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.
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.
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)
Table 1.
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.
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.
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.
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.
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 |
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.
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).
References
- 1.Parte A.C. LPSN—List of Prokaryotic names with Standing in Nomenclature (bacterio.net), 20 years on. Int J Syst Evol Microbiol. 2018;68:1825–1829. doi: 10.1099/ijsem.0.002786. [DOI] [PubMed] [Google Scholar]
- 2.Sakamoto M., Benno Y. Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. Int J Syst Evol Microbiol. 2006;56:1599–1605. doi: 10.1099/ijs.0.64192-0. [DOI] [PubMed] [Google Scholar]
- 3.Sakamoto M., Tanaka Y., Benno Y., Ohkuma M. Parabacteroides faecis sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. 2015;65:1342–1346. doi: 10.1099/ijs.0.000109. [DOI] [PubMed] [Google Scholar]
- 4.Sakamoto M., Kitahara M., Benno Y. Parabacteroides johnsonii sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. 2007;57:293–296. doi: 10.1099/ijs.0.64588-0. [DOI] [PubMed] [Google Scholar]
- 5.Sakamoto M., Suzuki N., Matsunaga N., Koshihara K., Seki M., Komiya H. Parabacteroides gordonii sp. nov., isolated from human blood cultures. Int J Syst Evol Microbiol. 2009;59:2843–2847. doi: 10.1099/ijs.0.010611-0. [DOI] [PubMed] [Google Scholar]
- 6.Lagier J.C., Armougom F., Million M., Hugon P., Pagnier I., Robert C. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect. 2012;18:1185–1193. doi: 10.1111/1469-0691.12023. [DOI] [PubMed] [Google Scholar]
- 7.Lagier J.C., Hugon P., Khelaifia S., Fournier P.E., La Scola B., Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev. 2015;28:237–264. doi: 10.1128/CMR.00014-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Lagier J.C., Khelaifia S., Alou M.T., Ndongo S., Dione N., Hugon P. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol. 2016;1:16203. doi: 10.1038/nmicrobiol.2016.203. [DOI] [PubMed] [Google Scholar]
- 9.Lagier J.C., Edouard S., Pagnier I., Mediannikov O., Drancourt M., Raoult D. Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev. 2015;28:208–236. doi: 10.1128/CMR.00110-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Fournier P.E., Lagier J.C., Dubourg G., Raoult D. From culturomics to taxonomogenomics: a need to change the taxonomy of prokaryotes in clinical microbiology. Anaerobe. 2015;36:73–78. doi: 10.1016/j.anaerobe.2015.10.011. [DOI] [PubMed] [Google Scholar]
- 11.Ramasamy D., Mishra A.K., Lagier J.C., Padhmanabhan R., Rossi M., Sentausa E. A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. Int J Syst Evol Microbiol. 2014;64:384–391. doi: 10.1099/ijs.0.057091-0. [DOI] [PubMed] [Google Scholar]
- 12.Seng P., Drancourt M., Gouriet F., La Scola B., Fournier P.E., Rolain J.M. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 2009;49:543–551. doi: 10.1086/600885. [DOI] [PubMed] [Google Scholar]
- 13.Morel A.S., Dubourg G., Prudent E., Edouard S., Gouriet F., Casalta J.P. 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. 2015;34:561–570. doi: 10.1007/s10096-014-2263-z. [DOI] [PubMed] [Google Scholar]
- 14.Sasser M. 2006. Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME), MIDI. Technical Note 101. Newark, DE: MIDI. [Google Scholar]
- 15.Dione N., Sankar S.A., Lagier J.-C., Khelaifia S., Michele C., Armstrong N. Genome sequence and description of Anaerosalibacter massiliensis sp. nov. New Microbe. New Infect. 2016;10:66–76. doi: 10.1016/j.nmni.2016.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Lo C.I., Sankar S.A., Fall B., Ba B.S., Diawara S., Gueye M.W. High-quality draft genome sequence and description of Haemophilus massiliensis sp. nov. Stand Genom Sci. 2016;11:31. doi: 10.1186/s40793-016-0150-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Zerbino D.R., Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–829. doi: 10.1101/gr.074492.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Luo R., Liu B., Xie Y., Li Z., Huang W., Yuan J. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience. 2012;1:18. doi: 10.1186/2047-217X-1-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.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. 2016;66:1100–1103. doi: 10.1099/ijsem.0.000760. [DOI] [PubMed] [Google Scholar]
- 22.Tan H.Q., Li T.T., Zhu C., Zhang X.Q., Wu M., Zhu X.F. Parabacteroides chartae sp. nov., an obligately anaerobic species from wastewater of a paper mill. Int J Syst Evol Microbiol. 2012;62:2613–2617. doi: 10.1099/ijs.0.038000-0. [DOI] [PubMed] [Google Scholar]