Following all experiments of EFV583 with phage, survivors were routinely tested for resistance to the phage. The survivors were plated and the resulting single colonies were collected and grown. The bacteria were then tested for susceptibility to the phage, by either spotting phages on them or by cross streak agar assay. For the spotting method, about 100–200 μl of bacterial culture was spread uniformly on a BHI agar plate and once it dried; 10 μl spots of phages were spotted, which was done in duplicates or triplicates. While the cross streak agar assays were performed by horizontally streaking the phage lysate in the middle of the plate and then vertically streaking the bacterial colonies over the phage in unidirectional streaks (Moore, 2011; Tiruvadi Krishnan et al., 2015).

Bacterial colonies that grew in presence of phage were considered to exhibit resistance to the phage. These colonies were then collected and grown at 37°C and checked numerous times for their resistant characteristics against the phage. Once their resistant property was confirmed, these bacteria were frozen with 25% glycerol at -80°C and store for future use.

Enterococcus faecalis V583 (ATCC 700802) VRE and EFDG1^r, EFDG1 phage-resistant bacterial strain were grown in brain heart infusion (BHI) broth (Difco, Detroit, MI, United States) aerobically in an incubator shaker with shaking at 220 RPM at 37°C. BHI agar (1.5%) was used for isolation streaks and isolations of phage. For top agar, 0.6% of agar (soft agar) was used, with BHI as nutrient supplement.

In order to combat the phage-resistant mutant strain, a new lytic phage was isolated from the sewage effluents collected from the “Nahal Sorek” decontamination facility located in West Jerusalem, as described previously (Khalifa et al., 2015a). The effluents were centrifuged using a 5430R centrifuge, and a fixed angle rotorFA-45-24-11HS; Eppendorf at 10,000 × g for 10 min, to pellet out the debris. The supernatant was filtered twice, initially through 0.45-μm-pore-size filters (Merck Millipore, Ltd., Ireland) to filter out larger particles and then through 0.22-μm-pore-size filters (Merck Millipore) to achieve lysate free from any residues. Different concentrations of the filtrates (between 0.1 and 1 ml) were then added to bacterial broth culture diluted 1:1000 in BHI. Once lysis was obtained, the lysate was centrifuged and filtered using the aforementioned method.

The phage lysate was then applied on bacterial lawn by the method of double layer agar to obtain clear plaques that symbolize the presence of phages. About 100 μl of bacteria was mixed with 100 μl of phage lysate and incubated for 20 min at 37°C to achieve better adsorption of the phage to the bacteria. The entire mixture was then added to 5 ml of melted 0.6% BHI top agar, poured evenly onto a BHI agar plate and incubated overnight at 37°C. The resultant single plaques were collected and added to 1 ml BHI and incubated at 4°C for diffusion. The liquid was centrifuged at 5000 RPM for 10 min and filtered through 0.22-μm-pore-size filters to obtain a clear phage lysate that was further used to grow large quantities of the phage. The phage was grown in BHI broth with EFV583 bacteria until a titer of 10⁹ or higher was achieved. The phage titer of the phage lysate was determined by counting the Plaque forming units per ml (PFU/ml). For this; the phage lysate was serially diluted in BHI broth and 5 μl of these dilutions were spotted onto an overlayer of molten 0.6% agarose with 100 μl of bacteria. This plate was incubated overnight at 37°C and the numbers of plaques were counted to finally calculate the PFU/ml. A large volume of EFLK1 phage lysate with a high titer (10¹⁰) was prepared and stored at 4°C.

To assess the phage effectiveness against planktonic bacteria, the growth kinetics were analyzed using a 96 well plate reader. The phage was added initially to both logarithmic cultures (10⁴ to 10⁵ CFU/ml) at MOI of 100–1000 (10⁸ PFU/ml) and stationary cultures (10⁸ to 10⁹ CFU/ml) of E. faecalis at MOI of 0.1–1 (10⁸ PFU/ml). Cell viability was also checked by determining CFU/ml for each well.

