Prophages carrying Zot toxins on different Vibrio genomes: A comprehensive assessment using multilayer networks.

Vibrios, a group of bacteria that are among the most abundant in marine environments, include several species such as Vibrio cholerae and Vibrio parahaemolyticus, which can be pathogenic to humans. Some species of Vibrio contain prophages within their genomes. These prophages can carry genes that code for toxins, such as the zonula occludens toxin (Zot), which contribute to bacterial virulence. Understanding the association between different Vibrio species, prophages and Zot genes can provide insights into their ecological interactions. In this study, we evaluated 4619 Vibrio genomes from 127 species to detect the presence of prophages carrying the Zot toxin. We found 2030 potential prophages with zot-like genes in 43 Vibrio species, showing a non-random association within a primarily modular interaction network. Some prophages, such as CTX or Vf33, were associated with specific species. In contrast, prophages phiVCY and VfO3K6 were found in 28 and 20 Vibrio species, respectively. We also identified six clusters of Zot-like sequences in prophages, with the ZOT2 cluster being the most frequent, present in 34 Vibrio species. This analysis helps to understand the distribution patterns of zot-containing prophages across Vibrio genomes and the potential routes of Zot-like toxin dissemination.

Genome-based characterization of a novel prophage of Vibrio parahaemolyticus, VPS05ph1, a novel member of Peduoviridae.

Vibrio parahaemolyticus is a globally important bacterium related to climate warming and health threat to human and marine animals. Yet, there is limited knowledge about its polylysogeny harboring multiple prophages and the genetic information. In this study, two prophages (VPS05ph1 and VPS05ph2) were identified in a V. parahaemolyticus isolate through genomic and transcriptional analyses. Both prophages were determined as HP1-like phages, located in a novel phylogenetic lineage of Peduoviridae. They shared a moderate genome-wide sequence similarity with each other and high synteny with the closest relatives, but showed low identities to the repressor counterparts of the representative phages within the family. In addition, no bacterial virulence genes, antibiotic resistance genes and known phage-encoded lytic proteins were identified on both prophage genomes. Moreover, the V. parahaemolyticus isolate was induced with mitomycin, which caused aberrant cellular morphology and nonviability of bacterial cells and excision of prophage VPS05ph1, accompanied by the respective inhibition and promotion of transcriptions of the cI-like and cox-like regulator genes for phage decision making. Results in this study provide the genetic context of polylysogeny in the V. parahaemolyticus isolate, support the diversity and prevalence of HP1-like phages in vibrios, and promote to explore interactions between the HP1-like prophage and its vibrio host.

Characterization and Genomic Analysis of Novel Vibrio parahaemolyticus Phage vB_VpaP_DE10.

In the present study, a novel lytic Vibrio parahaemolyticus phage, vB_VpaP_DE10, was isolated from sewage samples collected in Guangzhou city, China. Transmission electron microscopy revealed that phage vB_VpaP_DE10 has an icosahedral head (52.4 ± 2.5 nm) and a short non-contracted tail (21.9 ± 1.0 nm). Phage vB_VpaP_DE10 lysed approximately 31% (8/26) of the antibiotic-resistant V. parahaemolyticus strains tested. A one-step growth curve showed that phage vB_VpaP_DE10 has a relatively long latency time of 25 min and a burst size of ~19 PFU per cell. The genome of phage vB_VpaP_DE10 is a 42,871-bp-long dsDNA molecule with a G + C content of 49.19% and is predicted to contain 46 open reading frames, 26 of which are predicted to be related to functions such as phage structure, packaging, host lysis, and DNA metabolism. Sequence comparisons suggested that vB_VpaP_DE10 is a member of the genus Maculvirus within the family Autographiviridae. Morphological and genomic analysis indicated that vB_VpaP_DE10 is a novel V. parahaemolyticus phage.

Characterization and Genomic Analysis of ssDNA Vibriophage vB_VpaM_PG19 within Microviridae, Representing a Novel Viral Genus.

