Physiological and biochemical characteristics and virulence of Photobacterium damselae subsp. damselae mediated by a plasmid

Abstract:

[Background] Photobacterium damselae subsp. damselae (PDD) can infect a variety of marine animals to cause diseases. Comparative genome analysis revealed that PDD strains harboring a large plasmid pPDD1608 exhibit strong pathogenicity. Furthermore, gene annotation analysis unveiled the presence of virulence genes and secretion system-related genes in this plasmid. However, there is a dearth of studies examining the functions of this plasmid. [Objective] In this study, a highly pathogenic PDD strain was selected and its plasmid-deleted mutant was generated through induced mutagenesis. The phenotypic characteristics and pathogenicity were compared between the wild type and the mutant to reveal the role of this plasmid in the phenotype and pathogenicity of PDD. [Methods] The plasmid-deleted strain ΔpPDD1608 was successfully constructed by the temperature variation-SDS method. Then, the phenotypes of PDD1608 and ΔpPDD1608 cultured on the TSB plates were observed by scanning electron microscopy. The physiological and biochemical characteristics were measured by the microbiochemical tests and compared between PDD1608 and ΔpPDD1608. The antibiotic susceptibility of PDD1608 and ΔpPDD1608 was determined by the K-B disk diffusion method. The phospholipase and hemolytic activities of the two strains were measured by the indicator media in vitro. The pathogenicity of PDD1608 and ΔpPDD1608 was assessed through the artificial challenge test by injection in Sebastes schlegeli. [Results] The large plasmid of PDD1608 was effectively eliminated by the temperature variation-SDS method, and a gene-specific PCR method was established based on the whole genome sequence of PDD1608. The experimental results revealed no discernible disparities in antibiotic susceptibility or physiological and biochemical characteristics between PDD1608 and ΔpPDD1608. However, ΔpPDD1608 showed reduced hemolytic and phospholipase activities. Furthermore, the artificial challenge test demonstrated that PDD1608 displayed higher pathogenicity towards S.schlegeli, resulting in the death of all the tested fish within 24 h, whereas ΔpPDD1608 exhibited lower pathogenicity towards S.schlegeli, causing a 24-h mortality rate of 56.6%. [Conclusion] The comparison on hemolytic and phospholipase activities and pathogenicity between ΔpPDD1608 and PDD1608 confirmed that the plasmid pPDD1608 played a crucial role in mediating the high pathogenicity of PDD1608.