Genome sequences of copper resistant and sensitive Enterococcus faecalis strains isolated from copper-fed pigs in Denmark

Six strains of Enterococcus faecalis (S1, S12, S17, S18, S19 and S32) were isolated from copper fed pigs in Denmark. These Gram-positive bacteria within the genus Enterococcus are able to survive a variety of physical and chemical challenges by the acquisition of diverse genetic elements. The genome of strains S1, S12, S17, S18, S19 and S32 contained 2,615, 2,769, 2,625, 2,804, 2,853 and 2,935 protein-coding genes, with 41, 42, 27, 42, 32 and 44 genes encoding antibiotic and metal resistance, respectively. Differences between Cu resistant and sensitive E. faecalis strains, and possible co-transfer of Cu and antibiotic resistance determinants were detected through comparative genome analysis.


Introduction
Copper is an essential trace element with an ubiquitous cellular distribution and performs several biological functions [1]. It serves as an important structural component or catalytic co-factor for a wide range of different enzymes in various important biochemical pathways in bacteria, plants and animals [2]. Because Cu, among many other micronutrients, is beneficial for growth promotion and feed efficiency of farm animals [3,4], it is extensively used as an additive in swine feed. Normally, the concentration of Cu used in animal feed is in excess of the nutritional requirements of animals as it is used as an alternative to in-feed antibiotics for prevention of diarrheal disease [5]. Therefore, enteric bacteria, both commensal and pathogenic, in these animals have typically acquired several additional Cu resistance determinants to survive its toxicity [1,6,7].
Enterococci belong to the gastrointestinal flora of humans and animals, and have been known for more than a century for their pathogenicity to humans, causing urinary tract and surgical wound infections, bacteraemia and endocarditis [8]. Currently, more than 30 species within the genus Enterococcus have been described, and the two most studied enterococcal species are Enterococcus faecium and Enterococcus faecalis [9]. Over the last two decades, E. faecalis and E. faecium have become increasingly important nosocomial pathogens worldwide and are difficult treat due to their increasing multidrug resistance [10]. The intrinsic resistance of Enterococcus to many antibiotics and its acquisition of resistance determinants to other antimicrobial agents led to the emergence of Enterococcus as a nosocomial pathogen [11,12]. Recently, the co-selection of MDR isolates by antibiotics, metals and biocides has been reported [13,14], and the resistance of Enterococcus to both Cu and antibiotics has been established [15,16]. However, few studies have addressed gene transfer and the underlying molecular mechanisms of the various Cu resistance determinants in E. faecalis [17]. Herein, we present the genome sequences along with the main features of six E. faecalis strains showing the differences between Cu resistant and sensitive strains of E. faecalis, and suggesting possible co-transfer of Cu and antibiotic resistance determinants in these bacteria.

Classification and Features
Phylogenetic analysis was performed using the 16S rRNA gene sequences on the six strains S1, S12, S17, S18, S19 and S32 and related species. Sequences were aligned using Clustal W, and a phylogenetic tree was constructed using neighbor-joining (NJ) method implemented in MEGA version 6.0. The resultant tree topologies were evaluated by bootstrap analyses with 1,000 random samplings. Phylogenetic analysis based on 16S rRNA gene sequences showed that the six strains clustered together with E. faecalis ATCC 29212 and E. faecalis SFL with a high bootstrap value (100 %). All the E. faecalis are in a distinct branch with the other enterococci, such as E. casseliflavus, E. faecium, E. hirae and the another pig gut Firmicute, that is Streptococcus equinus NCDO 1037 (Fig. 1). The six strains could be classified as members of the genus Enterococcus based on their 16S rRNA gene phylogeny and phenotypic characteristics (Table 1).
E. faecalis is a Gram-positive, oval-shaped, and often highly pathogenic bacterium classified as a member of the genus Enterococcus (Table 1 and Fig. 2) [18,19]. It is a natural inhabitant of the mammalian gastrointestinal tract and is commonly found in soil, sewage, water and food [8]. E. faecalis is quite versatile and able to survive a variety of physical and chemical challenges by the acquisition of diverse genetic elements, which may contribute to their adaption to different hosts and environments [20,21]. They are able to grow in temperatures ranging from 0°C up to 50°C, and can survive in the presence of 6.5 % NaCl and in broth at pH 9.6 [22]. They can also be resistant to heavy and transition metals [17], as well as many different antibiotics [23][24][25], especially vancomycin [20,21].

