A novel phagomagnetic separation-ATP bioluminescence (PhMS-BL) for rapid and sensitive detection of viable Vibrio parahaemolyticus in aquatic product.

Detection of viable Vibrio parahaemolyticus (V. parahaemolyticus) is a major challenge due to its significant risk to food safety and human health. Herein, we developed a phagomagnetic separation-ATP bioluminescence (PhMS-BL) assay based on phage VPHZ6 for rapid and sensitive detection of viable V. parahaemolyticus. Phage as a recognition element was coupled to magnetic beads to capture and enrich V. parahaemolyticus, shortening detection time and improving method sensitivity. The intracellular ATP released by chemical lysis using CTAB was quantified using firefly fluorescein-adenosine triphosphate bioluminescence system to detect viable bacteria. So, PhMS-BL method was able to detect V. parahaemolyticus in a linear range of 2.3 × 102 to 1.3 × 107 CFU mL-1, with a detection limit of 78 CFU mL-1 within 15 min. It is successfully applied to detect V. parahaemolyticus in spiked lake water, lobster tail meat, and clam meat. The developed detection strategy can rapidly and sensitively detect viable V. parahaemolyticus in food matrixes.

Characterization of a Vibriophage Infecting Pathogenic Vibrio harveyi.

Bacterial diseases caused by Vibrio spp. are prevalent in aquaculture and can lead to high mortality rates among aquatic species and significant economic losses. With the increasing emergence of multidrug-resistant Vibrio strains, phage therapy is being explored as a potential alternative to antibiotics for biocontrol of infectious diseases. Here, a new lytic phage named vB_VhaS_R21Y (R21Y) was isolated against Vibrio harveyi BVH1 obtained from seawater from a scallop-farming area in Rongcheng, China. Its morphology, infection cycle, lytic profile, phage stability, and genetic features were characterized. Transmission electronic microscopy indicated that R21Y is siphovirus-like, comprising an icosahedral head (diameter 73.31 ± 2.09 nm) and long noncontractile tail (205.55 ± 0.75 nm). In a one-step growth experiment, R21Y had a 40-min latent period and a burst size of 35 phage particles per infected cell. R21Y was highly species-specific in the host range test and was relatively stable at pH 4-10 and 4-55 °C. Genomic analysis showed that R21Y is a double-stranded DNA virus with a genome size of 82,795 bp and GC content of 47.48%. Its high tolerance and lytic activity indicated that R21Y may be a candidate for phage therapy in controlling vibriosis in aquacultural systems.

A broad-host-range lytic phage vB_VhaS-R18L as a candidate against vibriosis.

Vibriosis is one of the most common bacterial diseases that cause high rates of mortality and considerable economic losses in aquaculture. Phage therapy has been considered as a promising alternative method to antibiotics in the biocontrol of infectious diseases. Genome sequencing and characterization of the phage candidates are prerequisites before field applications to ensure environmental safety. In this study, a lytic phage, named vB_VhaS-R18L (R18L), was isolated from the coastal seawater of Dongshan Island, China. The phage was characterized in terms of morphology, genetic content, infection kinetics, lytic profile, and virion stability. Transmission electronic microscopy indicated that R18L is siphovirus-like, comprising an icosahedral head (diameter 88.6 ± 2.2 nm) and a long noncontractile tail (225 × 11 nm). Genome analysis indicated R18L to be a double-stranded DNA virus with a genome size of 80,965 bp and a G + C content of 44.96%. No genes that encode known toxins or genes implicated in lysogeny control were found in R18L. A one-step growth experiment showed that R18L had a latent period of approximately 40 min and a burst size of 54 phage particles per infected cell. R18L showed lytic activity against a wide range of at least five Vibrio species (V. alginolyticus, V. cholerae, V. harveyi, V. parahemolyticus, and V. proteolyticus). R18L was relatively stable at pH 6-11 and at temperatures ranging from 4°C to 50°C. The broad lytic activity across Vibrio species and the stability in the environment make R18L a potential candidate for phage therapy in controlling vibriosis in aquaculture systems.

Characterization and genomic analysis of phage vB_ValR_NF, representing a new viral family prevalent in the Ulva prolifera blooms.

