Enterobacter timonensis sp. nov., a new bacterium isolated from a fresh human stool specimen

Abstract

Enterobacter timonensis strain mt20^T (= CSUR P2201^T, = DSM101775^T) is a new species isolated from a fresh human stool specimen.

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 use a taxonogenomics 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 it [5], [6].

Isolation and growth conditions

In 2016, we isolated from the stool specimen of a 38-month-old healthy girl from Senegal, the bacterial strain mt20^T. 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 two databases (Bruker and the constantly updated MEPHI databases (http://backup.mediterranee-infection.com/article.php?larub=280&titre=urms-database)). The study was validated by the ethics committee of the Institut Fédératif de Recherche IFR48 under number 09-022. Initial growth was obtained after 72 h of culture in a liquid medium containing 37 g of Difco Marine Broth (Becton Dickinson, Le Pont de Claix, France) per litre of sterile water at 37°C and on Columbia agar enriched with 5% sheep blood in strict aerobic conditions at 37°C.

Fig. 1.

MALDI-TOF MS reference mass spectrum. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Phenotypic characteristics

Colonies were brown and circular with a mean diameter of 8 mm. Bacterial cells were Gram-negative, bacillus-shaped with a mean diameter of 0.8 μm and a mean length of 2.5 μm (Fig. 2). Strain mt20^T showed catalase-positive and oxidase-positive activities. API ZYM was performed under strict aerobic conditions at 37°C as described in Table 1. Main characteristics of the strain are summarized in Fig. 3.

Fig. 2.

Scanning electron micrograph of Enterobacter timonensis using TM4000Plus microscope from Hitachi. Scale bar and acquisition settings are shown on the original micrograph.

Table 1.

Biochemical tests of Enterobacter timonensis (API ZYM)

Bacteria: Enterobacter timonensis
API ZYM
Test	Results (+/–)
Control	–
Alkaline phosphatase	+
Esterase (C4)	+
Esterase lipase (C8)	–
Lipase (C14)	–
Leucine arylamidase	+
Valine arylamidase	–
Cystine arylamidase	–
Trypsin	–
α-chymotrypsin	–
Acid phosphatase	+
Naphthalo-AS-BI-phosphohydrolase	+
α-galactosidase	+
β-galactosidase	+
β-glucuronidase	–
α-glucosidase	+
β-glucosidase	+
N-acetyl-β-glucosaminidase	+
α-mannosidase	–
α-fucosidase	–

Fig. 3.

Description of Enterobacter timonensis according to the digitalized protologue TA00949 on the www.imedea.uib.es/dprotologue website.

Strain identification

The 16S rRNA gene was sequenced to classify this bacterium. Amplification used the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequencing used 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 [8]. The 16S rRNA nucleotide sequences were assembled and corrected using CodonCode Aligner software (http://www.codoncode.com). Strain mt20^T exhibited a 98.74% sequence identity with Enterobacter cloacae strain ATCC 23373^T (GenBank Accession number NR_118011.1), the phylogenetically closest species with standing in nomenclature (Fig. 4). Based on the phylogenetic tree, we found that strain mt20^T do not reach the threshold of 16S required to identify a new species. For this reason, we investigated the genome sequence of this bacterial species and found that it is, nevertheless, a new species.

Fig. 4.

Phylogenetic tree showing the position of ‘Enterobacter timonensis’ strain mt20^T 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 0.2% nucleotide sequence divergence.

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 Mate Pair sample prep kit (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 data (MiSeq and Trimmomatic [13] softwares) or untrimmed data (only MiSeq software). 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 (number of scaffolds, N50, number of N). The genome of strain mt20^T is 4 199 690 bp long with a 56.8 mol% G+C content and 13 contigs. The degree of genomic similarity of mt20^T with closely related species was estimated using the OrthoANI software [14]. Values among closely related species (Fig. 5) ranged from 79.29%, between Enterobacter soli and Enterobacter kobei, to 91.59%, between Enterobacter asburiae and Enterobacter bugandensis. When the isolate was compared with these closely related species, values ranged from 82.03% with Enterobacter soli to 83.18% with Enterobacter asburiae. Based on the whole genome sequence, we consequently classify this strain as a member of a new species within the genus Enterobacter, family Enterobacteriaceae, phylum Proteobacteria.

Fig. 5.

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

Conclusion

After genome sequencing, strain mt20^T exhibited an average of nucleotide identity value < 95% with its phylogenetically closest species with standing in nomenclature, and is consequently proposed as the type strain of the new species Enterobacter timonensis sp. nov.

Nucleotide sequence accession number

The 16S rRNA gene and genome sequences were deposited in GenBank under Accession numbers LN906632 and FCOP00000000, respectively.

Deposit in culture collections

Strain mt20^T was deposited in the strain collection under number (= CSUR P2201^T, = DSM101775^T).

Conflict of interest

None to declare.

Funding sources

The study was supported by the Méditerranée Infection foundation, the National Research Agency under the programme ‘Investissements d′avenir’ (ANR-10-IAHU-03) and by Région Provence Alpes Côte d’Azur and European funding (FEDER PRIMI).