Bacterial strains and cultural conditions
E. faecalis strain EF3964 was recovered from a patient with urinary tract infections. It grows well on tryptic soy agars (TSA; Becton, Dickinson and company, MD, USA) and/or in tryptic soy broth (TSB; Becton, Dickinson and company, MD, USA) at 37° C for 16 h.
Phage isolation and purification
Phages against E. faecalis were isolated from sewages using E. faecalis strain EF3964 as an indicator by a double-layer plate methodology, as described previously . In briefly, sewage samples from the hospital sewage were centrifuged at 4,000 × g for 10 min. The supernatants were harvested and were filtered using a 0.22 µm membrane to remove bacteria. After that, 300 µl of the filtrates were mixed with 300 µl of the bacterial culture of EF3964 at mid-log phase, and the mixture incubated using a double-layer TSA plate 37 °C for 12 h to form the phage plaques. Presumptive single plaque was picked and was resuspended in 6 ml of sterile SM buffer (5.8 g of NaCl, 2.0 g of MgSO4·7H2O, 50 ml of Tris-HCl [pH7.4], 5.0 ml of 2% gelatin). The phage-containing SM buffer was then centrifuged at 12,000 × g for 30 s and the supernatant was filtered through a 0.22 µm pore size membrane. In the next, the phage preparations were given serial 10-fold dilutions with sterile SM buffer. Finally, the phage preparations were inoculated into the indicator bacteria at mid-log phase, which was then incubated using a double-layer TSA plate 37 °C to the next cycle. Phage isolation by the double-layer agar method was repeated four more times. The phages were purified by CsCl gradient ultra-centrifugation and then stored at 4 °C . The morphology of the phages was observed under transmission electron microscope (HITACHI H-7650, Japan).
Physical parameter of phage PHB08
For temperature sensitivity assay, 100 µl (109 Plaque Forming Units, PFU) of the purified phages were treated at different temperature (4, 20, 40, 50, 60, 70, and 80 °C) for 1 h. For acid-base sensitivity assay, 100 µl of phage (109 PFU) with 900 µl SM buffer of pH (3.0, 5.0, 7.0, 9.0, and 11.0), were treated at 37 °C for 1 h. Phage titer was determined using the double-layer plate method. This experiment was repeated three times.
One-step growth curve
The one-step growth curve of PHB08 is determined as described previously . In briefly, PHB08 with multiplicity of infection (MOI) of 0.01 was inoculated into the indicator bacteria at mid-log phase and the mixture was incubated at 37 °C for 5 min. After incubation, the mixture was centrifuged at 12,000 rotation per minute (rpm) for 30 s. The supernatant was discarded with equal volume TSB. The titer of phage was determined by double-layer plate method. This experiment was repeated three times.
Lytic Activity of PHB08
The lytic activity of phage PHB08 was analyzed in a 96-well microtiter plate by examining the optical density measurement method [26, 27]. Briefly, 100 µl of the mid-log phase E. faecalis EF3964 (6.6⋅107 Colony Forming Units, CFU) mixed with 100 µl of phage PHB08 of different MOI (0.001, 0.01, 0.1, 1.0, 10, 100, and 1000) was incubated at 37 °C (160 rpm). Wells with equal volume of TSB medium or Phosphate buffer saline (PBS) buffer added were used as controls. The absorbance value of resulting supernatant was measured at 590 nm using a multimode microplate reader (Tecan Spark 10M). This experiment was performed in triplicate.
Killing assay in vegetable module
The effect of phage PHB08 on host strain EF3964 in vegetable module was evaluate as previously described . Briefly, the vegetable was sterilized with sodium hypochlorite (100 µg/ml) for 5 min. After washing with sterile water, the vegetable sample was covered evenly host strain EF3964 (105 CFU/cm2) until sample dried naturally. Subsequently, phage PHB08 with different MOI values (1000, 100, 10, and 1.0) was sprayed on the vegetable leaves at 25 °C for 6, 12, and 24 h, respectively. The control group was added equal volume phosphate buffered saline (PBS; pH = 7.4). The survival of EF3964 was counted by 10-fold dilutions method. This experiment was repeated three times.
