Clinical strains of E. faecalis were isolated from the Department of Clinical Laboratory Medicine and subsequently preserved in our laboratory. To culture E. faecalis, brain heart infusion (BHI) medium was utilized and incubated aerobically with shaking at 37°C.

The bacteriophage was isolated from hospital sewage using the previously described method (Duerkop et al., 2016). Briefly, 2 mL of hospital sewage was centrifuged at 10,000 × g for 2 min, and the resulting supernatant was filtered through a 0.22 μm filter. The filtrate was mixed with log-phase bacteria EFS01 and cultured overnight with shaking at 37°C. The mixture was then centrifuged and filtered using a 0.22 μm aseptic filter. Next, 10 μL of the resulting supernatant was mixed with 100 μL of EFS01 host bacteria and added to 5 mL semi-solid BHI before being poured onto agar plates. The plates were then incubated overnight at 37°C until plaques formed. A single plaque was selected and purified by performing the plaque assay three times.

To determine the host range of SFQ1, the double-layer agar method was conducted on a panel of clinically isolated E. faecalis and E. faecium strains (Table 1). The strains were grown in BHI medium and their susceptibility to lysis by SFQ1 was determined by the formation of clear plaques. The appearance of clear single plaques indicated sensitivity to the phage and the ability of SFQ1 to lyse the strain.

Phage SFQ1 morphology was characterized by transmission electron microscopy (TEM) following previously established protocols (Chatterjee et al., 2019). In brief, the filtered phage lysate was loaded onto a copper grid and incubated for 10 min. The grid was then negatively stained with 10 μL of 2% phosphotungstic acid for an additional 5 min. After air-drying, the samples were visualized using a transmission electron microscope operating at 80 kV.

To determine the optimal ratio of bacteriophages to bacteria that could produce the highest yield of progeny, the multiplicity of infection (MOI) needs to be determined. In this study, we investigated the optimal MOI for phage SFQ1 by mixing the phage and host bacteria at MOIs of 0.001, 0.01, 0.1, 1 and 10. The mixture was incubated at 37°C with shaking at 220 rpm for 5 h. The lysate was then filtered using a 0.45 μm filter, and the phage titer in each experiment was determined using the double-layer agar method. The experiment was repeated three times to ensure accuracy.

The one-step phage growth process was conducted following the previously described method with some modifications. Initially, 5 mL of E. faecalis EFS01 in the exponential growth phase was mixed with phage SFQ1 at an MOI of 0.01. After 5 min of incubation, the mixtures were diluted by 200 times. The culture was further incubated at 37°C, and samples were collected at 10 min intervals. The phage titers in each sample were determined using the double-layer agar method.

The stability of phage SFQ1 was evaluated using various assays. Firstly, pH sensitivity was tested by mixing 10 μL of concentrated phage stock solution (2 × 10¹⁰ PFU/mL) with 990 μL of BHI medium, adjusted with HCl or NaOH to pH values ranging from 2.0 to 13.0. The mixture was then incubated at 37°C for 60 min, and the phage titer was determined using the double agar layer method. The thermal stability of phage SFQ1 was tested by incubating the phage at temperatures ranging from 4°C to 70°C for 1 h, followed by determining the survival phage titer using the double-layer agar method. Additionally, the chloroform sensitivity assay was conducted by incubating phage SFQ1 (10⁸ PFU/mL) with varying ratios of chloroform (10%, 30%, 50%, 70%, 90%) at 37°C with shaking for 10 min. The mixture was then centrifuged, and the phage titer in the upper layer was calculated using the double-layer agar method.

Extraction of the phage SFQ1 genome was conducted following a previously described method (Han et al., 2022). Initially, DNase I and RNase A were added to the phage stocks to attain a final concentration of 1 μg/mL, which facilitated the removal of contaminated DNA and RNA. The mixture was then incubated at 37°C for 60 min. Subsequently, the phage genomic DNA was extracted using the phenol and chloroform method (Duerkop et al., 2016). The DNA was finally dissolved in sterile water and preserved at a temperature of −20°C until genome sequencing.

The genome of phage SFQ1 was sequenced using the Illumina Hiseq 2500 platform. The reads were processed using Fastp to remove any errors and trimmed for quality control. Then, CLC software from QIAGEN (Germany) was applied to assemble the phage genome and annotated with RAST (Overbeek et al., 2014), an online tool that predicted the open reading frames (ORFs). Each ORF’s DNA and protein sequences were manually searched for homologs by BlastN and BlastP. Finally, the phage genome was visualized using the GraphPad Prism 8.0.2, and the sequence was deposited in NCBI under accession number OQ831052. The antibiotic resistance genes were predicted by the online prediction platform ResFinder ¹ and the virulence factors were predicted using the VirulenceFinder, ² respectively.

To investigate the genetic relationships between phage SFQ1 and other E. faecalis phages, a phylogenetic tree was constructed using complete genome sequences of 21 phages, including SFQ1. The analysis was carried out with MEGA 7 software, which allowed for the assessment of similarities and differences in their genetic traits. Additionally, intergenomic similarities between SFQ1 and other E. faecalis phages were calculated using VIRIDIC (Moraru et al., 2020) to gain a deeper understanding of their relationships.