Genome project history
The E. faecalis strains (S1, S12, S17, S18, S19 and S32) were isolated from Cu-fed pigs as part of the Danish Integrated Antimicrobial Resistance Monitoring (DAN-MAP) surveillance program [23]. The isolates were collected from healthy animals at or just prior to slaughter. Those whole-genome shotgun projects have been deposited in DDBJ/EMBL/GenBank under the accession number JTKS00000000, JTKT00000000, JTKU00000000, JTKV00000000, JTKW00000000 and JTKX00000000. Table 2 presents the project information and its association with MIGS version 2.0 compliance [26]. Cu resistant strains are E. faecalis strains S1, S18, S32, while the other three strains are Cu sensitive. Growth conditions and genomic DNA preparation E. faecalis were streaked on Slanetz agar (BD Difco) plates and grown for 48 h at 42°C. Each strain was inoculated separately into 25 ml of brain heart infusion broth at 37°C for 24 h. Genomic DNA was purified from the isolates using the Easy-DNA extraction kit (Invitrogen), and DNA concentrations were determined by the Qubit dsDNA BR assay kit (Invitrogen).

Genome annotation
The resulting contigs were uploaded onto the Rapid Annotation using Subsystem Technology server databases and the gene-caller GLIMMER 3.02 [27,28] to predict open reading frames. The predicted ORFs were translated and annotated by searching against clusters of orthologous groups using the SEED databases [29], as well as NCBI databases. RNAmmer 1.2 [30] and tRNAscan SE 1.23 [31] were used to identify rRNA genes and tRNA genes, respectively. CRISPR repeats were examined using CRISPR recognition tool (CRT) [32].

