Cellulomonas endometrii sp. nov.: a novel bacterium isolated from the endometrial microbiota

An isolate of a bacterium recovered from an endometrial biopsy failed to be identified by MALDI-TOF mass spectrometry and was subjected to 16S rRNA sequencing. The obtained sequence was compared by BLASTn against the NCBI database, which revealed that the most closely related species was Cellulomonas hominis and Cellulomonas pakistanensis, with 98.85% and 98.45% identity, respectively. Phenotypic characterisation and genome sequencing were performed. The isolate was facultative anaerobic, gram-positive, motile, non-spore forming, and rod-shaped. Cell wall fatty acid profiling revealed that 12-methyl-tetradecanoic acid was the most abundant fatty acid (36%). The genome size was 4.25 Mbp with a G + C content of 74.8 mol%. Genomic comparison of species closely related to this strain 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). Based on these data, we conclude that this isolate represents a new bacterial species belonging to the family Cellulomonadaceae and the phylum Actinomycetota. We propose the name Cellulomonas endometrii sp. nov. The type strain is Marseille-Q7820T (= CSUR Q7820 = CECT 30716). Supplementary Information The online version contains supplementary material available at 10.1007/s00203-023-03703-9.


Introduction
The endometrial microbiota was recently discovered and explored.It inhabits the lining of the uterus, previously thought to be sterile (Moreno and Franasiak 2017;Toson et al. 2022).Emerging evidence suggests that the endometrial microbiota plays an important role in reproductive health and may have implications for conditions, such as infertility, endometriosis, and miscarriage (Moreno et al. 2016;Toson et al. 2022).The composition and diversity of the endometrial microbiota have been found to vary significantly between women and may be influenced by factors, such as hormonal fluctuations, the menstrual cycle, and pregnancy (Benner et al. 2018;Toson et al. 2022).
However, much is still unknown about the endometrial microbiota and how it interacts with the host immune system and reproductive processes (Benner et al. 2018).Further research is needed to fully understand the role of the endometrial microbiota in health and disease and to develop potential diagnostic and therapeutic interventions (Toson et al. 2022).The emergence of culturomics, a powerful tool to study microbial diversity, has enabled the isolation and characterisation of many new bacterial species from various microbiota; however, this culture strategy had never been performed from an endometrial biopsy (Lagier et al. 2015;Lagier and Raoult 2016).
By applying this method, we succeeded in isolating a new member of the genus Cellulomonas from an endometrial biopsy designated Marseille-Q7820.Using a polyphasic taxonogenomic approach which integrates the annotated whole 377 Page 2 of 9 genome, proteomic information obtained from MALDI-TOF MS spectra, and phenotypic features, we provide a detailed description of this strain in pure culture.

Ethical approval, sampling, and isolation of the strain
An endometrial biopsy sampled for diagnostic purposes was sent to the clinical microbiology laboratory of Marseille Public University Hospitals (AP-HM, France) where it was retrospectively analysed using a culturomics strategy, as permitted by French law (Article L.1211-2 of the French Code on Public Health).The patient was informed of the possible reuse for research purposes of her sample and the personal data collected from her during her care.She could oppose this by reporting it to the data protection officer at the AP-HM.The patient did not express any objection.The personal data that were used for analysis were anonymised.Our independent local ethics committee (Agreement No. 2022-009) approved the clearance of ERC and compliance with data protection legislation.
Strain Marseille-Q7820 was thus obtained from an endometrial biopsy sample taken from a 34-year-old woman who had suffered from recurrent early miscarriage and chronic endometritis.The vaginal sample collected during the consultation revealed no bacterial vaginosis or sexually transmitted infections.The endometrial biopsy was performed after cleaning the cervix and vagina with sterile compresses soaked in Dakin®.The endometrial biopsy catheter tip (Pipelle ® , CDD laboratory, Paris, France) was inserted through the cervix into the uterine cavity to avoid contamination from the nearby tissues.The biopsy was then aspirated by suction.
To isolate strain Marseille-Q7820, the endometrial biopsy was pre-incubated in anaerobic blood culture vials (Becton Dickinson, Le Pont-de-Claix, France) supplemented with Difco Marine Broth (Becton Dickinson) at 37 °C for 1 day.Isolated colonies were then obtained through subculture on Columbia agar with 5% sheep blood (bioMérieux, Marcy l'Etoile, France) and were incubated at 37 °C in anaerobic conditions using AnaeroGen (bioMérieux) for 48 h.

