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. 2019 Sep 7;32:100602. doi: 10.1016/j.nmni.2019.100602

Parabacteroides massiliensis sp. nov., a new bacterium isolated from a fresh human stool specimen

S Bellali 1,2, CI Lo 2,3, S Naud 1,2, MDM Fonkou 1,2, N Armstrong 1,2, D Raoult 1,2, P-E Fournier 2,3, F Fenollar 2,3,
PMCID: PMC6796604  PMID: 31641517

Abstract

Parabacteroides massiliensis sp. nov., strain Marseille-P2231T (= CSURP2231 = DSM 101860) is a new species within the family Tannerellaceae. It was isolated from a stool specimen of a 25-year-old healthy woman. Its genome was 5 013 798 bp long with a 45.7 mol% G+C content. The closest species based on 16S rRNA sequence was Parabacteroides merdae strain JCM 9497T with 98.19% sequence similarity. Considering phenotypic features and comparative genome studies, we proposed the strain Marseille-P2231T as the type strain of Parabacteroides massiliensis sp. nov., a new species within the genus Parabacteroides.

Keywords: Bacteria, culturomics, human gut, Parabacteroides massiliensis, taxono-genomics

Introduction

Currently, the genus Parabacteroides includes eight valid species with standing in nomenclature [1]. Among them, Parabacteroides distasonis, Parabacteroides goldsteinii and Parabacteroides merdae previously belonged to the genus Bacteroides but were reclassified as members of the genus Parabacteroides since 2006 [2]. The species Parabacteroides faecis [3] and Parabacteroides johnsonii [4] (faeces) and Parabacteroides gordonii (blood) [5] were all isolated for the first time in humans. Culturomics is a concept developing different culture conditions to enlarge our knowledge of the human microbiota through the discovery of previously uncultured bacteria [6], [7], [8], [9]. Once it was isolated, we used a taxono-genomics approach including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phylogenetic analysis, main phenotypic description and genome sequencing, to describe this strain [10], [11]. Here we describe a new Parabacteroides massiliensis sp. nov., strain Marseille-P2231T (= CSURP2231 = DSM 101860) according the concept of taxono-genomics.

Isolation and growth conditions

In 2017, we isolated from a fresh stool sample of a 25-year-old healthy woman an unidentified bacterial strain. Screening was performed using MALDI-TOF MS on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously described [12]. The obtained spectra (Fig. 1) were imported into MALDI Biotyper 3.0 software (Bruker Daltonics) and analysed against the main spectra of the bacteria included in two databases (Bruker and the constantly updated MEPHI databases). The study was validated by the ethics committee of the IHU Méditerranée Infection under number 2016-010. Initial growth was obtained after 72 hours of culture in a Colombia agar enriched with 5% sheep's blood (bioMérieux, Marcy l’Etoile, France) in strict anaerobic conditions at 37°C and pH 7.5.

Fig. 1.

Fig. 1

MALDI-TOF MS reference mass spectrum of Parabacteroides massiliensis sp. nov. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Strain identification

The 16S rRNA gene was sequenced to classify this bacterium. Amplification was carried out using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequencing using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and ABI Prism 3130xl Genetic Analyzer capillary3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously described [13]. The 16S rRNA nucleotide sequences were assembled and corrected using CodonCode Aligner software (http://www.codoncode.com). Strain Marseille-P2231T exhibited a 98.19% sequence identity with Parabacteroides merdae strain JCM 9497T (GenBank accession number NR_041343), the phylogenetically closest species with standing in nomenclature (Fig. 2). We consequently classify this strain as a member of a new species within the family Tannerellaceae, phylum Bacteroidetes.

Fig. 2.

Fig. 3

Phylogenetic tree showing the position of Parabacteroides massiliensis strain Marseille-P2231T relative to other phylogenetically close neighbours. The respective GenBank accession numbers for 16S rRNA genes are indicated in parenthesis. Sequences were aligned using MUSCLE v3.8.31 with default parameters and phylogenetic inferences were obtained using the maximum likelihood method within MEGA 7 software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a majority consensus tree. The scale bar indicates a 2% nucleotide sequence divergence.

Phenotypic characteristics

Colonies were circular and smooth with a mean diameter of 1.2 mm. Bacterial cells were Gram-negative, rod-shaped, ranging in length from 1.27 to 2.46 μm and in width from 0.45 to 0.73 μm (Fig. 3). Strain Marseille-P2231T showed catalase-negative and oxidase-negative activities. Main phenotypic properties of strain Marseille-P2231T were studied by using the API 50 CH strips (Table 1), API ZYM strips (Table 2) and API 20A strips (Table 3). The main characteristics of strain Marseille-P2231T are summarized on digitalized protologue (www.imedea.uib.es/dprotologue) under the number TA00985. The biochemical and phenotypic features of strain Marseille-P2231T were compared with those of other close representative strains in the Porphyromonadaceae family (Table 4)

Fig. 3.

Fig. 2

Scanning electron micrograph of Parabacteroides massiliensis strain Marseille-P2231T using TM4000 microscope from HITACHI. Scale bar and acquisition settings are shown on the original micrograph.

