Abstract
Brachybacterium timonense strain Marseille-P4339T (=CSURP4339, =CECT9821) is a new species isolated from human sputum.
Keywords: Brachybacterium timonense, culturomics, new species, sputum, taxonogenomics
Introduction
Culturomics is the concept of 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 a bacterium is isolated, a taxonogenomic approach is used, including MALDI-TOF MS, phylogenetic analysis, main phenotypic description and genome sequencing, to describe it [5], [6].
Isolation and growth conditions
In 2017 we isolated from the human sputum an unidentified bacterial strain. The study was validated by the ethics committee of IHU Méditerranée Infection under number 2016-011. Screening was performed by MALDI-TOF MS on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously described [7]. 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 the database (Bruker database constantly updated with Microbes Evolution Phylogeny and Infections (MEPHI) database; http://www.mediterranee-infection.com/article.php?larub=280&titre=urms-database).The initial growth was obtained after 48 hours' culture on Columbia agar with 5% sheep's 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 reference spectrum generated.
Strain identification
The 16S rRNA gene was sequenced in order to classify this bacterium. Amplification was done by using the primer pair fD1 and rP2 (Eurogentec, Angers, France), and sequencing by 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 Brachybacterium timonense exhibited a 97.24% sequence identity with Brachybacterium faecium strain DSM 4810 (GenBank accession number NR_074655.2), the phylogenetically closest species with standing in nomenclature (Fig. 2). We consequently classified this strain as a member of a new species within the genus Brachybacterium, family Dermabacteraceae, phylum Actinobacteria.
Fig. 2.
Phylogenetic tree showing position of Brachybacterium timonense strain Marseille-P4339T relative to other phylogenetically close neighbours. Respective GenBank accession numbers for 16S rRNA genes are indicated in parentheses. Sequences were aligned using Muscle v3.8.31 with default parameters and phylogenetic inferences were obtained using maximum likelihood method within MEGA 7 software. Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 100 times to generate majority consensus tree. Scale bar indicates 5% nucleotide sequence divergence.
Phenotypic characteristics
Colonies were cocci with a mean diameter of 0.8 μm. Bacterial cells were Gram positive and round (Fig. 3). Strain Marseille-P4339T 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 (Table 2). By comparison with closely related species (Brachybacterium faecium Collins et al., 1988) [9], strain Marseile-P4339 has a similar phenotypic profile.
Fig. 3.
Electron micrograph of Brachybacterium timonense strain Marseille-P4339T was acquired with Hitachi TM4000Plus tabletop scanning electron microscope. Scale bar and acquisition settings are detailed on micrograph.
Table 1.
Description of Brachybacterium timonense according to digitalized protologue TA00881 (www.imedea.uib.es/dprotologue)
| Characteristic | Value |
|---|---|
| Taxonumber | TA00881 |
| Date of entry | 2019-04-19 |
| Draft number/date | 001 |
| Version | Submitted |
| Species name | Brachybacterium timonense |
| Genus name | Brachybacterium |
| Specific epithet | Brachybacterium timonense |
| Species status | sp. nov. |
| Species etymology | ti.mo.nen'se, N.L. masc. adj., timonense from Latin name of Hôpital de la Timone, hospital in Marseille, where strain Marseille-P4339 was isolated |
| Submitter | Kuete Yimagou Edmond |
| E-mail of submitter | edmondkuete@yahoo.fr |
| Designation of type strain | Marseille-P4339T |
| Strain collection numbers | CSURP4339 |
| 16S rRNA gene accession number | LT962482 |
| Genome accession number [EMBL] | OIWY00000000 |
| Data on origin of sample from which strain had been isolated | |
| Country of origin | France |
| Region of origin | Paca |
| Source of isolation | Sputum |
| Sampling date | 2016-08-14 |
| Geographic location | Marseille |
| Gram stain | Positive |
| Cell shape | Coccus |
| Motility | Nonmotile |
| Sporulation (resting cells) | None |
| Lowest temperature for growth | 25 |
| Highest temperature for growth | 45 |
| Temperature optimum | 37 |
| Oxidase | Positive |
| Catalase | Negative |
| Habitat | Human |
Table 2.
