Peptoniphilus Colimassiliensis sp. nov. and Peptoniphilus Urinimassiliensis sp. nov., Two New Species Isolated From Humans


 Strains Marseille-P3761 and Marseille-P3195 are representatives of two bacterial species isolated from human specimens. Strain Marseille-P3761 was isolated from the stool of a healthy volunteer, while strain Marseille-P3915 was cultivated from the urine of a kidney transplant recipient. Both strains are anaerobic Gram-positive cocci bacteria. Both are catalase-negative and oxidase-negative and grow optimally at 37°C in anaerobic conditions. They also metabolize carbohydrates such as galactose, glucose, fructose, and glycerol. The major fatty acids were hexadecanoic acid for both strains, Marseille-P3761 (38%) and Marseille-P3195 (31%). The highest DNA-DNA hybridization values of Marseille-P3761 and Marseille-P3195 strains when compared to their closest phylogenetic relatives were 52.3% and 56.4%, respectively. The morphological, biochemical, phenotypic and genomic characteristics strongly support that these strains are new members of the Peptoniphilus genus. Thus, we suggest that strains Marseille-P3761 (CSUR P3761 = CCUG71569) and Marseille-P3195 (CSUR P3195 = DSM 103468) are the type strains of two new Peptoniphilus species, for which we propose the names Peptoniphilus colimassiliensis sp. nov. and Peptoniphilus urinimassiliensis sp. nov., respectively.


Introduction
Understanding the role that bacterial diversity plays in diseases that affect humans is essential (Turnbaugh et al. 2007). The implementation of the Culturomics strategy, a concept which is based on diversi ed culture conditions in order to enlarge our knowledge of the human microbiota, has enabled the discovery of many previously uncultured bacteria (Lagier et al. 2016(Lagier et al. , 2018. In order to describe new bacterial isolates, we used the taxono-genomics approach that includes matrix-assisted laser desorption-ionisation time-of-ight (MALDI-TOF) mass spectrometry (MS) analysis, phylogenetic inference, description of the main phenotypic characteristics and genome sequencing and comparison to describe them (Fournier and Drancourt 2015;. The genus Peptoniphilus was described by Ezaki et al. as early as 2001 (Ezaki et al. 2001). Members of this genus are Gram-positive anaerobic cocci, non-motile, non-saccharolytic, and their major energy source results from the use of peptones and oligopeptides (Song, Liu and Finegold 2007). Peptoniphilus spp. have mostly been isolated from human clinical samples, notably vaginal discharges and peritoneal and gland abscesses (Ezaki et al. 2001). Currently, there are 20 species validly published with standing in the nomenclature (https://lpsn.dsmz.de/genus/peptoniphilus).
Herein, we report a broad description of two new Peptoniphilus species named Peptoniphilus urinimassiliensis sp. nov., strain Marseille-P3195 (previously reported) and Peptoniphilus colimassiliensis sp. nov., strain Marseille-P3761, both of which were isolated from human samples.

Strain isolation and identi cation
Strain Marseille-P3195 was isolated in a urine sample from a young man who had just received a kidney transplant for genetic focal segmental glomerulosclerosis (Brahimi et al. 2017), while strain Marseille-P3761 was isolated in a fresh stool sample from a volunteer living in France. These two bacterial strains were retrieved seven days after pre-incubation in an anaerobic blood culture bottle (Becton-Dickinson Diagnostics, Le Pont-de-Claix, France) supplemented with 5% sheep blood at 37°C. Individuals gave their free and informed consent for the project before sampling. The study was approved by the ethics committee of Institut Fédératif de Recherche IFR48 under number 2016-010 as part of a long term culturomics study of the human microbiota.
Attempts were made to identify bacterial colonies using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany), as previously reported (Lo et al. 2015). The obtained spectra were imported and analysed using Biotyper 3.0 software. They were then incremented in our local mass spectrometry database (https://www.mediterranee-infection.com/urms-data-base).
No identi cation was successful using MALDI-TOF MS. Therefore, sequencing of the 16S rRNA gene was performed for each of the studied strains using the primer pair fD1 and rP2 (Weisburg et al. 1991). They were sequenced using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and 3500xL Genetic Analyzer capillary3500xL sequencer (Thermo Fisher, Saint-Aubin, France), as previously described (Lo et al. 2016). The 16S rRNA nucleotide sequences were assembled and corrected using CodonCode Aligner software (http://www.codoncode.com).