Table 1.

Biochemical tests of Parabacteroides massiliensis (API 50 CH strips)

Tests Results Tests Results
Control Esculin +
Glycerol Salicin +
Erythrol d-cellobiose +
d-arabinose d-maltose +
l-arabinose d-lactose +
d-ribose d-melibiose +
d-xylose w d-saccharose +
l-xylose + d-trehalose +
d-adonitol Inulin
Methyl βd-xylopyranoside + d-melezitose +
d-galactose + d-raffinose w
d-glucose + Starch w
d-fructose + Glycogen
d-mannose + Xylitol
l-sorbose Gentibiose w
l-rhammose d-turanose +
Dulcitol d-lyxose
Inositol d-tagatose w
d-mannitol w d-fucose
d-sorbitol l-fucose
Methyl αd-mannopyranoside d-arabitol
Methyl αd-glucopyranoside w l-arabitol
N-acetylglucosamine + Potassium gluconate
Amygdalin + Potassium 2-ketogluconate
Arbutin Potassium 5-ketogluconate +

+, positive result; , negative result; w, weakly positive.

Table 2.

Biochemical tests of Parabacteroides massiliensis (API ZYM strips)

Tests Results
Alkaline phosphatase +
Esterase (C4)
Esterase Lipase (C8)
Lipase (C14)
Leucine arylamidase +
Valine arylamidase
Cystine arylamidase
Trypsin
α-chymotrypsin
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
α-galactosidase +
β-galactosidase +
β-glucuronidase +
α-glucosidase
β-glucosidase
N-acetyl- β-glucosaminidase +
α-mannosidase
α-fucosidase

+, positive result; , negative result.

Table 3.

Biochemical tests of Parabacteroides massiliensis (API 20A strips)

Tests Results
l-tryptophan +
Urea
d-glucose +
d-mannitol +
d-lactose +
d-saccharose +
d-maltose +
Salicin +
d-xylose +
l-arabinose +
Gelatin (bovine origin) +
Esculin ferric citrate +
Glycerol
d-cellobiose +
d-mannose +
d-melezitose +
d-raffinose
d-sorbitol
l-rhamnose +
d-trehalose +

+, positive result; , negative result.

Table 4.

Differential characteristics of 1, Parabacteroides massiliensis strain Marseille-P2231, compared with other closely related Porphyromonadaceae species: 2, Parabacteroides merdae[2]; 3, Parabacteroides johnsonii[4]; 4, Parabacteroides gordonii[5]; 5, Parabacteroides faecis strain 157T[3]; 6, Parabacteroides chartae NS31-3T[22]

Properties 1 2 3 4 5 6
Cell diameter (μm) 0.4–0.7 0.8–1.6 0.8 0.8 1.0 0.7–1.0
Oxygen requirement
Gram stain
Motility
Endospore formation
Acid phosphatase NA NA NA NA +
Catalase + variable +
Indole
Urease
Alkaline phosphatase + + + + + +
β-galactosidase + + + + + +
Mannose + + + + + +
Raffinose w + + + + +
Sucrose + + + + + +
Glucose + + + + + +
d-xylose + + + + + +
Maltose + + + + + +
Glycerol
Lactose + + + + + +
G+C content (mol%) 45.7 44.0 47.6 44.6 41.8 37.2
Habitat Human stool Human faeces Human faeces Human blood Human faeces Wastewater

+, positive result; −, negative result; w, weakly positive; NA, data not available.

Cellular fatty acid methyl ester analysis was performed by gas chromatography/mass spectrometry. Two samples were prepared with approximately 5 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as described by Sasser [14]. Gas chromatography/mass spectrometry analyses were performed as described elsewhere [15]. The most abundant fatty acid by far was 12-methyl-tetradecanoic acid (43%), followed by 3-hydroxy15-methyl-hexadecanoic acid (19%) and hexadecanoic acid (10%). Several branched structures and specific 3-hydroxy fatty acids were described. Minor amounts of unsaturated and other saturated fatty acids were also detected (Table 5).

Table 5.

Cellular fatty acid composition (%) of Parabacteroides massiliensis strain Marseille-P2231T

Fatty acids Name Mean relative % a
15:0 anteiso 12-methyl-Tetradecanoic acid 43.1 ± 1.1
17:0 3-OH iso 3-hydroxy-15-methyl-Hexadecanoic acid 18.5 ± 0.4
16:0 Hexadecanoic acid 9.5 ± 0.5
16:0 3-OH 3-hydroxy-Hexadecanoic acid 5.0 ± 0.2
15:0 Pentadecanoic acid 4.5 ± 0.3
15:0 iso 13-methyl-Tetradecanoic acid 3.5 ± 0.2
17:0 3-OH anteiso 3-hydroxy-14-methyl-Hexadecanoic acid 4.8 ± 0.8
18:2n6 9,12-Octadecadienoic acid 2.3 ± 0.1
5:0 iso 3-methyl-Butanoic acid 2.0 ± 0.2
18:1n9 9-Octadecenoic acid 1.9 ± 0.1
16:1n7 9-Hexadecenoic acid 1.1 ± 0.1
14:0 Tetradecanoic acid TR
17:0 3-OH 3-hydroxy-Heptadecanoic acid TR
17:0 anteiso 14-methyl-Hexadecanoic acid TR
17:0 iso 15-methyl-Hexadecanoic acid TR
14:0 iso 12-methyl-Tridecanoic acid TR
18:0 Octadecanoic acid TR
16:0 anteiso 13-methyl-Pentadecanoic acid TR
13:0 iso 11-methyl-Dodecanoic acid TR
17:0 Heptadecanoic acid TR
13:0 anteiso 10-methyl-Dodecanoic acid TR
a