Vibrio parahaemolyticus, a widespread marine bacterium, is responsible for a variety of diseases in marine organisms. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus is also known to cause acute gastroenteritis in humans. While numerous dsDNA vibriophages have been isolated so far, there have been few studies of vibriophages belonging to the ssDNA Microviridae family. In this study, a novel ssDNA phage, vB_VpaM_PG19 infecting V. parahaemolyticus, with a 5,572 bp ssDNA genome with a G+C content of 41.31% and encoded eight open reading frames, was isolated. Genome-wide phylogenetic analysis of the total phage isolates in the GenBank database revealed that vB_VpaM_PG19 was only related to the recently deposited vibriophage vB_VpP_WS1. The genome-wide average nucleotide homology of the two phages was 89.67%. The phylogenetic tree and network analysis showed that vB_VpaM_PG19 was different from other members of the Microviridae family and might represent a novel viral genus, together with vibriophage vB_VpP_WS1, named Vimicrovirus. One-step growth curves showed that vB_VpaM_PG19 has a short incubation period, suggesting its potential as an antimicrobial agent for pathogenic V. parahaemolyticus. IMPORTANCE Vibriophage vB_VpaM_PG19 was distant from other isolated microviruses in the phylogenetic tree and network analysis and represents a novel microviral genus, named Vimicrovirus. Our report describes the genomic and phylogenetic features of vB_VpaM_PG19 and provides a potential antimicrobial candidate for pathogenic V. parahaemolyticus.

Characterization of the Novel Phage vB_VpaP_FE11 and Its Potential Role in Controlling Vibrio parahaemolyticus Biofilms.

Vibrio parahaemolyticus causes aquatic vibriosis. Its biofilm protects it from antibiotics; therefore, a new different method is needed to control V. parahaemolyticus for food safety. Phage therapy represents an alternative strategy to control biofilms. In this study, the lytic Vibrio phage vB_VpaP_FE11 (FE11) was isolated from the sewers of Guangzhou Huangsha Aquatic Market. Electron microscopy analysis revealed that FE11 has a typical podovirus morphology. Its optimal stability temperature and pH range were found to be 20-50 °C and 5-10 °C, respectively. It was completely inactivated following ultraviolet irradiation for 20 min. Its latent period is 10 min and burst size is 37 plaque forming units/cell. Its double-stranded DNA genome is 43,397 bp long, with a G + C content of 49.24% and 50 predicted protein-coding genes. As a lytic phage, FE11 not only prevented the formation of biofilms but also could destroy the formed biofilms effectively. Overall, phage vB_VpaP_FE11 is a potential biological control agent against V. parahaemolyticus and the biofilm it produces.

Biochemical characterization of LysVpKK5 endolysin from a marine vibriophage.

Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.

Characterization of vB_VpaP_MGD2, a newly isolated bacteriophage with biocontrol potential against multidrug-resistant Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major foodborne pathogen and is also pathogenic to shrimp. Due to the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages have shown promise as antimicrobial agents that could be used for controlling antibiotic-resistant strains. Here, a V. parahaemolyticus phage, vB_VpaP_MGD2, was isolated from a clam (Meretrix meretrix) and further characterized to evaluate its potential capability for biocontrol. Podophage vB_VpaP_MGD2 had a wide host range and was able to lyse 27 antibiotic-resistant V. parahaemolyticus strains. A one-step growth curve showed that vB_VpaP_MGD2 has a short latent period of 10 min and a large burst size of 244 phages per cell. Phage vB_VpaP_MGD2 was able to tolerate a wide range of temperature (30 °C-50 °C) and pH (pH 3-pH 10). Two multidrug-resistant strains (SH06 and SA411) were suppressed by treatment with phage vB_VpaP_MGD2 at a multiplicity of infection of 100 for 24 h without apparent regrowth of bacterial populations. The frequency of mutations causing bacteriophage resistance was relatively low (3.1 × 10-6). Phage vB_VpaP_MGD2 has a double-stranded DNA with a genome size of 45,105 bp. Among the 48 open reading frames annotated in the genome, no lysogenic genes or virulence genes were detected. Sequence comparisons suggested that vB_VpaP_MGD2 is a member of a new species in the genus Zindervirus within the subfamily Autographivirinae. This is the first report of a member of the genus Zindervirus that can infect V. parahaemolyticus. These findings suggest that vB_VpaP_MGD2 may be a candidate biocontrol agent against early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) caused by multidrug-resistant V. parahaemolyticus in shrimp production.

