Journal Pre-proof Megasphaera vaginalis sp. nov., and Anaerococcus vaginimassiliensis sp. nov., new bacteria isolated from vagina of French woman with bacterial vaginosis Alexia Bordigoni, Cheikh Ibrahima Lo, Edmond Kuete Yimagou, Khoudia Diop, Bérangère Nicaise, Didier Raoult, Christelle Desnues, Florence Fenollar PII: S2052-2975(20)30058-5 DOI: https://doi.org/10.1016/j.nmni.2020.100706 Reference: NMNI 100706 To appear in: New Microbes and New Infections Received Date: 29 April 2020 Revised Date: 26 May 2020 Accepted Date: 29 May 2020 Please cite this article as: Bordigoni A, Lo CI, Kuete Yimagou E, Diop K, Nicaise B, Raoult D, Desnues C, Fenollar F, Megasphaera vaginalis sp. nov., and Anaerococcus vaginimassiliensis sp. nov., new bacteria isolated from vagina of French woman with bacterial vaginosis, New Microbes and New Infections, https://doi.org/10.1016/j.nmni.2020.100706. 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Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd. 1 Megasphaera vaginalis sp. nov., and Anaerococcus vaginimassiliensis sp. nov., new bacteria 2 isolated from vagina of French woman with bacterial vaginosis. 3 4 Alexia BORDIGONI1,2, Cheikh Ibrahima LO1,3, Edmond KUETE YIMAGOU1,2, Khoudia 5 DIOP1,3, Bérangère NICAISE1,3, Didier RAOULT1,2, Christelle DESNUES1,2 and Florence 6 FENOLLAR1,3* 7 8 1 Aix Marseille Univ, IRD, AP-HM, MEФI, Marseille, France 9 2 IHU-Méditerranée Infection, Marseille, France. 10 3 Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, Marseille, France 11 12 *Corresponding author : Pr. Florence FENOLLAR, Institut Hospitalo-Universitaire 13 Méditerranée-Infection, 19-21 Boulevard Jean Moulin, 13385, Marseille cedex 05, France ; 14 tel +33 413 732 401 ; fax : +33 413 732 402 ; e-mail: florence.fenollar@univ-amu.fr 15 Running title: Megasphaera vaginalis sp. nov., and Anaerococcus vaginimassiliensis sp. 16 nov. 17 Keywords: Megasphaera vaginalis sp. nov., and Anaerococcus vaginimassiliensis sp. nov.; 18 culturomics; taxonogenomics; vagina; bacteria. 1 Abstract: 2 Using culturomics method, two strains were isolated, identified and characterized following 3 the taxono-genomics concept. Megasphaera vaginalis sp. nov. strain Marseille-P4512 4 (=CSURP4512) and Anaerococcus vaginimassiliensis sp. nov. strain Marseille-P4857 5 (=CSURP4857) were isolated from a vagina of a French woman. The phylogenic tree, 6 phenotypic criteria and genomic analysis described here clearly show that these two bacteria 7 are different from previously known bacterial species with standing in nomenclature and new 8 members of Firmicutes phylum. 9 10 Introduction 11 Healthy vaginal microbiota is a complex dynamic ecosystem, mainly dominated by 12 Lactobacillus spp. and classified in five community state types (CST) depending on the 13 following majority species: CST I (L. crispatus), CST II (L. gasseri), CST III (L. iners) and 14 CST V (L. jensenii) [1, 2]. These beneficial bacteria are the first line of defense against 15 vaginal pathogens through competition and production of inhibitory compounds [3, 4]. 16 Bacterial vaginosis is a common infection due to an imbalance of the vaginal flora with an 17 increase in CST IV, which is represented by anaerobic pathogenic bacteria, such as 18 Atopobium sp, Gardnerella sp, Sneathia sp.. 19 During these last years, the method called culturomics, combined with taxono-genomic 20 analysis, has enabled the description of many bacterial species never known before [5, 6]. 21 Also, thanks to this strategy, our laboratory has characterized several new bacteria isolated 22 from the vagina [7-9]. 