Peptoniphilus genitalis sp. nov. and Mobiluncus massiliensis sp. nov.: Novel Bacteria Isolated from the Vaginal Microbiome

The strains Marseille-Q7072T (= CSUR Q7072T = CECT 30604 T) and Marseille-Q7826T (= CSUR Q7826T = CECT 30727 T) were isolated from vaginal samples. As MALDI-TOF mass spectrometry failed to identify them, their genomes were directly sequenced to determine their taxogenomic identities. Both strains are anaerobic without any oxidase and catalase activity. C16:0 is the most abundant fatty acid for both strains. Strain Marseille-Q7072T is non-spore-forming, non-motile, Gram-stain-positive, and coccus-shaped, while strain Marseille-Q7826T is non-spore-forming, motile, Gram-stain-variable, and curved rod-shaped. The genomic comparison of the Marseille-Q7072T and Marseille-Q7826T strains showed that all digital DNA–DNA hybridisation (dDDH) and mean orthologous nucleotide identity (OrthoANI) values were below published species thresholds (70% and 95–96%, respectively) with other closely related species with standing in nomenclature. Thus, we conclude that both strains are new bacterial species. Strain Marseille-Q7072T is a new member of the Bacillota phylum, for which the name Peptoniphilus genitalis sp. nov. is proposed, while the Marseille-Q7826T strain is a new member of the Actinomycetota phylum, for which the name Mobiluncus massiliensis sp. nov. is proposed. Supplementary Information The online version contains supplementary material available at 10.1007/s00284-023-03584-7.


Introduction
The vaginal microbiota is a complex ecosystem of microorganisms that inhabit the vagina and play a crucial role in women's genital health [1].This microbiota is dominated by lactobacilli, which are responsible for the production of lactic acid, thus maintaining an acidic vaginal pH that prevents colonisation by potentially harmful pathogens [1].An imbalance in the composition of the vaginal microbiota, known as bacterial vaginosis, is associated with an increased risk of sexually transmitted infections, obstetric complications, and pelvic inflammatory diseases [2].
Despite the importance of the vaginal microbiota for women's health, our understanding of its diversity and composition remains limited due to the difficulties in culturing and characterising the bacteria that make it up [3].Next-generation sequencing methods have led to significant advances in the study of the vaginal microbiota [4], but these approaches do not always allow for a complete characterisation of the microorganisms present.
In this context, culturomics-based approaches have emerged as a powerful complementary method for analysing the vaginal microbiota, allowing for a more in-depth characterisation of the cultivable microorganisms that compose it [5].As part of a complete characterisation of the vaginal microbiota by culturomics in women who gave birth prematurely and those who had repeated miscarriages, we isolated two new members of the genus Peptoniphilus and Mobiluncus from vaginal samples that did not match other species of their genus.These strains were designated Marseille-Q7072 T and Marseille-Q7826 T , respectively.
In accordance with the polyphasic taxonogenomic strategy combining annotated whole genome, proteomic information extracted from MALDI-TOF mass spectrometry spectra, and phenotypic aspects, we present here the description of these two strains.

Ethical Considerations and Sampling
Two vaginal specimens sampled during patient care and sent for diagnostic purposes to the clinical microbiology laboratory of our university hospital (AP-HM, located at the Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France) were retrospectively cultivated using a culturomics method as allowed by French law (Article L.1211-2 of the Public Health Code).Patients had been informed of the possible reuse for research purposes of their samples and their personal data collected during care.They had the possibility of opposing this by reporting it to the Data Protection Officer of the AP-HM.Neither patient had expressed any objection.The patient personal data used for analysis were anonymised.Our independent local ethics committee approved (Agreement Nos.2022-005 and 2022-009) the clearance of Ethics Review Committee and compliance with data protection legislation.
Strain Marseille-Q7072 T was isolated from a vaginal sample taken from a 41-year-old woman who had suffered from recurrent miscarriages.Strain Marseille-Q7826 T was isolated from a vaginal sample taken from a 30-year-old pregnant woman who was at 26 weeks of amenorrhoea.Neither patient had bacterial vaginosis or a sexually transmitted infection at the time of the sampling.

Strain Isolation
Two different strains, Marseille-Q7072 T and Marseille-Q7826 T , were cultured from vaginal samples using different pre-incubation methods.For Marseille-Q7072 T , a vaginal sample was pre-incubated at 37 °C in anaerobic blood culture vials (Becton Dickinson, Le Pont-de-Claix, France) supplemented with 40 ml of Tryptic Soy Broth (Sigma-Aldrich, St. Louis, USA) for seven days.For Marseille-Q7826 T , a vaginal sample was pre-incubated at 37 °C in anaerobic blood culture vials (Becton Dickinson) supplemented with 40 ml of Difco Marine Broth (Becton Dickinson) for 30 days.Subsequently, isolated colonies were subcultured on Columbia agar with 5% sheep blood (bioMérieux, Marcy l'Etoile, France) at 37 °C under anaerobic conditions using AnaeroGen (bioMérieux) after 48 h.

