Molecular characterization of lytic phages specific to antibiotic resistant isolates of Pseudomonas fluorescens infecting Labeo rohita and Clarias batrachus of Sub Himalayan region

Abstract

Aim : This investigation was aimed to develop antibacterial strategy to circumvent the problem of antibiotic resistant P. fluorescens infecting L rohita and Indian walking catfish, C. batrachus in Himalayan and Sub Himalayan regions. Methodology : Four lytic phages (PFPD, PFPK, PFPN and PFPC) were isolated against ten isolates of pathogenic P. fluorescens following overlay method using water and bottom sediments of Himalayan and Sub Himalayan regions. One step growth experiment was carried out to know the eclipse, latent periods and burst size of phages. Phage genomic DNA was extracted following chloroform: phenol: isoamyl method and digested using restriction enzymes (EcoRI and Hind III) (Bangalore genei, Bangalore) and isolation of phage protein was made as per Laemmli (1970) with slight modification. Results : Phages were identified as a member of family Siphoviridae (isometric head of 236.42nm and non-contractile lona tail of 521.22 nm) with havina ds DNA of 20-21 kbo and structural viral proteins of 22 -102 kDa. Phages exhibited minimum eclipse period (10 - 15 min), latent period (20 min) and highest burst size of 130. These phages conferred clear lytic plaques in the lawn of 8 of 10 (80%) host bacterium at 0.01 MOI, tolerated wide range of temperature (25 to 35°C), pH (6.5 to 8.0), yielded highest titer (108pfu) at 30 ±5 °C and exhibited highest (99%) in vitro lytic activities. The host bacterium did not develop resistance to all the phages studied during the entire period of the study. Interpretation : The profound in vitro lytic attributes of PFPD and PFPK phages and their tolerance to temperature and pH at par with aquaculture activities reveals that they may be a suitable option to alleviate transmission of even systematic P. fluorescens infection in hatchery and culture system. This information will provide more insight into the potential use of phage to mitigate incidences of Bacterial Haemorrhagic Septicaemia in aquaculture.