23 Megasphaera and Anaerococcus genera respectively belong to Veillonellaceae and 24 Peptoniphilaceae families within the Firmicutes phylum. At the time of writing and among 25 validly published name, 9 species were described in Megasphaera genus and 13 species in 26 Anaerococcus genus [10]. Members of the Megasphaera genus, described in 1971 by 27 Morrisson Rogosa [11], can, for some of them, be found in human fecal flora [12-13], 28 mammalian digestive tract [14] and brewery samples [15]. Some Anaerococcus spp. were 29 isolated from human clinical samples [16-17]. Among the 13 Anaerococcus species validly 30 published, 6 were isolated from vaginal discharge or ovarian abscess samples such as 31 Anaerococcus 32 Anaerococcus prevotii, Anaerococcus tetradius and Anaerococcus provencensis [16, 18]. hydrogenalis, Anaerococcus lactolyticus, Anaerococcus vaginalis 33 We report here the description of two new designated species, Megasphaera vaginalis sp. 34 nov. strain Marseille-P4857 and Anaerococcus vaginimassiliensis sp. nov. strain Marseille- 35 P4512, belonging to the Fimicutes phylum. 36 Materiel and methods 37 Strains isolation and identification 38 As part of a culturomic study investigating the human microbiome, we isolated two bacterial 39 strains from vaginal swabs of a French woman with bacterial vaginosis. These were strains 40 Marseille-P4857 and Marseille-P4512. The patients have endorsed an informed consent, 41 while the study was authorized by the ethics committee of the Institut Federatif de Recherche 42 IFR48 under the number 09-022. The vaginal swabs were directly seeded in petri dishes 43 containing 5% sheep blood agar (BioMérieux, Marcy l'Etoile, France) and incubated under 44 anaerobic condition (Thermo Scientific, Dardilly, France) at 37°C after 3 days. 45 Identification was performed with MALDI-TOF Mass Spectrometry (Bruker, Daltonics, 46 Bremen, Germany) as previously reported [19]. The spectra generated were analyzed by 47 Biotyper 3.0 software, which is regularly incremented with the local URMS database 48 (https://www.mediterranee-infection.com/urms-data-base). Misidentification with MALDI- 49 TOF MS led to amplification of the 16S rRNA gene using the primer pair fD1 and rP2 50 (Eurogentec, Angers, France) and then sequencing using the Big Dye® Terminator v1.1 51 Cycle Sequencing Kit and 3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint- 52 Aubin, France), as previously reported [20]. All 16S rRNA nucleotide sequences were 53 assembled and edited using CodonCode Aligner software (http://www.codoncode.com). Once 54 a consensus sequence is obtained, it will be submitted to the NCBI nucleotide database 55 (https://www.ncbi.nlm.nih.gov/nucleotide/) and a comparative analysis of nucleotides by 56 BLASTn 57 (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LI 58 NK_LOC=blasthome) is performed. Thus the sequences phylogenetically closest of the only 59 typical species are recovered to build the phylogenetic trees. 60 Phenotypic characterization 61 Different growth conditions were tested for strains in aerobic, microaerophilic and anaerobic 62 atmospheres (Thermo Scientific, Dardilly, France). The optimal temperature of growth was 63 assessed (28, 37, 45 and 55 °C) on 5% sheep blood-enriched Columbia agar medium 64 (BioMérieux, Marcy l’Etoile, France). According to the manufacturer’s recommendations, 65 API ZYM and API 50 CH strips (bioMérieux) were employed to assess the biochemical 66 characteristics of each strain. Phenotypic tests, such as Gram-staining, catalase and oxidase 67 were performed. Also, the spore-forming was searched for each strain as previously reported 68 [21]. The morphological structure of these two isolates was highlighted with a scanning 69 electron microscope (Hitachi High-Technologies, Tokyo, Japan) following the protocol 70 described by Belkacemi et al., 2019 [22]. 