Noncontiguous finished genome sequence and description of Enterococcus massiliensis sp. nov.

Accepted Manuscript Non-contiguous finished genome sequence and description of Enterococcus massiliensis sp. nov. Stéphanie Le Page, Teresa Cimmino, Amadou Togo, Matthieu Million, Caroline Michelle, Saber Khelaifia, Jean-Christophe Lagier, Didier Raoult, Jean-Marc Rolain PII:

S2052-2975(16)30025-7

DOI:

10.1016/j.nmni.2016.04.011

Reference:

NMNI 163

To appear in:

New Microbes and New Infections

Received Date: 4 February 2016 Revised Date:

19 April 2016

Accepted Date: 28 April 2016

Please cite this article as: Le Page S, Cimmino T, Togo A, Million M, Michelle C, Khelaifia S, Lagier JC, Raoult D, Rolain J-M, Non-contiguous finished genome sequence and description of Enterococcus massiliensis sp. nov., New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.04.011. 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.

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Taxonogenomics: reporting the genome of a new organism

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Full-length title: Non-contiguous finished genome sequence and description of Enterococcus

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massiliensis sp. nov.

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Short title (for the running head):

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Author list: Stéphanie Le Page1, Teresa Cimmino1, Amadou Togo1, Matthieu Million1, ,

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Caroline Michelle1, Saber Khelaifia1, Jean-Christophe Lagier1, Didier Raoult1 and Jean-Marc

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Rolain*1

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Affiliations: 1 Aix-Marseille Univ., URMITE UM 63 CNRS 7278 IRD 198 INSERM U1905,

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IHU Méditerranée Infection, Facultés de Médecine et de Pharmacie, 27 boulevard Jean

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Moulin, 13385 Marseille CEDEX 05, France

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* Corresponding author:

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Text word count: 1540 Words

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Abstract word count: 90 words

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Number of references: 19

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Number of tables: 3

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Number of figures: 3

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Key words: Enterococcus massiliensis, genome, taxono-genomics, culturomics

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ABSTRACT

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Enterococcus massiliensis strain sp. nov. (=CSUR P1927 = DSM 100308) is a new species

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within the genus Enterococcus. This strain was first isolated from a fresh stool sample of a

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man during culturomics study of intestinal microflora. Enterococcus massiliensis is a Gram-

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positive cocci, facultative anaerobic and motile. E. massiliensis is negative for mannitol and

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positive for beta-galactosidase contrary to E. gallinarum. The complete genome sequence is

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2,712, 841 bp in length with a GC content of 39.6% and contains 2,617 protein-coding genes

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and 70 RNA genes, including 9 rRNA genes.

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Abbreviations

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CSUR: Collection de souches de l’Unité des Rickettsies

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URMITE: Unité des Maladies Infectieuses et Tropicales Emergentes

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DSM: Deutsche Sammlung von Mikroorganismen

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MALDI-TOF MS: Matrix-Assisted laser-desorption/ionization time-of-flight mass

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spectrometry

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dDDH: digital DNA-DNA hybridization

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ACCEPTED MANUSCRIPT INTRODUCTION

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Enterococcus massiliensis sp. nov. strain AM1T (=CSUR P1927 = DSM 100308) belongs to

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the genus Enterococcus. This bacterium is a Gram-positive, facultative anaerobic, motile and

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unpigmented. It was isolated from a fresh stool sample of a human in Marseille as part of

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“culturomics” study (1). Currently, a polyphasic approach that combines proteomic by

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MALDI-TOF spectra analysis (2), genomic data with 16S rRNA sequence identity and

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phylogeny (3), genomic G + C content diversity and DNA-DNA hybridization (DDH) and

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phenotypic characterization is used to describe new bacterial species (4).

