Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis

Abstract

Enterococcus faecalis, a Gram-positive bacterium, poses a significant clinical challenge owing to its intrinsic resistance to a broad spectrum of antibiotics, warranting urgent exploration of innovative therapeutic strategies. This study investigated the viability of phage therapy as an alternative intervention for antibiotic-resistant E. faecalis, with a specific emphasis on the comprehensive genomic analysis of bacteriophage SAM-E.f 12. The investigation involved whole-genome sequencing of SAM-E.f 12 using Illumina technology, resulting in a robust dataset for detailed genomic characterization. Bioinformatics analyses were employed to predict genes and assign functional annotations. The bacteriophage SAM-E.f 12, which belongs to the Siphoviridae family, exhibited substantial potential, with a burst size of 5.7 PFU/infected cells and a latent period of 20 min. Host range determination experiments demonstrated its effectiveness against clinical E. faecalis strains, positioning SAM-E.f 12 as a precise therapeutic agent. Stability assays underscore resilience across diverse environmental conditions. This study provides a comprehensive understanding of SAM-E.f 12 genomic composition, lytic lifecycle parameters, and practical applications, particularly its efficacy in murine wound models. These results emphasize the promising role of phage therapy, specifically its targeted approach against antibiotic-resistant E. faecalis strains. The nuanced insights derived from this research will contribute to the ongoing pursuit of efficacious phage therapies and offer valuable implications for addressing the clinical challenges associated with E. faecalis infections.

Fig 1. Morphology of SAM-E.f 12 phage.

(A) Spot test showing plaques. (B) DLA method confirming lytic activity with abundant plaques and Bacteriophage Quantification. (C) Transmission electron micrograph of SAM-E.f 12 negatively stained with 2% w/v uranyl acetate, revealing a scale bar of 100 nm.

Fig 2. Annotated genome map of SAM-E.f 12 bacteriophage.

The map displays sixty-one open reading frames (ORFs) represented as arrows, indicating the direction of transcription. Proposed modules are based on hypothetical functions inferred from bioinformatic analysis.

Fig 3. Comparative analysis of phage genome alignment and AP heat map.

The Figure showcases the comparative analysis of SAM-E.f 12 genome alignment with Enterococcus phage NC_0422125. The alignment reveals a high query coverage of 98.3% and an Adjusted Percentage of Nucleotide Identity (APNI) of 90.16%. Despite minor misalignments, the accurate assembly of SAM-E.f 12’s distinct genomic makeup is evident, highlighting its validity.

Fig 4. Phage characteristics and environmental stability.

(A) MOI determination experiment of SAM-E.f 12 phage against E. faecalis. (B) One-step growth curve illustrating the replication dynamics of SAM-E.f 12 phage on E. faecalis. (C) Optimal lytic activity observed at 37°C, with a temperature range of -20°C to 40°C. Lytic activity declined beyond 50°C. (D) SAM-E.f 12 phage demonstrated optimal lytic activity between pH 4 and 10, peaking at pH 7. (E) Stability of SAM-E.f 12 phage under salt stress; the phage particle count reduced with increasing salt concentration, yet retaining lytic activity without reaching zero in tested samples. (F) Adsorption rate of SAM-E.f 12 phage on E. faecalis; more than 96% of phage particles were adsorbed by bacteria within 5 minutes.

Fig 5. Efficiency of phage therapy in E. faecalis-infected BALB/c mouse wounds.

The Fig demonstrates the notable enhancement of wound healing observed in the phage-treated group compared to the control group. The data provides evidence of the effectiveness of phage therapy in addressing E. faecalis infections in an in vivo BALB/c mouse wound model. The Fig presents the three groups: negative control (no bacterial infection), positive control (bacterial infection without phage treatment), and treatment group (infected with E. faecalis and treated with phage).

Fig 6

Histopathological Evaluation of Wound Healing Outcomes in Phage-Treated Group; (A): Phage-Treated; Experimental Group Treated with Bacteriophage, (B): Positive Control; Injured Mice with Bacterial Infection, (C): Negative Control (No Infection); Injured Mice without Bacterial Infection, (D): Healthy Mice; Control Group of Healthy Mice.