71 Genome characteristics 72 Genomic DNA extraction was performed with the EZ1 biorobot using the EZ1 DNA tissue kit 73 (Qiagen, Hilden, Germany), and sequencing was performed on the MiSeq instrument 74 (Illumina Inc, San Diego, CA, USA) using the Nextera Mate Pair and Nextera XT Paired End 75 (Illumina) sample preparation kit, as previously described [20]. The genomic assembly was 76 carried out using the three following softwares: Velvet [23], Spades [24] and Soap Denovo 77 [25]. MiSeq and Trimmomatic [26] softwares were used for trimmed or untrimmed 78 sequences. To reduce assembly gaps, GapCloser software [27] was used. Best assembly was 79 determined using different criteria, such as the number of scaffolds, N50 or number of N. 80 Scaffolds were deleted when their nucleotide number was lower than 800 (bp) and their depth 81 value lower than 25% of the mean depths. Genomes annotation of these two species was 82 performed as described elsewhere [28]. In addition, the Genome-to-Genome Distance 83 Calculator (GGDC) web server available online (http://ggdc.dsmz.de) made it possible to 84 assess the similarity between the genomes being compared and to replace the famous DNA– 85 DNA hybridization (DDH) by a digital DDH (dDDH) [29]. Average nucleotide identity 86 analysis was also evaluated using the OAT software [30]. 87 Results 88 Strain identification and phylogenetic analysis 89 Attempt to identify the strains cultivated on blood agar by mass spectrometry failed, thus 90 indicating that these isolates were not known from the MALDI-TOF database. Therefore, 91 their generated spectra were added to the local database. 16S rDNA-based similarity analysis 92 of strain Marseille-P4857 and strain Marseille-P4512 against GenBank exhibited highest 93 nucleotide sequence similarities of 95.12% with Megasphaera micronuciformis strain AIP 94 412.00 (accession number NR_025230.1) and 96.78% with Anaerococcus tetradius strain 95 CCUG 46590 (accession number NR_041941.1), being respectively the two phylogenetically 96 closest species. Since these similarity values were below the 98.65% threshold recommended 97 for the delimitation of new bacterial species [31-32], strain Marseille-P4857 and strain 98 Marseille-P4512 were considered potentially new species within the phylum Firmicutes. The 99 phylogenetic trees of Megasphaera spp. (Figure 1A) and Anaerococcus spp. (Figure 1B) have 100 shown positions concerning their respective closely related species with a validly published 101 name. In addition, the shape of each bacterium shown in Figure 2 was obtained from the 102 Hitachi TM4000 instrument. 103 Biochemical properties of the strains 104 The two strains grow strictly under anaerobic conditions with an optimal temperature at 37°C. 105 Strain Marseille-P4857 is Gram-negative anaerobic cocci with a mean cell diameter of 0.70 106 µm. Colonies of strain Marseille-P4857 were white to yellow, shiny, opaque and convex with 107 a diameter varying from 0.5 to 1mm on blood agar after 3 days of incubation. It presents 108 catalase-negative and oxidase-negative activities. Conversely, strain Marseille-P4512 is a 109 Gram-positive anaerobic bacterium. Cells are coccoid with a mean diameter of 1.08 µm. They 110 exhibit catalase-positive and oxidase-negative activities. Colonies of strain Marseille-P4512 111 are white with regular edges and a mean diameter of 2 mm. 112 Using the API ZYM strip, only acid phosphatase was positive for strain Marseille-P4857, 113 while alkaline phosphatase, leucine arylamidase and acid phosphatase were also positive for 114 strain Marseille-P4512. All remaining reactions were still