Enterococcus strains were collected from the Department of Clinical Laboratory Medicine and were grown aerobically on Brain-Heart Infusion Broth (BHI) broth at 37°C with shaking.

Bacteriophage was isolated from hospital sewage as previously described (Yang et al., 2016). Briefly, the sewage was pelleted, and the supernatant was filtered through a 0.22 μm-pore-size filter. Then, 500 μl of the sample was immediately mixed with 200 μl of bacterial culture, and 4 ml of molten BHI soft agar (0.4%) was added and poured onto BHI agar plates. After overnight culture, the formed plaque was picked, deposited in 1 ml of BHI, followed by a 10-fold dilution and double-layer agar assay to purify the phage. Then, one plaque from the third round of the purification process was picked for this study.

Phage particles were dropped on carbon-coated copper grids for 10 min. Then, the grids were stained with phosphotungstic acid (pH 7.0) for 15 s. The sample was examined under a Philips EM 300 electron microscope. The sizes of the phage were measured using AxioVision LE based on five randomly selected images.

The double-layer agar plate assay was used to calculate the phage titer. Briefly, 10-fold dilutions of phage solution were mixed with 200 μl of host bacteria, then mixed with 4 ml of molten BHI broth with 0.4% agar. Then, pour the mixture on a 1.5% agar plate. After overnight incubation at 37°C, the number of plaques was calculated as a plaque-forming unit (pfu). MOI experiments were performed by mixing log-phase bacteria (OD600 = 0.6) with a different number of phages, and the titer in the coculture was calculated using a double-layer agar plate assay after 5 h.

The one-step growth curve of efap05-1 was determined as described (Zhong et al., 2020). Briefly, 1 ml of log-phase bacteria and 1 ml of efap05-1 were mixed at an MOI of 10 and incubated at 37°C for 10 min. Then, the mixture was centrifuged for 1 min at a speed of 10,000 × g, and the pellet was resuspended in 6 ml of BHI medium. And samples were taken at the given time points, which are immediately pelleted and phage titer in the supernatant was measured immediately.

Bacteriophage adsorption assay with various time points was performed as previously described (Al-Zubidi et al., 2019). Briefly, the log phase bacterial cultures were pelleted and resuspended in medium to a final concentration of 3 × 10⁸ CFU/ml. Then, phage was added to a final titer of 3 × 10⁶ pfu/ml. The samples were cultured at 37°C for 10 min, and the phage titer in the supernatant were measured using the double-agar plating assays. The adsorption rate was calculated as (the original phage titer—the remaining phage titer)/the original phage titer.

Ten E. faecalis strains were selected to test the host range of efap05-1 through spot testing by dropping 1 μl of phage onto the double-layer soft agar premixed with the bacterial and cultured at 37°C for overnight. The formation of a clear plaque is considered as sensitive to phage efap05-1 infection.

Two microliter of serial 10-fold dilutions of phage efap05-1 were spotted on double layer agar plates containing a bacterial host. The number of plaques observed after overnight incubation were compared to the number obtained on the strain efa05.

The phage DNA extraction is performed as previously described (Khan et al., 2021). Then, phage genomic DNA was sequenced using an Illumina Hiseq 2,500 platform (~1 Gbp/sample). Fastp (Chen et al., 2018) was used for adapter trimming and filtering the raw reads. The data were assembled using the de novo assembly algorithm Newbler Version2.9 with default parameters, and the assembled genome was annotated using RAST (Overbeek et al., 2014). The DNA and protein sequences were checked for homologs with BLAST manually. The genome map was drawn by SnapGene 4.1.8. The sequence data is available in the NCBI under accession number OL505085.

The stability of phage under various conditions was tested by treating 10⁹ pfu of phage under different pH (pH 2–13), temperature (0°C, 30°C, 40°C, 50°C, and 60°C), or chloroform concentration (10%, 25%, 50%, 75%, and 95%) for 60 min, the then the titer of the phage was determined by double-layer agar assay.

The phage resistant mutants were selected as previously described (Shen et al., 2018). The log phase bacteria were mixed with phage efap05-1 and cultured until the bacteria was lysed. Then, the lysate was inoculated onto the BHI agar. After overnight incubation, the single colonies were checked for its resistance against phage using the double-layer agar assay, which confirmed that all the colonies on the plates are resistant to phage infection.