MALDI-TOF identification, 16S rRNA identification, and phylogenetic analysis
Strain Marseille-Q7820 was identified using a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) following the protocol described by Seng et al. (2013).The MALDI BioTyper software (version 2.0, Bruker) was used to analyse the spectra of the strain by standard pattern matching with default parameter settings.The resulting scores were interpreted as previously described by Hadjadj et al. (2016).

Genome extraction, sequencing, annotation, and comparison
Strain Marseille-Q7820 was subjected to mechanical treatment with acid-washed glass beads (G4649-500 g, Sigma-Aldrich, Saint-Quentin-Fallavier, France) using a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5 m/s) for 90 s, followed by a lysozyme incubation for two hours at 37 °C.DNA was then extracted using the EZ1 Robot and the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany).The DNA extracted was subjected to sequencing using a MiSeq sequencer (Illumina Inc., San Diego, CA, USA) and the Nextera Mate Pair sample preparation kit, along with the Nextera XT Paired End preparation kit (Illumina), following the methods previously outlined in Anani et al. (2019).
The resulting reads were assembled using SPAdes 3.13.1 software, excluding scaffolds below 800 bp and depth values below 25% of the average depths.The obtained genome was annotated using Prokka 1.14.5 (Seemann 2014;Zgheib et al. 2020) and compared with those of closely related species.
The 16S rRNA sequence was also analysed.The 16S rRNA sequences of the Marseille-Q7820 strain and their closely related species were aligned, and a phylogenetic tree was constructed with 1000 bootstrap replicates, based on the Neighbor-Joining method (Saitou and Nei 1987) and the Kimura 2-parameter methods (Kimura 1980), using the MEGA X software (Kumar et al. 2018).
Overall similarity among the genomes was evaluated using digital DNA-DNA hybridisation (dDDH) with the Genome-to-Genome Distance Calculator (GGDC) 2.1 web server (http:// ggdc.dsmz.de/ distc alc2.php) and mean nucleotide identity analysis with OrthoANI 1.2 software (Lee et al. 2016), respectively.The genome-based phylogenetic tree was automatically generated using TYGS for the Marseille-Q7820 strain and closely related species.The tree was deduced using FastME from GBDP distances calculated from genomic sequences.

Strain identification and phylogenetic analysis
Strain Marseille-Q7820 was isolated from an endometrial biopsy.After performing a comprehensive analysis using MALDI-TOF mass spectrometry, we failed to identify the isolate.The score obtained was less than 1.8, indicating that the species was not in the database and could potentially belong to an unknown species.
The phylogenetic tree in Fig. 1 presents the position of strain Marseillle-Q7820 in relation to other closely related species with a validly published name.

Phenotypic characterisation
The main characteristics of the Marseille-Q7820 strain are summarised in Supplementary Table 1.The Marseille-Q7820 strain showed optimal growth when cultured under anaerobic conditions at 37 °C for 2 days.Under these culture conditions, colonies are circular, pale yellow, opaque, and convex with an average diameter of 2 mm.Growth was also observed for the Marseille-Q7820 strain in a microoxic atmosphere.The Marseille-Q7820 strain is a gram-positive, motile (sliding), non-spore forming, and rod-shaped bacterium, positive for catalase and negative for oxidase.Using scanning electron microscopy, strain Marseille-Q7820 was determined to have an average diameter of 0.474 μm ± 0.075 μm and a length of 1.546 μm ± 0.283 μm (Fig. 2).

Genomic analysis
The Marseille-Q7820 strain exhibited a genome length of 4.25 Mbp, which was assembled into 39 contigs, with a G + C content of 74.8 mol% (Fig. 3).This strain was predicted to have 3922 genes, including 3864 protein-coding Fig. 2 Microscopy image of Cellulomonas endometrii sp.nov., strain Marseille-Q7820.The image was obtained using an SU5000 SEM genes, as well as 58 RNA-coding genes, comprised of six rRNA, 51 tRNA, and one tmRNA (Table 3).
Comparing the genomic features of this strain with other related species, the highest dDDH value obtained for the Marseille-Q7820 strain was 27.1% with Cellulomonas hominis (Table 4).Moreover, the OrthoANI values for strain Marseille-Q7820 ranged from 74.53 to 84.01%, further confirming its distinction from the other bacterial strains (Fig. 4).