Phenotypic characterization of Brachybacterium timonense based on biochemical tests
| Test | Result |
|---|---|
| API 50 CH | |
| Control | — |
| Glycerol | + |
| Erythrol | + |
| d-Arabinose | + |
| l-Arabinose | + |
| d-Ribose | + |
| d-Xylose | + |
| l-Xylose | + |
| d-Adonitol | + |
| Methyl-βd-xylopyranoside | + |
| d-Galactose | + |
| d-Glucose | + |
| d-Fructose | + |
| d-Mannose | + |
| l-Sorbose | + |
| l-Rhammose | + |
| Dulcitol | + |
| Inositol | + |
| d-Mannitol | + |
| d-Sorbitol | + |
| Methyl-αd-mannopyranoside | + |
| Methyl-αd-glucopyranoside | + |
| N-Acetylglucosamine | + |
| Amygdaline | + |
| Arbutine | + |
| Esculine | + |
| Salicine | + |
| d-Cellobiose | + |
| d-Maltose | + |
| d-Lactose | + |
| d-Melibiose | + |
| d-Saccharose | + |
| d-Trehalose | + |
| Inuline | + |
| d-Melezitose | + |
| d-Raffinose | + |
| Amidon | + |
| Glycogene | + |
| Xylitol | + |
| Gentibiose | + |
| d-Turanose | + |
| d-Lyxose | + |
| d-Tagatose | + |
| d-Fucose | + |
| l-Fucose | + |
| d-Arabitol | + |
| l-Arabitol | + |
| Potassium gluconate | + |
| Potassium 2-cetogluconate | — |
| Potassium 5-cetogluconate | + |
| API ZYM | |
| Control | — |
| Alkaline phosphatase | + |
| Esterase (C4) | + |
| Esterase lipase (C8) | + |
| Lipase (C14) | — |
| Leucine arylamidase | + |
| Valine arylamidase | — |
| Cystine arylamidase | + |
| Trypsine | + |
| α-Chymotrypsine | — |
| Acid phosphatase | — |
| Naphtalo-AS-BI-phosphohydrolase | + |
| α-Galactosidase | + |
| β-Galactosodase | + |
| β-Glucuronidase | — |
| α-Glucosidase | + |
| β-Glucosidase | + |
| N-Acetyl-β-glucosaminidase | + |
| α-Mannosidase | + |
| α-Fucosidase | — |
+, positive result; −, negative result.
Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France) with the EZ1 DNA tissue kit, then sequenced using MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera XT Paired end (Illumina), as previously described [10]. The assembly was performed with a pipeline incorporating different software (Velvet [11], Spades [12] and Soap Denovo [13]) on trimmed (Trimmomatic [14]) or raw data. 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 (four scaffolds, nine contigs).
The genome of strain Marseille-P4339 is 3<thinsp>092<thinsp>417 bp long with a 67.3 mol% G+C content and contains 2727 predicted genes. The degree of genomic similarity of strain Marseille-P4339 with closely related species was estimated by OrthoANI software [14]. Values among closely related species (Fig. 4) ranged from 73.98% between Brachybacterium nesterenkovii and strain Marseille-P4339 to 83.79% between Brachybacterium faecium and Brachybacterium saurashtrense. When the isolate was compared to these closely species, values ranged from 73.98% with Brachybacterium nesterenkovii to 75.53% with Brachybacterium muris.
Fig. 4.
Heat map generated with OrthoANI values calculated using OAT software between genus and species and other closely related species with standing in nomenclature.
Conclusion
Strain Brachybacterium timonense exhibited a 16S rRNA sequence divergence of <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 Brachybacterium timonense sp. nov.
Nucleotide sequence accession number
The 16S rRNA gene and genome sequences were deposited in GenBank under accession numbers LT962482 and OIWY00000000 respectively.
Deposit in culture collections
Strain Maseille-P4339 was deposited in two different strain collections under numbers CSURP4339 and CECT9821.
Conflict of interest
None declared.
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 (National Agency for Research; Méditerranée Infection 10-IAHU- 03). This work was also supported by ‘Région Provence Alpes Côte d’Azur’ and European funding ‘Fondo Europeo de Desarrollo Regional’ (FEDER). The authors thank Hitachi Corporation for providing the TM4000Plus Tabletop microscope. They also thank M. Lardière from IHU-Méditerranée Infection, Marseille for English-language review and A. Caputo from IHU-Méditerranée Infection, Marseille for submitting the genomic sequences to GenBank.
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