Complete genome sequence of bile-isolated Enterococcus avium strain 352

The Illumina data were used to evaluate the complexity of the genome and correct the PacBio long reads. First, we used ABySS to peform genome assembly with multiple-Kmer parameters and obtained optimal results for the assembly [8]. Second, canu (https://​github.​com/​marbl/​canu) was used to assemble the PacBio corrected long reads [9]. Finally, GapCloser software was subsequently applied to fill the remaining local inner gaps and correct the single base polymorphism for the final assembly results [10].

Gene annotation was determined by Annotation NCBI Prokaryotic Genome Annotation Pipeline [11]. Ribosomal RNA genes were detected by RNAmer 1.2 [12] and tRNA genes were recognized via tRNAscan SE v. 2.0 [13]. The circular genomic map was produced using CGView Server [14].

Phylogenetic analysis is based on orthologous genes. First, orthologous gene families were identified by the ORTHOMCL v2.0 program (reciprocal all-by-all BLASTP analysis) with an E-value of 10⁻⁵ [15]. Second, multiple alignments were generated with the MUSCLE v3.8.31 program, and the alignments were examined visually [16]. Third, the Maximum-likelihood (ML) methods were performed for the phylogenetic analyses using PhyML 3.0, and the model GTR + G was selected for ML analyses with 500 bootstrap replicates to calculate the bootstrap values [17]. The strains used for phylogenetic tree analysis included the E. avium strain ATCC 14025 (GCA000406965.1), the E. faecalis strain ATCC 19433 (GCA000392875.1), the E. faecium strain DO (GCA000174395.2), the E. gilvus strain ATCC BAA-350 (GCA_000394615.1), the E. pseudoavium strain CBA7133 (GCA 003386455.1), the E. sulfureus strain ATCC 49903 (GCA000407025.1), the E. raffinosus strain ATCC 49464 (GCA000393895.1), the E. gallinarum strain FDAARGOS_163 (GCA001558875.2), and the Vagococcus fluvialis strain DSM 5731 (GCA003337315.1). The putative virulence related genes were identified based on the whole genome of the E. avium strain 352 using the VFDB [18].

Total of 46,188,978 raw reads were obtained by Illumina HiSeq Sequencer, and 45,357,196 high quality reads were generated after quality control processes. In addition, 168,754 (1.26 Gb) high-quality reads with an average read length of 7500 bp and a 259-fold coverage were generated by PacBio sequencer. These sequences were used to assemble the genome of the E. avium strain 352 and we obtained a circular chromosome without gap. The complete genome is 4.79 Mb in size with a plasmid of 87.7 kb (Fig. 1) and the mean G + C content is 38.98%. This genome contains 4905 predicted genes as well as 18 rRNA and 68 tRNA genes, while there were 99 predicted genes in the plasmid.

The 16S rRNA gene sequence verified the taxonomic status of the E. avium strain 352 (data not shown). To further elucidate the phylogenetic relationships, whole genome DNA-sequence-based phylogenetic analysis was carried out (Fig. 2). The genome of a highly related and similar type of E. avium strain, E. avium strain ATCC 14025, was selected as standard. The dendrogram of phylogenetic trees illustrated that the E. avium strain 352 was most closely related to the E. avium strain ATCC 14025.

Bile salts have potent antimicrobial activity via damaging membranes and DNA. Thus, bacteria must have intrinsic adapted mechanisms to survive in bile and subsequently cause biliary tract infections [19]. Genomic analysis of the E. avium strain 352 showed the presence of numerous genes that may determine its bile resistance properties (Table 1). The presence of the genes sbcC, mutS and nifI involved in bile resistance in Gram-positive bacteria was identified [19]. It is interesting that there were two bsh genes encoding bile salt hydrolase with a protein sequence identity of 92.9% in the genome of the E. avium strain 352. This result indicated that the BSH might be play an important role in niche-specific adaptation for bile [20]. There were also some genes, including galU and hupB, involved in bile resistance in Gram-negative bacteria [19]. Further studies are needed to verify its genetic properties and evolution traits.

Further screening the genome of the E. avium strain 352 for putative virulence-associated genes was conducted by aligning gene sequences to the virulence factor database (Table 2). There are surface protein encoded genes including esp, fss1 and fss3. The E. avium strain 352 also contains the conservative heat shock protein genes clpC, clpE, and clpP [21]. The ecbA gene encoding a collagen binding MSCRAMM (acronym for microbial surface components recognizing adhesive matrix molecules) and gene lap encoding a listeria adhesion protein were found in the genome and might be contribute to adherence to the host tissue [22, 23]. The bsh gene encoding a bile salt hydrolase was also a virulence related factor in Listeria monocytogenes [24]. The clinical significance of this finding warrants further investigation.