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Enterococcus are classified in the genus of Streptococcus because of the presence of the D

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antigen until 1984 (Thiercelin and Jouhaud) (5) . But, after analysis of the genome of

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Streptococcus faecalis and S. faecium these strains have been transferred to the genus

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Enterococcus (6). Members of the genus Enterococcus are components of the intestinal flora

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of humans and animals. There are opportunistic pathogens with 2 principal strains:

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Enterococcus faecalis and Enterococcus faecium responsible for nosocomial infections (7,8).

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Here we present a summary classification and a set of features for E. massiliensis sp. nov.

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strain AM1T together with the description of the complete genome sequence and annotation.

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These characteristics support the circumscription of the species E. massiliensis.

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Organism information

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A stool sample was collected in 2015 from a voluntary patient as a negative control and

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isolated on Columbia agar supplemented with 5% sheep blood (bioMérieux, Marcy-l’Étoile,

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France) in aerobic and anaerobic condition using GasPak™ EZ Anaerobe Container System

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Sachets (BD) at 37°. Enterococcus massiliensis was sequenced as part of a culturomics study

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aiming to isolate all bacterial species colonizing the human gut (9). Enterococcus massiliensis

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strain AM1T (GenBank accession number LN833866) exhibited a 97% 16S rRNA nucleotide

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sequence similary with Enterococcus gallinarum (JF915769) , the phylogenetically-closest

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ACCEPTED MANUSCRIPT validly pubished bacterial species (Fig.1) after compararison with NCBI database. This value

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is lower than 98.7% 16S rRNA gene sequence similarity set as a threshold recommended by

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Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA

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hybridization.

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Growth occurred between 25°C and 37°C, but optimal growth was observed at 37°C, 24

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hours after inoculation. Colonies were smooth, withish and approximately 1 mm in diameter

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on 5% sheep blood-enriched agar (Biomerieux). Growth of the strain was tested under

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anaerobic and microaerophilic conditions using GasPak EZ Anaerobe pouch (Becton,

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Dickinson and Company) and CampyGen Compact (Oxoid) systems respectively, and in

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aerobic conditions, with or without 5% of CO2. Growth was achieved under aerobic (with and

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without CO2), microaerophilic and anaerobic conditions. Gram staining showed Gram-

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positive cocci without sporulation (Fig.2A). A motility test was positive and realized with

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API® M Medium (Biomerieux, Marcy l’Etoile, France) a semi-solid medium with an

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inoculation performed by swabbing one colony into the medium. After 24 hours of

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incubation, the growth of E. massiliensis was away from this stabbed line, characterisitic of

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the positive motility. Cells grown on agar exhibit a mean diameter of 0.5 µm and a mean

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length ranging from 1.1 to 1.3 µm (mean 1.2 µm) determined by negative staining

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transmission electron microscopy (Fig.2B).

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Differential phenotypic characteristics using API 50CH and API Zym system (Biomerieux)

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between E. massiliensis sp. nov. AM1T and others Enterococcus species (9) are presented in

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Table 1. Antibiotic Susceptibility testing was performed by the disk diffusion method on

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Mueller-Hinton agar with blood (Biomerieux, Marcy l’Etoile, France). E. massiliensis is

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susceptible to vancomycin, teicoplanine, linezolid, gentamicin, ciprofloxacin, doxycyclin,

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rifampicin and pristinamycin and resistant or intermediate to penicillin G, oxacillin,

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ACCEPTED MANUSCRIPT ceftriaxone, cefoxitin, trimethoprim/sulfamethoxazole, fosfomycin, erythromycin and

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clindamycin.

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Extended features descriptions

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Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis

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was carried out as previously described (2) using a Microflex spectrometer (Bruker Daltonics,

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Leipzig, Germany). Twelve distinct deposits were done for strain AM1T from 12 isolated

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colonies. Twelve distinct deposits were done for strain AM1T from 12 isolated colonies.