Conclusion
The 16S rRNA gene sequence identities between the strain Marseille-Q7820 and closely related species was 98.85% Cellulomonas hominis, 98.45% C. pakistanensis, and 98.16% C. taurus.Even if one of these values is higher than the 98.65% threshold commonly used for species delimitation (Kim et al. 2014;Stackebrandt and Goebel 1994), the strain is still considered a new bacterial species.Indeed, 16S rRNA gene sequences have shown their limitations in defining new bacterial species (Rossi-Tamisier et al. 2015).
Strain Marseille-Q7820 shows phenotypic differences from the closest species: (1) it does not use certain sugars, such as D-sucrose and D-mannose, unlike the others; (2) C18 fatty acids are also detected only in strain Marseille-Q7820.Additionally, dDDH and OrthoANI values between the compared genomes are also below established thresholds for species differentiation (70% and 95% to 96%, respectively) (Kim et al. 2014;Meier-Kolthoff et al. 2013).
Based on phenotypic, phylogenetic, and genomic analyses, we assert that strain Marseille-Q7820 is a new bacterium within the genera Cellulomonas in the family Cellulomonadaceae and the phylum Actinomycetota.Therefore, the name Cellulomonas endometrii sp.nov. is proposed.Although almost 70 species of the genus Cellulomonas are listed, only 38 species have currently a validly published name under the List of Prokaryotic names with Standing in Nomenclature (https:// lpsn.dsmz.de/ search?word= Cellu lomon as) (Parte et al. 2020).Cellulomonas spp.are mainly known as environmental pathogens, typically growing in decaying plant-rich soil, but they are also emerging rare human pathogens (Salas et al. 2014).Despite a number of isolates from human sources but of unknown clinical significance, bacteria of the genus Cellulomonas have only been isolated and implicated in active human infection in five cases in the literature to the best of our knowledge (Kimura et al. 2019).Chronic endometritis is currently defined as a continuous and subtle inflammation characterised by the infiltration of plasma cells in the stromal zone of the endometrium (Kimura et al. 2019).Although the impact of chronic endometritis has long been ignored, because symptoms are either subtle or absent, the potential adverse effects of chronic endometritis on fertility have recently been shown (Kimura et al. 2019).
Cellulomonas endometrii sp.nov.was isolated from an endometrial biopsy from a woman with chronic endometritis and recurrent miscarriages.The first question that should be asked is whether this strain is a contaminant.As the strain was not isolated from the patient's vaginal sample while being cultured in parallel with the same conditions, we can assume that Cellulomonas endometrii sp.nov.was part of the endometrial flora at the time of sampling.If this is the case, the other question that should be asked is whether this strain could potentially have a negative effect alone or in combination with other microorganisms.We do not currently have the elements to answer this, but the data underline the interest of exploring the endometrial microbiota by combining metagenomics and culturomics techniques.
Facultative anaerobic, Gram-positive, non-spore forming, motile, and rod-shaped bacterium.Cells are 1.546 μm ± 0.283 μm in length and 0.474 μm ± 0.075 μm in diameter and positioned in clusters.Catalase activity is positive, while oxidase activity is negative.Colonies are visible on Columbia agar with 5% sheep blood incubated anaerobically after 2 days of growth.They appear circular, pale yellow, opaque, and convex with a diameter of 2 mm.
The type strain Marseille-Q7820 T (= CSUR Q7820 = CECT 30716) was isolated from an endometrial sample taken from a 34-year-old woman suffering from recurrent early miscarriage and chronic endometritis.
The 16S rRNA and genome sequences have been deposited in GenBank under accession numbers OX458243 and CATKPC000000000, respectively.

Fig. 4
Fig. 4 Heat map of OrthoANI values between Cellulomonas endometrii sp.nov., strain Marseille-Q7820 and closely related species.The values were calculated using OAT software and displayed as a colour gradient

Table 3
Table summarising the genome properties of Cellulomonas endometrii sp.nov., strain Marseille-Q7820 and the other compared strains