Isolation of a lytic bacteriophage vB_EfaS_PHB08 and its endolysin lys08 against Enterococcus faecalis biofilm

Background: Enterococcus faecalis is an opportunistic pathogen can cause a variety of diseases, such as urinary tract infections and wound infections in human and animals. Recently, bacteriophages and their derivatives represent a good effect on fighting against bacterial infections. Methods: We isolated virulent bacteriophages of E. faecalis using the double-layer plate method. The bio-activities of the phage isolated were determined via one-step growth curve testing and bacterial killing assays. Illumina HiSeq sequencing was performed to determine the genetic characteristics and the lysins of the phage. Protein expression and antibiofilm assays were also performed to highlight the bio-activities of the phage lysins. Results: We isolated a virulent bacteriophage vB_EfaS_PHB08 (thereafter PHB08) from the sewage nearby hospital. PHB08 possessed a linear double-stranded DNA genome with 55,244 bp in length, which encoded 91 putative coding sequences (CDS). We found that PHB08 could inhibit the growth of host bacteria for 12 h. In vegetable models, PHB08 can reduce 1´10 5 Colony Forming Units (CFU) of E. faecalis per square centimeter at room temperature (25 °C) for 24 h. In addition, PHB08 and its endolysin can remove the biofilm formed by E. faecalis . Conclusions: A virulent phage and endolysin displayed a good effect on reducing and/or eradicating E. faecalis infection and biofilm.

different genotypes of E. faecalis play a role in spreading drug resistance genes, which further promotes the clinically difficult treatment of E. faecalis infections [12].
During the infection, E. faecalis strains always form single and mixed-species biofilms on both tissue and medical devices in the host, often under exposure to fluid flow, giving rise to infections that are recalcitrant to treatment [13]. Research showed that produce biofilm of E. faecalis is related with clinical disease [14]. Formation of biofilms confers the bacteria capacity to escape the killing of antibiotics and the elimination of the host immune system [15]. Therefore, eradication of biofilms formed by E. faecalis during the infection is beneficial for clinical treatment.
Bacteriophages (thereafter phages) are the natural predator of bacteria and they are probably the most abundant biological entities in nature [16]. Regarding their ability to kill pathogens with high specificity, phages have been proposed as promising therapeutic tools since their discovery in 1915 [17]. Recently, E. faecalis phages have been found to have the potential to specifically inhibit (or kill) the reproduction and survival of E. faecalis as a biological control agent [18][19][20]. Phages and their derivates such as the lysins have also displayed a good effect on reducing and/or eradicating bacterial biofilms in addition to their effective bactericidal activity [21][22][23]. In this study, we isolated a virulent vB_EfaS_PHB08 (thereafter PHB08) specific infect for E. faecalis and constructed a recombinant plasmid expressing endolysin lys08. Further studies revealed that PHB08 as a potential biological agent in lettuce model. In addition, PHB08 and its derivative exhibited effectively for removing E. faecalis biofilms.

Methods
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 [24]. 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 MgSO 4 ·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 ultracentrifugation and then stored at 4 °C [25]. The morphology of the phages was observed under transmission electron microscope (HITACHI H-7650, Japan).

One-step growth curve
The one-step growth curve of PHB08 is determined as described previously [24]. 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⋅10 7 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 [28]. 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 (10 5 CFU/cm 2 ) 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 [29]. 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 [32] and ARAGORN [33]. Phylogenetic tree was analyzed using the ClustalW program in MEGA 6.0 [34]. 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 (OD 600 ) 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 [35]. 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 [36]. 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⋅10 9 PFU/ml, 2.0⋅10 8 PFU/ml, 2.0⋅10 7 PFU/ml, and 2.0⋅10 6 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.

Statistical Analysis
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.

Microbiological characteristics of phage PHB08
The isolated phage PHB08 had a clear, translucent, uniform size plaque on a double-layer agar plate ( Fig. 1a). Under the observation of electron microscope, phage PHB08 had a rectangular head (length 124 mm ± 5, width 61 mm ± 5) and a long tail (158 mm ± 5) (Fig. 1b). Based on these morphological characteristics and according to the latest International Committee on Taxonomy of Viruses (ICTV) classification, PHB08 was determined as a member of the family Siphoviridae.
Phenotypic parameters of phage PHB08 Phage PHB08 was treated in different pH (3.0-11.0) at 37 °C for 1 h and treated in different temperature (4-80 °C for 1 h. The Acid-base tolerance results exhibited that the activity of PHB08 is relatively stable between 5.0-11.0 (Fig. 2a). Temperature tolerance results showed that the titer of phage PHB08 is quite stable between 4 °C and 60 °C (Fig. 2b). PHB08 was treated at 70 °C for 40 min or at 80 °C for 20 min, no titer was observed (Fig. 2b).The one-step growth curve of PHB08 showed that PHB08 has a latency of 20 min and a high-speed growth period of 40 min with an average burst size of 64 phage particles per infected (Fig. 2c). The host range assays indicated that phage PHB08 specifically infected 15 out of 19 E. faecalis clinical isolated, but not infecting other species including Enterococcus faecium (Table 1). Features of phage PHB08 genome The complete genome of phage PHB08 was linear double-stranded DNA genome of 55,244 bp, with a G + C content of 40% (Fig. 3). The genome of PHB08 was predicted 91 putative CDS (gene annotation for each CDS of PHB08 shown in were predicted [37]. Phylogenetic tree analysis the amino acid sequence of the major capsid protein (CDS71) and terminase large subunit (CDS75) indicated that PHB08 belongs to Saphexavirus, Siphoviridae family (Fig. 4).