The wide type strain efa05 and phage resistant mutant strain efa05R were selected for sequencing. Bacterial genomic DNA was extracted using UNlQ-10 Column Bacterial Genomic DNA Isolation Kit (sangon bitotec: SK1202), and then sent to Novogene Corporation for sequencing using the Illumina Hiseq 2,500 platform. Trimmomatic was used to remove adapter sequences and low-quality bases (Bolger et al., 2014). BWA was used to map clean reads to the reference genome sequence of efa05. Samtools (Li et al., 2009) was then used to prepare the data for use with the Integrative Genomics Viewer (IGV). DNA mutation locations were manually checked with IGV and SeqKit (Shen et al., 2016).

The ComEA gene and plasmid pMGP23:mCherry were amplified by PCR (the primers for the amplification of ComEA gene and the plasmid pMGP23:mCherry are listed in Table 1), and the PCR products were purified. The ComEA and plasmid were ligated by Gibson assembly to generate pMG-ComEA, and the constructed plasmid was comfired by sanger sequencing. The efa05R complementation strain was generated by electroporation of pMG-ComEA into strain efa05R followed by selection on BHI agar erythromycin (20ug/ml). The galE gene was complemented with the same protocol.

All the experiments were performed three times. The statistical analysis was performed using One-way ANOVA or t-test, and statistical significance was assumed if the value of p was <0.05.

An E. faecalis phage was isolated using plaque assay. It forms a clear plaque on the host strain efa05 in the double layer agar plates (Figure 1A). The phage particle was observed by transmission electron microscopy. It is a non-enveloped, head-tail structural particle. The prolate head is approximately 100 nm in length and 40 nm in width (Figure 1B). Thus, based on the morphology, phage vB_EfaS_efap05-1 belongs to Saphexavirus which is a member of Siphoviridae. The optimal multiplicity of infection (MOI) was 0.001, and the phage titer could reach approximately 8*10¹⁰ pfu/ml (Figure 1C). The one-step growth curve of efap05-1 indicates that this phage replicates quickly with a lysis period of about 20 min, and the phage titer approached plateau after 20 min (Figure 1D), and the burst size was estimated as about 20 pfu per bacterium.

The host range of efap05-1 was estimated by EOP (efficiency of plating) assays. Ten clinically isolated E. faecalis strains were tested and five strains could be lysed by efap05-1, indicating a modest host range (Table 2).

The stability of efap05-1 under various conditions was tested. It could maintain stability under pH 5–10, and other pH solutions could impair the viability of efap05-1 (Figure 2A). And efap05-1 is stable under 50°C because its titer was not changed after 60 min incubation at 50°C (Figure 2B), and is completely inactivated over 70°C. Besides, chloroform treatment did not affect the viability of phage efap05-1, indicating that it is a non-enveloped phage (Figure 2C).

Phage efap05-1 is a double-stranded (ds) DNA phage with a linear genome of 56,564 base pairs (bp). Its G + C content is 40% and encodes 99 ORFs and one tRNA (Figure 3), which are predicted by RAST (Overbeek et al., 2014) and visualized by SnapGene.

Most of the ORFs are functionally unknown, and 22 ORFs are functionally annotated, which can be categorized into several functional modules, including phage DNA replications, lysis, phage structural protein (Figure 3). However, efap05-1 did not encode any antibiotic-resistant gene or virulence gene, indicating that it is a safe candidate for phage therapy.

To study the phage resistant mechanism of E. faecalis efa05 against phage efap05-1, we mixed phage with host and cultured until the bacteria are lysed. And then inoculate the lysate on BHI agar plate. One phage-resistant mutant efa05R was selected, and its resistance against efap05-1 was confirmed by EOP experiment (Figure 4B).

Then, efa05R and wild-type strain efa05 was sent for whole-genome sequencing, and the mutation sites of these two strains were detected as Late competence protein ComEA and UDP-glucose 4-epimerase galE (Figure 4A). ComEA is a cell membrane protein that binds to the double-stranded DNA and initiates the DNA uptake process (Burghard-Schrod et al., 2021). And UDP-glucose 4-epimerase is involved in the biosynthesis of cell wall polysaccharides (Boels et al., 2001; Lee et al., 2014).

Usually, phage resistance is selected with one key mutation site (Li et al., 2018), the mutation of both genes detected in phage resistant mutant efa05R indicates that both genes are required for phage infection. As expected, the complementation of either ComEA or galE in efa05R could recover the phage sensitivity through enabling phage adsorption (Figures 4B,C). Thus, these data indicate that phage could bind to either polysaccharides or protein ComEA to initiate the phage infection cycle.