Phenotypic and biochemical characteristics
Bacterial colonies of strains Marseille-P3761 and Marseille-P3195 appear distinctly on 5% sheep blood-enriched Columbia (bioMérieux, Marcy l'Étoile, France). Phenotypic tests such Gram staining, sporulation, catalase and oxidase reactions were performed for each strain, as previously reported (Brahimi et al. 2017). The optimal temperature and pH for the growth of each strain was sought. Strains were inoculated on 5% sheep blood-enriched Columbia agar (bioMérieux) and incubated under different temperatures (20, 28, 32, 37, 45, and 56°C) and different pH levels (5, 6, 7, 7.5, 8, and 8.5). In addition, biochemical properties such as carbohydrate metabolism and enzymatic activities of these two strains were revealed using the API strips test (ZYM and 50 CH, bioMérieux). The morphology of the bacterial cells of each strain was highlighted using a TM4000 microscope (Hitachi Group, Krefeld, Germany). For each strain, 50 mg of bacterial biomass was collected from several culture plates in order to prepare the fatty acid methyl ester analysis, as previously described (Dione et al. 2018).

Genomes sequencing and analysis
The total DNA of the genomes was recovered using the EZ1 biorobot (Qiagen, Courtaboeuf, France) and the EZ1 DNA tissue kit. Sequencing was performed using MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT paired end (Illumina), as previously described (Morel et al. 2015). Several bioinformatics, including Velvet (Zerbino and Birney 2008), Spades (Bankevich et al. 2012), and SOAPdenovo (Luo et al. 2012) were used to assemble the genomes. GapCloser software (Xu et al. 2019) was used to reduce assembly gaps. Scaffolds which had fewer than 800 base pairs (bp) or had 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 each strain was evaluated by processing sequences using the Orthologous ANI Tool (OAT) software (Lee et al. 2016). Furthermore, the Genome-to-Genome Distance Calculator (GGDC) web server, which is available online (http://ggdc.dsmz.de), was used to calculate DNA-DNA hybridisation (DDH) values between the genomes of closest species, as previously described (Meier-Kolthoff et al. 2013).

Results And Discussion
Growth conditions of strains Different growth temperatures and pH levels were tested with these strains. They all grew optimally at 37°C in anaerobic conditions. The optimal pH was 7 for strain Marseille-P3761 and Marseille-P3195. Bacterial strains grew well, with distinct colonies on 5% sheep blood-enriched Columbia agar.

Morphology and biochemical characteristics
The colonies of the two bacterial strains are similar; they appear grey and circular on 5% sheep blood-enriched Columbia agar. The two strains are Gram-positive cocci, catalase and oxidase negative. They do not produce spores. Using API ZYM, esterase (C4), α-chymotrypsin, and naphthol-AS-  Supplementary Table S1.
The main biochemical characteristics of these strains were compared with those of other closely related Peptoniphilus species (Table 1). The cell morphology of each strain was revealed by scanning electron microscope. They are sphere-shaped bacteria and can aggregate in duplicate (Supplementary Figure S1). Hexadecanoic acid was detected as a major fatty acid for strains Marseille-P3761 (38%) and Marseille-P3195 (31%).
Minor amounts of unsaturated and other saturated structures were also detected ( Table 2).   sequence identity with Peptoniphilus coxii RMA 16757 (GenBank accession number NR_117556.1). The values obtained were below the threshold value of 98.65% recommended to delimit new prokaryotic species. Given these phylogenetic criteria, we consequently classi ed these strains as new members belonging to the genus Peptoniphilus within the phylum Firmicutes. In addition, the phylogenetic tree ( Figure 1) shows the positions of these two new species among related Peptoniphilus species with a validly published name.

Comparison and genomic speci cities
The genome size of Peptoniphilus colimassiliensis sp. nov., strain Marseille-P3761 was 1,986,843 bp long with a 48.6 mol% G+C content, whereas the genome of Peptoniphilus urinimassiliensis sp. nov., strain Marseille-P3195, was 1,822,830 bp with a 49.7 mol% G+C content (Figure 2).
The genome and 16S rRNA sequence were deposited in GenBank under accession numbers FTPC00000000 and LT598577, respectively. Figure 1 Phylogenetic trees highlighting the position of Peptoniphilus colimassiliensis sp. nov. and Peptoniphilus urinimassiliensis sp. nov. based on the 16S rRNA gene sequences relative to the most closely related type strains within the genus Peptoniphilus. Genbank accession numbers are indicated in parentheses. Sequences were aligned using MUSCLE with default parameters, phylogenetic inference were obtained using the maximum likelihood method and the MEGA 7 software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1,000 times to generate a majority consensus tree. The scale bar indicates a 1% nucleotide sequence.