The biofilm eradication efficiency of the phage was tested by adding it in a MOI of 0.1 (10⁸ PFU/ml) to a 2-week old stationary biofilm of E. faecalis. To obtain a 2-week old biofilm, overnight grown bacteria were diluted with BHI to obtain 10⁴ to 10⁵ CFU/ml of bacteria, which were then plated in a 96-well plate that was incubated for 2 weeks without any change of media or addition of new bacteria. Killing efficacy was validated using viable cell counting by calculating the CFU/ml of the biofilm as described previously (Khalifa et al., 2015a). Briefly, aspirated spent media was removed from the wells followed by washing the wells twice with sterile PBS (100 ul) carefully to not disrupt the biofilm. Sterile PBS (100 ul) was added and the biofilm was scraped and collected. The cells were sonicated in a water-bath for 5–10 min followed by several dilutions (1:10) and plating on BHI plates for CFU count.

We based our protocol on the one step growth method of Ellis and Delbrück (Ellis and Delbrück, 1939). Briefly, phages were added to stationary-phase bacteria (10⁹ CFU/ml) in a final concentration of 1 × 10⁷ PFU/ml followed by incubation in 37°C for 10 min. The samples were diluted at a ratio of 1:10⁴ in fresh BHI medium, followed again by incubation at 37°C for 10 more minutes. Starting at 20 min after the introduction of the bacteriophages to the bacterial cultures, the diluted mixtures were enumerated through a double-layered agar plaque assay. To this end, 3 ml of Soft BHI Agar (0.6% Agar) were mixed with 100 μL of the diluted mixture and with 100 μL of stationary-phase bacteria, according to the tested bacterial strain. The plaque assays were conducted in the following time points after the introduction of the bacteriophages: 20, 35, 50, 60, 70, 85, 100, 120 min, in biological duplicates. Latent time and burst size were calculated from this growth curve for each of the bacteriophages described, with each of the bacterial hosts.

To verify that the phage is indeed new, its DNA was isolated using the Norgen Biotek Phage DNA Isolation Kit (Cat. # 46800) and the resulting genome was sequenced and analyzed (Khalifa et al., 2015a,b). Analysis and comparison to other E. faecalis phages was performed using the GENIOUS 10.0.6 software and its plugins². Annotation of genes was carried out using PHAST³.

To observe the structure of the isolated phage accurately transmission electron microscopy (TEM) by the classic method of Gill as described in OpenWetWare⁴, was conducted following the procedure mention in our previous paper (Khalifa et al., 2015a). Briefly, 1 ml of phage lysate with 10⁹ PFU/ml was centrifuged at 19,283 × g (centrifuge 5430R, rotor FA-45-24-11HS; Eppendorf) for 2 h at room temperature. The supernatant was discarded, and the pellet was resuspended in 200 μl of 5 mM MgSO₄ and incubated overnight at 4°C. 30 μl of 5 mM MgSO₄ and 10 μl of the phage sample were mixed gently on a parafilm strip and 30 μl of 2% uranyl acetate was pipetted on it. On these drops of phage samples grids were then placed carefully using forceps, with the carbon side facing down. After about a minute the grids were dried and stored in the desiccator until further use. A TEM (Joel, TEM 1400 plus) with a charge-coupled device camera (Gatan Orius 600) was used to capture images.

The naïve and resistant E. faecalis were mixed in 1:1 ratio (v/v) and the efficacy of phages was checked individually and in cocktail against them. The experiment mirrored the one described above (see section “Characterization of Phage Activity against E. faecalis Naïve and Phage-Resistant Form in Planktonic and Biofilm Cultures”) for assessing phage activity on planktonic bacteria and on biofilm.

The in vitro fibrin clot model was prepared according to the protocol described by McGrath et al. (1994) and Entenza et al. (2009). Overnight cultures (10⁹ CFU/ml) of E. faecalis individually or in a mixed cultures of naïve and resistant bacteria were diluted 1:10 with citrated plasma. Clots were formed by triggering coagulation by adding 20 μl bovine thrombin (5000 U/ml) and 20 μl CaCl₂ (50 mmol). The resultant clots were then re-suspended in 500 μl of bacteriophage lysate of 10⁸ PFU/ml and for the control, 500 μl of BHI were added instead. The tubes were kept in an incubator-shaker for 6 h at 37°C. After incubation, the clots were washed with sterile PBS and lysed with 32 μl of 0.25% of Trypsin EDTA. After a 5-min centrifugation at 14,000 rpm, the cell pellet was re-suspended in 100 μl of PBS and used for calculating CFU/ml.