Expression and functional study of VpV262 Pol, a moderately halophilic DNA polymerase from the Vibrio parahaemolyticus phage VpV262.

Halophilic organisms are found widely in environments where the salt concentration is higher than 0.2 M. Halophilic proteins isolated from these organisms maintain structural integrity and function under high salt stress, whereas their non-halophilic homologs tend to aggregate and collapse. Here we report for the first time the expression and function of a DNA polymerase (DNAPol) VpV262 Pol, which belongs to DNAPol Family A from Vibrio parahaemolyticus phage VpV262. Enzymatic activity assay revealed that VpV262 Pol possessed 5'-3' polymerase activity as well as 3'-5' proofreading exonuclease activity. VpV262 Pol requires Mg2+ or Mn2+ to catalyze the polymerization reaction. Polymerization activity assay under a wide range of salt concentrations showed that VpV262 Pol maintains the highest polymerase activity with 0-0.3 M of NaCl/KCl and 0-0.5 M KAc (potassium acetate) /KGlc (potassium gluconate) when treated with 0-1 M corresponding salts, in contrast to significantly decreased activity of Phi29 Pol and Taq Pol above 0.2 M. Consistent with typical features of other halophilic proteins, negatively-charged amino acids are more frequently distributed on the surface of VpV262 Pol, contributing to highly solubility and enhanced halotolerance. While 3D-Structure of VpV262 Pol needs to be confirmed by experimental data further, this study here has added a member for the relatively small family of halotolerant DNA polymerase, and provides a valuable reference in isolation and characterization of DNA polymerases from halophilic organisms.

Isolation and characterization of Vibrio parahaemolyticus bacteriophage vB_VpaS_PG07.

A novel bacteriophage vB_VpaS_PG07 (hereafter designated PG07) that infects Vibrio parahaemolyticus was isolated. The bacteriophage was examined by transmission electron microscopy, and the result showed that PG07 belonged to family Siphoviridae, with an isometric polyhedral head (80 nm in diameter) and a long tail (175 nm in length). The one-step growth curve showed that the latent period and burst size were 10 min and 60 PFUs/infected cell, respectively. PG07 had double-stranded DNA genome of 112, 106 bp with 43.65 % G+C content. A total of 158 putative open reading frames (ORFs) were identified in the genome of PG07, including functional genes associated with integration, nucleotide metabolism and replication, structure and packaging and bacterial lysis. Sixteen tRNA genes were discovered, and no genes associated with pathogenicity and virulence were identified. The genome of PG07 showed very low similarity to phage genomes deposited in public databases (77.65 % nucleotide identity and 9 % query coverage). The newly sequenced PG07 could be considered as a novel T5-like virus. PG07 significantly reduced the mortality of shrimps challenged with V. parahaemolyticus, a bacterium causing acute hepatopancreatic necrosis disease (AHPND). The findings highlight the potential of PG07 as an effective antibacterial agent for phage prophylaxis and phage therapy in aquaculture.

Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus.

