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

Linda Abou Chacra, Marion Bonnet, Mégane Heredia, Gabriel Haddad, Nicholas Armstrong, Stéphane Alibar, Florence Bretelle, Florence Fenollar

<figure><img src="image_1.png" /></figure>

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Linda Abou Chacra, Marion Bonnet, Mégane Heredia, Gabriel Haddad, Nicholas Armstrong, et al. Peptoniphilus genitalis sp. nov. and Mobiluncus massiliensis sp. nov.: Novel Bacteria Isolated from the Vaginal Microbiome. Current Microbiology, 2024, 81 (4), pp.97. 10.1007/s00284-023-03584-7. hal-04543747

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

Linda Abou Chacra<sup>1,2</sup> · Marion Bonnet<sup>1,2</sup> · Mégane Heredia<sup>1,2</sup> · Gabriel Haddad<sup>2,3</sup> · Nicholas Armstrong<sup>3</sup> · Stéphane Alibar<sup>2</sup> · Florence Bretelle<sup>3,4</sup> · Florence Fenollar<sup>1,2</sup>

Received: 16 June 2023 / Accepted: 7 December 2023 / Published online: 19 February 2024
© The Author(s) 2024

**Abstract**

The strains Marseille-Q7072T (=CSUR Q7072T =CECT 30604T) and Marseille-Q7826T (=CSUR Q7826T =CECT 30727T) 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.

**Introduction**

The vaginal microbiota is a complex ecosystem of micro-
organisms 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 lac-
tic acid, thus maintaining an acidic vaginal pH that prevents
colonisation by potentially harmful pathogens [1]. An imbal-
ance 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 cul-
turing 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 characterisa-
tion 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 char-
acterisation 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 prema-
turely and those who had repeated miscarriages, we isolated
two new members of the genus *Peptoniphilus* and *Mobilun-
cus* from vaginal samples that did not match other species of
their genus. These strains were designated Marseille-Q7072T
and Marseille-Q7826T, respectively.

In accordance with the polyphasic taxonogenomic strat-
egy combining annotated whole genome, proteomic infor-
mation extracted from MALDI-TOF mass spectrometry
spectra, and phenotypic aspects, we present here the descrip-
tion of these two strains.

<footnote>☒ Florence Fenollar
florence.fenollar@univ-amu.fr</footnote>

<footnote n="1">¹ Aix-Marseille Université, IRD, AP-HM, SSA, VITROME, Marseille, France</footnote>

<footnote n="2">² IHU-Méditerranée Infection, Marseille, France</footnote>

<footnote n="3">³ Aix-Marseille Université, IRD, AP-HM, MEPHI, Marseille, France</footnote>

<footnote n="4">⁴ Department of Gynaecology and Obstetrics, Gynépole, La Conception, AP-HM, Marseille, France</footnote>
# Materials and Methods

## 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-Q7072T was isolated from a vaginal sample taken from a 41-year-old woman who had suffered from recurrent miscarriages. Strain Marseille-Q7826T 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-Q7072T and Marseille-Q7826T, were cultured from vaginal samples using different pre-incubation methods. For Marseille-Q7072T, 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-Q7826T, 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-Q7072T and Marseille-Q7826T. 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].

## Morphological Observation and Phenotypic Characterisation

By culturing each strain under different atmospheres, temperatures, pH, as well as salinity parameters, the optimum growth conditions for the two strains were identified. Different growth temperatures (room temperature, 28 °C, 37 °C, 42 °C, and 56 °C), various pH conditions ranging from 5.5 to 8.5, as well as different levels of salinity, including 0%, 5%, 7.5%, 10%, 15%, and 20%, were tested on Columbia agar with 5% sheep blood (bioMérieux) under aerobic, anaerobic (GENbag anaer, bioMérieux), and microaerophilic (GENbag Microaer, bioMérieux) conditions.

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].

For the biochemical properties, three API gallery systems (API® ZYM, API® 20A, and API® 50 CH [bioMérieux]) were evaluated according to the manufacturer's instructions. E-test gradient strips (bioMérieux) were used to determine antibiotic susceptibility according to EUCAST recommendations [10]. Finally, analysis of cellular fatty acid methyl esters (FAME) was conducted by gas chromatography/mass spectrometry (GC/MS), as previously reported [11, 12].

## 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

<figure><img src="image_3.png" /></figure>
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].

To evaluate the overall similarity between the compared genomes [18, 19], digital DNA-DNA hybridisation (dDDH) was carried out using the Genome-to-Genome Distance Calculator (GGDC) 2.1 web server (http://ggdc.dsmz.de/dists alc2.php). The average amino acid identity (AAI) calculation between the two paired strains was obtained through the CompareM packages (https://github.com/dparks1134/ CompareM). Average nucleotide identity (ANI) analysis was attempted using the OrthoANI 1.2 software [20]. The species thresholds for dDDH and AAI/ANI are 70% and 95–96%, respectively [19, 21].