negative with this API ZYM test. In 115 addition, using 50 CH strip, Megasphaera vaginalis strain Marseille-P4857 was positive for 116 glycerol, erythritol, arabinose, ribose, xylose, D-fructose, inositol, sorbitol, methyl αD- 117 glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, D-maltose, D-lactose, D- 118 melibiose, sucrose, inulin, D-melezitose, D-raffinose, glycogen, xylitol, gentiobiose, D- 119 lyxose, D-tagalose, fucose, potassium gluconate and potassium 5-ketogluconate. For 120 Anaerococcus vaginimassiliensis strain Marseille-P4512, glycerol, xylose, galactose, fructose, 121 glucose, methyl-αD-glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin 122 ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-trehalose, xylitol, gentiobiose, 123 potassium 5-ketogluconate were positive. A large phenotypic comparison of Marseille-P4857 124 and Marseille-P4512 with closely related species is displayed in Tables 1 and 2. The major 125 fatty acids found for Marseille-P4857 were C16:0 (22%) and C16:1n9 (15%). Concerning 126 Marseille-P4512, the major fatty acids were C16:0 (42%), C18:1n9 (25%) and C18:2n6 (19%). 127 Minor amounts of saturated fatty acids were also detected for both. 128 Genomic analysis 129 The size of the genomes of strains Marseille-P4857 and Marseille-P4512 were 2,206,375 and 130 1,836,452 bp long with 50.2 and 33.1 mol% G+C content respectively. The genomic 131 assembly was carried out into 17 contigs for Marseille-P4857 and into 1 scaffold for 132 Marseille-P4512. Indeed, 2,137 and 1,826 were assigned as predicted genes for Marseille- 133 P4857 and Marseille-P4512, respectively. In addition, 2,032 and 1,722 protein-coding genes 134 and 56 and 61 RNAs genes were found from respective genomes of Marseille-P4857 and 135 Marseille-P4512. The comparison of the genomes of M. vaginalis and A. vaginimassiliensis in 136 terms of size, G+C content as well as the number of genes compared to their phylogenetically 137 closest species is presented in Table 3. 138 Using dDDH analysis, values ranged from 17.7% between M. massiliensis and M. 139 paucivorans to 27.0% between M. micronuciformis and M. stantonii. At the end of the dDDH 140 analysis of Anaerococcus species used in this study, we obtained values ranging from 20.2% 141 between A. prevotii ACS-065-V-Col13 and A. mediterraneensis strain Marseille-P2765 to 142 33.6% between A. vaginalis ATCC 51170 and A. mediterraneensis strain Marseille-P2765. 143 These values are lower than the 70% threshold used for the delineation of prokaryotic species, 144 thus confirming that these three strains represent new species. The dDDH values obtained 145 from genomes analysis of species studied here are shown in Table 4. 146 In addition, OrthoANI analysis among closely related species (Figure 3) highlighted that 147 Megasphaera species had higher value of percentage of identity of 80.57% shared between M. 148 elsdenii and M. massiliensis. 68.58% was lowest value of similarity obtained between M. 149 elsdenii and M. paucivorans. Thus, OrthoANI analysis for Anaerococcus species revealed that 150 71.78% was the highest value of similarity that the M. vaginalis Marseille-P4857 strain shared 151 with M. stantonii. Analysis of Anaerococcus species revealed that OrthoANI values ranged 152 from 92.09% of similarity with A. prevotii and A. marasmi to 70.12% of similarity with A. 153 mediterraneensis and A. senegalensis. The highest percentage value obtained with strain 154 Marseille-P4512 was 78.23% of similarity with A. marasmi. 