The adsorption of phages to hosts is the first step of phage infection. Studies have shown that tailed phages use tail fibres or spikes to recognize bacterial receptors and mediate adsorption. However, whether other phage tail components can also recognize host receptors is unknown. To identify potential receptors, we screened a transposon mutagenesis library of the marine pathogen Vibrio parahaemolyticus and discovered that a vp0980 mutant (vp0980 encodes a predicted transmembrane protein) could not be lysed by phage OWB. Complementation of this mutant with wild-type vp0980 in trans restored phage-mediated lysis. Phage adsorption and confocal microscopy assays demonstrated that phage OWB had dramatically reduced adsorption to the vp0980 mutant compared to that to the wild type. Pulldown assays showed that phage tail tubular proteins A and B (TTPA and TTPB) interact with Vp0980, suggesting that Vp0980 is a TTPA and TTPB receptor. Vp0980 lacking the outer membrane region (aa 114-127) could not bind to TTPA and TTPB, resulting in reduced phage adsorption. These results strongly indicated that TTPA and TTPB binding with their receptor Vp0980 mediates phage adsorption and subsequent bacterial lysis. To the best of our knowledge, this study is the first report of a bacterial receptor for phage tail tubular proteins.

Genomic characterization of filamentous phage vB_VpaI_VP-3218, an inducible prophage of Vibrio parahaemolyticus.

The seawater temperature rise can promote the growth of potentially pathogenic Vibrio species. In the North Sea, V. parahaemolyticus strains have been isolated and characterized. These strains contain prophages that may contribute to the emergence of pathogenic strains in the marine environment. Here, we present the genome structure and possible biological functions of the inducible phage vB_VpaI_VP-3218, a novel filamentous phage carried by the V. parahaemolyticus strain VN-3218. Prophages of the strain VN-3218 were induced with mitomycin C and the DNA from the phage induction was sequenced. Two incomplete prophages were identified, only one complete phage genome with length of 11,082 bp was characterized. The phage vB_VpaI_VP-3218 belongs to the Inoviridae family and shows close homology to the Saetivirus genus. This phage can integrate into the chromosomal host genome and carries host-related regions absent in similar phage genomes, suggesting that this phage might integrate in other Vibrio host genomes from the environment. Furthermore, this phage might have a role in pathogenicity due to potential zonula occludens toxin genes. Based on its genomic similarity, the genome of vB_VpaI_VP-3218 phage probably integrates into the lysogen's chromosome and replicates as episome. This study complements prophage induction and bioinformatic studies applied to non-model species of potentially pathogenic Vibrio species. The characterization of this phage provides new insights with respect to the presence of filamentous phages in environmental V. parahaemolyticus strains, which might have a role in the emergence of new pathogenic strains in the North Sea.

Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture.

Vibrio parahaemolyticus is a major seafood-borne pathogen causing significant economic losses in aquaculture systems. Therefore, application of phage encoded enzymes, particularly endolysin, as a new strategy for effective biocontrol and therapeutic agent against bacterial diseases. In the present study, we synthesized endolysin gene (Vplys60) of bacteriophage qdv001 and biochemically characterized by expressing in Pichia pastoris X-33. In addition to, we also investigated the anti-biofilm and anti-vibriosis activity of Pichia-expressing Vplys60 against vibrio challenged in vivo aquaculture model, Artemia franciscana. The result indicated that the predicted molecular size of Pichia expressed Vplys60 was approximately 28 kDa as verified by SDS-PAGE and zymogram. Vplys60 manifested stable activity over broad range of pH (6-10), temperatures (37-75 °C) and salinity (100-600 mM NaCl). Biochemical and in silico analysis revealed that addition of calcium ion (Ca2+) enhanced the lytic activity of Vplys60 whereas other metal ions inhibited the activity. Additionally, calcium-dependent Vplys60 has showed a strong amidase activity by cleaving the peptidoglycan of V. parahaemolyticus. Our data also showed that Vplys60 (75 µg/ml) significantly inhibits biofilm formation (91.6%) and significantly reduced the bacterial population. The in vivo challenge study showed enhanced survival rate in combination with reduced vibrio load in Artemia after administration of Pichia-expressing Vplys60.

