# Anaerococcus urinimassiliensis sp. nov., a new bacterium isolated from human urine

Aurélie Morand, Mamadou Lamine Tall, Edmond Kuete Yimagou, Issa Isaac Ngom, Cheikh Ibrahima Lo, Florent Cornu, Michel Tsimaratos, Jean-Christophe Lagier, Anthony Levasseur, Didier Raoult, et al.

► To cite this version:

Aurélie Morand, Mamadou Lamine Tall, Edmond Kuete Yimagou, Issa Isaac Ngom, Cheikh Ibrahima Lo, et al.. Anaerococcus urinimassiliensis sp. nov., a new bacterium isolated from human urine. Scientific Reports, 2021, 11 (1), 10.1038/s41598-021-82420-z. hal-03215611

HAL Id: hal-03215611

https://amu.hal.science/hal-03215611v1

Submitted on 5 Jul 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Check for updates

OPEN

# Anaerococcus urinimassiliensis sp. nov., a new bacterium isolated from human urine

Aurélie Morand<fnref n="1" />,²✉, Mamadou Lamine Tall<fnref n="1" />, Edmond Kuete Yimagou<fnref n="1" />, Issa Isaac Ngom<fnref n="1" />, Cheikh Ibrahima Lo³, Florent Cornu⁴, Michel Tsimaratos⁴, Jean-Christophe Lagier<fnref n="1" />, Anthony Levasseur<fnref n="1" />,⁵, Didier Raoult<fnref n="1" /> & Pierre-Edouard Fournier³✉

To date there are thirteen species validly assigned to the genus *Anaerococcus*. Most of the species in this genus are anaerobic and of human origin. *Anaerococcus urinimassiliensis* sp. nov., strain Marseille-P2143T is member of family *Peptoniphilaceae*, which was isolated from the urine of a 17-year-old boy affected by autoimmune hepatitis and membranoproliferative glomerulonephritis using the culturomic approach. In the current study, a taxono-genomics method was employed to describe this new species. The strain Marseille-P2143T was gram positive cocci with translucent colonies on blood agar. Its genome was 2,189,509 bp long with a 33.5 mol% G + C content and exhibited 98.48% 16S rRNA similarity with *Anaerococcus provencensis* strain 9,402,080. When *Anaerococcus urinomassiliensis* strain Marseill-P2143T is compared with closely related species, the values ranged from 71.23% with *A. hydrogenalis* strain DSM 7454T (NZ_ABXA01000052.1) to 90.64% with *A. provencensis* strain 9402080T (NZ_HG003688.1). This strain has implemented the repertoire of known bacteria of the human urinary tract.

The genus *Anaerococcus* belonging to the phylum *Firmicutes*, was first described in 2001¹. Members of this bacterial genus are mainly anaerobic gram-positive cocci². They are mostly encountered in human vagina, but can also be detected in nostrils or skin³. *Anaerococcus* spp. were involved in human infections and were isolated from different sites of human body such as peritoneal, ovarian and cervical abscesses, an arthritic knee, bacteremia, foot ulcers, a sternal wound and vaginoses⁴⁻⁶. Actually, the genus *Anaerococcus* contains 13 species validly described with standing in nomenclature⁷. The culturomic concept has recently been developed in our laboratory as an alternative method to expand the human gut repertoire through the multiplication of culture conditions with a rapid identification method by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)⁸⁻¹¹. Isolation and identification of microorganisms by culturomic can be used for further studies¹² as well as for their diagnosis and/or therapeutic potential. The systematic description of new bacterial species recovered from patients may contribute to the description of emerging infections, but can also led to other discoveries. For example, the strain *Eubacterium limosum* isolated by culture in the gut sample has been used to test biotransformation's of specific pollutants, methoxychlor and dichlorodiphenyltrichloroethane (DDT) insecticides¹³. The studies conducted on probiotic *Escherichia coli* strain Nissle 1917, show that a visceral analgesic can be produced by bacterial strain, which could be the basis of the development of new visceral pain therapies¹⁴. Currently clinical trials using bacterial cocktails are being used to restore dybiosis by fecal transplantation.

We report here, through a taxono-genomics strategy¹⁵, the description of *Anaerococcus urinimassiliensis* strain Marseille-P2143 (= CSUR P2143 = DSM 103,473), a new bacterium isolated from the urine of a young boy affected by autoimmune hepatitis associated with membranoproliferative glomerulonephritis, and classified into *Peptoniphilaceae* family. This new bacterial species was shortly described in a new species announcement in 2016¹⁶.

<footnote n="1">¹Aix Marseille Université, IRD, AP-HM, MEΦI, Institut Hospitalo-Universitaire Méditerranée-Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France. ²Pédiatrie Spécialisée Et Médecine Infantile, Hôpital de La Timone, AP-HM, Marseille, France. ³Aix Marseille Université, IRD, AP-HM, SSA, VITROME, Institut Hospitalo-Universitaire Méditerranée-Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France. ⁴Pédiatrie Multidisciplinaire, Hôpital de La Timone, AP-HM, Marseille, France. ⁵Institut Universitaire de France (IUF), Paris, France. ✉email: aurelie.morand@ap-hm.fr; pierre-edouard.fournier@univ-amu.fr</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** MALDI-TOF MS reference spectrum of *Anaerococcus urinimassiliensis* Marseille-P2143T, the reference spectrum was generated by comparison of spectra from 12 individual colonies using the Biotyper 3.0 software.</figcaption></figure>

**Material and methods**

**Sample collection and ethical considerations.** In 2015, we isolated in the urine of a 17-year-old boy with autoimmune hepatitis and membranoproliferative glomerulonephritis, a bacterial strain belonging to the genus *Anaerococcus* that could not be identified at the species level. A signed and informed consent was collected from the patient and parents, and the study obtained approval from ethics committee of the Institut Fédératif de Recherche IFR48 under number 09-022. All the methods were carried out in accordance with relevant guidelines and regulations conformed to the Declaration of Helsinki.

