Isolation and Characterization of Bacteriophages and Their Potential to Control Bacterial Biofilm

Abstract

Biofilms are complex structures consisting of bacterial colonies encased in a mucilaginous protective coating, and represent major virulence factors contributing to the chronicity of many microbial infections. Biofilms are estimated to be involved in more than 60% of nosocomial infections and associated with about 80% of all chronic infections. The aim of the current study was to isolate and characterize bacteriophages infecting biofilm-forming multi-drug resistant E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter cloacae and Shigella dysenteriae, and to study the potential of those bacteriophages to control bacterial planktonic cells and biofilms. Clinical bacterial strains were selected on the basis of their biofilm-forming ability. Five bacteriophages were isolated from sewage water samples and named MJ1, AZ1, MJ2, Z and WZ1, where MJ1 infects E. coli, AZ1 infects P. aeruginosa, MJ2 infects E. cloacae, Z infects K. pneumoniae, and WZ1 infects S. dysenteriae. All phages had a narrow host range (only few bacterial strains). Adsorption rates of all phages to their hosts were significantly enhanced in the presence of MgCl2 or CaCl2. Each phage was classified into a viral family based on electron microscopy analysis, and assayed for heat- and pH-stability, latent period, burst size per cell, protein composition by SDS-PAGE, nucleic acid composition by agarose gel electrophoresis, and range of DNA bands upon EcoR enzyme restriction digestion. Phages MJ1 and WZ1 were assigned to the family Myoviridae, and displayed stability between 37-65 °C, and pH 5-11, with 21 and 24 minutes latent periods, and burst sizes of 300 and 430 phages per cell, respectively. MJ1 had a genome size of approximately 32 kb, with eleven proteins (12-110 kDa), and produced 2 DNA bands of upon digestion, whereas WZ1 had a genome of ~38 kb, with twelve proteins (22-150 kDa), and produced 3 bands upon digestion. Phages AZ1 and Z were assigned to the family Siphoviridae, and displayed stability between 37-70 °C, and pH 3-11 (for AZ1) or pH 5-11 (for Z), with a 33 min and 24 min latent periods, and burst sizes of 326 and 320 phages per cell, respectively. AZ1 had a Abstract xix genome size of ~50 kb, with seventeen proteins (12-110 kDa), and produced 9 DNA bands upon digestion, whereas Z had a genome of ~36 kb, with six proteins (18-65 kDa), and produced 2 bands upon digestion. Phage MJ2 was classified in the family Podoviridae, and displayed stability at 37-65°C, and at pH 5-11, with a 21 min latent period and a burst size of 350 phages per cell. MJ2 had a genome size of ~40 kb, with eleven proteins (12-150 kDa), and produced 2 DNA bands of upon digestion. The isolated phages were checked for their lytic activity against suspensions of their host bacteria. Phages MJ1, MJ2, AZ1, Z and WZ1 significantly reduced log-phase growth of bacterial cultures, showing no resistance within 5 hrs, and were effective in reducing the biofilm biomass of their respective hosts after 48 hrs, with more than 2-fold, 3-fold, 2-fold, 3-fold and 1.5-fold reduction, respectively. The susceptibility of the hosts to lysis by the specific phages were compared in both planktonic form (stationary phase) and in biofilm phenotype. Bacteria in biofilms and stationary planktonic phase were killed at a lower rate than log-phase planktonic bacteria. Additionally, E. cloacae and K. pneumoniae biofilm-formation was induced on stainless steel plates for 48 hrs, and tested for lysis by their respective phages. Significant biofilm reduction, but no total eradication, was detected for both bacteria under these conditions. Interestingly, the log-phase growth of P. aeruginosa and the 48 h biofilm biomass (up to 6-fold reduction) were significantly reduced by treatment with phage cocktail (MJ1, KH-49, AZ1) as compared to treatment with the single phages. In conclusion our findings suggest that waste water is a good source for finding bacteriophages against newly emerging antibiotic resistant bacteria. Phages can be used to control bacteria both in planktonic form and in biofilms. Single phage species may not be able to completely eradicate bacterial biofilm, but our present findings suggest that phage cocktails offer greater potential in eradication of bacteria (both in suspension and biofilms), and such cocktails can be further used for elaborated phage therapy studies.