Massilicoli timonensis sp. nov., a new bacterium isolated from the human microbiota.

Massilicoli timonensis sp. nov., strain Marseille-P3755T (= CSUR P3755 = DSM 103513) is a new bacterial species from the phylum Firmicutes and the family Clostridiales which was isolated from the human gut microbiota.

Introduction

Deciphering the bacterial diversity involved in normal and pathogenic functions appears fundamental [1]. In order to unveil the human gut microbial diversity, the culturomics approach, based on diversified culture conditions, was designed to isolate as yet uncultured species and to complement 16S ribosomal RNA (rRNA) metagenomics [2], [3], [4]. Furthermore, a new taxonomic strategy termed taxonogenomics was developed to include the analysis of complete genome sequences in combination with phenotypic characteristics [5]. Here we report a short description of strain Marseille-P3755T that was isolated from the human gut microbiota.

Isolation and growth conditions

As part of a culturomics study, a stool sample was collected from an 85-year-old Frenchwoman admitted in the Timone Hôpital Marseille in December 2016. A total of 0.3 g of faecal specimen was serially diluted in 900 μL of phosphate-buffered saline (Life Technologies, Carlsbad, CA, USA), and 50 μL of each dilution was seeded on 5% sheep's blood–enriched Columbia agar (bioMérieux, Marcy l’Etoile, France). After 3 days of incubation at 37°C in an anaerobic atmosphere generated by AnaeroGen (bioMérieux), several colonies grew and were isolated. The purified isolate obtained after three subcultures from a single colony could not be identified by MALDI-TOF MS. The screening was performed on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany), as previously reported [6]. Spectra obtained (Fig. 1) were imported and analysed by Biotyper 3.0 software against the Bruker database, which is continuously updated with information from the Microbes Evolution Phylogeny and Infections (MEPHI) database [1].

FIG. 1

MALDI-TOF MS reference spectrum of Massilicoli timonensis strain Marseille-P3755T. Reference spectrum was generated by comparison of spectra from 12 individual colonies.

This study was approved by the ethics committee of the Institut Fédératif de Recherche 48 under reference 2016-010. The patient provided written informed consent for participating in this study.

Phenotypic characteristics

The strain Marseille-P3755 colonies grown on Columbia agar plates after 3 days were circular and translucent, with a diameter of about 0.5 to 1 mm. Strain Marseille-P3755 is a strict anaerobic bacterium, has Gram-negative bacilli (0.3 μm × 2–3 μm), and is nonmotile and non–spore forming (Fig. 2). Strain Marseille-P3755 was negative for catalase and oxidase activities. Biochemical characteristics were investigated using API ZYM, API 50CH and API 20NE strips (bioMérieux). In API ZYM, enzymatic activities were observed for phosphatase acid and naphtol-AS-BI-phosphohydrolase. A slightly positive reaction was observed for phosphatase alkaline, esterase (C4) and esterase lipase (C8); the results of the other tests were negative. Using API 50CH strips, positive reactions were observed with: L-arabinose, D-ribose, D-xylose, L-xylose D-adonitol, D-glucose, D-fructose, dulcitol, inositol, D-mannose methyl-αD-mannopyranoside, N-acetyl-glucosamine amygdalin, arbutin, salicin, Dmaltose, D-saccharose, inulin glycogen, xylitol, gentiobiose, Larabitol and potassium 5-ketogluconate. Negative reactions were observed with: negative: glycerol, erythritol, D-arabinose, methyl-βD-xylopyranoside, D-galactose, D-mannose, L-sorbose, L-rhamnose, Dsorbitol, methyl-αD-glucopyranoside, esculin ferric citrate, Dcellobiose, D-lactose, D-melibiose D-trehalose D-melezitose Draffinose, amidone, D-turanose, D-lyxose, D-tagatose D-fucose, Lfucose, D-arabitol, potassium gluconate and potassium 2-ketogluconate. In API 20NE, all test results were negative, including nitrate reduction, indole formation, arginine dihydrolase and hydrolysis of esculin and gelatin.

FIG. 2

Scanning electron microscopy (SEM) of stained Massilicoli timonensis. sp. nov. Colony was collected from agar and immersed into a 2.5% glutaraldehyde fixative solution. Then a drop of suspension was directly deposited on a poly-l-lysine–coated microscope slide for 5 minutes and treated with 1% phosphotungstic acid (PTA) aqueous solution (pH 2.0) for 2 minutes to increase SEM image contrast. Slide was gently washed in water, air dried and examined with a tabletop SEM (Hitachi TM4000) approximately 60 cm high and 33 cm wide to evaluate bacteria structure. Scales and acquisition settings are shown.

Strain identification

In order to classify this bacterium, the 16S recombinant DNA (rDNA) gene was amplified using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequenced with the Big Dye Terminator v1.1 Cycle Sequencing Kit and the 3500xLGenetic Analyzer capillary sequencer (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [7]. The 16S rDNA nucleotide sequence was assembled and corrected using CodonCode Aligner software (https://www.codoncode.com/).