**Strain isolation and identification by MALDI-TOF MS.** The initial growth was obtained after 10 days of incubation in an anaerobic blood culture vial (Becton Dickinson, Le Pont-de-Claix, France) supplemented with 5 mL of 0.2 µm filtered rumen fluid. A pure culture of strain Marseille-P2143 was then obtained after 48 h of incubation at 37 °C on 5% sheep blood-Columbia agar medium (bioMérieux, Marcy l'Etoile, France) in anaerobic atmosphere generated using the GENbag Anaer system (bioMérieux) as previously described¹⁶. Strain Marseille-P2143T was not identified by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), after several attempts essayed as described elsewhere¹⁷. The screening was performed on a Microflex LT spectrometer (Bruker, Daltonics, Bremen, Germany) as previously reported¹⁸. The reference spectrum obtained (Fig. 1) was imported and analyzed using the Biotyper software (version 3.0)¹⁹ against the Bruker database, which was continually incremented with local URMS database (https://www.mediterranee-infection.com/urms-data-base/).

**Strain identification and phylogenetic tree.** In order to classify this bacterium, the 16S rRNA gene was amplified using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequenced using the Big Dye Terminator v1.1 Cycle Sequencing Kit and 3500xL Genetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France) as previously described¹². The 16S rRNA nucleotide sequence was assembled and corrected using CodonCode Aligner software (http://www.codoncode.com). For phylogenetic analysis, sequences of the phylogenetically closest species were obtained after performing a BLASTn search within the NCBI Blastn 16S rRNA Sequence Reference Base for closest related species to calculate sequence similarities of the 16S rRNA genes (refseq_rna) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK Loc=blasthome). "The All-Species Living Tree" Project of Silva²⁰. The alignment was performed using MUSCLE²¹. The evolutionary history was inferred using the Maximum Likelihood method based on the Tamura-Nei model²². The tree with the highest log likelihood (-5398.79) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic
search were automatically obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topol-
ogy with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number
of substitutions per site. The analysis involved 18 nucleotide sequences. The codon positions included were
1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total
of 1184 positions in the final dataset. Evolutionary analyses were conducted in MEGA software (version X)
(https://www.megasoftware.net/)23.

**Phenotypic characteristics and biochemical features.** The optimum growth condition of the strain was determined by culturing the strain under different temperatures, atmospheres, pH and salinity. The strains were cultured and incubated under aerobic, anaerobic (GENbag anaer, bioMérieux Limited, France) and micro-aerophilic (GENbag Microaer, bioMérieux Limited, France) conditions on Columbia agar enriched with 5% sheep blood (bioMérieux Limited, France) at the following temperatures: 25, 28, 37, 45, and 55 °C. The pH conditions used were 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and the salinity conditions used were the following: 0%, 5%,10%, 25%, 50%, 100%. The phenotypic characteristics of the strain such as Gram staining, motility, oxidase and catalase activities were determined using standard microbiological methods as previously described²³. These phenotypic and biochemical characteristics were tested for strain Marseille-P2143 incubated at 37 °C for 48 h. The use of carbon sources was assayed with API 50 CH strips. The API 50 CH strips were interpreted after incubation at 37 °C for 24 h. Antibiotic susceptibility was determined using disc diffusion plate method according to the instructions of the CA-SFM / EUCAST 2020 (https://www.eucast.org/clinical_breakpoints_and_dosing/eucast_setting_breakpoints/), in reference to the EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates²⁴. Antibiotic discs used were the following: erythromycin (15 µg/ml), penicillin G (10 UI), doxycycline (30 µg/ml), rifampicin (30 µg/ml), vancomycin (30 µg/ml), clindamycin (15 µg/ml), fosfomycin (50 µg/ml), amoxicillin (25 µg/ml), colistin (15 µg/ml), gentamycin (500 µg/ml), amoxicillin-clavulanic acid (30 µg/ml), ceftriaxone (30 µg/ml), colistin (50 µg/ml), trimethoprim-sulfamethoxazole (25 µg/ml), oxacillin (5 µg/ml), imipenem (10 µg/ml), tobramycin (10 µg/ml), and metronidazole (4 µg/ml). For fatty acids analysis, the bacterial strains cultivated on cos medium after 48 h in aerobic condition, were collected in triplicates in three tubes with approximately the same amount of biomass and were then weighed. We obtained an average of 130 mg of biomass per tube and cellular fatty acid methyl ester (FAME) analysis was performed by Gas Chromatography/Mass Spectrometry (GC/MS) as described by Sasser et al.²⁵. GC/MS analyses were carried out as previously described²⁶. Spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).

**Genome sequencing and assembly.** Genomic DNA was extracted using the EZ1 biorobot with the EZ1 DNA tissue kit (Qiagen, Hilden, Germany) and then sequenced on a MiSeq sequencer (Illumina Inc, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired End (Illumina), as previously described²⁷. The assembly was performed using a pipeline containing several softwares (Velvet²⁸, SPAdes²⁹ and SOAP Denovo³⁰) and trimmed (MiSeq and Trimmomatic³¹ softwares) or untrimmed data (only MiSeq software). GapCloser software³² was used to reduce assembly gaps. Scaffolds < 800 base pairs (bp) and scaffolds with a depth value lower than 25% of the mean depth were removed. The best assembly was selected using different criteria (number of scaffolds, N50, number of N). The degree of genomic similarity of strain Marseille-P2143T (NZ_FQRX0000000.1) with closely related species *Anaerococcus hydrogenalis* DSM 7454T (NZ_ABXA01000052.1), *Anaerococcus jeddahensis* strain SB3T (NZ_CWHU01000001.1), *Anaerococcus tetrad radius* ATCC 35,098T (NZ_GG666394.1), *Anaerococcus octavius* strain NCTC9810T (UFTA01000001.1), *Anaerococcus pacaensis* 9403502T (NZ_HG326663.1), *Anaerococcus prevotii* strain NCTC11806T (UFSY01000001.1), *Anaerococcus provencensis* strain 9402080T (NZ_HG003688.1), *Anaerococcus rubiinfantis* MT16T (FAVH01000001.1), *Anaerococcus senegalensis* JC48T (NZ_HE578907.1), was estimated using the OrthoANI software³³. These genomes were then aligned with scapper (https://github.com/tseemann/scapper). These aligned genomes were eventually used to build the phylogenetic tree using the Maximum likelihood method from MEGA X software³⁴.

