Physicochemical and genomic characterization of bacteriophage against Pseudomonas aerouginosa causing urinary tract infection; An apprach to biofilm reduction.

Abstract

Introduction: Pseudomonas aeruginosa is resistance to most of the antibiotics. This makes treatment of Pseudomonas aeruginosa difficult. The problem is further compounded by its ability to form biofilm. The aim of this study was to isolate lytic phage against antibiotic resistant Pseudomonas aeruginosa and to use it in the reduction of Pseudomonas aeruginosa biofilm. Methodology: Bacteriophage isolation was done by Double Layer Agar Assay method. Burst size and latent period of the phage was determined by one step growth curve experiment. Phage stability was also analyzed against different temperature and pH range. Phage cocktail was used to disrupt biofilm. The synergistic effect of phage and antibiotic in reducing biofilm was also analyzed. Effect of various external factors in phage stability was examined. Whole genome sequencing of phage DNA was done. Result: Lytic bacteriophage against Pseudomonas aeruginosa was isolated. The latent period of the phage TU_pse1B was 30 minute and burst size was 27 virion per bacterium. The optimum temperature for the phage TU_pse1B was 37:C and optimum pH was 9. Three distinct bands of phage proteins of size 35, 40 and 100KDa were observed after performing SDS PAGE. Phage DNA size was determined to be larger than 10 Kb from agarose gel electrophoresis. Whole genome sequencing of phage revealed its size to be 43,428 nucleotides (43 Kb) in length and the GC content of 62.16%. Calcium ion increased the phage adsorption. Phage showed stability against SDS and osmotic shock whereas it was susceptible to ethanol, acetone and CTAB. Phage TU_pse1B reduced the biofilm by 60.99% whereas Phage TU_pse1Bi reduced the biofilm by 60.37%. Synergism of phage and antibiotic was observed in reducing biofilm. Phage plus antibiotic reduced biofilm by further 26.67% than phage alone. Conclusion: Phage TU_pse1B showed good stability to various physiochemical factors as well as it was efficient in reducing Pseudomonas aeruginosa biofilm. So, this phage can be a good candidate for controlling antibiotic resistant Pseudomonas aeruginosa. Keywords: Antibiotic resistance, Biofilm, SDS, Phage cocktail, AST, Burst size.