155 Conclusion 156 Considering the phenotypic, biochemical and genomic analysis carried out on these bacteria, 157 strains Marseille-P4857 and Marseille-P4512 are proposed as new species. In addition, the 158 genomic evidences used in this study, such as the sequence similarity of the 16S rRNA gene 159 below the threshold value of 98.65% or OrthoANI values also <95% allowed us to formally 160 declare that Megasphaera vaginalis sp. nov., and Anaerococcus vaginimassiliensis sp. nov., 161 are new species within the phylum Firmicutes. 162 Description of Megasphaera vaginalis sp. nov. 163 Megasphaera vaginalis sp. nov. (va.gi.na'lis. L. n. fem. gen. vaginalis from the vagina which 164 is a female genital organ; vaginalis referring to the vagina). This bacterium is Gram-negative 165 anaerobic shell-shaped. Cells are 0.62 to 0.91 µm in diameter. Catalase and oxidase activities 166 are negative. Acid phosphatase activity is present. Colonies are white, shiny and convex with 167 a mean diameter of 0.5 mm on blood agar. The following tests were positive: glycerol, 168 erythritol, arabinose, ribose, D-xylose, D-fructose, inositol, D-sorbitol, methyl αD- 169 glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, sucrose, inulin, D- 170 maltose, D-lactose, D-melibiose, D-melezitose, D-raffinose, glycogen, xylitol, gentiobiose, D- 171 lyxose, D-tagalose, D-fucose, L-fucose, potassium gluconate and potassium 5-ketogluconate. 172 C16:0 (22.0%), C16:1n9 (14.8%), C12:0 (9.0%) and C14:0 3-OH (7.3%) were the major fatty acids 173 found with Megasphaera vaginalis sp. nov. The genome of strain Marseille-P4857 was 2.20 174 Mbp with 50.2 mol% of G+C content. The 16S rRNA and draft genome sequences are 175 deposited in the Genbank database under Accession numbers LT960586 and 176 OEQB00000000, respectively. The type strain of Megasphaera vaginalis sp. nov., strain 177 Marseille-P4857 was isolated from the vagina of a woman with bacterial vaginosis. 178 Description of Anaerococcus vaginimassiliensis sp. nov. 179 Anaerococcus 180 vaginimassiliensis: vagini refers to vagina and massiliensis to Massilia, the Latin name of 181 Marseille where the type strain was isolated). Gram‐staining is positive. It is coccus‐shaped 182 bacterium with a diameter ranged from 0.8 to 1.2 μm. A. vaginimassiliensis sp. nov., is a strict 183 anaerobic bacterium that grows preferentially at temperature 37°C. It has a catalase activity, 184 but not oxidase. Colonies are white with regular boundaries and have a mean diameter of 2 185 mm. A. vaginimassiliensis is able to ferment glycerol, xylose, D-galactose, D-glucose, D- 186 fructose, methyl αD-glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin 187 ferric citrate, salicin, trehalose, cellobiose, maltose, lactose, 188 potassium 5-ketogluconate. Alkaline phosphatase, leucine arylamidase and acid phosphatase 189 are positive. The major fatty acids were C16:0 (42%), C18:1n9 (25%) and C18:2n6 (19%). The 190 genome size of A. vaginimassiliensis strain Marseille-P4512 is 1.83 Mbp with 33.1 mol% 191 G+C content. The 16S rRNA and draft genome sequences of strain Marseille-P4512, are 192 available in Genbank database under accession numbers LT934505 and UZAS00000000, 193 respectively. The type strain is Marseille-P4512T, which was isolated from the vagina of a 194 woman with bacterial vaginosis. 195 vaginimassiliensis sp. nov. (va.gi.ni.mas.si.li.en’sis N.L. fem. adj. xylitol, gentiobiose and 196 References 197 1. Mitra A, MacIntyre DA, Lee YS, et al. Cervical intraepithelial neoplasia disease 198 progression is associated with increased vaginal microbiome diversity. Sci Rep. 2015; 199 5:16865. doi:10.1038/srep16865 200 2. Mitra A, MacIntyre DA, Marchesi JR, Lee YS, Bennett PR, Kyrgiou M. 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Towards a taxonomic coherence between average 302 nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of 303 prokaryotes [published correction appears in Int J Syst Evol Microbiol. 