**Genome annotation and analysis.** The prediction was performed using prodigal in the open reading frame (ORF)³⁵ with default parameters. Planned ORFs covering a sequencing gap region (containing N) were excluded. The bacterial proteome was predicted with BLASTP (E-value of 1e03, coverage of 0.7 and identity percentage of 30) against the database of orthologic group clusters (COG). If no matches were found, we searched the nr database³⁶ using BLASTP with an E-value of 1e03, a coverage of 0.7 and an identity percentage of 30. An E-value of 1e05 was used only if the sequence length was less than 80 amino acids (aa). The domains maintained by the PFAM (PFAM-A and PFAM-B domains) were searched on each protein using the hmmscan analysis tool. RNAmmer³⁷ and the tRNAScanSE tools³⁸ were used to find rRNA and tRNA genes. When BLASTP E-value was lower than 1e-03 for alignment length greater than 80 amino acids, ORFans are identified. However, when alignment lengths smaller than 80 amino acids were obtained, an E-value of 1e-05 was used. Artemis³⁹ was used for data management and visualization of genomic characteristics. The in-house MAGI software was used to analyze the average level of similarity of nucleotide sequences at the genome level. It calculated the average genomic identity of gene sequences (AGIOS) among the genomes compared¹⁵. This software combines Proteinortho software⁴⁰ to detect orthologic proteins in pairwise genomic comparisons. Then, the corresponding genes were recovered and the average percentage identity of nucleotide sequences among the orthological ORFs was determined using the Needleman–Wunsch global alignment algorithm. We also used the Genome-to-
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Phylogenetic tree highlighting the position of *Anaerococcus uriminassiliensis* Marseille-P2143T sp. nov., with regard to other closely related species. Genbank accession numbers of 16S rRNA are indicated in parentheses. Sequences were aligned using MUSCLE with default parameters, phylogenetic inference were obtained using the neighbor joining (A) and Maximum likelihood (B) method and the MEGA X software. Bootstrap values obtained by repeating the analysis 50 times to generate a majority consensus tree are indicated at the nodes. The scale bar indicates a 2% and 5% nucleotide sequence divergence.</figcaption></figure>

Genome Distance Calculator Web service to calculate DNA: Digital DNA hybridization estimates (dDDH) with confidence intervals according to recommended parameters (Formula 2, BLAST)$^{41}$.

The degree of genomic similarity of Marseille-P2143 with closely related species was estimated using the OrthoANI software$^{42}$.

**Ethical approval.** All the methods were carried out in accordance with relevant guidelines and regulations conformed to Declaration of Helsinki.

## Results and discussion

**Identification of Strain Marseille-P2143.** The mass spectrum of strain Marseille-P2143 was not present in the MALDI-TOF MS Bruker database. Thus, we were not able to identify this strain using this instrument. However, the 16S rRNA gene sequencing analysis indicated that it exhibited 98.48% sequence similarity with Anaerococcus provencensis strain 9402080T (Genbank accession number NR_133036.1), the phylogenetically closest species after blast in the NCBI database (Fig. 2). We consequently proposed to classify strain Marseille-P2143 Type strain as a new species within the genus *Anaerococcus*$^{43}$.

**Phenotypic characteristics and biochemical features.** The optimal growth of strain Marseille-P2143 was obtained after 5 day of culture at 37 °C under anaerobic conditions (anaeroGEN, Oxoid Ltd, Dardilly, France). Agar-grown colonies were small with a mean diameter of 50 µm and were translucent white. Bacterial cells were Gram-positive cocci ranging in diameter from 0.5 to 0.7 µm (Fig. 3). Catalase and oxidase activities were not observed. A brief description and characteristics of strain Marseille-P2143 are summarized in Table 1. Using API ZYM strip, positive reactions were observed for alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, cystine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, α-galactosidase and β-galactosidase. But negative reactions were noted with lipase, valine arylamidase, α-chymotrypsin, acid phosphatase, and β-glucuronidase. Using API 50 CH strip (bioMérieux), strain Marseille-P2143 was able to metabolize D-galactose, D-glucose, D-fructose, N-acetyl-glucosamine, salicin, D-maltose, D-lactose, and D-saccharose. Negative reactions were obtained for glycerol, D-mannose, methyl α-D-glucopyranoside, D-turanose, D-tagatose, erythritol, D-adonitol, D-mannose, mannitol, D-sorbitol, amygdalin, arbutin, esculin, D-melibiose, D-trehalose, inulin, D-melezitose, D-raffinose, starch, glycogen, D-arabitol, L-arabitol and Potassium 5-ketogluconate. When strain Marseille-P2143 was compared to *Anaerococcus provencensis* strain 9402080T the closest species with a validly published name, it differed in catalase activity (Table 2). The Antimicrobial susceptibility test according to the EUCAST showed that strain Marseille-P2143 was susceptible to Penicillin, Oxacillin, Amoxicillin, Amoxicillin-Clavulanic acid, Imipenem, Rifampin, Vancomycin, Clindamycin, Fosfomycin, and
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Morphology of *Anaerococcus urinimassiliensis* sp.nov. (A) Scanning electron microscopy of stained *Anaerococcus urinimassiliensis* sp. nov., Marseille-P2143<sup>T</sup>. (B) Gram staining of *Anaerococcus urinimassiliensis* sp. nov., Marseille-P2143<sup>T</sup>.</figcaption></figure>