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Spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed

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by standard pattern matching against 7,765 bacterial spectra including 92 spectra from 31

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Enterococcus species, in the BioTyper database. Interpretation of scores was as follows: a

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score > 2 enabled the identification at the species level, a score > 1.7 but < 2 enabled the

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identification at the genus level; and a score < 1.7 did not enable any identification (These

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scores were established by the manufacturer Bruker Daltonics). For strain AM1T, no

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significant MALDI-TOF score was obtained against the Bruker database thus suggesting that

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our isolate was a new species. We incremented our database with the spectrum from strain

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AM1T (Fig.3).

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Genome sequencing information

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Enterococcus massiliensis sp. nov. (GenBank accession number CVRN00000000 ) is the 54

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species described within Enterococcus genus.

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After DNA extraction by phenol-chloroform method, genomic DNA of E. massiliensis was

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sequenced on a MiSeq instrument (Illumina Inc, San Diego, CA, USA) using paired end and

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mate pair strategies.

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For genome annotation, Open Reading Frames (ORFs) were predicted using Prodigal (10)

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with default parameters but the predicted ORFs were excluded if they spanned a sequencing

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gap region. The predicted bacterial protein sequences were searched for against the GenBank

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ACCEPTED MANUSCRIPT database (11) and the Clusters of Orthologous Groups (COG) databases using BLASTP. The

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tRNAScanSE tool (12) was used to find tRNA genes, whereas ribosomal RNAs were detected

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using RNAmmer (13) and BLASTn against the GenBank database.

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The ARG-ANNOT database for acquired antibiotic resistance genes (ARGs) was employed

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for a BLAST search using the Bio-Edit interface (14). The assembled sequences were

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searched against the ARG database under moderately stringent conditions (e-value of 10−5 )

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for the in silico ARG prediction. E. massiliensis present Lsa gene, encoding a putative ABC

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protein Lsa with an identity to 72% with Lsa family ABC-F of E. faecalis in NCBI that

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confirm phenotypically the resistance to clindamycin.

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Analysis of presence of polyketide synthase (PKS) and Non-Ribosomal Polyketide Synthesis

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(NRPS) were performed by discriminating the gene with large size using a database realized

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in our laboratory, predicted proteins were compared against non redundant (nr) GenBank

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database using blastp and finally examined using antiSMASH (15). The analysis of genome

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revealed the absence of NRPKs and PKS. Lipoprotein signal peptides and number of

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transmembrane helices were predicted using SignalP (16) and TMHMM (17), respectively.

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ORFans were identified if their BLASTP E-value was lower than 10-3 for alignment length

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greater than 80 amino acids.

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Moreover, we used Genome-to-Genome Distance calculator (GGDC) web server available at

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(http://ggdc.dsmz.de) to estimate the overall similarity among the compared genomes and to

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replace the wet-lab DNA-DNA hydridization (DDH) by a digital DDH (dDDH) (18-19).

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GGDC 2.0 BLAST+ was chosen as alignment method and the recommended formula 2 was

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taken into account to interpret the results.

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We compared the genome of E. massiliensis with 9 other genomes of Enterococcus strains.

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The genome is 2,712,841 bp long (one chromosome, no plasmid) with a GC content of 39.6%

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(Table 2). The properties and statistics of the genome are summarized in Table 2. The draft

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ACCEPTED MANUSCRIPT genome of E. massiliensis is smaller than those of E. moraviensis, E. haemoperoxidus, E.

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caccae, E. casseliflavus, E. gallinarum and E. faecalis ( 3.60, 3.58, 3.56, 3.43, 3.16 and 2.96

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Mb respectively), but larger than those of E. saccharolyticus, E. columbae, E. cecorum and E.

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sulfureus ( 2.60, 2.58, 2.34 and 2.31 respectively). The G + C content of E. massiliensis is

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lower than those of E. casseliflavus and E. gallinarum ( 42.8 and 40.7) but greater than those

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of E. moraviensis, E. haemoperoxidus, E. caccae, E. saccharolyticus, E. columbae, E.