Strain Marseille-P3755T exhibited a 95.0% 16S rDNA similarity with Dielma fastidiosa strain JC13 (GenBank accession no. NR_125593.1), the phylogenetically closest species with standing in nomenclature (Fig. 3). We consequently proposed to classify this strain as a new genus named Massilicoli within the Firmicutes phylum. Massilicoli timonensis strain Marseille-P3755T is the species type.

FIG. 3

Phylogenetic tree highlighting position of Massilicoli timonensis sp. nov. with regard to other closely related species. GenBank accession numbers of 16S ribosomal RNA are indicated in parentheses. Sequences were aligned using MUSCLE with default parameters; phylogenetic inferences were obtained by maximum likelihood method and MEGA 7 software. Bootstrap values were obtained by repeating analysis 1000 times to generate majority consensus tree, indicated at nodes. Scale bar indicates 2% nucleotide sequence divergence.

Genome sequencing

Genomic DNA was extracted using the EZ1 biorobot with the EZ1 DNA tissue kit (Qiagen, Hilden, Germany), then sequenced on a MiSeq sequencer (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired End (Illumina), as previously described [8]. The assembly was performed using a pipeline containing several softwares (Velvet [9], Spades [10] and Soap Denovo [11]) on trimmed (MiSeq and Trimmomatic [12] softwares) or untrimmed data (only MiSeq software). GapCloser was used to reduce assembly gaps. Scaffolds <800 bp and scaffolds with a depth value lower than 25% of the mean depth were removed. The best assembly was selected by using different criteria (number of scaffolds, N50, number of N). The genome of strain Marseille-P3755T was 3 118 584 bp long with a 53.0 mol% G + C content. The degree of genomic similarity of strain Marseille-P3755T with closely related species was estimated with OrthoANI software [13]. OrthoANI values among closely related species (Fig. 4) ranged from 61.80% between Bulleidia extructa and Massilicoli timonensis to 70.36% between Clostridium innocuum and Eubacterium cylindroides. When M. timonensis was compared to these closely species, values ranged from 62.43% with Dielma fastidia to 70.36% with Clostridium innocuum.

FIG. 4

Heat map generated with OrthoANI values calculated using OAT software between Massilicoli timonensis sp. nov. and other closely related species with standing in nomenclature.

Conclusion

On the basis of unique phenotypic features, including MALDI-TOF MS spectrum, a 16S rRNA sequence divergence greater than >1.3% and an OrthoANI value < 95% with the phylogenetically closest species with standing in nomenclature, we formally propose the creation of the new genus ‘Massilicoli’ gen. nov., and Massilicoli timonensis sp. nov., strain Marseille-P3755T is the type strain.

Description of Massilicoli gen. nov.

Massilicoli (mas.si.li.co.li, N.L. masc. n., association of Massilia, the Latin name of Marseille, France, and colon, from which the type strain was isolated).

Description of Massilicoli timonensis strain Marseille-P3755T gen. nov., sp. nov.

Massilicoli timonensis (ti.mo.nen'sis, L. masc. adj., timonensis from Timone, the name of the university hospital in Marseille, France, where the strain type was isolated).

The characteristics of the species are listed in Table 1. The type strain is Marseille-P3755T (= CSUR P3755 = DSM 103513).

TABLE 1

Description of Massilicoli timonensis sp. nov.

Axonumber	TA00843
Species name	Massilicoli timonensis
Genus name	Massilicoli
Specific epithet	timonensis
Species status	sp. nov.
Species etymology	Massilicoli (mas.si.li.co.li, N.L. masc. n., association of Massilia, the Latin name of Marseille, France, and colon, from which the type strain was isolated)
	Massilicoli timonensis (ti.mo.nen'sis, L. masc. adj., timonensis from Timone, the name of university hospital in Marseille, France, where the strain type was isolated)
Designation of type strain	Strain Marseille-P3755
Strain collection numbers	(= CSUR P3755 = DSM 103513).
16S rRNA gene accession number	LT899395
Genome accession number (EMBL)	OEMR00000000
Genome status	Draft
Genome size	3 118 584 bp
GC mol%	53.0
Data on origin of sample from which strain had been isolated
Country of origin	France
Region of origin	Marseille
Source of isolation	Human stool
Gram stain	Negative
Cell shape	Rod
Motility	Motile
Colony morphology	On Columbia agar plates, colonies are circular and translucent, with diameter about 0.5 to 1 mm after 3 days of incubation at 37°C
Temperature optimum	37°C
pH optimum	7
Oxidase	Negative
Catalase	Negative

Data shown according to protologue TA00843 at Digital Protologue website (http://imedea.uib-csic.es/dprotologue/).

Nucleotide sequence accession number

The 16S rRNA gene and genome sequences were deposited in GenBank under accession numbers LT899395 and OEMR00000000 respectively.

Deposit in a culture collection

Strain Marseille-P3755T was deposited in the Collection de Souches de l’Unité des Rickettsies (CSUR) under P3755 and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under DSM 103513.

MALDI-TOF MS spectrum

The MALDI-TOF MS spectrum of ‘Massilicoli timonensis’ Marseille-P3755T is available online (http://backup.mediterranee-infection.com/article.php?larub=280&titre=urms-database).