Molecular Study of Broad Host Range Bacteriophage Against Colistin Resistant Salmonelly Typhi for Potential Use in Phage Therapy

Abstract

Introduction: Emergence of antibiotic resistance worldwide brings a major threat to treat the bacterial infections. In present context, resistance is seen almost all antibiotics even in colistin which is last line drugs. The common pathogens of colistin resistant are Salmonella spp and Escherichia coli mostly associated with animal infection. So, now it becomes an urgent need to investigate alternative treatment options. The use of Bacteriophages is an important alternative approach in the current era of drug-resistant pathogens. In this study, we aim to isolate lytic phages against colistin resistant Salmonella spp and characterize genetically for potential use in phage therapy. Methodology: Host bacterial strains were identified by biochemical test and 16S rRNA sequencing and antibiotic susceptibility test was performed by disc diffusion method. Bacteriophage isolation was done by Double Layer Agar Assay method. Burst size and latent period of phage was determined by one step growth curve experiment. Phage stability was also checked against pH and temperature. Multi host range property was also determined by spot assay and efficiency of plating method. Most potent phage was confirmed by Transmission Electron Microscopy and protein profiling was done by SDS PAGE. Whole genome sequence was done by Illumina platform to rule out the presence or absence of any virulence gene of bacterial origin. Results: Colistin resistant Salmonella Typhi was determined by antibiotic susceptibility test and host was identified from 16S rRNA sequencing and gene sequence submitted to NCBI GenBank. Nine different bacteriophages were isolated against three different strains of Salmonella Typhi. Isolated phages showed broad lytic ability against its host and other genus. Burst size of most potent phages, phage TU_sal2T and phage TU_sal5K were found to be 50 and 45 virion per bacterium with latent period of 20min at multiplicity of infection (MOI) 10. Optimum temperature and pH were determined as 37˚C and 8 respectively for both phages. Transmission electron micrograph revealed that the phage TU_sal2T belong to order Caudovirales and family Myoviridae. Whole genome sequencing revealed that phage TU_sal2T did not possess any virulent gene of bacterial origin, integrase gene and other toxic gene within their genome. Conclusion: Our finding showed phages were not extremely host specific, they evolved to achieve broader host range. Phage TU_sal2T could be a potential candidate for the therapeutics and biocontrol of multidrug resistant pathogens specifically for Salmonella spp.

Keywords: Bacteriophage, Colistin resistant, Host range, MDR, Phage Therapy