2014 304 May;64(Pt 5):1825]. Int J Syst Evol Microbiol. 2014; 64(Pt 2):346–351. 305 doi:10.1099/ijs.0.059774-0 306 307 308 309 Table 1: Different characteristics of 1, Megasphaera vaginalis sp. nov., strain MarseilleP4857; 2, Megasphaera micronuciformis strain AIP 412.00 [29]; 3, Megasphaera massiliensis strain NP3 [12]; 4, Megasphaera paucivorans strain DSM 16981 [15]. Properties Cell diameter (μm) Oxygen requirement Gram stain Motility Endospore formation α-glucosidase Catalase Oxidase Glycerol Erythritol D-arabinose L-arabinose D-ribose D-xylose D-galactose D-glucose D-fructose L-rhamnose Dulcitol Inositol D-mannitol D-sorbitol N-acetylglucosamine Esculin ferric citrate Salicin D-cellobiose D-maltose D-lactose D-melibiose D-trehalose D-melezitose D-raffinose Glycogen 310 311 1 0.6 to 0.9 + + + + + + + + + 2 0.4 to 0.6 NA NA - 3 0.8 + + w NA w + + + + + + + NA NA + + 4 1.2 to 1.5 NA NA NA + - + - + + + + + + + + + NA + NA + NA + + NA NA Human vaginal Human Human Spoiled Source swab stool stool beer +, positive reaction; -, reaction; NA, Not Available data; w, weak reaction. 312 313 314 Table 2 : Different characteristics of 1, Anaerococcus vaginimassiliensis strain MarseilleP4512; 2, Anaerococcus tetradius strain JCM 1964T [16]; 3, Anaerococcus prevotii strain ATCC 9321T [16]. 315 1 2 3 Properties 0.8 to 1.3 0.8 to 1.8 0.7 to 1.8 Cell diameter (μm) Oxygen requirement + + + Gram stain Motility + d Alkaline phosphatase + d d Leucine arylamidase + NA NA Acid phosphatase + α-galactosidase d β-galactosidase + d β-glucuronidase d d α-glucosidase + + β-glucosidase + d d Catalase NA NA Oxidase + NA Glycerol + D-ribose + Xylose + + d D-glucose + + d D-fructose + d d D-maltose + D-lactose Vaginal swab Vaginal discharges Vaginal discharges Source +, positive reaction; -, reaction; NA, Not Available data; d, strain-dependent. 316 317 Table 3: Genome comparison of closely related species to M. vaginalis strain Marseille-P4857T and A. vaginimassiliensis strain Marseille-P4512T Protein rRNA tRNA 2.21 3.24 2.91 1.77 2.50 2.65 2.74 2.88 G+C mol% 50.2 44.8 40.2 45.4 52.8 52.6 50.2 49.0 2 032 2 933 2 598 1 665 2 211 2 397 2 388 2 636 7 17 14 21 18 3 18 49 55 51 48 65 57 56 53 1.84 1.89 2.08 2.15 1.70 2.13 1.80 2.27 33.1 29.0 34.6 34.4 33.0 35.4 28.6 33.7 1 722 1 693 1 936 1 895 1 563 1 953 1 625 2 004 13 2 9 5 3 14 3 9 48 46 44 45 44 49 47 48 Species Size (Mb) Megasphaera vaginalis Megasphaera cerevisiae Megasphaera paucivorans Megasphaera micronuciformis Megasphaera elsdenii Megasphaera stantonii Megasphaera massiliensis Megasphaera hexanoica Anaerococcus vaginimassiliensis Anaerococcus vaginalis Anaerococcus mediterraneensis Anaerococcus tetradius Anaerococcus prevotii Anaerococcus marasmi Anaerococcus senegalensis Anaerococcus provencensis Other RNA 4 4 4 Gene Pseudogene 4 4 4 1 2 137 3 163 2 780 1 746 2 378 2 509 2 562 2 750 45 154 113 29 75 33 111 42 3 4 4 4 3 4 4 3 1 826 1 793 2 045 2 010 1 658 2 082 1 756 2 146 40 48 52 61 45 62 77 82 318 319 320 Table 4: Genomic comparison of Megasphaera vaginalis strain Marseille-P4857 and Anaerococcus vaginimassiliensis strain Marseille-P4512 between their closely related species using GGDC and formula 2 (dDDH estimates based on identities over HSP length). MEL MMI MCE MST MHE MPA MMA MVA AVG APR AVA ATE APA ASE AME APV 321 322 323 324 325 326 327 328 329 330 331 332 MEL 100 26.0 19.2 21.7 23.7 19.7 24.3 20.2 AVG 100 % Similarity of Megasphaera species MMI MCE MST MHE MPA 100 21.7 100 27.0 18.6 100 24.8 19.5 21.0 100 21.3 22.2 20.1 20.1 100 20.0 18.2 20.5 20.2 17.7 19.3 18.8 19.4 19.2 17.9 % Similarity of Anaerococcus species APR 