Anaerococcus ihuae sp. nov. and Mediannikoviicoccus vaginalis gen. nov., sp. nov., two new bacteria isolated from human vaginal samples

Strains Marseille-Q5893 (= CSUR Q5893 = CECT 30496) and Marseille-Q5883 (= CSUR Q5883 = CECT 30497) were isolated from vaginal samples using the culturomics approach. The 16S rRNA gene sequences of each strain were sequenced and then compared by BLASTn to the NCBI database. Strains Marseille-Q5893 and Marseille-Q5883 were most closely related to Anaerococcus obesiensis and Finegoldia magna, with identities of 98.5% and 90.0%, respectively. Strain Marseille-Q5893 is strictly anaerobic, while strain Marseille-Q5883 is facultative anaerobic. Both strains are Gram-positive, coccus-shaped, oxidase- and catalase-negative. The most abundant fatty acid for both strains is hexadecanoic acid, followed by 9-octadecenoic acid and tetradecanoic acid. Strain Marseille-Q5893 has a genome size of 1,831,271 bp with a G+C content of 29.4 mol%, whereas strain Marseille-Q5883 has a genome of 1,997,945 bp with a 33.6 mol% G+C content. The genomic comparison of closely related species with strains Marseille-Q5893 and Marseille-Q5883 showed that all digital DNA–DNA hybridization (dDDH) and orthologous average nucleotide identity (OrthoANI) values were lower than the published species thresholds (70% and 95–96%, respectively). Based on these data, we conclude that strain Marseille-Q5893 belongs to a new species in the family Peptoniphilaceae and strain Marseille-Q5883 belongs to a new genus in the family Peptostreptococcaceae. For these two new bacterial species, the names Anaerococcus ihuae sp. nov. and Mediannikoviicoccus vaginalis gen. nov., sp. nov., were proposed. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-022-03082-7.


Introduction
The vaginal ecosystem is defined as all the microorganisms present in the vagina. It is characterized by the predominance of Lactobacillus spp., which represents approximately 90-95% of vaginal bacteria (Ravel et al. 2011;Abou Chacra and Fenollar 2021). Bacterial vaginosis represents a unique disturbance of this complex vaginal ecosystem, with the disappearance of lactobacilli and the proliferation of anaerobic bacteria, probably due to hormonal, behavioral, or environmental factors (Abou Chacra et al. 2022). This can have psychological (Kenyon et al. 2013), gynecological (Norenhag et al. 2020;Soper 2020) and obstetrical (Fox and Eichelberger 2015; Beckers and Sones 2020) consequences, in particular prematurity (Fettweis et al. 2019;Bayar et al. 2020). Indeed, several studies have shown that an imbalance of the vaginal flora leads to a proliferation of pathogenic microorganisms from the cervix to the choriodecidual space that Communicated by Erko Stackebrandt.
Claudia Ly and Linda Abou Chacra are contributed equally. leads to inflammation, with fragility of the fetal membranes and shortening of the cervix (Côté and Pasquier 2018).
Using the "culturomics" approach, a culture technique consisting in incubating samples under different culture conditions and subsequently identifying the isolated species by matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Lagier and Raoult 2016), we isolated a new member of the genus Anaerococcus that did not correspond to the other species of this genus and a new genus Mediannikoviicoccus from vaginal samples. These strains are referenced as Marseille-Q5893 and Marseille-Q5883, respectively.
Herein, we reported the description of these two strains in pure culture, Marseille-Q5893 and Marseille-Q5883, according to the new polyphasic approach named taxonogenomic, which combines annotated whole genome and proteomic information obtained from MALDI-TOF MS spectra and phenotypic characteristics.

