Isolation And Biological Characteristic Of Two Lytic Enterococcus Bacteriophages Active Against Enterococcus Faecalis

In recent years, with widespread use of antibiotics and immunosuppressants and increasingly emergence of multi-drug resistant Enterococcus isolates, the occurrence of nosocomial infections caused by enterococci has progressively increased. In addition, AIDS patients, cancer individuals receiving chemotherapy, and people undergoing surgery and organ transplantation are easily at a risk of enterococcal infection. In worldwide, Enterococcus faecalis and Enterococcus faecium account for most infections. Recent data indicate that enterococci exhibit an intrinsic resistance to cephalosporins and increasing resistance to vancomycin, posing significant therapeutic challenges. Bacteriophages are viruses that infect and kill bacteria. The use of bacteriophages to control bacterial infection, phage therapy, has become the focus of renewed attention. Therefore, the comprehensive research on biological characteristics of Enterococcus phages and phage therapy against enterocococcal infection may be an importantly practical significance.A total of4phages (referred to as φEC-ZZ2, cpEc-ZZ3, φpEc-ZZ7and φpEc-ZZ8) were isolated from the hospital wastewater samples using20enterocococcal clinical strains including E. faecalis (12strains) and E. faecium (8strains). Morphological analysis revealed that these four lytic phages belong to Caudovirale order, Siphoviridae family. In this study, biological features of phage φEc-ZZ2and φEc-ZZ8were carried out, and the genome sequence of cpEc-ZZ2was determined. The results of these investigations indicate as following:(1) Both phage φEc-ZZ2and φEc-ZZ8were shown to have an isometric head of40nm in diameter and a filamentous tail of200nm in length.(2) Phage (pEc-ZZ2was found to lyse50%of enterocococcal strains. cpEc-ZZ8was shown to specifically lyse55%of enterocococcal strains.(3) Phage DNA of φEc-ZZ2and φEc-ZZ8was sensitive to several restriction endonucleases, including Hind III, EcoR V, Nde I and Xba I.(4) One-step growth assays were performed for determination of latent periods and burst size of phages. The latent period of cpEc-ZZ2was10min and the average burst size was100phage particles per infected cell. φEc-ZZ8is characterized with a longer latent period of15min and a bigger burst size of150phage particles per infected cell.(5) Proteomic patterns of phages were obtained by SDS-PAGE. A total of two major protein bands and five minor protein bands were observed in cpEc-ZZ2, and three major protein bands and eight minor protein bands were observed in cpEc-ZZ8.(6) Phage φEc-ZZ2and φEc-ZZ8exhibited a stably lytic activity at different pH ranging from4to10and after30min at60℃.(7) Sequencing of the genome of cpEc-ZZ2revealed that the phage had liner double-stranded DNA with a length of41,170bp. G+C content is34.59%. The genome of φEc-ZZ2contained59predicted ORFs. One scaffold, one contig and two minisatellite genes were found. Microsatellite genes, potential tRNA coding genes and rRNA coding genes are not found.In conclusion,4lytic phages of E. faecalis, based on morphological characteristics, belong to Caudovirale order, Siphoviridae family. cpEc-ZZ2and φEc-ZZ8were shown to have a shorter latent period, higher lytic activity, and more stable at60℃and at different pH values, suggesting φEc-ZZ2and cpEc-ZZ8as bioagents with a high potential to be applied for bacteriophage-based therapy against infections caused by E.faecalis strains. Sequencing of the genome of φEc-ZZ2revealed that the phage had liner double-stranded DNA with a length of41,170bp. G+C content is34.59%. The genome of φEc-ZZ2contained59predicted ORFs, with a length of35,760bp.