MALDI-TOF Identification
A Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) [6] was used to identify Marseille-Q7072 T and Marseille-Q7826 T .The MALDI BioTyper software (version 2.0, Bruker) was used to import and analyse the spectra of the two strains.Standard pattern matching was employed with default parameter settings, and the resulting scores were interpreted as described previously [7].
The phenotypic characteristics of the two strains including Gram staining, motility, sporulation, oxidase, and catalase activities were tested, as preliminarily reported by Ly and colleagues [8].In addition, their morphology was observed using a SU5000 scanning electron microscope (SEM, Hitachi High-Tech, Tokyo, Japan), as similarly described by Zgheib et al. [9].

Genome Extraction, Sequencing, Annotation, and Comparison
Mechanical lysis was performed on the two strains with acid-washed glass beads (G4649-500 g, Sigma-Aldrich) by a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5 m/s) for 90 s, followed by a two-hour lysozyme incubation at 37 °C.DNA extraction was then performed using the EZ1 instrument and the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany).
The DNA extract of both strains was sequenced using a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) via the Nextera Mate Pair sample preparation kit and the Nextera XT Paired End (Illumina), as previously mentioned [13].SPAdes 3.13.1 software was used at default settings to assemble the reads [14].Scaffolds with nucleotide counts < 800 bp and scaffolds with depth values < 25% of the average depths were suppressed.The resulting genome for each strain and the genomes of the closely related species were annotated with Prokka 1.14.5, as described previously [15,16], and compared.The 16S rRNA sequences of the two strains were extracted from their respective genomes and then blasted using BLASTn NCBI.The Clusters of Orthologous Groups (COG) database was used for bacterial protein sequences detection using BLASTP [17].
Furthermore, a phylogenetic tree was constructed from seven concatenated genes, namely dnaA, gyrA, gyrB, recA, rpoB, rplE, and rplD.This tree was built using the Iqtree software (v1.6.12)[22] and employed the maximum-likelihood method for phylogenetic inference.Subsequently, the sequences were aligned using MAFFT [23].Bootstrap values, derived from 1000 iterations of the analysis to create a majority consensus tree, are indicated at nodes [24].Only bootstrap values exceeding 70% are presented.Genomic sequences from various species under investigation were obtained from the NCBI database.These sequences were then annotated using the Prokka software [15].The target genes were extracted from the genomes and concatenated using the Emboss union server (https:// www.bioin forma tics.nl/ cgi-bin/ emboss/ union).

Strain Identification and Phylogenetic Analysis
Strains Marseille-Q7072 T and Marseille-Q7826 T were isolated from human vaginal samples.Systematic analysis using MALDI-TOF mass spectrometry failed to identify the two strains (Fig. 1).For both strains, the scores were lower than 1.8, indicating that the corresponding species were not found in the database and that they could potentially be an unknown species.
Based on these results, strains Marseille-Q7072 T and Marseille-Q7826 T were considered to be representative of two putative new species within the family Peptoniphilaceae in the phylum Bacillota and within the family Actinomycetaceae in the phylum Actinomycetota, respectively.

Phenotypic Characterisation
Optimal growth of Marseille-Q7072 T and Marseille-Q7826 T strains was obtained after two days of culture at 37 °C under anaerobic conditions.The main characteristics of the two strains are presented in Supplementary Table 1.
Strain Marseille-Q7072 T is an anaerobic and microaerophilic, Gram-stain-positive, non-spore-forming, non-motile, and coccus-shaped bacterium.Growth occurs under an anaerobic atmosphere within a temperature range of between 20 and 37 °C (optimum 37 °C), at pH 6-8.5 (optimum pH 7), and with 0-5% (w/v) NaCl (best < 5%), but also under microaerophilic conditions.Under optimal growing conditions, colonies appear circular, grey, opaque, and convex with a diameter of 2-3 mm.Catalase and oxidase activities are negative.As observed using electron microscopy, bacterial cells have a diameter of 0.609 μm ± 0.087 μm and are arranged in clusters (Fig. 2a).
Strain Marseille-Q7826 T is a facultative anaerobic, Gram-stain-variable, non-spore-forming, motile, and curved rod-shaped bacterium.Growth occurs under an anaerobic atmosphere in a temperature range of between 20 and 37 °C (optimum 37 °C), at pH 6-7.5 (optimum pH 7) and without adding NaCl.Under optimal growing conditions, colonies appear circular, colourless, translucent, and convex, with a diameter of 2-3 mm.Catalase and oxidase activities are negative.In electron microscopy, bacterial cells have a length of 1.216 μm ± 0.243 μm and a diameter of 0.381 μm ± 0.039 μm (Fig. 2b).