Killing of PHB08 in vitro
The effect of the PHB08 on host strain EF3964 was evaluated in liquid medium at 37 °C for 12 h. As shown in Fig. 5a, phage PHB08 showed a strong antibacterial ability at different MOI value (from 0.0001 to 100) for 12 h. After two hours, the curve of the OD 600nm value showed a downward trend, implying that the host bacteria were killed by phage PHB08. The effect of phage PHB08 on host strain was evaluated in lettuce as a vegetable model at room temperature (25 °C). With higher (MOI = 1000) phage-treated, the number of living bacteria EF3964 have a significantly reduction (P < 0.001) compared to control group. With phage-treated at MOI = 1, no significantly reduction of living bacteria EF3964 was observed within 24 h (Fig. 5b).

Antibiofilm activity
The recombinant protein lys08 was predicted to be 26.4 kDa, which was consistent with Polyacrylamide Gel Electrophoresis (PAGE-Gel) test results (Fig. 6a). The purified lys08 was detected by the spot method. The results showed 5 µl lys08 (5 µg) can form translucent halo on the plate, suggesting bactericidal activity of lys08 (Fig. 6b). The effect of the lys08 and phage on the biofilm was evaluated using 96-well microtiter plate method. In phage challenge groups, PHB08 was able to remove the formation of biofilm in co-culture 24 h at 37 °C (P < 0.001) (Fig. 6c). In lys08-treated groups, obviously reduction was observed (P < 0.001) (Fig. 6d).

Discussion
In recent years, the use of bacteriophages and their derivates fighting against bacterial infections and antimicrobial resistance have received more attentions [17]. In the present study, we isolated a virulent phage PHB08 from sewages using E. faecalis EF3964 as the indicator bacterium. The morphology and phylogenetic tree analysis further revealed that phage PHB08 belonged to the Siphoviridae family. The phenotypic parameters assays showed that PHB08 had relatively good stability at temperatures between 4 °C and 60 °C, and pH between 5.0 and 9.0, the average survival rate of phages during incubation is maintained at 40-50%. Our results were consistent with those of phages including Enterococcus phage vB_EfaS_HEf13 [38] and Enterococcus phage EF-P10 [39] when they were exposed to the same temperature (4-60 °C) and acid-base (5.0-9.0). It is worthy note that PHB08 can lyse 15 of the 19 E. faecalis strains, with a host range of 78.9%, which has a wider lytic range than the other E. faecalis bacteriophages (7%-70.5%) [38,[40][41][42]. The average burst of PHB08 was 64 phage particles per infected, in line with the generally reported estimate of about  phage particles per infected [43,44] .The genome sequence of phage PHB08 has the highest similarity (89-98%) with those phages (IME198, HEf13, Ef7.1, EF-P29, EF-P10, VD13, IME-EF1, SAP6, EF1c55, BC-611, Entf1, and Ef2.2) with ~ 40% G + C (Table 3). Phylogenetic analysis of the complete genome sequences of PHB08 and other representative the complete Saphexavirus genome. Our data showed that PHB08 was closely related to phage SAP6 ( Figure S1). The results indicated those phages, which were isolated from different countries (China, USA, Japan, South Korea, Poland, Russia) may have the complex evolutionary relationship. The harm caused by foodborne pathogens has become a serious challenge to humans [45].
Researchers began to use phages as natural antimicrobials in food to kill or inhibit foodborne pathogens, thus ensuring food safety [46,47]. A series of studies have shown that phages can be used in food safety. For example, application phages on Salmonella in cantaloupes resulted Salmonella significantly reduced [48]; With phage treated, 95% reduction the number of Campylobacter jejuni was observed, and also founded that Salmonella can be killed or inhibited by phage [49]. In our study, killing assay exhibited the host strain EF3964 can be inhibited or killed at different MOI for 12 h in medium. Potential bactericidal ability at low MOI was similar to those of the single phage 13076 and phage 14028 [26]. In vegetable model, PHB08 can kill 1⋅10 5 CFU/cm 2 E.
faecalis at 25 °C for 24 h. It can be inferred that phage-based biological control methods have great potential in improving the safety of food microorganisms. E.faecalis with biofilm formation can provide resistance to antibiotics [50], explore the new methods of against cell biofilms is currently one of the major problems in medicine [51]. Although some research have reported of phages fighting bacterial biofilms [52,53], only a few studies on the effects of E. faecalis phage endolysin on host bacteria biofilms. In our study, endolysin lys08 and phage PHB08 can effectively remove the host strain biofilm

Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request

Acknowledgments
We thanks to Lijun Yang 1 and Shuang Wang 1 for their critical suggestions on this article.

Author Contributions
DY and YC performed the data and drafted the main manuscript; DY, YC, ES, and LH planned and performed experiments; BW, HC, and ZP were responsible for experimental design, project management, and manuscript revision. All authors reviewed and agreed the publication of this manuscript.

Conflicts of interest
The authors declare that they have no competing interests. Figure 1 Morphological characteristics of phage PHB08. a. Appearance of phage PHB08 plaque on host strain EF3964. b. Electron microscopy of phage PHB08. Phage PHB08 has a rectangular head (length 124 mm ± 5, width 61 mm ± 5) and a long tail (158 mm ± 5). The scale in the right corner is 200 nm.  Circular genetic map of PHB08. The red part represents the distribution of the CDS region.

Figures
The black part represents the total content of GC (40%). The green part represents the GC skew +, which means the GC shift on the leading chain is positive and the purple is the GC skew -.

Supplementary Files
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