Characterization of bacteriophage VVP001 and its application for the inhibition of Vibrio vulnificus causing seafood-borne diseases.

Vibrio vulnificus is a major food-borne pathogen that causes septicemia and cellulitis with a mortality rate of >50%. However, there are no efficient natural food preservatives or biocontrol agents to control V. vulnificus in seafood. In this study, we isolated and characterized a novel bacteriophage VVP001. Host range and transmission electron microscopy morphology observations revealed that VVP001 belongs to the family Siphoviridae and specifically infects V. vulnificus. Phage stability tests showed that VVP001 is stable at a broad temperature range of -20 °C to 65 °C and a pH range from 3 to 11, which are conditions for food applications (processing, distribution, and storage). In vitro challenge assays revealed that VVP001 inhibited V. vulnificus MO6-24/O (a clinical isolate) growth up to a 3.87 log reduction. In addition, complete genome analysis revealed that the 76 kb VVP001 contains 102 open reading frames with 49.64% G + C content and no gene encoding toxins or other virulence factors, which is essential for food applications. Application of VVP001 to fresh abalone samples contaminated with V. vulnificus demonstrated its ability to inhibit V. vulnificus growth, and an in vivo mouse survival test showed that VVP001 protects mice against high mortality (survival rate >70% at a multiplicity of infection of 1000 for up to 7 days). Therefore, the bacteriophage VVP001 can be used as a good natural food preservative and biocontrol agent for food applications.

Bacteriophages Against Pathogenic Vibrios in Delaware Bay Oysters (Crassostrea virginica) During a Period of High Levels of Pathogenic Vibrio parahaemolyticus.

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.

Vibrio spp. infections.

Vibrio is a genus of ubiquitous bacteria found in a wide variety of aquatic and marine habitats; of the >100 described Vibrio spp., ~12 cause infections in humans. Vibrio cholerae can cause cholera, a severe diarrhoeal disease that can be quickly fatal if untreated and is typically transmitted via contaminated water and person-to-person contact. Non-cholera Vibrio spp. (for example, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus) cause vibriosis - infections normally acquired through exposure to sea water or through consumption of raw or undercooked contaminated seafood. Non-cholera bacteria can lead to several clinical manifestations, most commonly mild, self-limiting gastroenteritis, with the exception of V. vulnificus, an opportunistic pathogen with a high mortality that causes wound infections that can rapidly lead to septicaemia. Treatment for Vibrio spp. infection largely depends on the causative pathogen: for example, rehydration therapy for V. cholerae infection and debridement of infected tissues for V. vulnificus-associated wound infections, with antibiotic therapy for severe cholera and systemic infections. Although cholera is preventable and effective oral cholera vaccines are available, outbreaks can be triggered by natural or man-made events that contaminate drinking water or compromise access to safe water and sanitation. The incidence of vibriosis is rising, perhaps owing in part to the spread of Vibrio spp. favoured by climate change and rising sea water temperature.

Morphological characterization and biocontrol effects of Vibrio vulnificus phages against Vibriosis in the shrimp aquaculture environment.

The re-emerging field of phage therapy is the potential biocontrol agents for the transfer of virulence factor and to kill their bacterial hosts. In this study, the lytic Vibrio vulnificus phages were studied to provide a better understanding of phage-host interactions and development of phage therapy. Four new V. vulnificus phages were detected from shrimp aquaculture system, named VV1, VV2, VV3 and VV4. All lytic V. vulnificus phages are the Tectiviruses of the family Tectiviridae with typical double layered elongated icosahedral head and tailless morphology. Lytic V. vulnificus phages which infect other Vibrio isolates were further characterized by long term storage, enzymes treatment, organic solvents treatment, detergents treatment, pH stability, temperature stability, agar bioassay method and one-step growth experiment. The effects of chloroform, acetone, ethyl alcohol, methyl alcohol, ribonuclease (RNase), trypsin, protease, Triton-X100 treatments were not affected the growth of VV1, VV2, VV3 and VV4 phages. These phages (VV1-VV4) were inactivated completely with temperature (over 60 °C), pH (<3 or >12), lysozyme and sodium dodecyl sulfate (SDS) treatment. One-step growth experiments indicated that the latent period was at 3 h and burst size was at 37 °C. Agar bioassay method indicated that the percentage of inhibition was 75% (VV1) and 70% (VV2, VV3 & VV4), respectively. SDS-PAGE analysis of all V. vulnificus phages had similar protein patterns with molecular weight masses of 260, 249, 204, 148, 63, 59, 22 and 15 kDa. Hence, it could be concluded that V. vulnificus phage had a broad lytic spectrum and potential biocontrol of luminous Vibriosis in the shrimp aquaculture system.

Vibrio vulnificus phage PV94 is closely related to temperate phages of V. cholerae and other Vibrio species.

BACKGROUND: Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species. RESULTS: In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5'-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218. CONCLUSION: We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.

