High-Quality Draft Genome Sequences of 28 Enterococcus sp. Isolates

Abstract

The enterococci are low-GC Gram-positive bacteria that have emerged as leading causes of hospital-acquired infection. They are also commensals of the gastrointestinal tract of healthy humans and most other animals with gastrointestinal flora and are important for food fermentations. Here we report the availability of draft genome sequences for 28 enterococcal strains of diverse origin, including the species Enterococcus faecalis, E. faecium, E. casseliflavus, and E. gallinarum.

The enterococci are ecologically diverse, Gram-positive lactic acid bacteria that are found in the gastrointestinal consortia of humans, other mammals, reptiles, amphibians, birds, and insects and are utilized in production of fermented foods and probiotics (1, 2, 16). However, they have also emerged as leading causes of hospital-acquired infection at extraintestinal sites, including the heart, urinary tract, and surgical site wounds (9, 10). These infections have become a leading health care concern because of increasing antibiotic resistance mediated by mobile genetic elements (12). The ability of enterococci to colonize extraintestinal sites and cause infection involves both core and variable genetic traits (3, 15) and is not fully understood.

Enterococci exhibit diversity in metabolic capabilities (such as the ability of Enterococcus faecalis, but not E. faecium, to respire [11]) and in clinically relevant phenotypes such as toxin production (12) and biofilm formation (13). Enterococci can also be motile or nonmotile, and pigmented or nonpigmented (4), yet little is known about the mechanism and significance of these traits. Insights into basic enterococcal physiology are limited by a paucity of genome data. The genome sequences of the vancomycin-resistant bloodstream isolate E. faecalis V583 (14), the human oral isolate E. faecalis OG1RF (3), and the commercial probiotic strain E. faecalis Symbioflor (6) have been determined, and a draft genome sequence for E. faecium DO has been available in GenBank since 2002. Here we announce the availability of draft genome sequences of 28 additional enterococcal strains, including 16 E. faecalis strains, 8 E. faecium strains, 3 E. casseliflavus strains, and 1 E. gallinarum strain. In addition to expanding the Enterococcus nucleotide sequence in GenBank by approximately an order of magnitude, this is the first report of genome sequencing of the motile E. gallinarum strain and the motile, pigmented E. casseliflavus strains.

Most of the enterococcal strains utilized for our genome sequencing project are of clinical origin. The remaining strains are commensal isolates or were obtained from animals or insects. E. faecalis strains selected for this project were previously described in a study of E. faecalis strain diversity (12). To maximize the information yield from genome sequencing, we selected 16 E. faecalis strains that represent the deepest phylogenetic nodes in the E. faecalis multilocus sequence typing (MLST) dendrogram from the 106 strains previously examined (12). These strains include commensal isolates such as the human fecal isolate E1Sol, derived from a population with little exposure to antibiotics (8), and the fruit fly isolate Fly1 (5), as well as multiple antibiotic-resistant clinical isolates such as HIP11704, the strain implicated in the first documented interspecies transfer of vancomycin resistance genes to methicillin-resistant Staphylococcus aureus (7, 17). Six of the eight E. faecium strains and the E. casseliflavus and E. gallinarum strains utilized for genome sequencing were obtained from a clinical isolate repository (Eurofins Medinet) and were selected based on diversity in geographic isolation and antibiotic resistance phenotype. The remaining two E. faecium strains, Com12 and Com15, were isolated from the feces of healthy human volunteers. A full description of these strains will be included in a future report with the results of a comparative enterococcal genome analysis.

Genomes were sequenced to at least 15-fold coverage using 454 FLX pyrosequencing (Roche) with DNA fragment libraries and 3-kb paired-end reads according to the manufacturer's recommendations. Genomes were assembled using Newbler. Before assembly, quality of the 454 sequencing data was analyzed, and suspect libraries were removed. The runAssembly script was then used to assemble reads into contigs and scaffolds using the parameters −ar −rip −g. Each assembly was scanned for 16S rRNA sequence using the Ribosomal Database Project (RDP) to confirm the presence of one appropriately classified 16S rRNA sequence. Final assemblies were BLASTed to the NCBI nonredundant (NR) database, UniVecCore, and a mitochondrial database to remove any contaminating sequence. Annotation of assemblies was performed using a combination of ab initio and evidence-based approaches as described at the Broad Institute Enterococcus group database (http://www.broadinstitute.org/annotation/genome/enterococcus_faecalis/GeneFinding.html). A summary of gene finding data for each predicted locus can also be viewed at the Broad Institute Enterococcus group database.

Nucleotide sequence accession numbers.

GenBank accession numbers (in parentheses) for genomes within this project are as follows: E. casseliflavus EC10 (ACAL00000000), EC20 (ACAO00000000), and EC30 (ACAH00000000); E. faecalis ARO1/DG (ACAK00000000), ATCC 4200 (ACAG00000000), CH188 (ACAV00000000), D6 (ACAT00000000), DS5 (ACAI00000000), E1Sol (ACAQ00000000); Fly1 (ACAR00000000), HIP11704 (ACAN00000000), JH1 (ACAP00000000), Merz96 (ACAM00000000), T1 (ACAD00000000), T2 (ACAE00000000), T3 (ACAF00000000), T8 (ACOC00000000), T11 (ACAU00000000); and X98 (ACAW00000000); E. faecium 1,141,733 (ACAZ00000000), 1,230,933 (ACAS00000000), 1,231,408 (ACBB00000000), 1,231,410 (ACBA00000000), 1,231,501 (ACAY00000000), 1,231,502 (ACAX00000000), Com12 (ACBC00000000), and Com15 (ACBD00000000); and E. gallinarum EG2 (ACAJ00000000).