NEW METHODS FOR THE ISOLATION OF ENTEROCOCCUS FAECALIS PHAGE FROM THE MARINE ENVIRONMENT

Background: Enterococcus faecalis is a Gram-positive, commensal bacterium from the Enterococcaceae family. This bacterium is commonly associated with nosocomial infections, and increasing concentrations of enterococci in marine waters is an emerging threat. Moreover, the isolation of antibiotic resistant strains from natural environments is a growing research interest due to the critical need to develop alternative antibacterial therapies. Bacteriophage (phage) therapy is a promising alternative therapy for infections caused by a variety of bacteria, including E. faecalis and other enterococci. The isolation of a lytic phage could lead to the development of an effective counter measure, while the isolation of a temperate phage could advance our understanding of forces driving the evolution of antibiotic resistance, namely lysogenic conversion and transduction. The development of a new protocol for the isolation and characterization of a novel E. faecalis phage is an essential first step in phage therapy. Methods: The novel E. faecalis phage was isolated of the inflow water from the local treatment plant, potentially having elevated enterococci concentrations. Water samples were enriched following filtration through a 0.22 µm membrane; moreover the phage was enriched again in brain-hearth-infusion broth using E. faecalis (ATCC 49533) as the competent host. Phage was isolated by a double agar overlay plaque assay. The characterization of the novel phage began with extraction of the genomic DNA where a limited number of isolates were sent for sequencing. Transmission electron microscopy (TEM) was used to obtain a visual image of the isolated virus. Results: A novel working protocol for isolating E. faecalis phage was developed with use of an enrichment method. The isolated phage was inoculated from the 10^-3 plate of the serial dilution assay, and 3.97 X 10⁸ Plaque Forming Units (PFU) were calculated. A spot test was performed to ensure the lytic life cycle of the phage. The λ DNA ladder and 1kb ladder helped to establish an approximate genome size and concentration of 112.6 ng/µL of the novel phage DNA. TEM images allowed for initial visualization of the phage, showing it belongs to the Siphoviridae family of viruses with capsid diameter of ~ 50 nm and tail length of 240 nm. Conclusion: The newly developed protocol for isolating E. faecalis phage is optimized and stable, and resulted in isolation of the novel E. faecalis phage. Further characterization of the phage genome will be accomplished by annotating the novel E. faecalis phage DNA sequence.