Enhanced Host Range and Reduction of Biofilm by Synergistic Effect of Phage Cocktails Isolated from Rivers of Kathmandu, Nepal

Abstract

Introduction: The world is running out of antibiotics and search for new therapeutic tools to tackle this problem is a major concern worldwide. Using phages in therapeutics has great potential to fight antimicrobial resistance, which can be a solution to this global threat. Bacteriophages are viruses that specifically infect the bacterial host and lyse the host bacteria during their lytic cycle. Phages in therapeutics has been investigated from over a century, now it has been developed as a revitalized therapy. Narrow host range of phages possess both benefits and limitations; highly specific phages may not harm beneficial microbes while such phages cannot be effective to treat multi-bacterial infections. Combination of different phages with complementary features are often used to mitigate such issues. Concept of phage cocktails in treatment of infectious diseases is the most highlighted topic in phage therapy. The use of different phages combined in cocktails allows for the treatment of multiple pathogens, broadening the phages’ action spectrum. In this study, we aimed to test the efficacy of phage cocktails to enhance host range along with effective biofilm reduction. Methodology: Phages were isolated from the river water using different clinical strains of bacteria and were combined to make cocktails. Intraspecific and interspecific host range with both individual phages and cocktails phages was assessed by spot assay and liquid lysis assay/killing assay. Reduction of biofilm by individual phage and phage cocktails was determined by crystal violet assay. Results: From this research work, we found that Klebsiella phage cocktails were effective against E. coli pathogenic strains. On multi host range analysis with individual and cocktail Klebsiella phages, host range was enhanced by cocktails, which was confirmed by efficiency of plating assay. Morphological identification of phages by TEM showed all the Klebsiella phages were of Order Caudovirales and family Podoviridae. The maximum bacterial growth inhibition was at 4 hours of infection, with phage cocktails, showing the bacterial inhibition upto 50%. The bacteria used in our experiment were found to be moderate biofilm producers and there was substantial reduction in biofilm with phage cocktails as compared to individual phages. Conclusion: The results indicate that the application of the phages in the form of a cocktail have their potential to be used presumptively to control multi-bacterial infection. Phage cocktails of Klebsiella pneumoniae can effectively lyse E. coli bacterial strains, though different genus, than individual phages, along with effective biofilm reduction. With the extensive research, such phage therapy can treat infection in-vitro; as an application to treat multi-bacterial infections as well as such phage cocktails can be used as disinfectant to decontaminate hospital indwelling devices. Keywords: Antibiotic resistance, Bacteriophage, Biofilms, Host range, Phage cocktails