Abstract
Parabacteroides bouchesdurhonensis strain Marseille-P3763T (= CSURP3763) is a new species isolated from the stool of a heathy adult.
Keywords: Culturomics, new species, Parabacteroides bouchesdurhonensis, stool, taxono-genomics
Introduction
Culturomics is a concept developing different culture conditions in order to enlarge our knowledge of the human microbiota through the discovery of previously uncultured bacteria [[1], [2], [3], [4]]. Once an isolate is obtained, 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 (Table 1) and genome sequencing, to describe it [5,6].
Table 1.
Description of Parabacteroides bouchesdurhonensis according to the digitalized protologue TA00969 on the www.imedea.uib.es/dprotologue website
| TAXONUMBER | TA00969 |
|---|---|
| DATE OF THE ENTRY | 2019-05-28 |
| DRAFT NUMBER/DATE | 001 |
| VERSION | Submitted |
| TYPE OF DESCRIPTION | New Description |
| SPECIES NAME | Parabacteroides bouchesdurhonensis |
| GENUS NAME | Parabacteroides |
| SPECIFIC EPITHET | Parabacteroides bouchesdurhonensis |
| SPECIES STATUS | sp. nov. |
| SPECIES ETYMOLOGY | bou.ches.du.rho.nen'sis, N.L. neut. adj. bouchesdurhonensis, pertaining to Bouches du Rhône, the name of the French territory where strain Marseille-P3763 was isolated |
| SUBMITTER | KUETE YIMAGOU EDMOND |
| E-MAIL OF THE SUBMITTER | edmondkuete@yahoo.fr |
| DESIGNATION OF THE TYPE STRAIN | Marseille-P3763T |
| STRAIN COLLECTION NUMBERS | CSURP 3763 |
| 16S rRNA GENE ACCESSION NUMBER | LT722681 |
| GENOME ACCESSION NUMBER [RefSeq] | FYCK00000000 |
| GENOME ACCESSION NUMBER [EMBL] | |
| GENOME STATUS | Complete |
| GC mol % | 40.8 |
| DATA ON THE ORIGIN OF THE SAMPLE FROM WHICH THE STRAIN HAD BEEN ISOLATED | |
| COUNTRY OF ORIGIN | FRANCE |
| REGION OF ORIGIN | Bouches du Rhône |
| DATE OF ISOLATION | 2016-03-15 |
| SOURCE OF ISOLATION | STOOL |
| SAMPLING DATE | 2016-03-12 |
| GEOGRAPHIC LOCATION | MARSEILLE |
| SOURCE OF ISOLATION OF NON-TYPE STRAINS | GUT |
| GROWTH MEDIUM, INCUBATION CONDITIONS [Temperature, pH, and further information] USED FOR STANDARD CULTIVATION | 5% sheep's blood–enriched Columbia agar 37°C PH: 7.5 |
| GRAM STAIN | NEGATIVE |
| CELL SHAPE | Rod |
| MOTILITY | Non-motile |
| SPORULATION (resting cells) | None |
| LOWEST TEMPERATURE FOR GROWTH | 28°C |
| HIGHEST TEMPERATURE FOR GROWTH | 45°C |
| TEMPERATURE OPTIMUM | 37°C |
| HABITAT | HUMAN |
Isolation and growth conditions
In 2016, we isolated from human stool an unidentified bacterial strain. The study was validated by the ethics committee of IHU Méditerranée Infection under number 2016-011. A screening was performed using MALDI-TOF MS on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously described [7]. The spectra obtained (Fig. 1) were imported into MALDI Biotyper 3.0 software (Bruker Daltonics) and analysed against the main spectra of the bacteria included in the database (Bruker database constantly updated with MEPHI database: https://www.mediterranee-infection.com/urms-data-base/http://www.mediterraneeinfection.com/article.php?larub=280&titre=urms-database)). The initial growth was obtained after 48 h of culture on Columbia Agar with 5% sheep blood in anaerobic conditions at 37 °C and pH 7.5.
Fig. 1.
MALDI-TOF MS Reference mass spectrum. 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 done by 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 capillary sequencer (Thermofisher, Saint-Aubin, France), as previously described [8]. The 16S rRNA nucleotide sequences were assembled and corrected using CodonCode Aligner software (http://www.codoncode.com). Strain Parabacteroides bouchesdurhonensis exhibited a 96.68% sequence identity with Parabacteroides chinchillae strain JCM 17104 (GenBank accession number NR_113208.1, the phylogenetically closest species with standing in nomenclature (Fig. 2). We consequently classify this strain as a member of a new species within the genus Parabacteroides, family Tannerellaceae, phylum Bacteroidetes.
Fig. 2.
Phylogenetic tree showing the position of Parabacteroides bouchesdurhonensis strain Marseille-P3763T 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 100 times to generate a majority consensus tree. The scale bar indicates a 5% nucleotide sequence divergence.
Phenotypic characteristics
Colonies were beige in colour and circular in shape with a mean diameter of 1 mm. Bacterial cells were Gram-negative, rod-shaped, ranging in length from 0.4 to 0.8 μm and in width from 0.7 to 1.2 μm and non-motile (Fig. 3). Strain Marseille-P3763T showed catalase-positive and oxidase-negative activities. Characteristics of the strain are summarized in Table 1. API 50CH and API ZYM tests were performed at 37 °C under anaerobic conditions and the results are summarized in Table 2.
Fig. 3.