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.bioinformatics.nl/cgi-bin/emboss/union).

<figure><img src="image_4.png" /><figcaption>**Fig. 1** MALDI-TOF MS reference spectra of *a* *Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072T and *b* *Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826T. The reference spectrum was generated</figcaption></figure>

# Results

## Strain Identification and Phylogenetic Analysis

Strains Marseille-Q7072T and Marseille-Q7826T 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.

Strains Marseille-Q7072T and Marseille-Q7826T had 16S rRNA sequences that shared 99% similarity with *Peptoniphilus harei* strain NCTN13077 (LR134524.1) and 99.5% similarity with *Mobiluncus curtisii* strain ATCC 35241 T (GL385912.1), respectively. However, the dDDH values for the Marseille-Q7072T and Marseille-Q7826T strains were highest, respectively, with *P. coxii* (46.9%) and *M. curtisii* (29.5%), two bacterial species listed on the website of List of Prokaryotic names with Standing in Nomenclature (LPSN) (https://lpsn.dsmz.de/, accessed 22 November 2023), but remained below the threshold used to distinguish prokaryotic species (70%). Similarly, the highest OrthoANI values of the Marseille-Q7072T and Marseille-Q7826T strains were 88% with *P. harei* and 86% with *M. curtisii*, respectively, but were below the 95% threshold.

Based on these results, strains Marseille-Q7072T and Marseille-Q7826T 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.

<figure><img src="image_5.png" /></figure>

by comparison of spectra from 6 individual colonies using the Biotyper 3.0 software
## Phenotypic Characterisation

Optimal growth of Marseille-Q7072T and Marseille-Q7826T 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-Q7072T 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-Q7826T 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).

For strain Marseille-Q7072T, using an API ZYM strip, positive reactions were observed for leucine arylamidase, naphtol-AS-BI-phosphohydrolase, and acid phosphatase. Using an API 20A strip, strain Marseille-Q7072T exhibited positive reactions for gelatine, esculin, D-trehalose, and D-glucose. Moreover, using API 50 CH strip, strain Marseille-Q7072T was positive for esculin, salicin, lactose, melezitose, xylitol, L-fucose, L-arabitol, and 5-keto-gluconate. These results were compared to those of *Peptoniphilus coxii* RMA 16757 [25], *Peptoniphilus harei* DSM 10020 [26], and *Peptoniphilus timonensis* JC401 [27] (Table 1).

For strain Marseille-Q7826T, using an API ZYM strip, positive reactions were detected for leucine arylamidase, naphtol-AS-BI-phosphohydrolase, β-galactosidase, D-glucosidase, esterase, phosphatase acid, β-glucosidase, and D-mannosidase. Using an API 20A strip, strain Marseille-Q7826T exhibited positive reactions for D-glucose, D-lactose, D-maltose, salicin, D-xylose, esculin, and D-mannose. Finally, using an API 50 CH strip, strain Marseille-Q7826T

<figure><img src="image_6.png" /><figcaption>**Fig. 2** Scanning electron microscopy of *a* *Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072T and *b* *Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826T</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<table><caption>**Table 1** Comparison of *Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072T and *Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826T with their phylogenetically closest species with a validly published name</caption><thead><tr><th>Properties</th><th>Pgen</th><th>Pcox</th><th>Pharei</th><th>Ptim</th><th>Mobmass</th><th>Mobcurt</th><th>Mobhom</th><th>Mobmul</th></tr></thead><tbody><tr><td>Cell size</td><td>0.6 µm</td><td>&lt;0.7 µm</td><td>0.5–1.5 µm</td><td>0.91 µm</td><td>1.2 µm</td><td>1.7 µm</td><td>1.7 µm</td><td>2.9 µm</td></tr><tr><td>O<sub>2</sub> requirement</td><td>Anaerobic and microaerophilic</td><td>Anaerobic</td><td>Anaerobic</td><td>Anaerobic</td><td>Anaerobic</td><td>Anaerobic</td><td>Anaerobic</td><td>Anaerobic</td></tr><tr><td>Gram stain</td><td>+</td><td>+</td><td>+</td><td>+</td><td>Variable</td><td>Variable</td><td>Variable</td><td>Variable</td></tr><tr><td>Mobility</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>+</td><td>—</td><td>+</td></tr><tr><td>Catalase</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Production of:</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Acid phosphatase</td><td>+</td><td>NA</td><td>—</td><td>—</td><td>+</td><td>w</td><td>NA</td><td>w</td></tr><tr><td>Alkaline phosphatase</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>w</td><td>w</td><td>NA</td></tr><tr><td>NAphthol-AS-BI-phosphohydrolase</td><td>+</td><td>NA</td><td>—</td><td>—</td><td>—</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Valine arylamidase</td><td>—</td><td>NA</td><td>—</td><td>—</td><td>—</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>α-glucosidase</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>+</td><td>w</td><td>+</td></tr><tr><td>β-glucosidase</td><td>—</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td><td>w</td><td>—</td></tr><tr><td>β-glucuronidase</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>NA</td><td>—</td></tr><tr><td>Utilization of:</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>D-glucose</td><td>+</td><td>NA</td><td>—</td><td>—</td><td>+</td><td>w</td><td>—</td><td>w</td></tr><tr><td>D-sucrose</td><td>—</td><td>NA</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>D-mannose</td><td>—</td><td>+</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>G + C content (mol%)</td><td>34.3</td><td>44.62</td><td>34.4</td><td>30.7</td><td>57</td><td>51–52</td><td>NA</td><td>49–50</td></tr><tr><td>Habitat</td><td>Human vagina</td><td>Human specimens</td><td>Human sacral ulcer</td><td>Human stool</td><td>Human vagina</td><td>Human vagina</td><td>Human vagina</td><td>Human vagina</td></tr></tbody></table>

