Isolation, identification, and characterization of Vibrio parahaemolyticus phage Vpas_PP24

Abstract:

[Background] Vibrio parahaemolyticus is a major pathogen of Penaeus vannamei. Posing a threat to food safety, environment, and sustainable development, conventional antibiotic therapy is no longer effective. Biocontrol of this bacteria seems to inevitable for the sustainable development of P. vannamei aquaculture. Phage, a natural safe antibacterial, is highly specific and typically only infects or kills an individual species of bacteria with high efficiency. [Objective] To identify virulent phage that can efficiently lyse V. parahaemolyticus so that the set of methodology was established to explore the phage agents for the control of V. parahaemolyticus. [Methods] Double-layer agar technique was used to enrich phages from sewage samples of seafood market with 28 strains of V. parahaemolyticus derived from diseased shrimps. The lytic spectra of phages were determined through spot test. The one with broad spectrum was characterized by transmission electron microscopy, biological characterization, and whole-genome sequencing. [Results] Vpas_PP24, a virulent phage against V. parahaemolyticus was screened out. It had an icosahedral head with about 92 nm in length and around 46 nm in width and a long tail of about 147 nm in length, which was thus identified as a member of Siphoviridae in Caudovirales. The full-length genome of Vpas_PP24 was 83 482 bp, and it harbored 118 open reading frames (ORFs), with 13 known protein-coding genes, and no non-coding RNA, virulence genes, or resistance genes. Genome alignment suggested that Vpas_PP24 may be a new phage species against Vibrio. The phage could lyse 15 (54%) of the 28 V. parahaemolyticus strains and 18 (16%) of 116 other Vibrio species. The optimal multiplicity of infection (MOI) of Vpas_PP24 was 0.000 1 and titer was 3.0×10¹⁰ PFU/mL. One-step growth curve indicated that it had a latent period of 10 min, rise period of 150 min, and burst size of 30 PFU/cell. The optimal temperature and pH for the phage growth were<50 ℃ and pH 4.0–11.0, respectively. The phage was tolerant to chymotrypsin, papain, and shrimp hepatopancreas extract but sensitive to proteinase K. Vpas_PP24 was also sensitive to ultraviolet radiation. The emergence frequency of Vpas_PP24-insensitive mutants was 2×10^-5. [Conclusion] A virulent phage Vpas_PP24 with new genotype was screened out. It has the potential to be further developed into novel anti- V. parahaemolyticus agent, thanks to its broad lytic spectrum, biological properties, as well as its promising stability.