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cecorum, E. sulfureus and E. faecalis (39.6, 36.1, 35.7, 35.8, 36.9, 36.6, 36.4, 38.0 and 37.5

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respectively). Of the 2687 predicted chromosomal genes, 2617 were protein-coding genes and

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70 were RNAs including 61 tRNAs and 9 rRNAs (5S = 4, 23S = 2, 16S = 3). A total of 1889

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genes (72.2%) were assigned to a putative function described in Fig.3 and Table 3. 71 genes

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were identified as ORFans (2.71%) and the remaining genes were annotated as hypothetical

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proteins. The distribution of genes into COGs functional categories is presented in Table 3.

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CONCLUSION/PERSPECTIVES

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On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the

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creation of Enterococcus massiliensis sp. nov. AM1T. This strain was isolated in Marseille,

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France.

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Taxonomic and nomenclatural proposals

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Description of Enterococcus massiliensis sp. nov.

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Enterococcus massiliensis (massiliensis because this strain was isolated in Massilia , the Latin

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name of Marseille, where the strain was sequenced).

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ACCEPTED MANUSCRIPT Colonies were whitish and approximately 1 mm diameter on 5% sheep blood-enriched agar.

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Cells are Gram-positive, non haemolytic,facultative anaerobic with a mean length of 1.2 µm

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and a mean diameter of 0.6 µm. Growth occurred between 25°C to 37°C, but optimal growth

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was observed at 37°C. Alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine

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arylamidase, acid phosphatase, β-galactosidase and N-acetyl-β-glucosaminidase activities

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were present. Esculine activity was also positive but catalase, oxydase, β-galactosidase and N-

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acetyl-β-glucosaminidase were negative. Positive reaction were obtained for D-ribose, D-

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glucose, D-fructose, D-mannose and N-acetylglucosamine. E. massiliensis was susceptible to

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vancomycin, teicoplanine, linezolid, gentamicin, ciprofloxacin, doxycyclin, rifampicin and

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pristinamycin, but resistant to for trimethoprim/sulfamethoxazole, fosfomycin, erythromycin

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and clindamycin.

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The G+C content of the genome is 39.6%. The 16S rRNA and genome sequences are

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deposited in GenBank under accession numbers LN833866 and CVRN00000000,

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respectively. The type strain AM1T (=CSUR P1927 = DSM 100308) was isolated from a fresh

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stool sample of a patient in Marseille, France.

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ACKNOWLEDGMENTS

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We are very grateful to Xegen company for automating the genome annotation process.

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CONFLICT OF INTEREST AND FINANCIAL DISCLOSURE

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No potential conflict of interest or financial disclosure for all authors.

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FIGURE LEGEND

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Fig. 1. A consensus phylogenetic tree highlighting the position of Enterococcus

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massiliensis relative to other type strains within the genus Enterococcus by 16S. In

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brackets are indicated the GenBank accession numbers. Sequences were aligned using

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CLUSTALW, and phylogenetic inferences obtained using the maximum-likehood method in

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the MEGA6 software package. Numbers at the nodes are percentages of bootstrap values

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from 1000 replicates that support the nodes. Streptococcus pneumoniae and Staphylococcus

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aureus were used as outgroups. The scale bar represents 1% nucleotide sequence divergence.

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Fig. 2. 2(A). Gram of Enterococcus massiliensis and 2(B). Transmission electron micrograph

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of E. massiliensis strain, taken using a Technai G20 Cryo (FEI) at an operating voltage of 200

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kV with magnification 1000x.

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Fig. 3. Graphical circular map of chromosome. From outside to center: genes on forward

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strand (colored by COGs categories explain in Table 3), genes on reverse strand (colored by

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COGs categories), RNA genes (tRNAs green, rRNAs red), G+C content, G+C skew.

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ACCEPTED MANUSCRIPT TABLE LEGENDE

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Table 1. Differential characteristics of Enterococcus massiliensis sp AM1, E. faecalis, E.