<table><caption>**Table 1.** Description and characteristics of *Anaerococcus urinimassiliensis* Marseille-P2143<sup>T</sup>.</caption><thead><tr><td>Property</td><td>Terms</td></tr></thead><tbody><tr><td>Genus name</td><td>Anaerococcus</td></tr><tr><td>Species name</td><td>Anaerococcus urinimassiliensis</td></tr><tr><td>Specific epithet</td><td>Urinimassiliensis</td></tr><tr><td>Species status</td><td>sp. nov</td></tr><tr><td>Designation of the type strain</td><td>Marseille-P2143<sup>T</sup></td></tr><tr><td>Strain collection numbers</td><td>CSUR P2143, DSM 103,473</td></tr><tr><td>16S rRNA gene accession number</td><td>LN898272</td></tr><tr><td>Genome accession number</td><td>FQRX01000001</td></tr><tr><td>Genome size</td><td>2,190,108 bp</td></tr><tr><td>G + C (mol %)</td><td>33.47</td></tr><tr><td>Origin</td><td>Marseille, France</td></tr><tr><td>Date of isolation</td><td>2015</td></tr><tr><td>Source of isolation</td><td>Human urine sample</td></tr><tr><td>Conditions used for standard cultivation</td><td>Columbia agar + with 5% sheep blood for 48 h of incubation</td></tr><tr><td>Gram stain</td><td>Positive</td></tr><tr><td>Cell shape</td><td>Cocci</td></tr><tr><td>Cell size (diameter)</td><td>0.5–0.7 (µm)</td></tr><tr><td>Motility</td><td>Nonmotile</td></tr><tr><td>Colony morphology</td><td>Translucent white</td></tr><tr><td>Temperature optimum</td><td>37 °C</td></tr><tr><td>pH range</td><td>5.5–8</td></tr><tr><td>Relationship to O<sub>2</sub></td><td>Anaerobe</td></tr><tr><td>Oxidase</td><td>Negative</td></tr><tr><td>Catalase</td><td>Negative</td></tr><tr><td>Salinity range</td><td>&lt; 5%</td></tr></tbody></table>

Tobramycin but resistant to Ceftriaxone, Erythromycin, Doxycycline, Gentamicin, Colistin and Trimethoprim-Sulfamethoxazole. Hexadecanoic acid was the most abundant fatty acid (57%). Unsaturated fatty acids 9-Octadecenoic acid and 9,12-Octadecadienoic were also detected with significant amounts. Tetradecanoic acid and Octadecanoic acid were minor fatty acids were quantified. By comparing strain Marseille-P2143 with related close species (Table 3), we obtain mainly a similar profile between species with C<sub>16:0</sub> as the abundant fatty acid (> 50%).
<table><caption>**Table 2.** Differential phenotypic characteristics of *A. urinimassiliensis* sp. nov, Marseille-P2143T and related species of the genus *Anaerococcus*. 1, *A. urinimassiliensis* sp. nov, Marseille-P2143T; 2, *A. provencensis* 9402080T; 3, *A. prevotii* DSM 20548T; 4, *A. pacaensis* 9403502T. +: positive reaction; -: negative reaction; na: not available.</caption><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>Cell diameter (μm)</td><td>0.5-0.7</td><td>0.9-1.3</td><td>0.6-1.5</td><td>0.9-1.4</td></tr><tr><td>Oxygen requirement</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></tr><tr><td>Motility</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Production of:</td><td></td><td></td><td></td><td></td></tr><tr><td>Alkaline phosphatase</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>β-galactosidase</td><td>+</td><td>+</td><td>-</td><td>na</td></tr><tr><td>Indole</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Catalase</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Acid form:</td><td></td><td></td><td></td><td></td></tr><tr><td>Mannose</td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>Glucose</td><td>+</td><td>+</td><td>+/-</td><td>-</td></tr><tr><td>Lactose</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Raffinose</td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>G+C content (mol%)</td><td>33.47</td><td>33.48</td><td>33</td><td>35.05</td></tr><tr><td>Origin</td><td>Human urine</td><td>Human cervical abscess</td><td>Human plasma</td><td>Human blood</td></tr></tbody></table>

<table><caption>**Table 3.** Cellular fatty acid composition (%) of *Anaerococcus urinimassiliensis* sp. nov, Marseille-P2143T compared with other *Anaerococcus* species. TR trace amounts < 1%, ND not detected.</caption><thead><tr><th>Fatty acids</th><th>Name</th><th>A. urinimassiliensis</th><th>A. jeddahensis</th><th>A. rubiinfantis</th><th>A. vaginalis</th><th>A. senegalensis</th></tr></thead><tbody><tr><td>16:00</td><td>Hexadecanoic acid</td><td>56.8</td><td>51.7</td><td>52.7</td><td>59.2</td><td>54.6</td></tr><tr><td>18:1n9</td><td>9-Octadecenoic acid</td><td>13.6</td><td>13.6</td><td>28.2</td><td>18.8</td><td>17.6</td></tr><tr><td>18:2n6</td><td>9,12-Octadecadienoic acid</td><td>13.5</td><td>16.4</td><td>10.1</td><td>6.5</td><td>9.2</td></tr><tr><td>14:00</td><td>Tetradecanoic acid</td><td>5.4</td><td>7.7</td><td>2.1</td><td>6.9</td><td>4.4</td></tr><tr><td>18:00</td><td>Octadecanoic acid</td><td>4.7</td><td>2.6</td><td>3.6</td><td>5.7</td><td>11.5</td></tr><tr><td>18:1n7</td><td>11-Octadecenoic acid</td><td>2.4</td><td>1.5</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>15:00</td><td>Pentadecanoic acid</td><td>TR</td><td>1.2</td><td>TR</td><td>1.0</td><td>TR</td></tr><tr><td>16:1n7</td><td>9-Hexadecenoic acid</td><td>TR</td><td>TR</td><td>1.1</td><td>TR</td><td>ND</td></tr><tr><td>12:00</td><td>Dodecanoic acid</td><td>TR</td><td>1.1</td><td>TR</td><td>TR</td><td>TR</td></tr><tr><td>10:00</td><td>Decanoic acid</td><td>TR</td><td>2.8</td><td>ND</td><td>1.2</td><td>TR</td></tr></tbody></table>