Strains: Pgen Peptoniphilus genitalis sp. nov., strain Marseille-Q7072; Pcox Peptoniphilus coxii RMA 16757 [25]; Pharei Peptoniphilus harei DSM 10020 [26]; Ptim Peptoniphilus timonensis JC401 [27]; Mobmass Mobiluncus massiliensis sp. nov., strain Marseille-Q7826; Mobcurt Mobiluncus curtisii BV345-16 [28]; Mobhom Mobiluncus homelsii BV376-6 [28]; Mobmul, Mobiluncus mulieris BV64-5 [28]

+ Positive, — Negative, w Weakly positive, NA Not available

was positive for D-xylose, esculin, starch, gluconate, and 5-ketogluconate. These results were compared to those of *Mobiluncus curtisii* BV345-16 [28], *Mobiluncus homelsii* BV376-6 [28] and *Mobiluncus mulieris* BV64-5 [28] (Table 1).

For strain Marseille-Q7072T, the most abundant fatty acid was C16:0 (46%), followed by C18:1n9 (15%), and C14:0 (11%). For strain Marseille-Q7826T, the most abundant fatty acid was C16:0 (54%), followed by C18:1n9 (18%), and C18:2n6 (15%). Minor amounts of unsaturated, branched, and saturated fatty acids were also described (Table 2).

The antimicrobial susceptibilities of Marseille-Q7072T and Marseille-Q7826T strains were evaluated against various antibiotics. For the strain Marseille-Q7072T, the minimum inhibitory concentrations were as follows: <0.016 µg/mL for penicillin G, 0.023 µg/mL for amoxicillin, 0.47 µg/

mL for ceftriaxone, 0.19 µg/mL for ceftazidime, 0.004 µg/mL for imipenem, 1.5 µg/mL for ciprofloxacin, 0.5 µg/mL for azithromycin, 0.5 µg/mL for clindamycin, 0.016 µg/mL for daptomycin, 1.5 µg/mL for doxycycline, 8 µg/mL for fosfomycin, 1.5 µg/mL for gentamicin, 24 µg/mL for tobramycin, 0.5 µg/mL for metronidazole, 0.75 µg/mL for nitrofurantoin, 0.002 µg/mL for rifampicin, 0.75 µg/mL for linezolid, <0.016 µg/mL for teicoplanin, and 0.064 µg/mL for vancomycin. Amikacin and trimethoprim-sulfamethoxazole showed no activity against strain Marseille-Q7072T.