Introduction: Vibrio is an important bacterial genus containing many pathogenic species. Although more and more Vibrio phages were isolated, the genome, ecology and evolution of Vibrio phages and their roles in bacteriophage therapy, have not been fully revealed. Methods: Novel Vibrio phage vB_ValR_NF infecting Vibrio alginolyticus was isolated from the coastal waters of Qingdao during the Ulva prolifera blooms, Characterization and genomic feature of phage vB_ValR_NF has been analysed using phage isolation, sequencing and metagenome method. Results and Discussion: Phage vB_ValR_NF has a siphoviral morphology (icosahedral head 114±1 nm in diameter; a tail length of 231±1 nm), a short latent period (30 minutes) and a large burst size (113 virions per cell), and the thermal/pH stability study showed that phage vB_ValR_NF was highly tolerant to a range of pHs (4-12) and temperatures (-20 - 45 °C), respectively. Host range analysis suggests that phage vB_ValR_NF not only has a high inhibitory ability against the host strain V. alginolyticus, but also can infect 7 other Vibrio strains. In addition, the phage vB_ValR_NF has a double-stranded 44, 507 bp DNA genome, with 43.10 % GC content and 75 open reading frames. Three auxiliary metabolic genes associated with aldehyde dehydrogenase, serine/threonine protein phosphatase and calcineurin-like phosphoesterase were predicted, might help the host V. alginolyticus occupy the survival advantage, thus improving the survival chance of phage vB_ValR_NF under harsh conditions. This point can be supported by the higher abundance of phage vB_ValR_NF during the U. prolifera blooms than in other marine environments. Further phylogenetic and genomic analysis shows that the viral group represented by Vibrio phage vB_ValR_NF is different from other well-defined reference viruses, and can be classified into a new family, named Ruirongviridae. In general, as a new marine phage infecting V. alginolyticus, phage vB_ValR_NF provides basic information for further molecular research on phage-host interactions and evolution, and may unravel a novel insight into changes in the community structure of organisms during the U. prolifera blooms. At the same time, its high tolerance to extreme conditions and excellent bactericidal ability will become important reference factors when evaluating the potential of phage vB_ValR_NF in bacteriophage therapy in the future.

Characterization of vB_ValM_PVA8, a broad-host-range bacteriophage infecting Vibrio alginolyticus and Vibrio parahaemolyticus.

Phage therapy was taken as an alternative strategy to antibiotics in shrimp farming for the control of Vibrio species of Vibrio parahaemolyticus and Vibrio alginolyticus, which cause substantial mortality and significant economic losses. In this study, a new Vibrio phage vB_ValM_PVA8 (PVA8), which could efficiently infect pathogenic isolates of V. alginolyticus and V. parahaemolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. The phage was characterized to be a member of the Straboviridae family with elongated head and contractile tail by transmission electron microscopy. Genome sequencing showed that PVA8 had a 246,348-bp double-stranded DNA genome with a G + C content of 42.6%. It harbored totally 388 putative open reading frames (ORFs), among them 92 (23.71%) assigned to functional genes. Up to 27 transfer RNA (tRNA) genes were found in the genome, and the genes for virulence, antibiotic resistance, and lysogeny were not detected. NCBI genomic blasting results and the phylogenetic analysis based on the sequences of the large terminase subunits and the DNA polymerase indicated that PVA8 shared considerable similarity with Vibrio phage V09 and bacteriophage KVP40. The phage had a latent period of 20 min and a burst size of 309 PFUs/infected cell with the host V. alginolyticus, and it was stable over a broad pH range (4.0-11.0) and a wide temperature span (-80°C to 60°C), respectively, which may benefit its feasibility for phage therapy. In addition, it had the minimum multiplicity of infection (MOI) of 0.0000001, which revealed its strong multiplication capacity. The shrimp cultivation lab trials demonstrated that PVA8 could be applied in treating pathogenic V. parahaemolyticus infection disease of shrimp with a survival rate of 88.89% comparing to that of 34.43% in the infected group, and the pond application trails confirmed that the implementation of PVA8 could rapidly yet effectively reduce the level of the Vibrio. Taken together, PVA8 may be potential to be explored as a promising biological agent for Vibrio control in aquaculture farming industry.

Isolation, characterization, and complete genome sequence of vibrio phage KIT04, a novel lytic phage of the subfamily ermolyevavirinae.