The lytic phage EFDG1 was isolated against vancomycin-resistant strains of E. faecalis V583 planktonic and biofilms cultures (Khalifa et al., 2015a). When strain EFDG1^r (resistant to EFDG1 phage) evolved during the experiments (Figure 1A), we screened various sewage effluent samples containing potential anti-E. faecalis phages. Within several weeks, a new lytic phage was isolated, EFLK1, from a sample collected from the sewage water of the “Nahal Sorek” decontamination facility. The new phage exhibited clear plaques on double-layered agar lawn of EFDG1^r. It also prevented the growth of EFDG1^r, as observed both in optical density growth curve (Figure 1A) and colony forming units (CFUs) count (Figure 1B). In agreement, a one-step growth experiment revealed that phage EFLK1 propagated on an EFDG1^r while EFDG1 phage did not (Figure 1C).

Despite the similarity between the two phages (32.187%), the efficiency and kinetics of EFLK1 phage infectivity against the parental strain V583 was distinct from that of EFDG1 phage. The lysis caused by EFDG1 phage during logarithmic growth was observed within 5 h (Figure 1A) and it reduced the viable cells number by four logs (Figure 2A) and (Khalifa et al., 2015a). In these conditions, EFLK1 phage was markedly slower; its lysis started after 12 h (Figure 2A) and it reduced cell viability by three logs (Figure 2C). In contrast, EFLK1 phage faired better against stationary cultures of V583 strain: EFLK1 phage lysis was observed after 10 h compared to much less significant lysis by EFDG1 phage (Figure 2B). These differences in lysis are reflected in the reduction of cell viability from 10¹¹ to ∼10⁵ by EFLK1 phage and only to ∼10⁸ by EFDG1 phage (Figure 2D).

Since we aim to use EFDG1 and EFLK1 phages as therapeutic agents, we tested various combinations of phage cocktails, initially on naïve E. faecalis V583. To this end, we combined EFDG1 and EFLK1 phages in various ratios: 1:1 (cocktail#1), 1:2 (cocktail#2), 2:1 (cocktail#3), 1:3 (cocktail#4), and 3:1 (cocktail#5) correspondingly (Figure 2). The best cocktail was found to be Cocktail#1 (1:1) as it killed the logarithmic culture of E. faecalis V583 as efficiently as EFDG1 phage (Figures 2A–C), and the stationary culture like EFLK1 phage (Figures 2B–D). Thus, cocktail#1 equaled the better phage in each case and outperformed the other cocktails against the challenging biofilm of E. faecalis V583 (Figure 2E).

When cocktail#1 was used against EFDG1^r mutants, it was found to be as effective as EFLK1 phage against logarithmic, stationary and biofilm cultures of the resistant mutants (Figure 3).

Finally, in order to simulate the evolution of resistant mutants within a culture, we mixed the WT E. faecalis V583 with its resistant mutant EFDG1^r and treated them with individual phages and cocktail #1, separately (Figure 4). We found that cocktail #1 and EFLK1 phage were effective against the bacterial mixture while EFDG1 phage was not.

A recognized tool for the study of bacterial endocarditis (Hershberger et al., 2000), we used the in vitro fibrin-clot infection model (McGrath et al., 1994; Entenza et al., 2009) to assess the efficacy of the cocktail and the individual phages EFDG1 and EFLK1 against E. faecalis V583 and EFDG1^r (Figure 5). CFU/ml results following a 6-h incubation of E. faecalis V583 with EFLK1 phage and cocktail#1 showed a CFU reduction of three logs with EFDG1 phage and six logs with EFLK1 phage and cocktail#1. EFLK1 phage and the cocktail also reduced the CFU by five and six logs respectively, against EFDG1^r, while, EFDG1 phage failed as expected.