For the strain Marseille-Q7826T, the minimum inhibitory concentrations were as follows: 0.16 µg/mL for penicillin G, 0.016 µg/mL for amoxicillin, 0.75 µg/mL for ceftriaxone, 0.38 µg/mL for ceftazidime, 0.002 µg/mL for imipenem, <0.016 µg/mL for clindamycin, 0.38 µg/mL for azithromycin, 0.19 µg/mL for daptomycin, 8 µg/
<table><caption>**Table 2** Cellular fatty acids composition (%) of strains Marseille-Q7072<sup>T</sup> (*Peptoniphilus genitalis* sp. nov.), *Peptoniphilus harei* DSM 10020, Marseille-Q7826<sup>T</sup> (*Mobiluncus massiliensis* sp. nov.), and *Mobiluncus curtisii* BV345-16</caption><thead><tr><th>Fatty acids</th><th>Name</th><th>Peptoniphilus genitalis sp. nov., Marseille-Q7072</th><th>Peptoniphilus harei DSM 10020</th><th>Mobiluncus massiliensis sp. nov., Marseille-Q7826</th><th>Mobiluncus curtisii BV345-16<sup>a</sup></th></tr></thead><tbody><tr><td>C<sub>16:0</sub></td><td>Hexadecanoic acid</td><td>46.1</td><td>32.1</td><td>54.0</td><td>38.2</td></tr><tr><td>C<sub>18:1n9</sub></td><td>9-octadecenoic acid</td><td>15.6</td><td>17.0</td><td>18.6</td><td>18.8</td></tr><tr><td>C<sub>14:0</sub></td><td>Tetradecanoic acid</td><td>11.5</td><td>4.4</td><td>0.8</td><td>2.1</td></tr><tr><td>C<sub>18:2n6</sub></td><td>9,12-octadecadienoic acid</td><td>10.6</td><td>17.0</td><td>15.1</td><td>20.9</td></tr><tr><td>C<sub>18:0</sub></td><td>Octadecanoic acid</td><td>6.6</td><td>7.2</td><td>6.4</td><td>15.7</td></tr><tr><td>C<sub>16:1n7</sub></td><td>9-Hexadecenoic acid</td><td>6.0</td><td>1.0</td><td>ND</td><td>2.1</td></tr><tr><td>C<sub>18:1n7</sub></td><td>11-Octadecenoic acid</td><td>1.9</td><td>1.9</td><td>TR</td><td>ND</td></tr><tr><td>C<sub>15:0</sub></td><td>Pentadecanoic acid</td><td>1.1</td><td>ND</td><td>TR</td><td>ND</td></tr><tr><td>C<sub>17:0</sub></td><td>Heptadecanoic acid</td><td>TR</td><td>ND</td><td>TR</td><td>1.3</td></tr><tr><td>C<sub>17:0</sub> anteiso</td><td>14-Methyl-hexadecanoic acid</td><td>ND</td><td>4.2</td><td>ND</td><td>1.3</td></tr><tr><td>C<sub>14:0</sub></td><td>Tetradecanoic acid</td><td>ND</td><td>4.4</td><td>TR</td><td>ND</td></tr><tr><td>C<sub>17:1n7</sub></td><td>10-Heptadecenoic acid</td><td>ND</td><td>ND</td><td>4.1</td><td>ND</td></tr></tbody></table>

TR trace amounts < 1%, ND Not detected

<sup>a</sup>Percentage values were corrected from source by removing non fatty acid structures (DMA and unknown

mL for doxycycline, 0.094 µg/mL for gentamicin, 1.5 µg/mL for amikacin, 0.25 µg/mL for tobramycin, 0.0125 µg/mL for nitrofurantoin, < 0.002 µg/mL for rifampicin, 0.19 µg/mL for linezolid, 0.023 µg/mL for teicoplanin, and 0.125 µg/mL for vancomycin. Ciprofloxacin, fosfomycin, and metronidazole showed no activity against strain Marseille-Q7826<sup>T</sup>.

## Genomic Analysis

For strain Marseille-Q7072<sup>T</sup>, the number of reads was 879694. The genome length was 2040803 bp, assembled into 33 contigs, with a G+C content of 34.3 mol% (Fig. 3a). Strain Marseille-Q7072<sup>T</sup> had 1947 predicted genes, including 1911 protein-coding genes. Strain Marseille-Q7072<sup>T</sup> also had 36 RNA-coding genes, including four rRNA, 31 tRNA, and one tmRNA.

<figure><img src="image_8.png" /><figcaption>**Fig. 3** Graphical circular map of genomes of *a Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072<sup>T</sup> and *b Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826<sup>T</sup></figcaption></figure>

<figure><img src="image_9.png" /></figure>
For strain Marseille-Q7826<sup>T</sup>, the number of reads was 1732 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<sup>T</sup> had 1908 predicted genes, including 1855 protein-coding genes. Strain Marseille-Q7826<sup>T</sup> 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<sup>T</sup> and Marseille-Q7826<sup>T</sup> strains according to the COGs database is also presented in Table 3. Figure 4 shows two phylogenetic trees based on concatenated sequences of 7 housekeeping genes, highlighting the position of each strain in relation to other closely related species. Strain Marseille-Q7072<sup>T</sup> 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<sup>T</sup> is most closely related to *M. curtisii* ATCC 35241<sup>T</sup> and *M. holmesii* ATCC 35242<sup>T</sup> 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<sup>T</sup>, 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<sup>T</sup> 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<sup>T</sup>, the highest dDDH value is 29.5% with *M. curtisii* (Table 5),