Ethical approval and isolation of strains
Strain Marseille-Q5893 was isolated from a vaginal sample of a 30-year-old non-pregnant woman, whereas strain Marseille-Q5883 was isolated from a vaginal sample from a 23-year-old pregnant woman. These two women had no bacterial vaginosis or sexually transmitted infection at the time of the consultation. The study was approved by the local ethics committee of the Institut Hospitalo-Universitaire Méditerranée Infection (Marseille, France) under agreement number 2021-016. The patients provided signed informed consent.
The culture of strains Marseille-Q5893 and Marseille-Q5883 was achieved after pre-incubation of the vaginal samples at 37 °C in anaerobic blood culture vials (Becton Dickinson, Le Pont-de-Claix, France) supplemented with 40 mL of Difco Marine Broth (Becton Dickinson) for 7 days and 21 days, respectively. Then, isolated colonies were obtained by subculture on 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l'Etoile, France) at 37 °C in an anaerobic atmosphere using AnaeroGen (bioMérieux) after 48 h.

Identification of strains and phylogenetic analysis
Identification of strains Marseille-Q5893 and Marseille-Q5883 was carried out using a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) (Seng et al. 2013). The spectra of the two strains were imported into the MALDI Biotyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings). Interpretation of the scores was performed as previously described (Hadjadj et al. 2016).
The 16S rRNA gene of strains Marseille-Q5893 and Marseille-Q5883 was sequenced using the fD1 and rP2 universal primers (Eurogentec, Angers, France) as previously described (Drancourt et al. 2000), using an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Thermo Fisher, Saint-Aubin, France). If the 16S rRNA sequence similarity with closely related species was between 95 and 98.65%, the strain was suggested as belonging to a new species (Stackebrandt and Goebel 1994;Kim et al. 2014). If the similarity was below 95%, the strain would be considered a new genus (Tindall et al. 2010;Rossi-Tamisier et al. 2015).
Using the MEGA-X (Kumar et al. 2018), the resulting 16S rRNA gene sequences were aligned and a phylogenetic tree was obtained with 1000 bootstrap replicates, based on the maximum likelihood (ML) and the Kimura 2-parameter methods (Kimura 1980).

Morphological and phenotypic characterization
The morphology of the both species was observed via a SU5000 scanning electron microscope (SEM; Hitachi High-Technologies, Tokyo, Japan) as previously described (Zgheib et al. 2021). The phenotypic characteristics of both strains such as Gram staining, motility, oxidase, and catalase activities were determined after incubation on 5% sheep blood-enriched Columbia agar (bioMérieux) at 37 °C in anaerobic atmosphere for 48 h. Gram staining of cells was carried out using a Color Gram 2 kit (bioMérieux). Catalase activity was determined by observing bubble production after the application of 3% (v/v) hydrogen peroxide solution. Oxidase activity was evaluated via the oxidation of 1% (w/v) p amino-dimethylaniline oxalate. The spore formation test was also performed on strains after a shock for 20 min at 80 °C.
The biochemical properties were evaluated using rapid API ZYM, API 20A, and API 50CH strips (bioMérieux) according to the manufacturer's instructions. Antibiotic susceptibility was determined using E-test gradient strips (bioMérieux) according to the EUCAST recommendations (Matuschek et al. 2014). Finally, cellular fatty acid methyl ester (FAME) analysis was performed by chromatography/ mass spectrometry GC/MS as previously reported (Sasser 2006;Dione et al. 2016).
The sequencing of strains Marseille-Q5893 and Marseille-Q5883 was performed using a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) via the Nextera Mate Pair sample prep kit and Nextera XT Paired End (Illumina) as previously reported (Anani et al. 2019). SPAdes 3.13.1 software was used with default parameters to assemble the reads (Bankevich et al. 2012). Scaffolds with a nucleotide number < 800 bp and scaffolds with a depth value < 25% of the mean depths were removed. The resulting genome for each strain and the genomes of closely related species were annotated with Prokka 1.14.5 as previously described (Seemann 2014; Zgheib et al. 2020). The genome as well as 16S rRNA sequences extracted from the genomes were compared to closely related species (Table 2).
Furthermore, the CRISPRCasFinder program was used to easily detect clustered regularly interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes in both strains (Grissa et al. 2007). The PathogenFinder 1.1 program was also applied to identify gene families that correlate with pathogenicity (Cosentino et al. 2013).