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casseliflavus, E. gallinarum, E. haemoperoxidus, E. cecorum, E. sulfureus and E. caccae.

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Table 2. Nucleotide content and gene count levels of the genome

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Table 3. Number of genes associated with the 25 general COG functional categories.

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E. casseliflavus

E. gallinarum

E. haemoperoxidus

E. cecorum

E. sulfureus

E. caccae

Facultative anaerobic Positive Motile -

Facultative anaerobic Positive -

Facultative anaerobic Positive Motile +

Facultative anaerobic Positive Motile -

Facultative anaerobic Positive -

Facultative anaerobic Positive -

Facultative anaerobic Positive +

Facultative anaerobic Positive -

+ +

-

+

+

na na na +

+ -

na na na +

na na na +

+ + + + 39.6 Human stool

+ + + 37.3 Intestines mammals

+ + + + + 40.7 Intestines mammals

+ + 35.8 Water

+ + 36.3 Commensal chicken

+ + 37.8 Plants

+ 35.8 Human stool

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+ + V + + + 42.7 Intestines mammals

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E. faecalis

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Gram stain Motility Pigment Production of Alkaline phosphatase Catalase Oxydase β-glucuronidase α-galactosidase β-galactosidase N-acetyl-glucosamine Acide form Mannitol Sorbose L-arabinose Sorbitol D-Rafinose Xylose D-Trehalose G + C content (%) Habitat

E. massiliensis

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Properties Cell diameter (µm) Oxygen requirement

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Table 1. Differential characteristics of Enterococcus massiliensis sp AM1, E. faecalis, E. casseliflavus, E. gallinarum, E. haemoperoxidus, E. cecorum, E. sulfureus and E. caccae.

+ : positive result ; - : negative results ; v : variable, na : data not available.

ACCEPTED MANUSCRIPT Table 2. Nucleotide content and gene count levels of the genome % of totala 100 39.6 88.77 100 2.60 97.39 72.18 71.19 9.55 24.07

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Value 2,712,841 1,075,567 2,408,151 2687 70 2617 1889 1863 250 630

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Attribute Genome size (bp) DNA G + C content (bp) DNA coding region (bp) Total genes RNA genes Protein-coding genes Genes with function prediction Genes assigned to COGs Genes with peptide signals Genes with transmembrane helices

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The total is based on either the size of the genome in base paris or the total number of protein coding genes in the annotated genome.

ACCEPTED MANUSCRIPT Table 3. Number of genes associated with the 25 general COG functional categories.

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description Rna processing and modification Chromatin structure and dynamics Energy production and conversion Cell cycle control, cell division, chromosome partitioning Amino acid transport and metabolism Nucleotide transport and metabolism Carbohydrate transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Translation, ribosomal structure and biogenesis Transcription Replication, recombination and repair Cell wall/membrane/envelope biogenesis Cell motility Posttranslational modification, protein turnover, chaperones Inorganic ion transport and metabolism Secondary metabolites biosynthesis, transport and catabolism General function prediction only Function unknown Signal transduction mechanisms Intracellular trafficking, secretion, and vesicular transport Defense mechanisms Extracellular structures Nuclear structure Cytoskeleton Not in COGs

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% agea 0 0 2.98 0.84 6.38 2.56 9.48 1.76 2.03 5.88 7.15 5.98 3.40 0.19 2.06 3.97 0.76 9.94 7.26 2.25 0.91 2.29 0 0 0 28.81

the total is based on the total number of protein coding genes in the annotated genome.

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Value 0 0 78 22 167 67 248 46 53 154 187 155 89 5 54 104 20 260 190 59 24 60 0 0 0 754

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Color of COG class COG class A B C D E F G H I J K L M N O P Q R S T U V W Y Z -

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Fig. 1

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Fig. 2 B

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Fig. 3