A novel Vibrio alginolyticus phage and its therapy application in Portunus trituberculatus larvae

Ubiquitous pathogenic marine Vibrio alginolyticus is a very important pathogenic factor causing the outbreaks of sever vibriosis in marine-cultured animals broadly, an opportunistic pathogen for human, and a major pathogen associated with seafood. Antimicrobial drug resistances increased considerably, making the development of alternative therapeutics urgently needed. Isolation and characterization of V. alginolyticus phage vB_Va_Val-yong3 was performed. vB_Va_Val-yong3 contains an icosahedral head and a flexible tail. The diameter of the head is 62 +/- 4 nm. The length and width of the tail is 110 +/- 9.5 nm and 8 +/- 1 nm, respectively. Its burst size is 87.5 PFU/cell. vB_Va_Val-yong3 is euryhaline, chloroform-tolerant, relatively UV-tolerant, stable at 4-50 degrees C and pH 5-12. Its genome size and GC content are 42,534 bp and 45.6%. It shared the highest nucleotide sequence similarity of 29.67% and the highest intergenomic similarity of 16.3% with all the other phages in PASC scanning and VIRIDIC scanning. Both the values were far below the threshold (> 70%) for genus definition, which illustrated that vB_Va_Val-yong3 revealed a novel phage genus. In the proteomic tree, vB_Va_Val-yong3 and three unclassified Vibrio phages (HY01, ST2-1pr and Seahorse) formed a clade, which clustered with two other clades representing the Mesyanzhionvvidriae family and the Casjensvidriae family respectively. VirClust analysis revealed that 27.7% of genes were shared between Vibrio phages vB_Va_Val-yong3, HY01 and ST2-1pr, which was higher than the family demarcation criteria (> 17%). Far <17% of genes shared between the three Vibrio phages and other viruses (including phage Seahorse and the phages of Mesyanzhionvvidriae and Casjensvidriae family). We advocate the establishment of a new phage family harboring Vibrio phages vB_Va_Val-yong3, HY01 and ST2-1pr. Phage therapy tests on swimming crab Portunus trituberculatus larvae were performed via immersion. vB_Va_Val-yong3 increased the survival rate of the larvae from 33.33% to 51.67% (p < 0.01). Very satisfactorily, the survivors of the infected larvae treated with vB_Va_Val-yong3 could molt and grow normally, while the survivors of the untreated larvae infected with V. alginolyticus all had the problems of molting and slow growth. This brings the very valuable results: all the survivors of the control groups were dwarf, while that of the experimental groups were normal. Food processing tests were also performed using vB_Va_Val-yong3 to eliminate V. alginolyticus in sashimi. The relative bacteriostatic rate of vB_Va_Val-yong3 in sashimi was 97.58% (p < 0.01). Results suggest that vB_Va_Val-yong3 has good potential to be developed as therapeutic agent against V. alginolyticus. This study enriches knowledge about Vibrio phages and lays a foundation for the application of the V. alginolyticus phage.