Mean peak area percentage; TR, trace amounts <1%.

Genome sequencing

Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France) with the EZ1 DNA tissue kit and then sequenced using MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit (Illumina), as previously described [16]. The assembly was performed with a pipeline incorporating different software (Velvet [17], Spades [18] and Soap Denovo [19]), and trimmed data (MiSeq and Trimmomatic [20] software) or untrimmed data (only MiSeq software). GapCloser was used to reduce assembly gaps. Scaffolds <800bp in length and scaffolds with a depth value <25% of the mean depth were removed. The best assembly was selected using different criteria (number of scaffolds, N50, number of N). The genome of strain Marseille-P2231T is 5 013 798 bp long (23 scaffolds, 27 contigs, 762 401 N50) with a 45.7 mol% G+C content and contains 4 195 predicted genes. The degree of genomic similarity of Marseille-P2231T with closely related species was estimated using the OrthoANI software [21]. Values among closely related species (Fig. 4) ranged from 70.20% between Parabacteroides massiliensis and Parabacteroides chartae to 91.01% between P. merdae and P. johnsonii. When the isolate was compared with these closely related species, values ranged from 70.20% with P. chartae to 88.73% with P. merdae.

Fig. 4.

Fig. 4

Heatmap generated with OrthoANI values calculated using the OAT software between Parabacteroides massiliensis and other closely related species with standing in nomenclature.

Conclusion

Strain Marseille-P2231T exhibiting a 16S rRNA sequence divergence <98.7% and an OrthoANI value <95% with its phylogenetically closest species with standing in nomenclature, is consequently proposed as the type strain of the new species Parabacteroides massiliensis sp. nov.

Description of Parabacteroides massiliensis sp. nov.

Parabacteroides massiliensis (mas.si.li.en'sis, L. fem. adj., massiliensis, ‘of Massilia’, the Latin name of Marseille, where this strain was isolated). Cells are obligate anaerobic, Gram-negative, non-motile and non-spore-forming. Catalase and oxidase activities are negative. Cells have a length of 1.27–2.46 μm and a width of 0.45–0.73 μm. Colonies grown at 37°C on 5% sheep-blood-enriched Columbia agar (bioMérieux), and were circular and smooth after 72 hours of incubation under anaerobic conditions. They had a mean diameter of 1.2 mm on agar. Strain Marseille-P2231 reacts positively with leucine arylamidase, alkaline phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-d-glucosaminidase, d-glucose, d-fructose, d-mannose, esculin, salicin, lactose, melibiose, sucrose and potassium 5-ketogluconate. Negative reactions were observed with esterase, lipase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase, α-mannosidase, α-fucosidase, glycerol, ribose, d-adonitol, rhammose, sorbitol, inulin, glycogen, xylitol, fucose, arabitol, arabitol and potassium 2-ketogluconate. The most abundant fatty acid by far was 12-methyl-tetradecanoic acid (43%) followed by 3-hydroxy 15-methyl-hexadecanoic acid (19%) and hexadecanoic acid (10%). The genome is 5 013 798 bp long and its G+C content is 45.7 mol%. Strain Marseille-P2231T, isolated from a fresh stool sample of a 26-year-old healthy woman, was deposited in the CSUR and DSMZ collections under accession numbers CSURP2231 and DSM 101860, respectively. The 16S rRNA and genome sequences are available in the GenBank database under accession numbers LN899828 and FTLH00000000, respectively.

Nucleotide sequence accession number

The 16S rRNA gene and genome sequences were deposited in GenBank under accession number LN899828, and FTLH00000000, respectively.

Deposit in culture collections

Strain Marseille-P2231T or strain SN4T was deposited in strain collection under number (= CSURP2231T = DSM 101860).

Acknowledgements

This work was also supported by Région Provence Alpes Côte d’Azur and European funding FEDER PRIMI. The authors thank the Hitachi Corporation for providing the TM4000Plus Tabletop microscope. They also thank Aurelia Caputo for submitting the genomic sequences to GenBank.

Conflict of interest

None to declare.

Funding sources

This work was funded by the IHU Méditerranée Infection (Marseille, France) and by the French Government under the Investissements d'avenir (Investments for the Future) programme managed by the Agence Nationale de la Recherche (reference: Méditerranée Infection 10-IAHU-03).

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