**Genome properties.** The genome of Marseille-P2143 was 2,189,509 bp long with 33.5 mol% G+C content (Fig. 4). It was composed of 10 scaffolds (11 contigs). Of the 2077 predicted genes, 1952 were protein-coding genes and 61 were RNAs (4 genes are 5S rRNA, 3 genes are 16S rRNA, 5 genes are 23S rRNA, 46 genes are tRNA genes) 64 were pseudo genes.

**Genome comparison.** The draft genome sequence of *Anaerococcus urinimassiliensis* (2,189,509 bp) was larger than that of *A. prevotii* strain NCTC11806T (UFSY01000001.1) (2,004,977 bp), *A. pacaensis* strain 9403502T (NZ_HG326663.1) (845,487 bp) and *A. provencensis* strain 9402080T (NZ_HG003688.1) (29,418). The G+C content of *A. urinimassiliensis* strain Marseille-P2143T (33.5 mol%) was smaller than those of *A. provencensis* strain 9402080T (NZ_HG003688.1) (29,418) (33.7%), *A. pacaensis* strain 9403502T (NZ_HG326663.1) (34.1%) and *A. prevotii* strain NCTC11806T (UFSY01000001.1) (35.6%). The gene content of *A. urinimassiliensis* strain Marseille-P2143 (2077 genes) was smaller than those of *A. provencensis* strain 9402080T (NZ_HG003688.1) (2146) and *A. pacaensis* strain 9403502T (NZ_HG326663.1) (2223), but larger than those of *A. prevotii* strain NCTC11806T (UFSY01000001.1) (1894) respectively (Table 4). OrthoANI values among closely related species (Figs. 5) ranged from 69.52% between *A. pacaensis* strain 9403502T (NZ_HG326663.1) and *A. rubiinfantis* strain MT16T (FAVH01000001.1) to 92.77% between *A. jeddahensis* strain SB3T (NZ_CWHU01000001.1) and *A. rubiinfantis* strain MT16T (FAVH01000001.1). When *Anaerococcus urinimassiliensis* strain Marseill-P2143is compared with closely species, the values ranged from 71.23% with *A. hydrogenalis* strain DSM 7454T (NZ_ABXA01000052.1) to 90.64% with *A. provencensis* strain 9402080T (NZ_HG003688.1). The phylogenetic tree highlighting the position of the genome of strain Marseille-P2143 in relation to other closely related species with a validly published name is presented in (Fig. 6). Genes with putative function (by COGs) were 1796
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Graphical circular map of the chromosome of *Anaerococcus urinimassiliensis* sp.nov. From outside to the center: genes on the forward strand colored by COG categories (only genes assigned to COG), genes on the reverse strand colored by COG categories (only gene assigned to COG), RNA genes (tRNAs green, rRNAs red), GC content and GC skew.</figcaption></figure>

<table><caption>**Table 4.** Comparison of genome size, G + C mol% content and gene count of Marseille-P2143<sup>T</sup> with other species within the family *Peptoniphilaceae*.</caption><thead><tr><td>Species</td><td>Size (bp)</td><td>G + C (%mol)</td><td>Total of genes</td></tr></thead><tbody><tr><td>Anaerococcus provencensis strain 9402080<sup>T</sup></td><td>29,418</td><td>33.7</td><td>2146</td></tr><tr><td>Anaerococcus prevotii strain NCTC11806<sup>T</sup></td><td>2,004,977</td><td>35.6</td><td>1894</td></tr><tr><td>Anaerococcus pacaensis strain 9403502<sup>T</sup></td><td>845,487</td><td>34.14</td><td>2223</td></tr><tr><td>Anaerococcus urinimassiliensis strain Marseille-P2143</td><td>2,189,509</td><td>33.5</td><td>2077</td></tr></tbody></table>

for *Anaerococcus urinimassiliensis* strain Marseille-P2143<sup>T</sup> (81%), ,903 for *A.provencensis* strain 9402080<sup>T</sup>(NZ_HG003688.1) (82%), 1539 for *A.prevotii* strain NCTC11806<sup>T</sup> (UFSY01000001.1) (86) and 1867 for *Anaerococcus pacaensis* strain 9403502<sup>T</sup>(NZ_HG326663.1) (78%). Finally, 414, 426, 253 and 538 genes (19%,18%, 14% and 22%) were annotated as hypothetical proteins for *Anaerococcus urinimassiliensis* strain Marseille-P2143, *A.provencensis* strain 9402080<sup>T</sup>(NZ_HG003688.1), *A.prevotii* strain NCTC11806<sup>T</sup> (UFSY01000001.1) and *A.pacaensis* strain 9403502<sup>T</sup>(NZ_HG326663.1), respectively Table 5. Analysis of the Clusters of Orthologous Groups (COGs) categories shows that the mobile elements of the Marseille-P2143 genome appear to be more numerous than those of the genomes of *A.provencensis* strain 9402080<sup>T</sup>(NZ_HG003688.1), *A.prevotii* strain NCTC11806<sup>T</sup> (UFSY01000001.1), *A.pacaensis* strain 9403502<sup>T</sup>(NZ_HG326663.1) (82, 45, 16 and 48 in category [X], respectively).
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Heatmap generated with OrthoANI values calculated using the OAT software between *Anaerococcus urinimassiliensis* Marseille-P2143T sp. nov. and other closely related species with standing in nomenclature.</figcaption></figure>

The 16S rRNA gene sequence obtained by sanger sequencing was identical to that obtained within the sequenced genome (Locus Taq: CJIAFGEE_00528, contic: FQRX01000011.1, Per. Ident: 99.51% using Blast).

