The Novel Enterococcus Phage vB_EfaS_HEf13 Has Broad Lytic Activity Against Clinical Isolates of Enterococcus faecalis

Abstract

Enterococcus faecalis is a Gram-positive, facultative anaerobic bacterium frequently found in the gastrointestinal tract, oral cavity, and periodontal tissue. Although it is considered a commensal, it can cause bacteremia, endocarditis, endodontic infections, and urinary tract infections. Because antibiotics are cytotoxic not only to pathogens, but also to health-beneficial commensals, phage therapy has emerged as an alternative strategy to specifically control pathogenic bacteria with minimal damage to the normal flora. In this study, we isolated a novel phage, Enterococcus phage vB_EfaS_HEf13 (phage HEf13), with broad lytic activity against 12 strains of E. faecalis among the three laboratory strains and 14 clinical isolates of E. faecalis evaluated. Transmission electron microscopy showed that phage HEf13 has morphological characteristics of the family Siphoviridae. Phage HEf13 was stable at a wide range of temperature (4-60 • C) and showed tolerance to acid or alkaline (pH 3-12) growth conditions. Phage HEf13 had a short latent period (25 min) with a large burst size (approximately 352 virions per infected cell). The lytic activity of phage HEf13 at various multiplicities of infection consistently inhibited the growth of diverse clinical isolates of E. faecalis without any lysogenic process. Moreover, phage HEf13 showed an effective lytic activity against E. faecalis on human dentin ex vivo infection model. Whole genome analysis demonstrated that the phage HEf13 genome contains 57,811 bp of double-stranded DNA with a GC content of 40.1% and 95 predicted open reading frames (ORFs). Annotated functional ORFs were mainly classified into four groups: DNA replication/packaging/regulation, phage structure, host cell lysis, and additional functions such as RNA transcription. Comparative genomic analysis demonstrated that phage HEf13 is a novel phage that belongs to the Sap6virus lineage. Furthermore, the results of multiple sequence alignment showed that polymorphism of phage infection protein of E. faecalis (PIP EF) contributes to determine the host specificity of phage HEf13 against various E. faecalis strains. Collectively, these results suggest that phage HEf13 has characteristics of a lytic phage, and is a potential therapeutic agent for treatment or prevention of E. faecalis-associated infectious diseases.

Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis

“…Therefore, we suggest that further molecular characterization of replication enzymes may be of special interest and can lead to discoveries of novel properties of these proteins, especially since our knowledge of the variability and diversity of phage-encoded DNA polymerases is relatively poor. In fact, recent reports on the characterization of other enterococcal phages indicated that this group of viruses may have interesting and unique features, pointing to their high biodiversity [ 39 , 41 , 42 , 43 , 44 , 45 , 62 , 63 , 64 , 65 ].…”

The Influence of Enterococcus faecalis as a Dental Root Canal Pathogen on Endodontic Treatment: A Systematic Review

“…In this systematic review, three studies discussed some treatment options to reduce or remove E. faecalis from the root canal system and the periradicular area [23][24][25]. Antibiotic medications were widely used for the treatment and prevention of E. faecalis caused by many pathogenic bacteria.…”

“…Other studies discussed novel treatment techniques using newly isolated phages for targeting E. faecalis strains from the oral cavity. Lee D et al found that phage HEf13 has high lytic activity against human dentin, which suggests the effectiveness of using phage HEf13 as a dental therapeutic agent against E. faecalis-related apical periodontitis [24]. A recent study conducted by Ghorbanzadeh A et al in 2020 found three effective disinfection methods for the partial elimination of E. faecalis biofilm [25].…”

Characterization of a Lytic Bacteriophage vB_EfaS_PHB08 Harboring Endolysin Lys08 against Enterococcus faecalis Biofilms

“…Morphological characterization revealed that PHB08 had a rectangular head (length 124 mm ± 5, width 61 mm ± 5) and a long tail (158 mm ± 5) ( Figure 1 a). These morphological characteristics were similar to Enterococcus phage HEf13 [ 55 ] and phage IME-EF1 [ 56 ]. The phylogenetic analysis showed that PHB08 was closely related to phage SAP6 (isolated from South Korea) ( Figure 3 c).…”

“…Potential bactericidal ability at low MOI was similar to those of the single phage 13076 and phage 14028 [ 36 ]. Characterizations on the temperature and pH stabilities showed that PHB08 had relatively excellent stability at temperatures between 4 and 60 °C, and pH between 5.0 and 9.0, which displays similar characteristics to those of the other E. faecalis phages, such as Enterococcus phage vB_EfaS_HEf13 [ 55 ], and Enterococcus phage EF-P10 [ 57 ]. One-step growth curve revealed that PHB08 had a latency of 10 min and a high-speed growth period of 50 min with an average burst size of 64 phage particles per infected cell after 70 min at 37 °C ( Figure 1 d), which is in agreement with the generally reported average burst of phages (about 30-122 phage particles per infected) [ 58 , 59 ].…”