Draft genome sequence of Enterococcus faecium strain LMG 8148

Enterococcus faecium, traditionally considered a harmless gut commensal, is emerging as an important nosocomial pathogen showing increasing rates of multidrug resistance. We report the draft genome sequence of E. faecium strain LMG 8148, isolated in 1968 from a human in Gothenburg, Sweden. The draft genome has a total length of 2,697,490 bp, a GC-content of 38.3 %, and 2,402 predicted protein-coding sequences. The isolation of this strain predates the emergence of E. faecium as a nosocomial pathogen. Consequently, its genome can be useful in comparative genomic studies investigating the evolution of E. faecium as a pathogen.


Introduction
Enterococci commonly reside in the gastro-intestinal tract of a wide variety of invertebrate and vertebrate hosts, including humans. Since they produce bacteriocins, Enterococcus spp. are widely used as starter cultures for food fermentations or probiotic supplements [1]. Since the 1970s however, they have enigmatically progressed from commensal organisms of little clinical interest to leading nosocomial pathogens causing infections of the urinary tract, bloodstream, and surgical wounds, among others [2]. The large majority of human enterococcal infections are caused by two species: E. faecalis and E. faecium. Worryingly, acquired antibiotic resistance against a multitude of drugs is increasingly being reported in these organisms [3].
Here, we report the draft genome of E. faecium LMG 8148, a strain of human origin isolated in 1968 in Gothenburg, Sweden [4].

Classification and features
Enterococcus is a large genus of Gram-positive, nonsporulating, facultative anaerobic, round-shaped, lactic acid-producing bacteria (Table 1) [5]. E. faecium belongs to the family Enterococcaceae, order Lactobacillales, class Bacilli, and phylum Firmicutes. Microscopically, enterococci are often observed as pairs or short chains of cells ( Fig. 1) [5]. They were classified as group D streptococci until assigned a separate genus in 1984 [6]. E. faecalis and E. faecium are the two most prominent species within the genus. Enterococci can grow in a wide range of environmental conditions, including temperature (5-50°C), pH (4.6-9.9), 40 % (w/v) bile salts, and 6.5 % NaCl [7]. To investigate evolutionary relationships with other Enterococcus species and E. faecium strains, a phylogenetic tree was constructed using 16S rDNA sequences (Fig. 2). As expected, E. faecium LMG 8148 forms a cluster with the other E. faecium strains.

Genome project history
The strain LMG 8148 was isolated from a human in Gothenburg (Sweden) in 1968 [4]. The strain was obtained through the Belgian Coordinated Collection of Microorganisms. DNA samples were sequenced at the EMBL GeneCore facility (Heidelberg, Germany) and assembled using CLC Genomics Workbench (version 7.5.1). The draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline. This draft whole-genome sequence has been deposited at DDBJ/ ENA/GenBank under the accession LOHT00000000.
The project information, and its association with MIGS version 2.0 [8], is summarised in Table 2.

Growth conditions and genomic DNA preparation
Bacterial cultures were inoculated from single colonies on lysogeny broth agar in 5 ml of lysogeny broth and grown overnight at 37°C, with 200 rpm orbital shaking. The DNeasy Blood&Tissue Kit (Qiagen) was used for DNA isolation, following the manufacturer's instructions and pre-treatment protocol for Gram-positive bacteria. Concentration and purity of isolated DNA was determined spectrophotometrically using the Nanodrop ND-1000 and fluorometrically using Qubit analysis (ThermoFisher Scientific).

Genome sequencing and assembly
100 bp paired-end sequencing was performed on an Illumina HiSeq 2000 machine at the EMBL GeneCore facility in Heidelberg (Germany). The total number of paired reads was 9,317,630. Sequencing data was analysed with the Qiagen CLC Genomics workbench version 7.5.1. After a trimming step for quality (score limit: 0.05) and ambiguous nucleotides (maximum 2 ambiguities), reads were assembled de novo using a mismatch cost of 2, a deletion cost of 3, an insertion cost of 3, length fraction 0.5, and similarity fraction 0.8. The assembly yielded 366 contigs (minimum length 200 bp) with an average coverage of 317× and an average contig length of 7,370 bp (N50 length of 41,184 bp). The total length of the draft genome is 2,697,490 bp with a GC-content of 38.3 %.

Genome annotation
All contigs were annotated using NCBI's Prokaryotic Genome Annotation Pipeline. Pfam domains [9] in the predicted protein sequences were identified using the Batch Web CD-Search Tool from NCBI [10]. Predicted proteins were classified into COG [11] functional categories using the WebMGA web server for metagenomic analysis [12]. For further characterization of the predicted genes, CRISPRFinder [13], the SignalP 4.1 server [14], and the TMHMM server [15] were used to predict  Evidence codes -IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature); NAS: Non-traceable VAuthor Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [26] CRISPR repeats, signal peptides, and transmembrane domains, respectively. For the CRISPRFinder tool, only confirmed CRISPRs and not questionable CRISPRs were taken into account.

Genome properties
The properties of this draft genome are summarised in Table 3. Assembly yielded 366 contigs containing 2,697,490 bp with a 38.3 % GC-content. The total number of 2,772 genes predicted by PGAP includes 2,402 protein coding genes (totalling 2,136,945 base pairs), 303 pseudo genes, and 67 RNA genes (56 tRNA and 11 rRNA genes). For 19.37 % of the proteincoding genes, no putative function was assigned, and these were annotated as hypothetical proteins. Further characteristics of the predicted genes are given in Table 3, and classification into functional COG categories is shown in Table 4.   . Lactobacillus plantarum was included as an outgroup. Genbank accession numbers of the aligned sequences are indicated between brackets. 16S rDNA sequences were aligned using MUSCLE, and the phylogenetic tree was determined using the neighbourjoining algorithm with the Kimura 2-parameter distance model in MEGA (version 7) [27]. A gamma distribution (shape parameter = 1) was used for rate variation among sites. The optimal tree with the sum of branch lengths = 0.1983 is shown, and nodes that appeared in more than 50 % of replicate trees in the bootstrap test (1000 replicates) are marked with their bootstrap support values

Conclusions
The presented genome sequence is from a strain isolated in 1968, and thus precedes the emergence of enterococci as important causative agents of hospital-acquired infections in the 1970s and 1980s [2]. Consequently, this genome could be useful for comparative genomic studies looking to solve the remarkable recent emergence of E. faecium as a notorious nosocomial pathogen. The total is based on the total number of protein coding genes in the genome