Strain identification and phylogenetic analysis
Strains Marseille-Q5893 and Marseille-Q5883 could not be identified by our MALDI-TOF MS instrument, as the score was lower than 1.8, suggesting that the corresponding species was not referenced in the database and could be an unknown species (Fig. S1). Additionally, strain Marseille-Q5893 revealed a 98.5% 16S rRNA sequence similarity with Anaerococcus obesiensis strain FDAARGOS_989 (CP067016.1), the phylogenetically closest species with standing in the nomenclature. As this value was below the 98.65% threshold for defining a new bacterial species, strain Marseille-Q5893 was considered as a representative of a putatively new species within the family Peptoniphilaceae in the phylum Firmicutes. Similarly, strain Marseille-Q5883 revealed a 90.0% 16S rRNA sequence similarity with Finegoldia magna strain FDAARGOS_1556 (CP085957.1), the phylogenetically closest species with standing in the nomenclature. As this value was below the 95% threshold for defining a new bacterial genus, strain Marseille-Q5883 was considered as a representative of putatively new genus within the family Peptostreptococcaceae in the phylum Firmicutes. The phylogenetic trees highlighting the position of each of the two strains relative to other closely related species with a validly published name are shown in Fig. 1.

Phenotypic characterization
The optimal growth of strains Marseille-Q5893 and Marseille-Q5883 was obtained after 2 days of culture at 37 °C under anaerobic conditions (anaeroGEN, Oxoid Ltd, Dardilly, France). In these culture conditions, strains Marseille-Q5893 formed circular, white, and opaque colonies with a diameter of 2-2.5 mm. Colonies from strain Marseille-Q5883 appear circular, white, and translucent, with a diameter of 1.5-2 mm. With the strain Marseille-Q5883, growth occurs also under a micro-aerophilic atmosphere but not for strain Marseille-Q5893.
The characteristics of strain Marseille-Q5893 and strain Marseille-Q5883 are summarized in supplementary  Table S1.

Production of
Acid phosphatase also had 57 RNA-coding genes, including 12 rRNA, 44 tRNA, and 1 tmRNA. In addition, the genomic characteristics of these new strains are statistically compared with other related species (Table 2). The most elevated value of dDDH for strain Marseille-Q5893 against the accessible genomes for type strains of species, with standing in the nomenclature, is 53% with Anaerococcus rubeinfantis (Table 3). For strain Marseille-Q5883, the most elevated value of dDDH is 54.9% with Peptoniphilus obesi (Table 3). These estimations are less than 70% of the cutoff utilized for delineating prokaryotic species, consequently affirming that these strains represent two new species, distinct from these other bacterial strains.
OrthoANI values of strain Marseille-Q5893 ranged between 65.9 and 93.4%, confirming that this strain is different from the other tested bacterial strains (Table 3). In the aggregate, the above information supports strain Marseille-Q5893 as representing a novel species in the family Peptoniphilaceae, for which the name Anaerococcus ihuae sp. nov., is proposed. For strain Marseille-Q5883, OrthoANI values ranged from 63.49 to 72.73%, affirming again that this strain is different from the other tested bacterial strains (Table 3). Overall, the above information  also supports that strain Marseille-Q5883 represents a new genus in the family Peptostreptococcaceae, for which the name Mediannikoviicoccus vaginalis gen. nov. is proposed.
Using the CRISPRCasFinder program, only the Marseille-Q5883 strain had a genomic structure corresponding to a CRISPR with a very high level of evidence. Indeed, a conserved region consisted of a 28-bp length sequence "GTT GTT CCT GCA TGC AGG GGT GAT CC" repeated 20 times in a 1,189-bp fragment and separated by 19 unique sequence of similar size (spacers) was detected. Moreover, the cas genes cluster (Type IE) was also evidenced.
Using the PathogenFinder 1.1 program, the strain Marseille-Q5893 was predicted as a human pathogen with a 0.7 probability, matched to 6 pathogenic protein families (4 hypothetical conserved protein and 1 putative transposon integrase from Finegoldia magna as well as 1 hypothetical conserved protein from Streptococcus pyogenes). Likewise, the strain Marseille-Q5883 was predicted as a human pathogen with a 0.8 probability, matched to 15 pathogenic protein families (1 putative peptidase, 1 putative chimeric erythrocyte-binding protein, 1 DNA topoisomerase III, 1 putative transposon integrase, 1 ABC transporter permease protein, 4 conserved hypothetical protein from Finegoldia magna as well as 1 Superfamily II DNA and RNA helicase, 1 site-specific recombinase and 3 conserved hypothetical protein from Streptococcus pyogenes, and 1 conserved hypothetical protein Streptococcus pneumoniae Taiwan19F-14).