Vibrio vulnificus bacteriophage SSP002 as a possible biocontrol agent.

A novel Vibrio vulnificus-infecting bacteriophage, SSP002, belonging to the Siphoviridae family, was isolated from the coastal area of the Yellow Sea of South Korea. Host range analysis revealed that the growth inhibition of phage SSP002 is relatively specific to V. vulnificus strains from both clinical and environmental samples. In addition, a one-step growth curve analysis and a bacteriophage stability test revealed a latent period of 65 min, a burst size of 23 ± 2 PFU, as well as broad temperature (20°C to 60°C) and pH stability (pH 3 to 12) ranges. A Tn5 random transposon mutation of V. vulnificus and partial DNA sequencing of the inserted Tn5 regions revealed that the flhA, flhB, fliF, and fleQ mutants are resistant to SSP002 phage infection, suggesting that the flagellum may be the host receptor for infection. The subsequent construction of specific gene-inactivated mutants (flhA, flhB, fliF, and fleQ) and complementation experiments substantiated this. Previously, the genome of phage SSP002 was completely sequenced and analyzed. Comparative genomic analysis of phage SSP002 and Vibrio parahaemolyticus phage vB_VpaS_MAR10 showed differences among their tail-related genes, supporting different host ranges at the species level, even though their genome sequences are highly similar. An additional mouse survival test showed that the administration of phage SSP002 at a multiplicity of infection of 1,000 significantly protects mice from infection by V. vulnificus for up to 2 months, suggesting that this phage may be a good candidate for the development of biocontrol agents against V. vulnificus infection.

Complete genome sequence of Vibrio vulnificus bacteriophage SSP002.

Vibrio vulnificus phages are abundant in coastal marine environments, shellfish, clams, and oysters. SSP002, a V. vulnificus-specific bacteriophage, was isolated from oysters from the west coast of South Korea. In this study, the complete genome of SSP002 was sequenced and analyzed for the first time among the V. vulnificus-specific bacteriophages.

Sharing of prey: coinfection of a bacterium by a virus and a prokaryotic predator.

UNLABELLED: Rarely, if ever, has a single bacterial cell been confirmed to simultaneously host two fundamentally different predators. Two such predators are viruses and the predatory prokaryotes known as Bdellovibrio and like organisms. Viruses or bacteriophage are particles requiring prey cells in an active metabolic state to complete their life cycle. The Bdellovibrio and like organisms, unlike viruses, are bacteria that can efficiently infect and grow in prey which are in stationary phase. In this study, electron microscopic examination revealed an unprecedented coinfection by the two agents of Vibrio vulnificus, introducing a new bacterial predation paradigm. Rather than the viruses and Bdellovibrio and like organisms competing for a single prey cell, both can survive in the same cell and successfully reproduce themselves. This is an especially valuable mechanism when the prey is in short supply, and the survival of the predators may be at stake. IMPORTANCE: This article describes the coinfection of a prokaryotic prey or host cell by both a bacteriophage (phage) and the predatory bacterium of the group Bdellovibrio and like organisms (BALOs). Such coinfection has not been previously reported and therefore introduces a new paradigm for predation of bacteria. This finding invites new studies on the interactions of BALOs, phage, and prey in predation. Predation is an important mechanism in nature for helping to keep bacterial populations in check and also plays a major role in the cycling of nutrients through the microbial loop. How dual infection by phage and BALOs imposes on these and other functions of predation is fertile ground for future studies and serves as a keystone reference on bacterial predation and mortality.

Characterization of a new plasmid-like prophage in a pandemic Vibrio parahaemolyticus O3:K6 strain.

Vibrio parahaemolyticus is a common food-borne pathogen that is normally associated with seafood. In 1996, a pandemic O3:K6 strain abruptly appeared and caused the first pandemic of this pathogen to spread throughout many Asian countries, America, Europe, and Africa. The role of temperate bacteriophages in the evolution of this pathogen is of great interest. In this work, a new temperate phage, VP882, from a pandemic O3:K6 strain of V. parahaemolyticus was purified and characterized after mitomycin C induction. VP882 was a Myoviridae bacteriophage with a polyhedral head and a long rigid tail with a sheath-like structure. It infected and lysed high proportions of V. parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae strains. The genome of phage VP882 was sequenced and was 38,197 bp long, and 71 putative open reading frames were identified, of which 27 were putative functional phage or bacterial genes. VP882 had a linear plasmid-like genome with a putative protelomerase gene and cohesive ends. The genome does not integrate into the host chromosome but was maintained as a plasmid in the lysogen. Analysis of the reaction sites of the protelomerases in different plasmid-like phages revealed that VP882 and PhiHAP-1 were highly similar, while N15, PhiKO2, and PY54 made up another closely related group. The presence of DNA adenine methylase and quorum-sensing transcriptional regulators in VP882 may play a specific role in this phage or regulate physiological or virulence-associated traits of the hosts. These genes may also be remnants from the bacterial chromosome following transduction.