<table><caption>**Table 3** Number of genes associated with the 25 general COG functional categories of Peptoniphilus genitalis sp. nov., strain Marseille-Q7072<sup>T</sup> and Mobiluncus massiliensis sp. nov., strain Marseille-Q7826<sup>T</sup></caption><thead><tr><th rowspan="2">Code</th><th colspan="2">Peptoniphilus genitalis</th><th colspan="2">Mobiluncus massiliensis</th><th rowspan="2">Description</th></tr><tr><th>Value</th><th>% of total</th><th>Value</th><th>% of total</th></tr></thead><tbody><tr><td>[J]</td><td>136</td><td>7.12</td><td>132</td><td>7.12</td><td>Translation</td></tr><tr><td>[A]</td><td>0</td><td>0</td><td>1</td><td>0.05</td><td>Rna processing and modification</td></tr><tr><td>[K]</td><td>96</td><td>5.02</td><td>61</td><td>3.29</td><td>Transcription</td></tr><tr><td>[L]</td><td>99</td><td>5.18</td><td>89</td><td>4.80</td><td>Replication, recombination and repair</td></tr><tr><td>[B]</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Chromatin structure and dynamics</td></tr><tr><td>[D]</td><td>19</td><td>0.99</td><td>22</td><td>1.19</td><td>Cell cycle control, mitosis and meiosis</td></tr><tr><td>[Y]</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Nuclear structure</td></tr><tr><td>[V]</td><td>72</td><td>3.77</td><td>40</td><td>2.16</td><td>Defense mechanisms</td></tr><tr><td>[T]</td><td>36</td><td>1.88</td><td>35</td><td>1.89</td><td>Signal transduction mechanisms</td></tr><tr><td>[M]</td><td>56</td><td>2.93</td><td>85</td><td>4.58</td><td>Cell wall/membrane biogenesis</td></tr><tr><td>[N]</td><td>0</td><td>0</td><td>15</td><td>0.81</td><td>Cell motility</td></tr><tr><td>[Z]</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Cytoskeleton</td></tr><tr><td>[W]</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Extracellular structures</td></tr><tr><td>[U]</td><td>17</td><td>0.89</td><td>27</td><td>1.46</td><td>Intracellular trafficking and secretion</td></tr><tr><td>[O]</td><td>50</td><td>2.62</td><td>53</td><td>2.86</td><td>Posttranslational modification, protein turnover, chaperones</td></tr><tr><td>[X]</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Mobilome: prophages, transposons</td></tr><tr><td>[C]</td><td>84</td><td>4.40</td><td>87</td><td>4.69</td><td>Energy production and conversion</td></tr><tr><td>[G]</td><td>32</td><td>1.67</td><td>103</td><td>5.55</td><td>Carbohydrate transport and metabolism</td></tr><tr><td>[E]</td><td>103</td><td>5.39</td><td>102</td><td>5.50</td><td>Amino acid transport and metabolism</td></tr><tr><td>[F]</td><td>48</td><td>2.51</td><td>51</td><td>2.75</td><td>Nucleotide transport and metabolism</td></tr><tr><td>[H]</td><td>46</td><td>2.41</td><td>59</td><td>3.18</td><td>Coenzyme transport and metabolism</td></tr><tr><td>[I]</td><td>39</td><td>2.04</td><td>22</td><td>1.19</td><td>Lipid transport and metabolism</td></tr><tr><td>[P]</td><td>92</td><td>4.81</td><td>53</td><td>2.86</td><td>Inorganic ion transport and metabolism</td></tr><tr><td>[Q]</td><td>13</td><td>0.68</td><td>5</td><td>0.27</td><td>Secondary metabolites biosynthesis, transport and catabolism</td></tr><tr><td>[R]</td><td>157</td><td>8.22</td><td>137</td><td>7.39</td><td>General function prediction only</td></tr><tr><td>[S]</td><td>131</td><td>6.86</td><td>90</td><td>4.85</td><td>Function unknown</td></tr><tr><td>–</td><td>99</td><td>5.18</td><td>157</td><td>8.46</td><td>Not in COGs</td></tr></tbody></table>
<figure><img src="image_10.png" /><figcaption>**Fig. 4** Phylogenetic trees based on concatenated sequences of 7 housekeeping genes **a Peptoniphilus genitalis** sp. nov., strain Marseille-Q7072<sup>T</sup> and **b Mobiluncus massiliensis** sp. nov., strain Marseille-Q7826<sup>T</sup> (Bold), and closely related species</figcaption></figure>

<table><caption>**Table 4** Summary of genome properties for *Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072<sup>T</sup>, *Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826<sup>T</sup> compared with their phylogenetically closest species with a validly published name</caption><thead><tr><th>Strains</th><th>Accession</th><th>Size (bp)</th><th>G+C (%)</th><th>Total genes</th><th>Protein-coding genes</th><th>rRNAs</th><th>tRNAs</th></tr></thead><tbody><tr><td>Pgen</td><td>CATKPB000000000</td><td>2,040,803</td><td>34.3</td><td>1,947</td><td>1,911</td><td>4</td><td>31</td></tr><tr><td>Pharei</td><td>CP068103</td><td>1,929,566</td><td>33.9</td><td>1,828</td><td>1,777</td><td>9</td><td>41</td></tr><tr><td>Mobmass</td><td>OX458329</td><td>2,216,029</td><td>57.0</td><td>1,908</td><td>1,855</td><td>6</td><td>46</td></tr><tr><td>Mobcurt</td><td>GL385912</td><td>2,137,068</td><td>55.3</td><td>1,871</td><td>1,823</td><td>0</td><td>47</td></tr></tbody></table>