The methodology for the biofilm assay was adapted from a previous study (Goodarzi et al., 2022). To establish a biofilm, fill each well of a 96-well plate with 100 μL of BHI medium containing an overnight culture of E. faecalis. The plates were incubated at 37°C for 24 h, 48 h, or 72 h to allow the biofilm to form. After removing the planktonic cells, 100 μL of phage with a titer of 10⁸ PFU/mL was added to each well, while control wells were added with 100 μL of SM buffer. Incubation was continued at 37°C for 6 h. Then, plates were washed with PBS and the biofilm biomass was stained with crystal violet for 15 min. To quantify the results, crystal violet was solubilized in 0.2 mL of 95% ethanol, and the OD₆₀₀ was determined using a SpectraMax M3 multimode microplate reader. Each sample was examined in triplicate, and BHI medium alone served as the negative control.

Each experiment was repeated three times to ensure the reliability of the results. The statistical analysis was carried out using GraphPad Prism 8.0.2, and student’s t-test was used where applicable. A p-value less than 0.05 was considered statistically significant.

Bacteriophage that specifically targets E. faecalis strain EFS01 was obtained from the sewage of the hospital. Phage plaques were first observed on a top agar lawn containing the clinical strain EFS01. The plaque was purified three times, resulting in the isolation of phage SFQ1. This phage formed clear plaques that were approximately 3 mm in diameter on a double-layered agar plate, as shown in Figure 1A. Electron microscopy revealed that phage SFQ1 had an icosahedral head with a diameter of around 50 nm and a long tail (Figure 1B). Thus, phage SFQ1 is a siphovirus and was designated vB_EFaS_SFQ1.

Phage SFQ1’s stability was evaluated under different pH, temperature, and chloroform treatments. The results revealed that SFQ1 remained viable within a pH range of 5 to 11 (Figure 1C). Additionally, SFQ1 was able to survive at 50°C but rapidly became inactivated at 60°C (Figure 1D). However, after chloroform treatment, the titer of SFQ1 is not changed using various concentrations of chloroform, indicating that it is completely resistant to chloroform (Figure 1E). These findings suggest that SFQ1 can withstand moderate acid and alkali conditions, high temperatures, and chloroform treatment.

The optimal MOI was determined by mixing phage with bacteria at different ratios, and when the MOI was 0.01, it produced the maximum titer of particles (1.44 × 10¹⁰ PFU/mL), indicating that 0.01 is the optimal MOI of SFQ1 (Figure 2A).

To further characterized phage SFQ1, a one-step growth curve experiment was conducted, which showed that phage SFQ1 had a 10 min latent period and plateaued 30 min after phage infection (Figure 2B). Thus, the phage lysis time is about 30 min, and the burst size is about 110 PFU/cell based on the curve.

The host range of SFQ1 was estimated by plaque assays. Ten clinically isolated E. faecalis strains and five E. faecium strains were tested for their sensitivity against SFQ1. Six E. faecalis strains could be lysed by SFQ1, indicating a modest host range, while none of the E. faecium strains could be infected by SFQ1 (Table 1).

The genome structure of SFQ1, a newly isolated E. faecalis phage, was investigated in this study. The phage genome was shown to be linear double-stranded DNA with a length of 40,787 base pairs and a G + C content of 35%. The genomic similarity of SFQ1 to E. faecalis Phage (NC_042126.1) was found to be 94.2%, based on sequence alignment. The SFQ1 genome encodes 63 predicted open reading frames (ORFs), with 17 of them considered functional genes and the remaining 46 annotated as hypothetical proteins (Figure 3). The complete genome sequence of SFQ1 is available at GenBank (accession no. OQ831052).

The annotated genes in SFQ1’s genome were genes involved in DNA replication and modification, phage genome packaging, structural proteins, and host lysis proteins modules. ORF2 was predicted to encode the HNH homing endonuclease, which is involved in phage DNA packing by cooperation with terminase, which is encoded by ORF3 and ORF4. While the phage structural proteins, such as tail fiber and capsid, are encoded in a specific region. ORF13 encodes the phage major tail protein, ORF15 encodes the phage tail tape measure protein, ORF17 encodes the phage tail assembly protein. The phage lysin, which includes a N-acetylmuramoyl-L-alanine amidase, is also predicted close to the tail fiber gene. But, most of the genes are unknown proteins, such as the genes that are used to control host metabolism are not annotated. And none of the ORFs encoded virulence factors or antibiotic resistance genes, and the genome of SFQ1 did not encode lysogenic modules, such as integrase or repressor proteins. These data indicate that SFQ1 is a strictly lytic phage and can be safely used to treat E. faecalis infections.

To better understand the evolution and relationship of phage SFQ1 with other E. faecalis phages, the genome of SFQ1 was compared with that of 20 E. faecalis phages, which were downloaded from NCBI that have homology with SFQ1.

Based on the whole genome sequences, the phylogenetic tree showed that IME278 was grouped into a clade with siphovirus, which is consistent with the results of the whole genome similarity analysis (Figure 4). Phage vB_EfaS_AL3 was highly similar to phage SFQ1. Then, we used VIRIDIC to calculate the intergenomic similarities, showing the similarity between phages SFQ1 and NC_042126.1 was 84.2%, while that with NC_042023.1 was 80.1% (Figure 5).

The efficiency of phage SFQ1 in destroying existing biofilms of E. faecalis EFS01 was assessed under various biofilm-forming conditions (Figure 6). Biofilm experiments were conducted thrice with duplicate readings, and the biofilm was grown to three-time points of 24 h, 48 h, and 72 h. At each stage, phage SFQ1 significantly reduced the E. faecalis EFS01 biofilm within 6 h, and the reduction was statistically significant (p < 0.05) (Figure 6), indicating that SFQ1 can disrupt the biofilm efficiently.