Looking for the hidden: Characterization of lysogenic phages in potential pathogenic Vibrio species from the North Sea.

The incidence of potentially pathogenic Vibrio species in the marine environment around Europe, is correlated with the increase of surface seawater temperature. Despite their importance, little is known about the trigger factors of potential outbreak-causing strains in this region. As prophages may compose a major reservoir of virulence traits in marine ecosystems, this study aims to identify and characterize the genomes of lysogenic Vibrio phages exemplarily from the North Sea. Therefore, 31 isolates from potentially pathogenic Vibrio species from the North Sea were screened for inducible prophages with mitomycin C. From them, one V. cholerae isolate and 40% V. parahaemolyticus isolates carried inducible prophages. Three lysogenic phages were selected for genomic characterization. The phage vB_VpaM_VP-3212 (unclassified Myoviridae) has a genome with a length of 36.81 Kbp and 55 CDS were identified. This lysogenic phage of V. parahaemolyticus contains genes related to replicative transposition mechanism, such as transposase and mobile elements similar to Mu-like viruses. The phage vB_VpaP_VP-3220 (Podoviridae, unclassified Nona33virus) has a genome length of 58,14 Kbp and contains 63 CDS. This V. parahaemolyticus phage probably uses a headful (pac) packaging replication mechanism. The phage vB_VchM_VP-3213 (unclassified Myoviridae) has a genome with a length of 41 Kbp and 63 CDS were identified, including integrase and Xer system for lysogenic recombination. This lysogenic phage of V. cholerae has similar genomic features as lambdoid phages. Although no pathogenicity genes were identified, their similarity among other phage genomes indicates that these phages can affect the development of pathogenic Vibrio strains in marine environments.

Genome characterization of novel lytic Myoviridae bacteriophage ϕVP-1 enhances its applicability against MDR-biofilm-forming Vibrio parahaemolyticus.

A pathogen of significance in the aquaculture sector, the Gram-negative marine bacterium Vibrio parahaemolyticus causes gastroenteritis associated with consumption of improperly prepared seafood. This bacterium can be controlled using lytic bacteriophages as an alternative to antibiotics. Ï•VP-1 is a lytic phage of V. parahaemolyticus that was isolated from an aquafarm water sample with the aim of assessing its potential as a bio-control agent and determining its physicochemical properties and genomic sequence. Morphological analysis by transmission electron microscopy and phylogenetic analysis based on the large terminase subunit gene showed that this phage belongs to the family Myoviridae. It could infect multiple-drug-resistant (MDR) V. parahaemolyticus and V. alginolyticus strains of mangrove and seafood origin. With a maximum adsorption time of 30 min, ϕVP-1 has a short latent period of 10 min with burst size of 44 particles/cell. Whole-genome sequencing was done using the Illumina platform, and annotation was done using GeneMarkS and Prodigal. The 150,764bp genome with an overall G+C content of 41.84% had 203 putative protein-encoding open reading frames, one tRNA gene, and 66 predicted promoters. A number of putative DNA replication and regulation, DNA packaging and structure, and host lysis genes were identified. Comparison of the ϕVP-1 genome sequence to those of known Vibrio phages indicated little discernible DNA sequence similarity, suggesting that ϕVP-1 is a novel Vibrio phage. Sequence analysis revealed the presence of 64 potential ORFs with a T4-like genomic organization. In silico analysis suggested an obligate lytic life cycle and showed the absence of lysogeny or virulence genes. The complete sequence of Ï•VP-1 was annotated and deposited in the GenBank database (accession no. MH363700). The genetic features of this novel phage suggest that it might be applicable for phage therapy against pathogenic strains of V. parahaemolyticus.

Genome sequence analysis of Vibrio parahaemolyticus lytic phage Vp_R1 with a C3 morphotype.