Strains: *Pgen Peptinophilus genitalis* Marseille-Q7072, *Pharei Peptoniphilus harei* FDAARGOS_1136, *Mobmass Mobiluncus massiliensis* Marseille-Q7826, *Mobcurt Mobiluncus curtisii* ATCC 35241

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<sup>T</sup> 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<sup>T</sup> 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<sup>T</sup> represents a new species in the family *Actinomycetaceae*, for which the name *Mobiluncus massiliensis* sp. nov. is proposed. Strain Marseille-Q7072<sup>T</sup> 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.
<table><caption>**Table 5** dDDH values of *Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072T and *Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826T with other closely related species with standing in nomenclature</caption><thead><tr><th>Query strain</th><th>Subject strain</th><th>dDDH (in %)</th><th>G+C content<br>difference (in %)</th></tr></thead><tbody><tr><td rowspan="12">Q7072</td><td>"Peptoniphilus septimus"</td><td>69.3</td><td>0.32</td></tr><tr><td>Peptoniphilus coxii</td><td>46.9</td><td>10.48</td></tr><tr><td>Peptoniphilus harei</td><td>35.1</td><td>0.4</td></tr><tr><td>Peptoniphilus ivorii</td><td>33.5</td><td>18.96</td></tr><tr><td>Peptoniphilus asaccharolyticus</td><td>30.2</td><td>1.87</td></tr><tr><td>Peptoniphilus lacrimalis</td><td>29.2</td><td>0.49</td></tr><tr><td>Peptoniphilus duerdenii</td><td>28.5</td><td>0.02</td></tr><tr><td>Peptoniphilus indolicus</td><td>28.2</td><td>2.68</td></tr><tr><td>Peptoniphilus timonensis</td><td>26.1</td><td>3.58</td></tr><tr><td>Peptoniphilus gorbachii</td><td>24.8</td><td>2.86</td></tr><tr><td>Peptoniphilus tyrrelliae</td><td>24.3</td><td>2.23</td></tr><tr><td>Peptoniphilus porci</td><td>22.6</td><td>3.06</td></tr><tr><td>Peptoniphilus lacydonensis</td><td>22</td><td>4.36</td></tr><tr><td>Peptoniphilus faecalis</td><td>21.9</td><td>3.85</td></tr><tr><td>Peptoniphilus ovalis</td><td>20</td><td>3.61</td></tr><tr><td>Peptoniphilus stercorisuis</td><td>17.2</td><td>6.39</td></tr><tr><td rowspan="11">Q7826</td><td>Schaalia turicensis</td><td>30</td><td>0.1</td></tr><tr><td>Mobiluncus curtisii</td><td>29.5</td><td>1.37</td></tr><tr><td>Mobiluncus holmesii</td><td>29.4</td><td>1.4</td></tr><tr><td>Mobiluncus mulieris</td><td>27.3</td><td>1.96</td></tr><tr><td>Schaalia meyeri</td><td>26.9</td><td>8.55</td></tr><tr><td>Trueperella pyogenes</td><td>24.5</td><td>2.67</td></tr><tr><td>Schaalia odontolytica</td><td>24.1</td><td>8.89</td></tr><tr><td>Mobiluncus porci</td><td>20.5</td><td>0.73</td></tr><tr><td>Trueperella bernardiae</td><td>19.3</td><td>9.19</td></tr></tbody></table>

## 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 taxonomic identity of a bacterium.

Strain Marseille-Q7072T 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=Peptoniphilus, 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 [33–35].

Strain Marseille-Q7826T is a new member of *Actinomyceseta*, 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=Mobiluncus, 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
<figure><img src="image_11.png" /><figcaption>**Fig. 5** Phylogenetic tree based on 16S rRNA of *a Peptoniphilus genitalis* sp. nov., strain Marseille-Q7072<sup>T</sup> and *b Mobiluncus massiliensis* sp. nov., strain Marseille-Q7826<sup>T</sup> (Bold), and closely related species</figcaption></figure>

Marseille-Q7072<sup>T</sup> and Marseille-Q7826<sup>T</sup> represent new bacterial species. If we take into account all the sequences available in the databases, the genomic data for Marseille-Q7072<sup>T</sup> 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.

**Description of Peptoniphilus genitalis sp. nov. (Earlier synonym "Peptinophilus septimus")**

*Peptoniphilus genitalis* (ge.ni.ta’lis. L. masc. adj. *genitalis*, belonging to the genital system). Cells are anaerobic and

microaerophilic, Gram-stain-positive, non-spore-forming, non-motile, and coccus-shaped. Bacterial cells are 0.609 µm ± 0.087 µm in diameter and positioned in clusters. 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%).