Conclusion
The 16S rRNA gene sequence identities between the two strains Marseille-Q5893 and Marseille-Q5883 and closely related species were less than 98.65% and 95%, respectively, the threshold delimiting a new bacterial species and a new bacterial genus, respectively. The dDDH values between the compared genomes were all below the recommended threshold of 70%. Therefore, according to phenotypic, phylogenetic, and genomic analyses, we state that the 2 new strains are new members belonging to Firmicutes group for which Anaerococcus ihuae sp. nov. and Mediannikoviicoccus vaginalis gen. nov. are proposed as their names.
Cells are strictly anaerobic, Gram-positive, non-sporeforming, non-motile, and coccus-shaped. Bacterial cells are nearly 0.75 μm in diameter and disposed in clusters. Catalase and oxidase activities are negative. After 48 h incubation on Columbia agar supplemented with 5% sheep blood, colonies appear circular, white, and opaque with a diameter of 2-2.5 mm. Growth occurs only under an anaerobic atmosphere in a temperature range of 28-42 °C (optimum 37 °C), at pH 6-7.5 (optimum pH 7), and with 0-20% (w/v) NaCl (optimum 15-20%). Using API ZYM strips, only leucine arylamidase and acid phosphatase enzyme activities are positive. Using API 50 CH strips, acid is not produced from glucose or other sugars. All remaining reactions were still negative with API 20E strips. The major fatty acids are hexadecanoic acid (61%), 9-octadecenoic acid (20%), and tetradecanoic acid (6%). The size of genome is 1.09 Mbp and its G+C content is 29.4 mol%.
The type strain Marseille-Q5893 T (= CSUR Q5893 = CECT 30,496) was isolated from a vaginal sample of a 30-year-old healthy woman at day 16 of the menstrual cycle.
The 16S rRNA and genome sequences are deposited in GenBank under accession numbers OM728648 and CAK-MRU010000001, respectively.
Cells are facultative anaerobic, Gram-positive, nonspore-forming, non-motile, and coccus-shaped. Catalase and oxidase activities are negative. The major FAMEs (> 8%) are hexadecenoic, 9-octadecenoic acid and tetradecanoic acid. The genome size is 1.95 Mbp and its G+C content is 33.6 mol%. This genus is a member of the family Peptoniphilaceae with Mediannikoviicoccus vaginalis as the type species.
Cells are facultative anaerobic, Gram-stain-positive, nonspore-forming, non-motile, and coccus-shaped. Bacterial cells are nearly 0.63 μm in diameter and occur in pairs or short chains. Catalase and oxidase activities are negative. After 48 h incubation on Columbia agar supplemented with 5% sheep blood, colonies are circular, white, and translucent with a diameter of 1.5-2 mm. Growth occurs under an anaerobic and micro-aerophilic atmosphere in a temperature range of 28-42 °C (optimum 37 °C), at pH 6-7.5 (optimum pH 7) and with 0-20% (w/v) NaCl (optimum 15-20%).
Using API ZYM strips, positive results were obtained for leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Using API 50CH strips, acid is not produced from glucose or other sugars. The most abundant fatty acid by far was hexadecanoic acid (62%), followed 9-octadecenoic acid (15%), and tetradecanoic acid (8%). The genome size of strain Marseille-Q5883 is 1.95 Mbp and its G+C content is 33.6 mol%.
The type strain Marseille-Q5883 T (= CSUR Q5883 = DSM 30497) was isolated from a vaginal sample of a 23-year-old pregnant woman with threatened premature delivery with no identified etiology.
The 16S rRNA and genome sequences are deposited in GenBank under accession numbers OM728652 and CAK-MRI010000001, respectively.