Vibrio phage KIT04 was isolated from muscle tissue samples collected from a local market in Vietnam. KIT04 is a lytic phage that is specific to Vibrio parahaemolyticus. The one-step growth curve determined the burst size and latent period of 0.01 multiplicity of infection KIT04 in V. parahaemolyticus as approximately 156 plaque-forming units/bacterium and 45 min, respectively. Vibrio phage KIT04 has an approximately 76.4 ± 4.5 nm diameter icosahedral head and a tail length of approximately 159.5 ± 16.6 nm long tail. KIT04 significantly reduced V. parahaemolyticus ATCC 17802 in vitro. Complete genome analysis showed that KIT04 had a 114,933 bp dsDNA genome with 40.24% G + C content and 160 open reading frames (ORFs). However, the phage genome contained 24 tRNAs and no lysogeny-related genes. Moreover, five of the 160 ORFs encoded unique hypothetical proteins, indicating that KIT04 is a novel phage. Genomic comparison indicated that KIT04 is closely related to the Vibrio phages pVp-1 and VPT02. Further, phylogenetic analysis of the major tail proteins and whole genome supported the KIT04 classification into the subfamily Ermolyevavirinae. Our study describes a new candidate phage that could be used as a bioagent for controlling Vibrio pathogens.

Isolation and Characterization of a Newly Discovered Phage, V-YDF132, for Lysing Vibrio harveyi

A newly discovered lytic bacteriophage, V-YDF132, which efficiently infects the pathogenic strain of Vibrio harveyi, was isolated from aquaculture water collected in Yangjiang, China. Electron microscopy studies revealed that V-YDF132 belonged to the Siphoviridae family, with an icosahedral head and a long noncontractile tail. The phage has a latent period of 25 min and a burst size of 298 pfu/infected bacterium. V-YDF132 was stable from 37 to 50 °C. It has a wide range of stability (pH 5-11) and can resist adverse external environments. In addition, in vitro the phage V-YDF132 has a strong lytic effect on the host. Genome sequencing results revealed that V-YDF132 has a DNA genome of 84,375 bp with a GC content of 46.97%. In total, 115 putative open reading frames (ORFs) were predicted in the phage V-YDF132 genome. Meanwhile, the phage genome does not contain any known bacterial virulence genes or antimicrobial resistance genes. Comparison of the genomic features of the phage V-YDF132 and phylogenetic analysis revealed that V-YDF132 is a newly discovered Vibrio phage. Multiple genome comparisons and comparative genomics showed that V-YDF132 is in the same genus as Vibrio phages vB_VpS_PG28 (MT735630.2) and VH2_2019 (MN794238.1). Overall, the results indicate that V-YDF132 is potentially applicable for biological control of vibriosis.

Characterization and Genomic Analysis of a Bacteriophage with Potential in Lysing Vibrio alginolyticus

Vibrio alginolyticus is one of the major pathogens causing vibriosis to a variety of aquatic animals as well as bringing about severe food safety concerns. Nowadays, phage therapy has received increasing attention as an alternative to the antibiotics that have being limited for use in aquaculture industries. In this work, a potent bacteriophage, vB_ValM_PVA23 (PVA23), which efficiently infects pathogenic strains of V. alginolyticus, was isolated from sewage water and characterized by microbiological and genomic analyses. Based on the transmission electronic observation, the phage was characterized to be the Myoviridae family. It has a latent period of 10 min and a burst size of 203 PFUs/infected bacterium, and was stable over a broad pH range (5.0−11.0) and a wide temperature span (−80 °C to 60 °C), respectively. Genome sequencing results show that PVA23 has a 246,962-bp double-stranded DNA with a G + C content of 41.25%. The lab and plant shrimp farming trials demonstrated that phage preparation derived from PVA23 out-performed the chemical disinfectant iodine treatment in the prevention of V. alginolyticus propagation, and the phage application could rapidly yet significantly reduce the level of V. alginolyticus in the pond within 12 h, with negligible rebound observed. These results suggests that phage PVA23 has the potential to be used as an anti-V. alginolyticus agent in aquaculture industries.

Isolation and characterization of a podovirus infecting the opportunist pathogen Vibrio alginolyticus and Vibrio parahaemolyticus.

