Isolation and characterization of novel broad-host range Vibrio phages for phage-based biocontrol formulation for the treatment of early mortality syndrome in shrimps
Kitiya Vongkamjan Auranda,*, Wichanan Wannasrichana, Haemarat Khongkhaia, Ubolsree Leartsakulpanichb, Warangkhana Songsungthongb , Kittiya Showpanishb , Sakunrat Ubonprasertb,Wachiraporn Wachiradusitband Kallaya Dangtipb
aDepartment of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok, Thailand
bNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand
Early Mortality Syndrome (EMS) causing acute infections in shrimps mostly caused by Vibrio spp., is undermining the aquatic industry around the world. Bacteriophages, viruses that infect bacteria are becoming a drug of choice for EMS treatment because of their chemical-free properties.This study aims to isolate bacteriophages and characterize their potential as the candidates against Vibrio spp. for EMS treatment.
Over 400 samples were collected from shrimp farms, mangrove forests, and fresh markets, and shellfish-aquatic animals for phage isolation. Isolated phages were tested host range with disease-causing strains of Vibrio spp. Stabilities of phages under various conditions, including pH, temperature, salinity were tested. Transmission electron microscopy (TEM) and whole-genome sequencing of phage candidates were analyzed to confirm safety and suitability of biocontrol application.
Across all samples, over 200 phages were isolated, and we successfully identified 4 candidate phages from aquatic wastewater named APT10, APR17, ASP27, and APT49. These were classified by TEM as podoviruses. Phages showed high lysis ability of 60% with 40 tested strains, especially V. parahaemolyticus. All phages showed reduced survivability to acidic pH 3 in 24 hours, while survived over pH 9, and 10 without significant PFU reduction. At 45°C, phages could tolerate with a slight decrease in titer. Phages ASP27 and APT10 survived at 60°C for 24 hours as indicating by countable titer of 8 × 105 PFU/mL. Furthermore, all phages could also tolerate various concentrations of salt (up to 15% NaCl). Genome analysis showed that their genome sizes range from 41-43 kb classified under Autoscriptoviridae family; Maculvirus genus. Sequence similarity analysis between these phages revealed that they were different in species. Overall, antibiotic resistant, toxic, or lysogenic-associated genes were not found in their genomes, indicating that these phages were suitable as candidates for further development of phage-based biocontrol for further studies.
Phages APT10, APR17, ASP27, and APT49 were characterized as promising candidates against Vibrio spp. for EMS biocontrol, suggested by their broad host range against Vibrio spp., stability across various environmental conditions, and absence of harmful genomic elements support their suitability for further development as phage-based therapeutics in aquaculture.