**Conclusion**

On the basis of unique phenotypic features, including the MALDI-TOF spectrum, a 16S rRNA sequence similarity lower than < 98.65% and, an OrthoANI and a DDH values lower than 95% and 70% respectively with the phylogenetically closest species with standing in nomenclature, we formally propose strain Marseille-P2143T as the type strain of *Anaerococcus urinimassiliensis* sp. nov., a new species within the genus *Anaerococcus*. *Anaerococcus urinimassiliensis* (u.ri.ni.mas.si.li.en'sis. N.L adj.masc. urinimmassiliensis, composed of urini, from the latin urina, urine and massiliensis, from Massilia, the roman name of Marseille, France, where the strain Marseille-P2143 was first isolated. The colonies are thin, translucent and 50 µm in diameter. Cells are Gram-positive and anaerobic cocci. Cells have a diameter ranging from 0.5 to 0.7 µm. They do not produce catalase and oxidase, but exhibit alkaline phosphatase, leucine arylamidase, esterase lipase, α-galactosidase, naphthol-AS-BI-phosphohydrolase and β-galactosidase. D-glucose, D-galactose, D-maltose, D-lactose, D-fructose, N-acetyl-glucosamine, salicin, and sucrose are metabolized. The genome of strain Marseille-P2143 is 2,189,509 bp long with a 33.5 mol% G+C content. Its 16S rRNA gene sequence and whole-genome sequence are deposited in GenBank under accession numbers LN898272.1 and NZ_FQRX00000000.1, respectively. The type strain Marseille-P2143T (=CSUR P2143 = DSM 103,473) was isolated from the urine of a 17-year-old boy suffering from autoimmune hepatitis and membranoproliferative glomerulo-nephritis. This new species implements the repertoire of human urinary tract known bacteria⁴⁴.

**Description of Anaerococcus urinimassiliensis** sp. nov. *Anaerococcus urinimassiliensis* (u.ri.ni.mas.si.li.en'sis. L. gen. masc. urini, of Urine and massiliensis referring to the Latin name of Marseille where strain Marseille-P2143 was cultivated). Cells are Gram-positive and non-motile, but they are negative for catalase and oxidase activities. They had a mean diameter of 0.6 µm. On blood agar after 48 h of incubation at 37 °C, colonies of strain Marseille-P2143 appear translucent white. The optimum growth is observed at pH 7.5. Major cellular fatty acid was Hexadecanoic acid (57%), while unsaturated fatty acids such as 9-Octadecenoic acid and
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Phylogenetic tree highlighting the position of *Anaerococcus urinimassiliensis* Marseille-P2143<sup>T</sup> sp. nov., with regard to other closely related species considering the genome. Genbank accession numbers of genome are indicated in parentheses. Sequences were aligned using scraper with default parameters, phylogenetic inference were obtained using the Maximum likelihood method and the MEGA X software. Bootstrap values obtained by repeating the analysis 50 times to generate a majority consensus tree are indicated at the nodes. The scale bar indicates a 1% nucleotide sequence divergence.</figcaption></figure>

9,12-Octadecadienoic were also detected with strain Marseille-P2143. The enzymes activities of alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, cystine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, α-galactosidase and β-galactosidase were positive using the API ZYM. Also, fructose, galactose, glucose, N-acetyl-glucosamine, salicin, maltose, lactose, and saccharose are fermented but not for mannose, methyl α-D-glucopyranoside, melibiose, turanose, erythritol, tagatose, adonitol, mannose, mannitol, arbutin, esculin, trehalose, sorbitol, inulin, melezitose and amygdalin. 16S rRNA and genome sequences of this new species are deposited in GenBank under accession numbers LN898272 and FQRX00000000, respectively. The genome size is 2.19 Mb with a G+C content at 33.47%. The type strain Marseille-P2143<sup>T</sup> (=CSUR P2143 =DSM 103,473). The Marseille-P2143 strain was isolated in the urine of a young boy suffering from autoimmune hepatitis membranoproliferative glomerulonephritis.