Bacterial infections have a negative impact on both animal husbandry industry and medicine, and increasing bacterial drug resistance exacerbates this adverse impact. Phages show promise as an alternative to drugs against drug-resistant bacteria. In this study, a novel virulent bacteriophage (phage) vB_ValP_IME234 against Vibrio alginolyticus and Vibrio parahaemolyticus was isolated from freshwater in Beijing, China. Phage vB_ValP_IME234 had an isometric head (59 nm in diameter) and a short tail (10 nm long), belonging to Podoviridae family. Its complete genome is liner double-stranded DNA (dsDNA) with a GC content of 41.6% while encoding 61 putative proteins. Three transfer RNA (tRNA) and no lysogenic gene was detected. vB_ValP_IME234 had a polyvalent infectivity, a burst of 390 PFU/cell, and is stable under different temperatures (4 °C to 50 °C) and pH (6.0 to 10.0) values. Host range test showed that vB_ValP_IME234 has the ability to infect seven strains of Vibrio in total. Phylogenetic analyses based on terminase and capsid suggested that this phage had a close relationship with Vibrio phages. These results indicate that vB_ValP_IME234 could be used as a potential biocontrol agent against V. alginolyticus strains.

Isolation and Characterization of the Novel Phages vB_VpS_BA3 and vB_VpS_CA8 for Lysing Vibrio parahaemolyticus.

Accumulating evidence has indicated that the multiple drug resistant Vibrio parahaemolyticus may pose a serious threat to public health and economic concerns for humans globally. Here, two lytic bacteriophages, namely vB_VpS_BA3 and vB_VpS_CA8, were isolated from sewage collected in Guangzhou, China. Electron microscopy studies revealed both virions taxonomically belonged to the Siphoviridae family with icosahedral head and a long non-contractile tail. The double-stranded DNA genome of phage BA3 was composed of 58648 bp with a GC content of 46.30% while phage CA8 was 58480 bp with an average GC content of 46.42%. In total, 85 putative open reading frames (ORFs) were predicted in the phage BA3 genome while 84 were predicted in that of CA8. The ORFs were associated with phage structure, packing, host lysis, DNA metabolism, and additional functions. Furthermore, average nucleotide identity analysis, comparative genomic features and phylogenetic analysis revealed that BA3 and CA8 represented different isolates but novel members of the family, Siphoviridae. Regarding the host range of the 61 V. parahaemolyticus isolates, BA3 and CA8 had an infectivity of 8.2 and 36.1%, respectively. Furthermore, ∼100 plaque-forming units (pfu)/cell for phage BA3 and ∼180 pfu/cell for phage CA8 were determined to be the viral load under laboratory growth conditions. Accordingly, the phage-killing assay in vitro revealed that phage CA8 achieved approximately 3.65 log unit reductions. The present results indicate that CA8 is potentially applicable for biological control of multidrug resistant V. parahaemolyticus.

Analysis of 19 Highly Conserved Vibrio cholerae Bacteriophages Isolated from Environmental and Patient Sources Over a Twelve-Year Period.

The Vibrio cholerae biotype "El Tor" is responsible for all of the current epidemic and endemic cholera outbreaks worldwide. These outbreaks are clonal, and it is hypothesized that they originate from the coastal areas near the Bay of Bengal, where the lytic bacteriophage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) specifically preys upon these pathogenic outbreak strains. ICP1 has also been the dominant bacteriophage found in cholera patient stools since 2001. However, little is known about the genomic differences between the ICP1 strains that have been collected over time. Here, we elucidate the pan-genome and the phylogeny of the ICP1 strains by aligning, annotating, and analyzing the genomes of 19 distinct isolates that were collected between 2001 and 2012. Our results reveal that the ICP1 isolates are highly conserved and possess a large core-genome as well as a smaller, somewhat flexible accessory-genome. Despite its overall conservation, ICP1 strains have managed to acquire a number of unknown genes, as well as a CRISPR-Cas system which is known to be critical for its ongoing struggle for co-evolutionary dominance over its host. This study describes a foundation on which to construct future molecular and bioinformatic studies of these V. cholerae-associated bacteriophages.

A Review of Phage Therapy against Bacterial Pathogens of Aquatic and Terrestrial Organisms.

Since the discovery of bacteriophage in the early 1900s, there have been numerous attempts to exploit their innate ability to kill bacteria. The purpose of this report is to review current findings and new developments in phage therapy with an emphasis on bacterial diseases of marine organisms, humans, and plants. The body of evidence includes data from studies investigating bacteriophage in marine and land environments as modern antimicrobial agents against harmful bacteria. The goal of this paper is to present an overview of the topic of phage therapy, the use of phage-derived protein therapy, and the hosts that bacteriophage are currently being used against, with an emphasis on the uses of bacteriophage against marine, human, animal and plant pathogens.