Genomic Analysis
For strain Marseille-Q7072 T , the number of reads was 879 694.The genome length was 2 040 803 bp, assembled into 33 contigs, with a G + C content of 34.3 mol% (Fig. 3a).Strain Marseille-Q7072 T had 1947 predicted genes, including 1911 protein-coding genes.Strain Marseille-Q7072 T also had 36 RNA-coding genes, including four rRNA, 31 tRNA, and one tmRNA.For strain Marseille-Q7826 T , the number of reads was 1 732 034.The genome length was 2 216 029 bp, assembled into one contig, with a G + C content of 57 mol% (Fig. 3b).Strain Marseille-Q7826 T had 1908 predicted genes, including 1855 protein-coding genes.Strain Marseille-Q7826 T also had 53 RNA-coding genes, including six rRNA, 46 tRNA, and one tmRNA.
The distribution of functional classes of predicted genes for strains Marseille-Q7072 T and Marseille-Q7826 T strains according to the COGs database is also presented in Table 3. Figure 4 shows two phylogenetic trees based on concatened sequences of 7 housekeeping genes, highlighting the position of each strain in relation to other closely related species.Strain Marseille-Q7072 T is most closely related to "Peptoniphilus septimus" SAHP1, a bacterium recently reported in a case of blood mono-infection in a cervical cancer patient receiving chemotherapy, not well described, not available in a strain collection and not officially recognized by nomenclature [29] and P. harei FDAARGOS_1136 within the genus Peptoniphilus.
Strain Marseille-Q7826 T is most closely related to M. curtisii ATCC 35241 T and M. holmesii ATCC 35242 T within the genus Mobiluncus.
Furthermore, the genomic features of these two new strains are statistically compared with other related species (Table 4).For strain Marseille-Q7072 T , if we include in the analysis exclusively bacterial species currently validated by the nomenclature, the highest dDDH value is 46.9% with P. coxii (Table 5), the OrthoANI values ranged from 69 to 88% (Supplementary Fig. 1) and the highest AAI value is 88% with P. harei (Supplementary Table 2).These estimates are therefore below the thresholds required to distinguish prokaryotic species (dDDH < 70% and AAI/ ANI < 95 ~ 96%), indicating that strain Marseille-Q7072 T is a new bacterial species.However, if we include in the analysis all sequences available in current databases, the highest dDDH value is 69.3% with "P.septimus" (Table 5) and the highest AAI value is 95.94% with "P.septimus" (Supplementary Table 2).For strain Marseille-Q7826 T , the highest dDDH value is 29.5% with M. curtisii (Table 5), the OrthoANI values ranged from 71 to 86% (Supplementary Fig. 2) and the highest AAI value is 89.24% with M. holmesii (Supplementary Table 2), confirming that this strain is different from other bacterial strains.It should also be noted that the Marseille-Q7072 T strain showed 16S rRNA sequence similarities of 99.67% with the "P.septimus" SAHP1 strain (CP097885.1)and 99% with the P. harei NCTN13077 strain (LR134524.1)(Fig. 5).The Marseille-Q7826 T strain showed 99.54% 16S rRNA sequence similarity with M. curtisii ATCC 35241 T (GL385912.1),the closest species phylogenetically (Fig. 5).These identity values were therefore above the threshold of 98.65% for the definition of a new bacterial species [21,30].However, it has now been clearly demonstrated that the thresholds, originally designed to standardise the use of 16S rRNA gene sequences in taxonomy, do not apply to several genera [31].Taken as a whole, the above information supports the proposals that strain Marseille-Q7826 T represents a new species in the family Actinomycetaceae, for which the name Mobiluncus massiliensis sp.nov. is proposed.Strain Marseille-Q7072 T represents a species in the family Peptoniphilaceae.Genomic analyses indicate that it corresponds to "P. septimus" SAHP1, a strain that has not been accurately described, is not available in any strain collection and has therefore not been officially validated within ICNP.Thus, the proposed name for this species is Peptoniphilus genitalis sp.nov.and "Peptoniphilus septimus" SAHP1 is an earlier synonym.

