenetic analysis
All species from the same order of the new species were retrieved and 16S sequences were download from NCBI, by parsing NCBI eUtils results and the NCBI taxonomy page. Sequences were aligned using CLUSTALW, with default parameters and phylogenetic inferences obtained using the neighbor-joining method with 500 bootstrap replicates, within MEGA6 software.
Oxidase and catalase tests, Gram-stain, motility, and sporulation were performed using standard procedures (Murray, Baron, Jorgensen, Landry, & Pfaller, 2007). To observe cell morphology, they were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer for at least 1 hr at 4°C. A drop of cell suspension was then deposited for approximately 5 min on glow-discharged formvar carbon film on 400 mesh nickel grids (FCF400-Ni, EMS). The grids were dried on blotting paper and cells were negatively stained for 10 s with 1% ammonium molybdate solution in filtered water at RT. Electron micrographs were acquired using a Tecnai G20 Cryo (FEI) transmission electron microscope operated at 200 keV.

| Biochemical and antibiotic susceptibility tests
Biochemical tests were performed using API ZYM, API 20A, and API 50CH strips (bioMérieux) according to the manufacturer's instructions. The strips were incubated for 4, 24, and 48 hr respectively.
Cellular fatty acid methyl ester (FAME) analysis was performed using Gas Chromatography/Mass Spectrometry (GC/MS). Strain Marseille-P2341 T was grown on Columbia agar enriched with 5% sheep's blood (bioMérieux). Two samples were then prepared with approximately 50 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as described by Sasser (Sasser, 2006). GC/MS analyses were carried out as previously described (Dione et al., 2016). In brief, fatty acid methyl esters were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500-SQ 8 S, Perkin Elmer, Courtaboeuf, France).
A spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).
Antibiotic susceptibility was tested using the disc diffusion method (Le Page et al., 2015). The results were read using Scan 1200 (Interscience, Saint-Nom-la-Bretèche, France).

| Genomic DNA preparation
Genomic DNA (gDNA) of strain Marseille-P2341 T was extracted in two steps: a mechanical treatment was first performed using acid-washed glass beads (G4649-500 g Sigma) and a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5) for 3 × 30 s.
Then after 2 hr of lysozyme incubation at 37°C, DNA was extracted on the EZ1 biorobot (Qiagen, Hilden, Germany) using the EZ1 DNA tissue kit. The elution volume was 50 μl. The gDNA was quantified by a Qubit assay using the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 103 ng/μl.  In total, 9.2 Gb of information was obtained from a 1042 K/mm 2 cluster density with a cluster passing quality control filters of 91.6%

| Genome sequencing and assembly
(18,078,000 passing filter paired reads). Within this run, the index representation for strain Marseille-P2341 T was determined to 13.14%.
The 2,375,075 paired reads were trimmed then assembled in a scaffold.    (Figure 2a) was then added to our database and compared to other known species of the family Peptoniphilaceae (Johnson, Whitehead, Cotta, Rhoades, & Lawson, 2014). Their differences are shown in the gel view which was obtained ( Figure 2b).

| Phenotypic characteristics
Only grown in anaerobic conditions, strain Marseille-P2341 T grows at temperatures between 25 to 42°C, with optimal growth at 37°C after 48 hr of incubation. It needs NaCl concentrations lower than 5 g/L and a pH ranging from 6.5 to 8.5. After 2 days of incubation at 37°C under anaerobic conditions on Columbia agar (bioMérieux), colonies are circular, white, and opaque with a diameter of 2-2.5 mm.
Gram-staining shows a Gram-positive coccus. Individual cells show a diameter ranging from 0.6 to 0.8 μm under an electron microscope ( Figure 3). Nonmotile and nonspore-forming, strain Marseille-P2341 T exhibited positive oxidase activity. Nevertheless, catalase activity was negative and nitrate was not reduced.

| Genome properties
The genome measures 1,671,491 bp long and has 49.48 mol% of G+C content (Table 4, Figure 4). It is composed of one scaffold composed of one contig. Of the 1,501 predicted genes, 1,446 were protein-coding genes and 55 were RNAs (two genes were 5S rRNA, two genes were 16S rRNA, two genes were 23S rRNA, 49 genes were tRNA genes). A total of 1,056 genes (73.03%) were assigned a putative function (by cogs or by NR blast). 56 genes were identified as ORFans (3.87%). The remaining 292 genes were annotated as hypothetical proteins (20.19%).
Genome statistics are summarized in Table 4 and the distribution of the genes in COGs functional categories is presented in Table 5.

| Genomic comparison
The comparison of the genome of our species with the closest related species ( than other compared species ( Figure 5). Moreover, the AGIOS analysis shows that strain Marseille-P2341 T shares between 509 and 542 orthologous genes with closely related species (Table 7) and analysis of the average percentage of nucleotide sequence identity ranged from 50.8% to 56.4% with P. micra and H. sueciensis, respectively (Table 7). In addition, the digital DNA-DNA hybridization (dDDH) of strain Marseille-P2341 T and its closest species varied between 22.40% to 36% with 22.40, 23.60, 23.70, 25.50, 25.90, and 36% for H. kunzii, A. hydrogenalis, P. micra, P. coxii, H. sueciensis, and P. indolicus, respectively. Unfortunately, M. asaccharolytica was not included in this comparison because its genome was not sequenced.  (Kim et al., 2014;Stackebrandt & Ebers, 2006). Created A polyphasic taxono-genomic strategy based on the combination of phenotypic and genomic analyses (Fournier & Drancourt, 2015; was used to describe the new species whose strain Marseille-P2341 T is the type strain.  The numbers of proteins per genome are indicated in bold. The G+C content of strain Marseille-P2341 T and its phylogeneticallyclosest species ranges from 28.40 to 49.48 mol% and, as previously demonstrated, the difference in the G+C content is, at most, 1% in a species. Thus, overall, these values justify the strain Marseille-P2341 T being classified as a distinct species. The AGIOS and GGDC values also confirm it belongs to a new species (Klenk, Meier-Kolthoff, & Göker, 2014).