Using API ZYM, 20A, and 50 CH strips, positive reactions were obtained for leucine arylamidase, Naphtol-AS-BI-phosphohydrolase, acid phosphatase, gelatine, esculin, D-trehalose, D-glucose, salicin, lactose, melezitose, xylitol, L-fucose, L-arabitol, and 5-keto-gluconate.

<figure><img src="image_12.png" /></figure>
The most abundant fatty acid was C₁₆:₀ followed by C₁₈:₁n₉ and C₁₄:₀. The size of the genome is 2.04 Mbp and its G+C content is 34.3 mol%.

The type strain Marseille-Q7072T (=CSUR Q7072T =CECT 30604T) 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.

**Description of Mobiluncus massiliensis sp. nov.**

*Mobiluncus massiliensis* (mas.si.li.en'sis. N.L. masc. adj. massiliensis, from “Massilia”, the Latin name of Marseille).

Cells are facultative anaerobic, Gram-stain-variable, non-
spore-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, trans-
lucent and convex, with a diameter of between 2 and 3 mm.
Growth occurs under an anaerobic atmosphere in a tempera-
ture range of between 20 and 37 °C (optimum 37 °C), at pH
6–7.5 (optimum pH 7) and without adding NaCl.

Using API ZYM, 20A, and 50 CH strips, positive reactions were obtained for leucine arylamidase, naphtol-ASBI-phosphohydrolase, β-galactosidase, D-glucosidase, esterase, phosphatase acid, β-glucosidase, D-mannosidase, D-glucose, D-lactose, D-maltose, salicin, D-xylose, esculin, D-mannose, starch, gluconate, and 5-ketogluconate.

The most abundant fatty acid by far was C₁₆:₀ followed by C₁₈:₁n₉ and C₁₈:₂n₆. The size of the genome is 2.21 Mbp and its G+C content is 57 mol%.

The type strain Marseille-Q7826T (=CSUR Q7826T =CECT 30727T) 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.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00284-023-03584-7.

**Acknowledgements** We thank Ludivine Brechard for sequencing the genome and Aurelia Caputo for submitting the genomic sequence to GenBank. This work received (partial) support from the Hitachi High-Tech Corporation.

**Author Contributions** FB and FF conceived the study. LAC, MB, MH, NA, GH, and SA performed the analyses. LAC, MB, MH, NA, GH, SA, FB, and FF interpreted the data. FB and FF reviewed the manuscript. All authors read and approved the final manuscript.

**Funding** This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection and the French National Research Agency as part of the “Investissements d’avenir” (investments in the future) programme, reference ANR-10-IAHU-03.

**Data Availability** The datasets presented in this search are available in online repositories. The names of the repository(s) and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/nuccore/CATKPB00000000; https://www.ncbi.nlm.nih.gov/nuccore/OX458314; https://www.ncbi.nlm.nih.gov/nuccore/OX458329; https://www.ncbi.nlm.nih.gov/nuccore/OX458307

**Declarations**

**Conflict of interest** The authors have no conflicts of interest to declare. Funding sources had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review, or approval of the manuscript.

**Ethical Approval** The study was approved by the local ethics committee of the Institut Hospitalo-Universitaire Méditerranée Infection (Marseille, France) under agreement Nos. 2022-005 and 2022-009.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

**References**

1. Abou Chacra L, Fenollar F (2021) Exploring the global vaginal microbiome and its impact on human health. Microb Pathog 160:105172

2. Abou Chacra L, Fenollar F, Diop K (2022) Bacterial vaginosis: what do we currently know? Front Cell Infect Microbiol. https://doi.org/10.3389/fcimb.2021.672429

3. Wang WL, Xu SY, Ren ZG et al (2015) Application of metagenomics in the human gut microbiome. World J Gastroenterol 21:803

4. Van De Wijgert JHHM, Borgdorff H, Verhelst R et al (2014) The vaginal microbiota: what have we learned after a decade of molecular characterization? PLoS ONE 9:e105998

5. Lagier JC, Hugon P, Khelaifia S et al (2015) The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev 28:237–264

6. Seng P, Abat C, Rolain JM et al (2013) Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 51:2182–2194

7. Hadjadj L, Rathored J, Keita MB et al (2016) Non contiguous-finished genome sequence and description of *Microbacterium gorillae* sp. nov. Stand Genomic Sci 11:1–9

8. Ly C, Abou Chacra L, Birsal E et al (2022) *Anaerococcus ihuae* sp. nov. and *Mediannikoviicoccus vaginalis* gen. nov., sp. nov.,
two new bacteria isolated from human vaginal samples. Arch Microbiol 204:508

