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

Accepted Manuscript Enterobacter timonensis sp. nov., a new bacterium isolated from a fresh human stool specimen Tatsuki Takakura, Hussein Anani, Amaël Fadlane, Anthony Fontanini, Didier Raoult, Jacques Yaacoub Bou Khalil PII:

S2052-2975(19)30083-6

DOI:

https://doi.org/10.1016/j.nmni.2019.100586

Article Number: 100586 Reference:

NMNI 100586

To appear in:

New Microbes and New Infections

Received Date: 17 May 2019 Revised Date:

17 June 2019

Accepted Date: 26 June 2019

Please cite this article as: Takakura T, Anani H, Fadlane A, Fontanini A, Raoult D, Bou Khalil JY, Enterobacter timonensis sp. nov., a new bacterium isolated from a fresh human stool specimen, New Microbes and New Infections, https://doi.org/10.1016/j.nmni.2019.100586. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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

Tatsuki Takakura1,2, Hussein Anani3, Amaël Fadlane2, Anthony Fontanini2, Didier Raoult2, 3

Hitachi High-Technologies Corporation, Analytical & Medical Solution Business Group, 882

Ichige Hitachinaka-shi, Ibaraki-ken, 312-8504, Japan

Institut Hospitalo-Universitaire Méditerranée-Infection, 19-21 Boulevard Jean Moulin,

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13005, Marseille, France 3

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Jacques Yaacoub Bou Khalil2*

Aix-Marseille Université, Institut de Recherche pour le Developpement (IRD), UMR

Microbes Evolution Phylogeny and Infections (MEPHI), 19-21 Boulevard Jean Moulin,

Corresponding author: Jacques Yaacoub Bou Khalil, MEPHI, Institut Hospitalo-

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13005, Marseille, France

Universitaire Méditerranée Infection, 19-21 Boulevard Jean Moulin 13005 Marseille

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Cedex 05, France. Phone: + 33 (0) 4 13 73 24 01. Fax: + 33 (0) 4 13 73 24 02. E-mail address: [email protected]

Running title: Enterobacter timonensis sp. nov.

Keywords: Culturomics, taxono-genomics, Enterobacter timonensis

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Abstract:

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Enterobacter timonensis strain mt20T (= CSUR P2201T, = DSM101775T) is a new species

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isolated from a fresh human stool specimen.

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ACCEPTED MANUSCRIPT Introduction

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Culturomics is a concept developing different culture conditions in order to enlarge our

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knowledge of the human microbiota through the discovery of previously uncultured bacteria

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(1-4). Once it was isolated, we used a taxono-genomics approach including matrix-assisted laser

time-of-flight

mass

spectrometry

(MALDI-TOF

MS),

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phylogenetic analysis, main phenotypic description and genome sequencing, to describe it (5-

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6).

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Isolation and growth conditions

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In 2017, we isolated from the stool specimen of a 38-month-old healthy girl from Senegal, the

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bacterial strain mt20T. A screening have been made by MALDI-TOF MS on a Microflex LT

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spectrometer (Bruker Daltonics, Bremen, Germany) as previously described (7). The obtained

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spectra (Figure 1) were imported into MALDI Biotyper 3.0 software (Bruker Daltonics) and

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analyzed against the main spectra of the bacteria included in two databases (Bruker and

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constantly

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infection.com/article.php?larub=280&titre=urms-database)). The study was validated by the

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ethics committee of "Institut Fédératif de Recherche IFR48" under number 09-022. The initial

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growth was obtained after 72h of culture in a liquid medium containing 37 g of Difco Marine

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Broth (Becton Dickinson, Le Pont de Claix, France) per liter of sterile water at 37°C and on

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5% sheep-blood-enriched Columbia agar in strict aerobic conditions at 37°C.

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Phenotypic characteristics

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Colonies were brown and circular with a mean diameter of 8 mm. Bacterial cells were Gram-

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negative, bacilli-shaped with a mean diameter of 0.8 µm and a mean length of 2.5 µm (Figure

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2). Strain mt20T showed catalase-positive and oxidase-positive activities. API ZYM was

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MEPHI

databases

(http://www.mediterranee-

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ACCEPTED MANUSCRIPT performed under strict aerobic conditions at 37°C as described in Table 1. Main

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characteristics of the strain are summarized in Figure 3.

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Strain identification

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The 16S rRNA gene was sequenced in order to classify this bacterium. Amplification was

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done by using the primer pair fD1 and rP2 (Eurogentec, Angers, France ) and sequencing

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using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and ABI Prism 3130xl Genetic

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Analyzer capillary3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin,

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France ), as previously described (8). The 16S rRNA nucleotide sequences were assembled

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and corrected using CodonCode Aligner software (http://www.codoncode.com ). Strain mt20T

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exhibited a 98.74 % sequence identity with Enterobacter cloacae strain ATCC 23373T

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(Genbank accession number NR_118011.1), the phylogenetically closest species with

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standing in nomenclature (Figure 4). Actually, based on the phylogenetic tree, we found that

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strain mt20T do not respect the threshold of 16S to identify a new species. For that, we

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investigated the genome sequence of this bacterial species and found that it is nevertheless a

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new species.