**Deposit in culture collections.** Strain Marseille-P2143<sup>T</sup> was deposited in the French culture collection center, *Collection des Souches de l'Unité des Rickettsies* (CSUR), under the number CSUR P2143. And in the DSM collection under the number 103473.
<table><caption>**Table 5.** Number of genes associated with the 25 general clusters of orthologous group (COG) functional categories of strain Marseille-P2143 with other species within the family *Peptoniphilaceae*.</caption><thead><tr><td>Code</td><td>Anaerococcus urinimassiliensis</td><td>Anaerococcus provencensis</td><td>Anaerococcus prevotii</td><td>Anaerococcus pacaensis</td><td>Description</td></tr></thead><tbody><tr><td>J</td><td>188</td><td>184</td><td>176</td><td>191</td><td>Translation, ribosomal structure and biogenesis</td></tr><tr><td>A</td><td>0</td><td>0</td><td>0</td><td>0</td><td>RNA processing and modification</td></tr><tr><td>K</td><td>128</td><td>152</td><td>115</td><td>142</td><td>Transcription</td></tr><tr><td>L</td><td>112</td><td>107</td><td>98</td><td>110</td><td>Replication, recombination and repair</td></tr><tr><td>B</td><td>1</td><td>1</td><td>1</td><td>1</td><td>Chromatin structure and dynamics</td></tr><tr><td>D</td><td>30</td><td>31</td><td>30</td><td>31</td><td>Cell cycle control, cell division, chromosome partitioning</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>93</td><td>96</td><td>72</td><td>111</td><td>Defense mechanisms</td></tr><tr><td>T</td><td>77</td><td>100</td><td>70</td><td>74</td><td>Signal transduction mechanisms</td></tr><tr><td>M</td><td>90</td><td>88</td><td>71</td><td>72</td><td>Cell wall/membrane/envelope biogenesis</td></tr><tr><td>N</td><td>8</td><td>11</td><td>7</td><td>10</td><td>Cell motility</td></tr><tr><td>Z</td><td>1</td><td>1</td><td>1</td><td>2</td><td>Cytoskeleton</td></tr><tr><td>W</td><td>3</td><td>5</td><td>4</td><td>4</td><td>Extracellular structures</td></tr><tr><td>U</td><td>21</td><td>23</td><td>15</td><td>21</td><td>Intracellular trafficking, secretion, and vesicular transport</td></tr><tr><td>O</td><td>76</td><td>75</td><td>71</td><td>83</td><td>Posttranslational modification, protein turnover, chaperones</td></tr><tr><td>X</td><td>82</td><td>45</td><td>16</td><td>48</td><td>Mobilome: prophages, transposons</td></tr><tr><td>C</td><td>90</td><td>102</td><td>84</td><td>109</td><td>Energy production and conversion</td></tr><tr><td>G</td><td>164</td><td>207</td><td>133</td><td>126</td><td>Carbohydrate transport and metabolism</td></tr><tr><td>E</td><td>94</td><td>110</td><td>95</td><td>124</td><td>Amino acid transport and metabolism</td></tr><tr><td>F</td><td>58</td><td>62</td><td>58</td><td>61</td><td>Nucleotide transport and metabolism</td></tr><tr><td>H</td><td>68</td><td>75</td><td>74</td><td>83</td><td>Coenzyme transport and metabolism</td></tr><tr><td>I</td><td>58</td><td>64</td><td>43</td><td>65</td><td>Lipid transport and metabolism</td></tr><tr><td>P</td><td>102</td><td>100</td><td>97</td><td>117</td><td>Inorganic ion transport and metabolism</td></tr><tr><td>Q</td><td>16</td><td>15</td><td>8</td><td>21</td><td>Secondary metabolites biosynthesis, transport and catabolism</td></tr><tr><td>R</td><td>151</td><td>158</td><td>122</td><td>168</td><td>General function prediction only</td></tr><tr><td>S</td><td>85</td><td>91</td><td>78</td><td>93</td><td>Unknown function</td></tr><tr><td>-</td><td>414</td><td>426</td><td>253</td><td>538</td><td>Not in cog</td></tr></tbody></table>

Received: 12 January 2020; Accepted: 20 October 2020

Published online: 29 January 2021

## References

1. Ludwig, W., Schleifer, K. H. & Whitman, W. B. Revised road map to the phylum Firmicutes. In *Bergey's Manual of Systematic Bacteriology* Vol. 2 (eds De Vos, P., Garrity, G. M., Jones, D. *et al.* ) 1–13 (Springer, New York, 2009).

2. Ezaki, T. *et al.* Proposal of the genera *Anaerococcus* gen. nov., *Peptoniphilus* gen. nov. and *Gallicola* gen. nov. for members of the genus Peptonstreptococcus. *Int. J. Syst. Evol. Microbiol.* **51**, 1521–1528 (2001).

3. Song, Y., Liu, C. & Finegold, S. M. *Peptoniphilus gorbachii* sp. nov., *Peptoniphilus olsenii* sp. nov., and *Anaerococcus murdochii* sp. nov. isolated from clinical specimens of human origin. *J. Clin. Microbiol.* **45**, 1746–1752 (2007).

4. Jain, S., Bui, V., Spencer, C. & Yee, L. Septic arthritis in a native joint due to *Anaerococcus prevotii*. *J. Clin. Pathol.* **61**, 775–776 (2008).

5. La Scola, B., Fournier, P. E. & Raoult, D. Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era. *Anaerobe* **17**, 106–112 (2011).

6. Pépin, J. *et al.* The complex vaginal flora of West African women with bacterial vaginosis. *PLoS ONE* **6**, e25082 (2011).

7. Parte, A. C. LPSN-list of prokaryotic names with standing in nomenclature. *Nucleic Acids Res.* **42**, D613–D616 (2014).

8. Lagier, J. C. *et al.* Microbial culturomics: paradigm shift in the human gut microbiome study. *Clin. Microbiol. Infect.* **18**, 1185–1193 (2012).

9. Lagier, J. C. *et al.* The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. *Clin. Microbiol. Rev.* **28**, 237–264 (2015).

10. Lagier, J. C. *et al.* Culture of previously uncultured members of the human gut microbiota by culturomics. *Nat. Microbiol.* **1**, 16203 (2016).

11. Lagier, J. C. *et al.* Non-contiguous-finished genome sequence and description of *Anaerococcus senegalensis* sp. nov. *Stand Genomic Sci.* **6**, 116–125 (2012).

12. Morel, A. S. *et al.* Complementarity between targeted real-time specific PCR and conventional broad-range 16S rDNA PCR in the syndrome-driven diagnosis of infectious diseases. *Eur. J. Clin. Microbiol. Infect. Dis.* **34**, 561–570 (2015).

