Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin.

Currently, effective options are needed to fight vancomycin-resistant Enterococcus faecalis (VRE). The present study shows that combinations of phage and vancomycin are highly efficient against VRE, despite being resistant to the antibiotic. Vancomycin-phage EFLK1 (anti-E. faecalis phage) synergy was assessed against VRE planktonic and biofilm cultures. The effect of the combined treatment on VRE biofilms was determined by evaluating the viable counts and biomass and then visualized using scanning electron microscopy (SEM). The cell wall peptidoglycan was stained after phage treatment, visualized by confocal microscopy and quantified by fluorescence activated cell sorting (FACS) analysis. The combined treatment was synergistically effective compared to treatment with phage or antibiotic alone, both in planktonic and biofilm cultures. Confocal microscopy and FACS analysis showed that fluorescence intensity of phage-treated bacteria increased eight-fold, suggesting a change in the peptidoglycan of the cell wall. Our results indicate that with combined treatment, VRE strains are not more problematic than sensitive strains and thus give hope in the continuous struggle against the current emergence of multidrug resistant pathogens.

Phages in a thermoreversible sustained-release formulation targeting E. faecalis in vitro and in vivo.

INTRODUCTION: Enterococcus faecalis is a key pathogen recovered from root canals when conventional treatment fails. Phage therapy has generated new interest in combating pathogens. A sustained-release formulation using specific phages against E. faecalis may offer an alternative approach. OBJECTIVES: To evaluate the efficacy of anti-E. faecalis phages formulated in a thermo- sustained-release system against E. faecalis in vitro and in vivo. METHODS: EFDG1 and EFLK1 phages were formulated with poloxamer P407. Gelation time, phage survival, activity and toxicity were evaluated. Lytic activity was evaluated in vitro against E. faecalis at various growth phases, including anti-biofilm activity. Methods included viable bacterial count (CFU/mL), biofilm biomass determination and electron microscopy (live/dead staining). Further evaluation included infected incisors in an in vivo rat model. Anti-E. faecalis phage-cocktail suspension and sustained-release phage formulation were evaluated by viable bacterial count (CFU/mL), histology, scanning electron microscopy (SEM) and 16S genome sequencing of the microbiota of the root canal. RESULTS: Gelation time for clinical use was established. Low toxicity and a high phage survival rate were recorded. Sustained-release phages reduced E. faecalis in logarithmic (4 logs), stationary (3 logs) and biofilm (4 logs) growth phases. Prolonged anti-biofilm activity of 88% and 95% reduction in biomass and viable counts, respectively, was recorded. Reduction of intracanal viable bacterial counts was observed (99% of enterococci) also seen in SEM. Phage treatment increased Proteobacteria and decreased Firmicutes. Histology showed reduced periapical inflammation and improved healing following phage treatment. CONCLUSION: Poloxamer P407 formulated with phages has an effective and long-lasting effect in vitro and in vivo targeting E. faecalis.

Defeating Antibiotic- and Phage-Resistant Enterococcus faecalis Using a Phage Cocktail in Vitro and in a Clot Model.

The deteriorating effectiveness of antibiotics is propelling researchers worldwide towards alternative techniques such as phage therapy: curing infectious diseases using viruses of bacteria called bacteriophages. In a previous paper, we isolated phage EFDG1, highly effective against both planktonic and biofilm cultures of one of the most challenging pathogenic species, the vancomycin-resistant Enterococcus (VRE). Thus, it is a promising phage to be used in phage therapy. Further experimentation revealed the emergence of a mutant resistant to EFDG1 phage: EFDG1r. This kind of spontaneous resistance to antibiotics would be disastrous occurrence, however for phage-therapy it is only a minor hindrance. We quickly and successfully isolated a new phage, EFLK1, which proved effective against both the resistant mutant EFDG1r and its parental VRE, Enterococcus faecalis V583. Furthermore, combining both phages in a cocktail produced an additive effect against E. faecalis V583 strains regardless of their antibiotic or phage-resistance profile. An analysis of the differences in genome sequence, genes, mutations, and tRNA content of both phages is presented. This work is a proof-of-concept of one of the most significant advantages of phage therapy, namely the ability to easily overcome emerging resistant bacteria.

Phage Therapy: A New Horizon in the Antibacterial Treatment of Oral Pathogens.

Dental diseases are perhaps the most prevalent infection-related diseases in humans. Biofilm is involved in almost every infectious disease compromising oral health, notably caries, periodontal disease, gingivitis, endodontic infections and peri-implantitis. Current therapies of biofilm-derived oral infections lack sensitivity; they are not species-specific and kill pathogenic species as well as commensal species, which are protective against the formation of pathogenic biofilms. Moreover, antibiotics have a limited effect on biofilm and are almost unused in oral diseases. A promising alternative approach is bacteriophage (phage) therapy. Phages play a key role in the natural balance in a predator-prey relationship with bacteria and thus have the potential to be efficient anti-bacterial agents. Phages are highly efficient against biofilm, strain specific and easy to isolate and manipulate. Thus, like in many other medicinal fields, phage therapy offers new horizons to dentistry, both therapeutics and research. The present review presents the etiology of common oral diseases, characterization of the infection and the treatment challenges of phage therapy in dentistry. Recent findings and development in the use of phages for prevention, control, and treatment of oral infections as well as possibilities of engineering the oral microbiome are discussed.

Phage therapy against Enterococcus faecalis in dental root canals.

Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals.

Complete Genome Sequence of Enterococcus Bacteriophage EFLK1.

We previously isolated EFDG1, a lytic phage against enterococci for therapeutic use. Nevertheless, EFDG1-resistant bacterial strains (EFDG1(r)) have evolved. EFLK1, a new highly effective phage against EFDG1(r) strains, was isolated in this study. The genome of EFLK1 was fully sequenced, analyzed, and deposited in GenBank.