Electron micrograph of Parabacteroides bouchesdurhonensis strain Marseille-P3763T was acquired with a Hitachi TM4000Plus tabletop scanning electron microscope.
Table 2.
Phenotypic characterization of Parabacteroides bouchesdurhonensis based on the biochemical tests: Profile Index: (A) API 50 CH, (B) API ZYM
| Bacteria: Parabacteroides bouchesdurhonensis | |||
|---|---|---|---|
| Test | Results (+/–) | Test | Results (+/-) |
| (A) API 50 CH | |||
| Control | – | Esculine | - |
| Glycerol | + | Salicine | + |
| Erythrol | + | d-cellobiose | - |
| d-arabinose | + | d-maltose | + |
| l-arabinose | + | d-lactose | + |
| d-ribose | + | d-melibiose | + |
| d-xylose | + | d-saccharose | + |
| l-xylose | + | d-trehalose | + |
| d-adonitol | + | Inuline | + |
| Methyl-βd-xylopyranoside | + | d-melezitose | + |
| d-galactose | + | d-raffinose | + |
| d-glucose | + | Amidon | + |
| d-fructose | + | Glycogene | + |
| d-mannose | + | Xylitol | + |
| l-sorbose | + | Gentibiose | + |
| l-rhammose | + | d-turanose | + |
| Dulcitol | + | d-lyxose | + |
| Inositol | + | d-tagatose | + |
| d-mannitol | + | d-fucose | + |
| d-sorbitol | + | l-fucose | + |
| Methyl-αd-mannopyranoside | + | d-arabitol | + |
| Methyl-αd-glucopyranoside | + | l-arabitol | + |
| N-acetylglucosamine | + | Potassium gluconate | + |
| Amygdaline | + | Potassium 2-cetogluconate | – |
| Arbutine | + | Potassium 5-cetogluconate | + |
| Bacteria: Parabacteroides bouchesdurhonensis | |
|---|---|
| Test | Results (+/–) |
| (B) API ZYM | |
| Control | – |
| Alkaline phosphatase | + |
| Esterase (C 4) | + |
| Esterase lipase (C 8) | + |
| Lipase (C 14) | – |
| Leucine arylamidase | + |
| Valine arylamidase | – |
| Cystine arylamidase | + |
| Trypsine | – |
| α-chymotrypsine | – |
| Acid phosphatase | + |
| Naphthalo-AS-BI-phosphohydrolase | + |
| α-galactosidase | + |
| β-galactosidase | + |
| β-glucuronidase | – |
| α-glucosidase | + |
| β-glucosidase | – |
| N-acetyl-β-glucosaminidase | + |
| α-mannosidase | – |
| α-fucosidase | + |
Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France) with the EZ1 DNA tissue kit and then sequenced on the MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera XT Paired end (Illumina), as previously described [9]. The assembly was performed with a pipeline incorporating different softwares (Velvet [10], Spades [11] and Soap Denovo [12]) on trimmed (Trimmomatic [13]) or raw data. GapCloser was used to reduce assembly gaps. Scaffolds <800 bp and scaffolds with a depth value <25% of the mean depth were removed. The best assembly was selected by using different criteria (17 scaffolds, 19 contigs). The genome of strain Marseille-P3763T is 3.7321 Mb long with a 40.8 mol% G + C content and contains 3024 predicted genes. The degree of genomic similarity of strain Marseille-P3763T with closely related species was estimated using the OrthoANI software [14]. Values among closely related species (Fig. 4) ranged from 69.68% between Parabacteroides distasonis and Parabacteroides chartae to 90.65% between Parabacteroides johnsonii and Parabacteroides merdae. When the isolate was compared with these closely related species, values ranged from 70.54% with Parabacteroides chartae to 78.30% with Parabacteroides chinchillae.
Fig. 4.
Heatmap generated with OrthoANI values calculated using the OAT software between genus species and other closely related species with standing in nomenclature.
Conclusion
Strain Parabacteroides bouchesdurhonensis exhibits a 16S rRNA sequence divergence <98.65% and an OrthoANI value <95% with its phylogenetically closest species with standing in nomenclature, together with unique phenotypic features. It is consequently proposed as the type strain of the new species: Parabacteroides bouchesdurhonensis sp. nov.
Nucleotide sequence accession number
The 16S rRNA gene and genome sequences were deposited in GenBank under accession number LT722681 and FYCK00000000, respectively.
Deposit in culture collections
Strain Marseille-P3763T was deposited in the collections under number CSURP3763.
Conflict of interest
None to declare.
Acknowledgements
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 (ANR, fr: National Agency for Research) (reference: Méditerranée Infection 10-IAHU- 03). The authors thank the Hitachi Corporation for providing the TM4000 Plus Tabletop microscope. They also thank Magdalen Lardière from IHU-Méditerranée Infection, Marseille for reviewing the English and Aurelia Caputo from IHU-Méditerranée Infection, Marseille for submitting the genomic sequences to GenBank.
References
- 1.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]
- 2.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]
- 3.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] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 4.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]
- 5.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]
- 6.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]
- 7.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]
- 8.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]
- 9.Diop A., Khelaifia S., Armstrong N., Labas N., Fournier P.-E., Raoult D. Microbial culturomics unravels the halophilic microbiota repertoire of table salt: description of Gracilibacillus massiliensis sp. nov. Microb Ecol Health Dis. 2016;27:32049. doi: 10.3402/mehd.v27.32049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.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]
- 11.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]
- 12.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]
- 13.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]
- 14.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]