13. Yim, Y. *et al.* Reductive dechlorination of methoxychlor and DDT by human intestinal bacterium *Eubacterium limosum* under anaerobic conditions. *Arch. Environ. Contam. Toxicol.* **54**, 406–411 (2008).

14. Pérez-Berezo, T. *et al.* Identification of an analgesic lipopeptide produced by the probiotic *Escherichia coli* strain Nissle 1917. *Nat. Commun.* **8**(1), 1314 (2017).
15. Ramasamy, D. et al. A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. *Int. J. Syst. Evol. Microbiol.* **64**, 384–391 (2014).

16. Morand, A. et al. *Anaerococcus urinomassiliensis* sp. nov., isolated from a urine sample of a 17-year-old boy affected by autoimmune hepatitis and membranoproliferative glomerulonephritis. *New Microbes New Infect.* **6**(13), 56–58 (2016).

17. Lo, C. I. et al. MALDI-TOF mass spectrometry: a powerful tool for clinical microbiology at Hôpital Principal de Dakar, Senegal (West Africa). *PLoS ONE* **10**, e0145889 (2015).

18. Fall, B. et al. The ongoing revolution of MALDI-TOF mass spectrometry for microbiology reaches tropical Africa. *Am. J. Trop. Med. Hyg.* **92**, 641–647 (2015).

19. Nagy, E., Becker, S., Kostrzewa, M., Barta, N. & Urbán, E. The value of MALDI-TOF MS for the identification of clinically relevant anaerobic bacteria in routine laboratories. *J. Med. Microbiol.* **61**, 1393–1400 (2012).

20. Yilmaz, P. et al. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks. *Nucleic Acids Res.* **42**, D643–D648 (2014).

21. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. *Nucleic Acids Res.* **32**(5), 1792–1797 (2004).

22. Tamura, K. & Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. *Mol. Biol. Evol.* **10**(3), 512–526 (1993).

23. Jousimies-Somer, H. et al. *Wadsworth-KTL Anaerobic Bacteriology Manual* 6th edn. (Star Publishing, Belmont, CA, 2002).

24. Nagy, E. et al. Development of EUCAST disk diffusion method for susceptibility testing of the *Bacteroides fragilis* group isolates. *Anaerobe* **31**, 65–71 (2015).

25. Sasser, M. *Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME)* (MIDI, Technical Note, Newark, NY, 2006).

26. Dione, N. et al. Genome sequence and description of *Anaerosalibacter massiliensis* sp. nov. *New Microbes New Infect.* **10**, 66–76 (2016).

27. Lo, C. I. et al. High-quality draft genome sequence and description of *Haemophilus massiliensis* sp. nov. *Stand Genomic Sci.* **11**, 31 (2016).

28. Zerbino, D. R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. *Genome Res.* **18**, 821–829 (2008).

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

30. Luo, R. et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. *Gigascience.* **1**, 18 (2012).

31. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. *Bioinformatics* **30**, 2114–2120 (2014).

32. Xu, G. C. et al. LR_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly. *Gigascience*. https://doi.org/10.1093/gigascience/giy157 (2019).

33. Lee, I., Ouk Kim, Y., Park, S. C. & Chun, J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. *Int. J. Syst. Evol. Microbiol.* **66**, 1100–1103 (2016).

34. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. *Mol. Biol. Evol.* **35**(6), 1547–1549 (2018).

35. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. *BMC Bioinform.* **11**, 119 (2010).

36. Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Sayers, E. W. GenBank. *Nucleic Acids Res.* **44**, D67–72 (2016).

37. Lagesen, K. et al. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. *Nucleic Acids Res.* **35**, 3100–3108 (2007).

38. Lowe, T. M. & Chan, P. P. tRNAscan-SE on-line: integrating search and context for analysis of transfer RNA genes. *Nucleic Acids Res.* **44**, W54–57 (2016).

39. Carver, T., Harris, S. R., Berriman, M., Parkhill, J. & McQuillan, J. A. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. *Bioinformatics* **28**, 464–469 (2012).

40. Lechner, M. et al. Proteinortho: detection of (co-)orthologs in large-scale analysis. *BMC Bioinform.* **12**, 124 (2011).

41. Meier-Kolthoff, J. P., Göker, M., Spröer, C. & Klenk, H. P. When should a DDH experiment be mandatory in microbial taxonomy?. *Arch. Microbiol.* **195**, 413–418 (2013).

42. Jain, C. et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. *Nat. Commun.* **9**, 5114 (2018).

43. Kim, M. et al. 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**(2), 346–351 (2014).

44. Morand, A. et al. Human bacterial repertoire of the urinary tract: a potential paradigm shift. *J. Clin. Microbiol.* **57**, e00675–e718 (2019).

## Acknowledgements

This work has benefited from support of the French State, managed by the "Agence Nationale pour la Recherche" including the "Programme d'Investissement d'Avenir" under the reference Méditerranée Infection 10-IAHU-03. We acknowledge Professor Florence Fenollar, Doctor Matthieu Million, Doctor Grégory Dubourd and Professor Chabrol for their significant help.

## Author contributions

D.R., M.T., J.C.L. and P.E.F. conceived and designed the experiments, M.T. and F.C. actively participated in the specimen collection, A.M., L.T., E.K.Y., I.I.N., C.I.L., F.C. and A.L. performed the experiments, A.M., L.T., A.L. and P.E.F. analysed the data, A.M., L.T., C.I.L. and P.E.F. wrote the manuscript. All authors read and approved the final manuscript.

## Competing interests

The authors declare no competing interests.

## Additional information

Correspondence and requests for materials should be addressed to A.M. or P.-E.F.

Reprints and permissions information is available at www.nature.com/reprints.

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
<figure><img src="image_9.png" /></figure>

**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/.

© The Author(s) 2021