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Genome sequencing

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Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France ) with the

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EZ1 DNA tissue kit and then sequenced on the MiSeq technology (Illumina, San Diego, CA,

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USA) with the Nextera Mate Pair sample prep kit (Illumina), as previously described (9). The

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assembly was performed with a pipeline incorporating different softwares (Velvet (10),

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Spades (11) and Soap Denovo (12)), on trimmed (MiSeq and Trimmomatic (13) softwares) or

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untrimmed data (only MiSeq software). GapCloser was used to reduce assembly gaps.

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Scaffolds < 800 base pairs (bp) and scaffolds with a depth value lower than 25% of the mean

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depth were removed. The best assembly was selected by using different criteria (number of

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ACCEPTED MANUSCRIPT scaffolds, N50, number of N). The genome of strain mt20T is 4,199,690 bp long with a 56.8

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mol% G+C content and 13 contigs. The degree of genomic similarity of mt20T closely related

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species was estimated using the OrthoANI software (14). Values among closely related

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species (Figure 5) ranged from 79.29% between Enterobacter soli and Enterobacter kobei to

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91.59% between Enterobacter asburiae and Enterobacter bugandensis. When the isolate was

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compared to these closely species, values ranged from 82.03% with Enterobacter soli to

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83.18% with Enterobacter asburiae. Based on the whole genome sequence, we consequently

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classify this strain as a member of a new species within the genus Enterobacter , family

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Enterobacteriaceae , phylum Proteobacteria.

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Conclusion

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After genome sequencing, strain mt20T exhibited an average of nucleotide identity value < 95

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% with its phylogenetically closest species with standing in nomenclature, and is

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consequently proposed as the type strain of the new species Enterobacter timonensis sp. nov..

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Nucleotide sequence accession number. The 16S rRNA gene and genome sequences were

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deposited in Genbank under accession number LN906632 and FCOP00000000, respectively.

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Deposit in culture collections. Strain mt20T was deposited in strain collection under number

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(= CSUR P2201T, = DSM101775T).

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Conflict of interest:

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None to declare.

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Acknowledgements:

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ACCEPTED MANUSCRIPT We sincerely thank Sakazume Taku, Takashi Irie, Kyoko Imai, Shigeki Matsubara, Akiko

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Hisada, Yusuke Ominami, and all Hitachi Team in Japan for the collaborative study we are

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conducting together between HHT and IHU, and for the installation and services on the

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TM4000Plus microscope in our facility.

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Funding sources:

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The study was supported by the Méditerranée Infection foundation, the National Research

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Agency under the program “Investissements d’avenir”, reference ANR-10-IAHU-03 and by

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Région Provence Alpes Côte d’Azur and European funding FEDER PRIMI.

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Figure 1: MALDI-TOF MS Reference mass spectrum. Spectra from 12 individual colonies

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were compared and a reference spectrum was generated.

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Intens. [a.u.]

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ACCEPTED MANUSCRIPT Figure 2: Scanning electron micrograph of Enterobacter timonensis using TM4000Plus

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microscope from HITACHI. Scale bar and acquisition settings are shown on the original

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micrograph.

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ACCEPTED MANUSCRIPT Figure 3: Description of Enterobacter timonensis according to the digitalized protologue

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TA00949 on the www.imedea.uib.es/dprotologue website.

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ACCEPTED MANUSCRIPT Figure 4: Phylogenetic tree showing the position of “Enterobacter timonensis” strain mt20T

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relative to other phylogenetically-close neighbors.

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The respective GenBank accession numbers for 16S rRNA genes are indicated in parenthesis.

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Sequences were aligned using Muscle v3.8.31 with default parameters and phylogenetic

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inferences were obtained using the maximum likelihood method within MEGA 7 software.

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Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis

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1000 times to generate a majority consensus tree. The scale bar indicates a 0.2 % nucleotide

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sequence divergence.

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ACCEPTED MANUSCRIPT Figure 5: Heatmap generated with OrthoANI values calculated using the OAT software

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between Enterobacter timonensis and other closely related species with standing in

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nomenclature.

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Table 1: Biochemical tests of Enterobacter timonensis (API ZYM). Bacteria: Enterobacter timonensis API ZYM

Control

-

Alkaline phosphatase

+

Esterase (C 4)

+

Esterase Lipase (C 8)

-

Lipase (C 14)

-

Leucine arylamidase

+

Valine arylamidase

-

Cystine arylamidase

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Trypsine

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Acid phosphatase

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Naphtalo-AS-BI-phosphohydrolase

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α-galactosidase

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β-galactosodase

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β-glucuronidase

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α-glucosidase

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β-glucosidase

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N-acetyl-β-glucosaminidase

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α-mannosidase

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α-fucosidase

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