Insights from the genome sequence
All of the six strains contain a four gene operon, copY-ZAB, encoding a Cu resistance determinant (Table 5), which was initially observed in the Gram-positive bacterium E. hirae [33]. CopA and CopB are P-type ATPases responsible for ATP-dependent Cu + transport across the cytoplasmic membranes. The Cu chaperone CopZ binds two Cu + atoms in a solvent accessible manner, presumably to facilitate their transfer to the transcriptional regulator CopY. Upon binding Cu + , CopY undergoes a conformational change and is released from the copA operator allowing expression of the copYZAB operon [1]. A gene encoding the cytoplasmic Cu homeostasis protein CutC was identified in all six strains (Table 5), and CutC has been demonstrated to be involved in Cu homeostasis in E. faecalis [34]. In addition, another possible gene encoding a putative Cu + -translocating P-type ATPase, was identified in all six strains named ctpA in this study ( Table 5). The genome comparisons of the six E. faecalis strains using E. faecalis S32 as the reference strain by CGview comparison tool [35] indicated that S1 and S18 were more similar to the reference strain S32 than the other three strains (Fig. 3).
The tcrYAZB operon was initially identified on the pA17sv1 plasmid in E. faecium, which also carried genes encoding resistance to erythromycin (ermB) and vancomycin (vanA) [17,36]. High toxic Cu levels could be tolerated due to the presence of tcrB in E. faecium or Fig. 3 Graphical circular map of the genome comparison of E. faecalis S32 with the other five strains. Labeling from the outside to the inside circle: ring 1 and 4 show the protein coding genes on the forward/reverse strand (colored by COG categories); ring 2 and 3 show the denote genes on the forward/reverse strand; ring 5, 6, 7, 8 and 9 show the CDS vs CDS BLAST results of E. faecalis S32 with S1, S18, S12, S19 and S17, respectively; ring 10 shows the G + C content (peaks out/inside the circle indicate values higher or lower than the average G + C content, respectively); ring 11 shows GC skew (calculated as (G -C)/(G + C), peaks out/inside the circle indicates values higher or lower than 1, respectively). Ring 5-9 were arranged based on the CDS BLAST results, with the similarity rank from high to low, that is S1 and S18 were more similar to the reference strain S32 than the other three strains E. faecalis which encodes a Cu + -translocating P-type ATPase homologous to CopB encoded on copYZAB operon [37]. Comparing these six E. faecalis strains against others previously identified with increased Cu resistance, the tcrYAZB operon and adjacent cueO encoding a multicopper oxidase were only identified in E. faecalis S1, S18 and S32 ( Table 5). Blasting of the tcrYAZB operon against the contigs of the other three strains verified that they were indeed lacking Cu resistance genes. The cueO gene identified in putative copper resistant strains encodes a multicopper oxidase that is transported across the cytoplasmic membrane and oxidizes Cu(I) to Cu(II) and so aids protection from high Cu concentrations in Enterococcus [9] or other Grampositive strains [16]. The approximate 20-gene copper pathogenicity/fitness island present in E. faecalis S1, Table 5 Copper and antibiotic resistance genes in E. faecalis strains. S1, S18 and S32 represent the three Cu resistant E. faecalis strains, and S12, S17 and S19 represent the three Cu sensitive E. faecalis strains Genes Strain name S1 S18 S32 S12 S17 S19 copYABZ copper resistance genes in sensitive strains (For S1, S18 and S32, one of the copY is on the Cu resistant island, and the other is on the chromosome.); tcrYABZ copper resistance genes in resistant strains; ctpA: copper resistance genes; cueO: multicopper oxidase genes; cutC: genes encoding cytoplasmic copper homeostasis protein; tetM: tetracycline resistance genes; vanA: vancomycin resistance genes; Streptothricin acetyltransferase gene: streptothricin resistance genes Fig. 4 Cu pathogenicity island in E. faecalis S1, S18 and S32. a: prolipoprotein diacylglyceryl trPropertyansferase, b: intergral membrane protein, c: chaperone, d: hypothetical protein, e:transposase, f: disrupted P-type ATPase, g: integrase, h: adenylate kinase, i: resolvase, copY: CopY family transcriptional regulator, cueO: multicopper oxidase, cusR: Cu(I)-sensing regulator, cusS: Cu(I)-sensing sensor, tcrY: tcrYAZB operon regulator, tcrA: putative copper-efflux CPx-type ATPase, tcrB: Cu + -translocating CPx-type ATPase, tcrZ: putative chaperone S18 and S32, show cueO is located in close vicinity of tcrYAZB and probably regulated by an adjacent twocomponent regulator system (Cu(I)-sensing regulator (cusR) and Cu(I)-sensing sensor (cusS)) ( Fig. 4). Transposase and mobile element protein genes were also identified on this pathogenicity/fitness island next to tcrYAZB, indicating mobility. Moreover, genes encoding prolipoprotein diacylglyceryl transferase, which is responsible for oxidative stress tolerance potentially also caused by Cu + , could be identified on these potential pathogenicity and/or fitness islands as well. For the other three Cu sensitive E. faecalis S12, S17 and S19, tcrYAZB, cueO, cusR, cusS or genes encoding a prolipoprotein diacylglyceryl transferase could not be detected. The antibiotic resistance gene tetM (resistance to tetracycline) could be identified in the three Cu resistant E. faecalis S1, S18, S32, and Cu sensitive E. faecalis S12; vanA (encoding vancomycin resistance) was identified only in Cu resistant E. faecalis S32; streptothricin acetyltransferase gene was identified in the Cu resistant E. faecalis S1, S18, S32; and aminoglycoside adenylyltransferase gene was identified in two Cu resistant E. faecalis S1 and S18 (Table 5).

Conclusions
Since the co-transfer of genes encoding antibiotic resistance along with Cu tolerance genes in one transconjugant has been demonstrated [14], the results in this study might provide valuable information corroborating the co-transfer of genes encoding additional Cu resistance and genes encoding numerous antibiotic resistances. Also, the identified antibiotic resistance gene tetM in all the Cu resistant strains is consistent with the MDR Enterococcus strains observed in the environment [13][14][15][16].