DNA extraction and analysis of genome sequence
The phages’ genomic DNA extracted using the phenol-chloroform protocol was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH = 8.0]) and was sequenced on an Illumina HiSeq 2,500 sequencer with 2 × 100 bp read length. The short reads were assembled into the genome by means of SOAPdenovo . Open reading frames (ORFs) were predicted using Glimmer [30, 31]. The final assembled sequence was searched against the current protein and nucleotide databases (http://www.ncbi.nlm.nih.gov/) by means of the basic local alignment search tool (BLAST). Protein BLAST (BLASTP) was used to identify putative homologies and proteins sharing similarities with the predicted phage proteins. The genomes were scanned for tRNAs using tRNA scan-SE  and ARAGORN . Phylogenetic tree was analyzed using the ClustalW program in MEGA 6.0 . The complete genome sequence of PHB08 was deposited in GenBank under the accession number MK570225.
Cloning, expression and activity identification of lys08
The putative lysin gene lys08 of phage PHB08 was amplified by polymerase chain reaction (PCR) with specific primers (5’-CGTGTGTCACATACCTGAATTG-3’; 5’-GCAGTAACAGCCATTCATCTATG-3’), and then was cloned into the expression vector pET-28a, generating pET-lys08, which was finally transformed into the expressed strain BL21 (DE3). Single colonies of BL21 were picked and inoculated into TSB medium containing 50 µg/ml of kanamycin, incubated overnight at 37 °C. After that, bacterial culture was transferred into fresh TSB containing 50 µg/ml of kanamycin and the mixture was cultured at 37 °C until Optical Density (OD600) value reaches 0.6 ~ 0.8. The expression of protein Lys08 was induced by the addition of 0.6 mM/L IPTG in the bacterial culture and further incubation at 25 °C for 16 h. Lys08 was purified by Ni-nitrilotriacetic acid column as previously described . The purified protein lys08 was dialyzed in a protein preservation solution (50 mmoL/l Tris, 0.3 mol/l NaCl, pH 8.0) in 0.45 µm membrane concentrated. Purified protein was quantified by the Bradford Protein Assay Kit (Thermo Fisher Scientific) and stored at -80 °C until uses.
Antibiofilm by phage PHB08 and its endolysins lys08
Biofilm formation was detected using a 96-well microtiter plate as previously described . In briefly, 100 µl of the overnight cultured bacterial strain EF3964 was added to each well and was incubated at 37 °C for 24 h, 48 h, or 72 h, respectively. Equal volumes of PBS (pH = 7.4) was included as controls. Each well was washed three times with sterile PBS buffer. Subsequently, 100 µl phage PHB08 (2.0⋅109 PFU/ml, 2.0⋅108 PFU/ml, 2.0⋅107 PFU/ml, and 2.0⋅106 PFU/ml) with TSB medium or 100 µl lys08 (50 µg and 100 µg) was added to every well at 37 °C for 4 h. After incubation, 1% crystal violet solution was added 100 µl to each well at 37 °C for 15 min, then washed three times with sterile PBS buffer until the liquid has no more crystal violet color. Finally, 150 µl of 33% acetic acid added to each well. The absorbance value of resulting supernatant was measured at 600 nm as mentioned above. This experiment was repeated three times.
Student’s unpaired t-test was used for statistical significance when comparing results for two groups; while ordinary one-way analysis of variance (ANOVA) with post-hoc analysis by Dunnett’s test was used when comparing the results of more than two groups. Data are present as “Mean ± SD”. Differences were considered statistically significant if P < 0.05 (∗). All statistical analyses were performed using GraphPad Prism software.