To explain the phenotypic differences between EFLK1 and EFDG1 phages, their genomes were compared (Khalifa et al., 2015a). In addition to EFLK1 phage, three additional E. faecalis phages of the Spounavirinae subfamily of the Myoviridae phage family of lytic phages (Lavigne et al., 2009), were described. The highest resemblance (∼45%) was found with EFDG1 phage, previously isolated by us (Khalifa et al., 2015a), and less with the other two phages phiEF24c and ECP3 (Figures 6A–C). All four phages share 60 core genes which are ∼28% of their genes (Figure 6A).

The most significant difference between EFDG1 and EFLK1 phages genome sequences found was the number of tRNA genes (Figure 6D): EFDG1 phage contains 24 of them, EFLK1 phage carries none and the other two phages (phiEF24c and ECP3) contain five each which are all clustered in one region as in EFDG1 phage (Figure 6D). An analysis of the distribution of tRNA genes among all 2,053 fully sequenced tailed phages (NCBI, Caudovirales, taxid: 28883) revealed that nearly 60% (1,227) lacked tRNA genes as well (Figure 6D). Of the remainder, the majority carried only 1–5 genes, just like ECP3 and phiEF24c (Figure 6D). The highest number of tRNA genes (33) was found in the Streptomyces phage Jay2Jay (accession: KM652554.1). The cassette of five tRNAs in phiEF24c is identical to that in ECP3. EFDG1 phage contains the same five tRNAs and 19 more, all in a different order. Blast of these cassettes shows that they are unique and do not correspond to the ones on the E. faecalis genome.

One explanation for the presence of tRNA genes in phages is variance in codon usage between their own genome and that of the host. Consequently, one would expect EFDG1 phage codon usage to correlate less with that of its E. faecalis host than the codon usage of the other phages and the large amount of tRNAs to compensate for that extra bias. However, COR function (R Bioconductor package) analysis disproved this theory, as no significant differences were found between the codon usage of the phages and E. faecalis. EFDG1 has 0.921 ECP3; 0.89522, EFLK1; 0.8954, and phiEF24c share 0.8948 correlation with the E. faecalis codon usage (Supplementary Table S1).

Next, we looked at the specific tRNA genes carried by these phages in accordance with their codon usage (Supplementary Table S1). The three anti-E. faecalis Spounavirinae phages with tRNA’s (EFDG1, ECP3, and phiEF24c) carry tRNA genes corresponding to codons CTA, AGA, GAC, and TGG of leucine, arginine, aspartic acid, and tryptophan respectively. Besides these, EFDG1 phage encodes for two while ECP and phiEF24c phages for single tRNA corresponding to the initiation and sole methionine codon AUG. Indeed, in the three phages, the usage of all these codons is higher than their usage in E. faecalis (Supplementary Table S1), perhaps justifying the corresponding tRNA gene’s existence. Surprisingly, the codon usage of these five codons is also higher in EFLK1 phage, which does not have any tRNA genes (Supplementary Table S1). Moreover, the codon usage should lead the four phages to carry the tRNA gene corresponding to codon TAC (tyrosine) while EFLK1, ECP3, and phiEF24c phages should carry tRNA’s for AAG (lysine), AAC (asparagine), ACA (threonine) and GTA (valine) like EFDG1 phage, however this is not the case. On the other hand, EFDG1 phage carries several tRNA genes, such as TTA (leucine) which, according to the codon usage, seems to be not required. Thus, the question of the effect of codon usage on the presence of these tRNA and its relation to the phenotypic differences remains open.

We also analyzed the P2 mutation described by Uchiyama et al. (2011) in phage phiEF24c. The P2 mutation, located in Orf31, a conserved putative tail fiber, dramatically improves the ability of phiEF24c to adsorb and lyse the cells (Uchiyama et al., 2011). Nevertheless, EFDG1, EFLK1, and ECP3 phages harbor homologs for Orf31 but none of them has the P2 mutant allele (Figure 6E).