21.4 100 AVA 27.1 21.6 100 ATE 21.9 21.5 28.8 100 APA 25.9 20.5 36.0 24.6 100 ASE 21.7 21.8 29.4 32.3 25.7 100 MMA MVA 100 20.4 100 AME 23.4 20.2 33.6 21.2 25.5 24.3 100 APV 22.6 20.5 26.2 22.2 21.7 21.6 23.9 100 MEL, Megasphaera elsdenii 14-14 (NZ_CP009240.1); MMI, Megasphaera micronuciformis F0359 (NZ_AECS00000000.1); MCE, Megasphaera cerevisiae DSM 20462 (FUXD01000000); MST, Megasphaera stantonii DSM 106750 (NZ_CP029462.1); MHE, Megasphaera hexanoica MH (CP011940.1); MPA, Megasphaera paucivorans DSM 16981 (NZ_FNHQ00000000.1); MMA, Megasphaera massiliensis NP3 (CAVO000000000.1) and MVA, Megasphaera vaginalis MarseilleP4857 (NZ_OEQB00000000.1). AVG, Anaerococcus vaginimassiliensis Marseille-P4512 (UZAS00000000); APR, Anaerococcus prevotii ACS-065-V-Col13 (NC_013171.1); AVA, Anaerococcus vaginalis ATCC 51170 (NZ_CAGU00000000.1); ATE, Anaerococcus tetradius ATCC 35098 (ACGC00000000.1); APA, Anaerococcus pacaensis 9403502 (CAJJ000000000.2); ASE, Anaerococcus senegalensis JC48 (NZ_CAEK00000000.1); AME, Anaerococcus mediterraneensis Marseille-P2765 (NZ_LT635772.1); APV, Anaerococcus provencensis 9402080 (NZ_CAJU000000000.2). 94 100 Megasphaera indica strain NMBHI-10 (NR 134080.1) Megasphaera elsdenii DSM 20460 (NR 102980.1) Megasphaera massiliensis strain NP3 (NR 133027.1) Megasphaera hexanoica strain MH (NR 157635.1) Megasphaera paucivorans strain VTT E-032341 (NR 043657.1) 100 Megasphaera sueciensis strain VTT E-97791 (NR 043656.1) (A) Megasphaera cerevisiae DSM 20462 (NR 113307.1) 100 Megasphaera vaginalis strain Marseille-P4857 (LT960586.1) Megasphaera micronuciformis strain AIP 412.00 (NR 025230.1) Negativicoccus massiliensis strain Marseille-P2082 (NR 144729.1) Dialister micraerophilus strain JCM 17567 (NR 113356.1) Dialister invisus DSM 15470 strain E7.25 (NR 025680.1) 91 73 Allisonella histaminiformans strain MR2 (NR 028862.1) 0.02 Anaerococcus prevotii DSM 20548 strain CCUG 41932 (NR 041939.1) 76 Anaerococcus tetradius strain CCUG 46590 (NR 041941.1) Anaerococcus vaginimassiliensis strain Marseille-P4512 (LT934505.1) 100 Anaerococcus vaginalis ATCC 51170 strain JCM 8138 (NR 114314.1) Anaerococcus mediterraneensis strain Marseille-P2765 (NR 147392.1) (B) Peptoniphilus asaccharolyticus strain JCM 1765 (NR 113382.1) 91 Peptoniphilus methioninivorax strain NRRL B-23883 (NR 117515.1) Peptoniphilus lacrimalis strain GIFU 7667 (NR 115505.1) Peptoniphilus timonensis strain JC401 (NR 118307.1) 96 100 333 Peptoniphilus tyrrelliae strain RMA 19911 (NR 117555.1) 0.02 334 Figure 1: Phylogenetic trees displaying the position of Megasphaera vaginalis strain 335 Marseille-P4857T (A) and Anaerococcus vaginimassiliensis strain Marseille-P4512T (B) 336 relative to their closest phylogenetically species. The respective GenBank accession numbers 337 for 16S rRNA genes are indicated in parenthesis. Sequence alignment and phylogenetic 338 inferences were obtained using the maximum likelihood method within MEGA 7 software. 339 The numbers at the nodes are percentages of bootstrap values obtained by repeating the 340 analysis 1000 times to generate a majority consensus tree. 341 342 343 Figure 2: Scanning electron micrograph of Megasphaera vaginalis strain Marseille-P4857T 344 and Anaerococcus vaginimassiliensis strain Marseille-P4512T using the Scanning Electron 345 Microscope TM4000 from Hitachi. Scale bar and acquisition settings are presented on the 346 pictures. 347 348 349 Figure 3: Heatmap generated with OrthoANI values calculated using the OAT software for 350 Megasphaera vaginalis sp. nov., strain Marseille-P4857 (A) and Anaerococcus 351 vaginimassiliensis strain Marseille-P4512 (B) with their respective closely related species 352 with standing in nomenclature.