Phage Vp_R1 belongs to the family Podoviridae and has a C3 morphotype, with an elongated head with a diameter of 190 ± 1.1 nm and an ultrashort tail with a length of 9 ± 1.2 nm. The double-stranded DNA genome is 112.1 kb long, has a mol% G + C content of 40.3, contains 129 ORFs, and encodes four tRNAs. Phylogenetic analysis suggests that phage Vp_R1 is a novel member of the genus Kuravirus.

Genome characterization of the novel lytic Vibrio parahaemolyticus phage vB_VpP_BA6.

A lytic bacteriophage, designated Vibrio phage vB_VpP_BA6, was isolated from sewage collected in Guangzhou, China. The double-stranded DNA genome of phage BA6 is composed of 50,520 bp with a G+C content of 41.77%. It possesses 64 open reading frames relating to phage structure, packaging, host lysis, DNA metabolism, and additional functions. Three tRNAs genes (encoding Pro, Ile and Trp) were detected. Comparison of its genomic features and phylogenetic analysis revealed that phage BA6 is a novel member of the family Podoviridae. This phage may represent a potential therapeutic agent against multidrug-resistant Vibrio parahaemolyticus.

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.

Characterization of a lytic vibriophage VP06 of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a human enteropathogenic bacterium and is also pathogenic to shrimp and finfish. In a search for a biocontrol agent for V. parahaemolyticus and other pathogenic Vibrio species, a lytic phage VP06 was isolated from oyster using V. parahaemolyticus as the host. VP06 is a Siphoviridae phage with a polyhedral head and a long tail. The genome sequence of VP06 was 75,893 nucleotides in length and the G + C content was 49%; a total of 101 CDSs were identified in VP06, of which 39 exhibited functional domains/motifs. The genomic sequence of VP06 is similar to those of a lytic Vibrio vulnificus phage SSP002 and a temperate V. parahaemolyticus phage vB_VpaS_MAR10, although VP06 has distinct features in the CDS arrangement and 14 unique CDSs. Phylogenetic analysis revealed that VP06, SSP002 and vB_VpaS_MAR10 belong to a novel genus cluster of Siphoviridae phages. This phage lysed 28.1% of various Vibrio strains, and the efficiency of plating method revealed that VP06 was highly effective in lysing strains of Vibrio alginolyticus, Vibrio azureus, Vibrio harveyi and V. parahaemolyticus. The properties of VP06, including its broad range of hosts and resistance to environmental stresses, indicate that it may be a candidate biocontrol agent.

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.

Complete Genome of a Novel Lytic Vibrio parahaemolyticus Phage VPp1 and Characterization of Its Endolysin for Antibacterial Activities.

Vibrio parahaemolyticus is an important foodborne pathogen that is generally transmitted via raw or undercooked seafood. Endolysins originating from bacteriophages offer a new way to control bacterial pathogens. The objectives of this study were to sequence a novel lytic V. parahaemolyticus phage VPp1 and determine the antibacterial activities of the recombinant endolysin (LysVPp1) derived from this phage. The complete VPp1 genome contained a double-stranded DNA of 50,431 bp with a total G+C content of 41.35%. The genome was predicted to encode 67 open reading frames (ORFs), which were organized as nucleotide metabolism, replication, structure, packaging, lysis, and some additional functions. Two tRNAs were encoded to carry anticodons UGG and CCA. Among the functional proteins, ORF33 was deduced to encode endolysin, whereas no holin/antiholin or Rz/Rz1 lysis gene equivalents were found in the VPp1 genome. ORF33 was cloned and expressed. The endolysin LysVPp1 could lyse 9 of 12 V. parahaemolyticus strains, showing its relatively broader host spectrum than phage VPp1, which lysed only 3 of 12 V. parahaemolyticus strains. Furthermore, for EDTA-pretreated bacterial cells, the optical density of the LysVPp1 treatment group decreased by 0.4 at 450 nm, compared with less than 0.1 in control groups, demonstrating enhanced hydrolytic properties. These results contribute to the potential for development of novel enzybiotics for controlling V. parahaemolyticus.