9. Zgheib R, Ibrahim A, Anani H et al (2021) *Neglectibacter timon-ensis* gen. nov., sp. nov. and *Scatolibacter rhodanostii* gen. nov., sp. nov., two anaerobic bacteria isolated from human stool samples. Arch Microbiol 204:45

10. Matuschek E, Brown DFJ, Kahlmeter G (2014) Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clin Microbiol Infect 20:0255–0266

11. Sasser M (2006) Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME). Microbial ID, Newark

12. Dione N, Sankar SA, Lagier JC et al (2016) Genome sequence and description of *Anaerosalibacter massiliensis* sp. nov. New Microbes New Infect 10:66–76

13. Anani H, Abou Abdallah R, Chelkha N et al (2019) Draft genome and description of *Merdibacter massiliensis* gen. nov., sp. nov., a new bacterium genus isolated from the human ileum. Sci Rep 9:1–10

14. Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477

15. Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069

16. Zgheib R, Anani H, Raoult D, Fournier P-E (2020) Draft genome sequence of *Salirhabdus euzebyi* strain Q1438. Microbiol Resour Announc 9:e00246-e320

17. Elsawi Z, Togo AH, Beye M et al (2017) *Hugonella massiliensis* gen. nov., sp. nov., genome sequence, and description of a new strictly anaerobic bacterium isolated from the human gut. Microbiologyopen 6:e00458

18. Auch AF, von Jan M, Klenk HP, Göker M (2010) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2:117–134

19. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform 14:1–14

20. Lee I, Kim YO, Park SC, Chun J (2016) OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103

21. Kim M, Oh HS, Park SC, Chun J (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64:346–351

22. Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274

23. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780

24. Russo CAM, Selvatti AP (2018) Bootstrap and rogue identification tests for phylogenetic analyses. Mol Biol Evol 35:2327–2333

25. Citron DM, Tyrrell KL, Goldstein EJC (2012) *Peptoniphilus coxii* sp. nov. and *Peptoniphilus tyrrelliae* sp. nov. isolated from human clinical infections. Anaerobe 18:244–248

26. Diop K, Diop A, Michelle C et al (2019) Description of three new *Peptoniphilus* species cultured in the vaginal fluid of a woman diagnosed with bacterial vaginosis: *Peptoniphilus pacaensis* sp. nov., *Peptoniphilus raoultii* sp. nov., and *Peptoniphilus vaginalis* sp. nov. Microbiologyopen 8:e00661

27. Mishra AK, Lagier JC, Robert C et al (2012) Non contiguous-finished genome sequence and description of *Peptoniphilus timon-ensis* sp. nov. Stand Genomic Sci 7:1–11

28. Spiegel CA, Roberts M (1984) *Mobiluncus* gen. nov., *Mobiluncus curtisii* subsp. *curtisii* sp. nov., *Mobiluncus curtisii* subsp. *holm-essii* subsp. nov., and *Mobiluncus mulieris* sp. nov., curved rods from the human vagina. Int J Syst Bacteriol 34:177–184

29. Wang H, Yang JL, Chen C et al (2022) Identification of *Peptoniphilus vaginalis*-like bacteria, *Peptoniphilus septimus* sp. nov., from blood cultures in a cervical cancer patient receiving chemotherapy: case and implications. Front Cell Infect Microbiol 12:954355

30. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Evol Microbiol 44:846–849

31. Rossi-Tamisier M, Benamar S, Raoult D, Fournier PE (2015) Cautionary tale of using 16S rRNA gene sequence similarity values in identification of human-associated bacterial species. Int J Syst Evol Microbiol 65:1929–1934

32. Aujoulat F, Mazuet C, Criscuolo A et al (2021) *Peptoniphilus nemausensis* sp. nov. A new Gram-positive anaerobic coccus isolated from human clinical samples, an emended description of the genus Peptoniphilus and an evaluation of the taxonomic status of Peptoniphilus species with not validly published names. Syst Appl Microbiol 44:126235

33. Althaqafi A, Munshi A, Altayib H et al (2023) Septic abortion secondary to *Peptoniphilus asaccharolyticus* complicated by bacteremia: a case report and review of literature. Cureus 15:e35978

34. Wan X, Wang S, Wang M et al (2021) Identification of *Peptoniphilus harei* from blood cultures in an infected aortic aneurysm patient: case report and review published literature. Front Cell Infect Microbiol 11:1243

35. Brown K, Church D, Lynch T, Gregson D (2014) Bloodstream infections due to *Peptoniphilus* spp.: report of 15 cases. Clin Microbiol Infect 20:O857–O860

36. Wylensek D, Hitch TCA, Riedel T et al (2020) A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity. Nat Commun 11:6389

37. Arries C, Ferrieri P (2022) *Mobiluncus curtisii* bacteremia: case study and literature review. Infect Dis Rep 14:82–87

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