Figure 1.
Characterization of isolated bacteriophage IME-EF1.
A. Observing the morphology of bacteriophage IME-EF1 with Transmission Electronic Microscope. Magnification: ×30.0 k, bar length of 40 nm. B. One-step growth kinetics of phage IME-EF1 was determined in its host strain of E. faecalis 002.
Figure 2.
Genomic annotation and comparison of IME-EF1.
A. The comparison of phage genomes from IME-EF1, EFAP-1, phiFL1-3, EFRM31, EfaCPT1, EF24c, EF11, SAP6, and BC-611 was performed using software Mauve2.3.1. B. Genomic annotation of IME-EF1 was conducted with RAST and its arrangement was illustrated using CLC Genomic Bench.
Figure 3.
A. Amino acid sequence of IME-EF1 endolysin was aligned and phylogenized with the submitted, confirmed, or putative endolysin from six strains of E. faecalis bacteriophage in GenBank. B. SDS-PAGE and Western blot were used to identify the expression of endolysin in E. coli M15. Lanes 1 and 4 indicate the negative control with the cells transformed with the empty vector pColdI. Lanes 2 and 5 show the cells transformed with recombinant pColdI containing IME-EF1 endolysin gene, whereas Lane 3 was pre-stained with the protein marker. Lanes 1 to 3 were stained with Coomassie brilliant blue, whereas Lanes 4 and 5 were transferred to the PVDF membrane and blotted with anti-His antibody. The black arrow indicates the expressed endolysin.
Figure 4.
Endolysin lytic activity and binding assay.
A. The OD600 descent is used to evaluate the lytic activity of IME-EF1 endolysin on the isolated E. faecalis, E. faecium, and S. aureus. B. Assay of IME-EF1 endolysin binding to sensitive bacterial strains. The expressed IME-EF1 endolysin was incubated with sensitive bacteria E. faecalis 002 and E. faecium 010, as well as the insensitive bacteria E. faecium 81 and S. aureus for 10 min, and the bacteria was then pelleted and the remaining supernatant endolysin was measured with ELISA at OD490. “Input” means no bacteria were added. Significance was determined using independent t test (* P<0.05).
Figure 5.
Bactericidal evaluation in mice.
After intraperitoneal inoculation with E. faecalis 002, the phage or endolysin was administrated at 30 min or 4 h intraperitoneally, respectively. A. Mice survival rate was evaluated using Kaplan–Meier analysis. B. The tail blood samples were collected at different time points, and the bacterial number in blood was counted on BHI agar plate.