Discussion
The cut-off values used to classify bacterial strains at the genus and species level, established under the assumption that the level of 16S rRNA sequence variation between species was homogeneous among genera, proved inadequate for many genera.For example, Rossi-Tamisier et al. have shown that in only 17 of the 158 genera in their study (10.8%), all species respected the 95% and 98.7% thresholds for genus and species levels, respectively [31].Thus, given the limitations of using 16S rRNA sequence analysis to classify bacterial species, whole genome sequencing remains the more advanced and accurate strategy to determining the taxonogenomic identity of a bacterium.Strain Marseille-Q7072 T is a new member of Bacillota, for which the name Peptoniphilus genitalis sp.nov. is proposed.Currently, the genus Peptoniphilus contains 21 species with validly published names and thirteen species without validly published names (https:// lpsn.dsmz.de/ search?word= Pepto niphi lus, accessed 22 November 2023).Growth is mainly strict anaerobic but some bacteria of the genus Peptoniphilus such as Peptoniphilus genitalis sp.nov., Peptoniphilus lacydonensis, and Peptoniphilus nemausensis have the ability to grow under microaerophilic conditions [32].Most species of Peptoniphilus have been detected in human clinical specimens including the vagina [26,33,34].They are part of the human commensal flora, but they are also opportunistic pathogens, particularly in the context of polymicrobial infections [33][34][35].
Strain Marseille-Q7826 T is a new member of Actinomycetota, for which the name Mobiluncus massiliensis sp.nov. is proposed.The genus Mobiluncus is currently composed of four species: Mobiluncus curtisii, Mobiluncus holmesii, Mobiluncus mulieris, and Mobiluncus porci (https:// lpsn.dsmz.de/ search?word= Mobil uncus, accessed 22 November 2023).With the exception of M. porci, which was isolated from pig intestines [36], all other species have most often been isolated from human genital sites, mainly the vagina [37], and especially in cases of bacterial vaginosis.However, their role in bacterial vaginosis remains poorly understood and unclear.

Conclusion
In conclusion, based on the results of phenotypic, phylogenetic and genomic analyses, and taking into account only bacterial species validated according to nomenclature, strains Marseille-Q7072 T and Marseille-Q7826 T represent new bacterial species.If we take into account all the sequences available in the databases, the genomic data for Marseille-Q7072 T indicate that it corresponds to "P. septimus" SAHP1.However, as the strain of "P.septimus" SAHP1 is not available, it is not possible to go any further in comparing these two strains.These data underline the importance of at least systematically depositing every new bacterial strain in a collection, in order to make it accessible to the scientific community.
Catalase and oxidase activities are negative.After 48 h of incubation on Columbia agar with 5% sheep blood, colonies appear circular, grey, opaque, and convex, with a diameter of between 2 and 3 mm.Growth occurs under an anaerobic atmosphere in a temperature range of 20-37 °C (optimum 37 °C), at pH 6-8.5 (optimum pH 7) and with 0%-5% (w/v) NaCl (optimum < 5%).
The type strain Marseille-Q7072 T (= CSUR Q7072 T = CECT 30604 T ) was isolated from a vaginal sample of a 41-year-old woman who had suffered recurrent miscarriages.
The 16S rRNA and genome sequences were deposited in GenBank under accession numbers OX458314 and CAT-KPB000000000, respectively.
Cells are facultative anaerobic, Gram-stain-variable, nonspore-forming, motile, and curved rod-shaped.Bacterial cells have a length of 1.216 μm ± 0.243 μm and a diameter of 0.381 μm ± 0.039 μm.Catalase and oxidase activities are negative.After 48 h of incubation on Columbia agar with 5% sheep blood, colonies appear circular, colourless, translucent and convex, with a diameter of between 2 and 3 mm.Growth occurs under an anaerobic atmosphere in a temperature range of between 20 and 37 °C (optimum 37 °C), at pH 6-7.5 (optimum pH 7) and without adding NaCl.
The most abundant fatty acid by far was C 16:0 followed by C 18:1n9 and C 18:2n6 .The size of the genome is 2.21 Mbp and its G + C content is 57 mol%.
The type strain Marseille-Q7826 T (= CSUR Q7826 T = CECT 30727 T ) was isolated from a vaginal sample taken from a 30-year-old pregnant woman at 26 weeks of amenorrhoea.
The 16S rRNA and genome sequences were deposited in GenBank under accession numbers OX458307 and OX458329, respectively.

Table 3
Number of genes associated with the 25 general COG functional categories of Peptoniphilus genitalis sp.nov., strain Marseille-Q7072 T and Mobiluncus massiliensis sp.nov., strain Marseille-Q7826 T