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.

MALDI-TOF MS reference spectra of a Peptinophilus genitalis sp. nov., strain Marseille-Q7072T and b Mobiluncus massiliensis sp. nov., strain Marseille-Q7826T. The reference spectrum was generated by comparison of spectra from 6 individual colonies using the Biotyper 3.0 software

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.

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).

Scanning electron microscopy of a Peptoniphilus genitalis sp. nov., strain Marseille-Q7072T and b Mobiluncus massiliensis sp. nov., strain Marseille-Q7826T

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, ß-galactosidase, D-glucosidase, esterase, phosphatase acid, ß-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 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/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-Q7826T.

For strain Marseille-Q7072T, 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-Q7072T had 1947 predicted genes, including 1911 protein-coding genes. Strain Marseille-Q7072T also had 36 RNA-coding genes, including four rRNA, 31 tRNA, and one tmRNA.

Graphical circular map of genomes of a Peptoniphilus genitalis sp. nov., strain Marseille-Q7072T and b Mobiluncus massiliensis sp. nov., strain Marseille-Q7826T

For strain Marseille-Q7826T, 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-Q7826T had 1908 predicted genes, including 1855 protein-coding genes. Strain Marseille-Q7826T 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-Q7072T and Marseille-Q7826T 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-Q7072T 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.

Phylogenetic trees based on concatened sequences of 7 housekeeping genes a Peptoniphilus genitalis sp. nov., strain Marseille-Q7072T and b Mobiluncus massiliensis sp. nov., strain Marseille-Q7826T (Bold), and closely related species

Strain Marseille-Q7826T 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-Q7072T, 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-Q7072T 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-Q7826T, 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-Q7072T 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-Q7826T 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].

Phylogenetic tree based on 16S rRNA of a Peptoniphilus genitalis sp. nov., strain Marseille-Q7072T and b Mobiluncus massiliensis sp. nov., strain Marseille-Q7826T (Bold), and closely related species

Taken as a whole, the above information supports the proposals that strain Marseille-Q7826T represents a new species in the family Actinomycetaceae, for which the name Mobiluncus massiliensis sp. nov. is proposed. Strain Marseille-Q7072T 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.