Isolation, characterization and application of a lytic phage vB_VspM_VS1 against Vibrio splendidus biofilm

Vibrio splendidus is a common pathogen in the ocean that infects Apostichopus japonicus, Atlantic salmon and Crassostrea gigas, leading to a variety of diseases. In this study, a virulent phage vB_VspM_VS1, which infects V. splendidus, was isolated from aquaculture ponds in Dalian, China, and it belongs to the family Straboviridae in the order Caudoviricetes. vB_VspM_VS1 had an adsorption rate of 96% in 15 min, a latent period of 65 min, and a burst size of 140 ± 6 PFU/cell. The complete genome of phage vB_VspM_VS1 consists of a linear double-stranded DNA that is 248,270 bp in length with an average G + C content of 42.5% and 389 putative protein-coding genes; 116 genes have known functions. There are 4 tail fiber genes in the positive and negative strands of the phage vB_VspM_VS1 genome. The protein domain of the phage vB_VspM_VS1 tail fibers was obtained from the Protein Data Bank and the SMART (http://smart.embl.de) database. Bacterial challenge tests revealed that the growth of V. splendidus HS0 was apparently inhibited (OD600 < 0.01) in 12 h at an MOI of 10. In against biofilms, we also showed that the OD570 value of the vB_VspM_VS1-treated group (MOI = 1) decreased significantly to 0.04 ± 0.01 compared with that of the control group (0.48 ± 0.08) at 24 h. This study characterizes the genome of the phage vB_VspM_VS1 that infects the pathogenic bacterium V. splendidus of A. japonicus.


Introduction
Vibrio splendidus is a gram-negative ubiquitous inhabitant pathogen of brackish water and marine environments that is associated with epizootics, including Apostichopus japonicus, crustaceans and fishes [1][2][3].However, at present, there is no good drug treatment for V. splendidus.Li et al. reported that persister cells of V. splendidus were formed in culture with 10 minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin, persister cells are difficult to eradicate by antibiotic [4].In addition, antibiotic-resistant bacteria are frequently found in aquaculture [5,6].Therefore, it is necessary to develop the application potential of phages against aquaculture pathogens.
Phages are viruses that can infect bacteria, spirochetes and actinomycetes [7]; they are highly host-specific and do not infect humans or animals.They parasitize only host cells that are susceptible to infection, so they can be used as ideal antibacterial biological agents to replace antibiotics [8].With the use of antibiotics, the problem of resistant bacteria is becoming increasingly serious; therefore, there is an urgent need for phage-derived products (endolysins) and phages to replace antibiotic drugs [9,10].A number of phages PVS-1, PVS-2, PVS-3 and vB_VspP_pVa5, that infect V. splendidus have been reported, and their possess the efficacy to inactivate V. splendidus, the cultures infected with PVS-1, PVS-2, PVS-3 and vB_VspP_pVa5 at a MOI of 10 started to show significant decreases in optical density (OD 600 ) compared with the control culture at 3 h after infection (P < 0.05) [11].However, information on the genomes of V. splendidus phages has not been reported.Recently, many studies have reported that phages are effective in the treatment of V. splendidus or other bacteria important for aquaculture, Wu et al. reported that novel Vibrio harveyi phage vB_VhaS_PcB-1G exhibited a broad host range against V. harveyi (33/54) and large burst sizes (210 PFU/cell).Zhu et al. found that phage XC31 could kill Vibrio mediterranei 117-T6 and significantly decrease the Vibrio density in the infection system (P < 0.05).Rasmussen et al. found that combining phage KVP40 and probiont Phaeobacter inhibens DSM17395 resulted in a lower level of fish pathogenic Vibrio anguillarum than using the probiont alone [12][13][14].
Biofilms are highly resistant to antibiotics, desiccation, heat and acidic conditions [15].Many bacteria in biofilms are approximately 10 to 1000 times less susceptible to antibiotics than culture bacteria for the reason of the extracellular polymeric substances of the biofilm prevent contact with antibiotics [16].This makes the complete elimination of biofilms in the food industry, the clinic nearly and animal husbandry impossible [17].
Phages show high strain specificity to host bacteria, moreover, host bacteria are highly adaptable to phages.In a phage-host arms race, phage mutation occurs while the phage is infecting resistant bacteria [18][19][20].Phage cocktails are effective against pathogenic bacteria that mutate and adapt.Hence, the continued screening of new phages is of great significance.Sequence analysis of the phage genome provides important resources for the development and utilization of phages.In this study, we identified a phage vB_VspM_VS1 that infects V. splendidus, and characterized its genome.

Bacterial strains and growth conditions
Wild-type V. splendidus HS0 was isolated from diseased A. japonicus and stored in glycerol at -80 ˚C at Ningbo University, China, and used as the host bacterium.V. splendidus was grown aerobically on 2216E plates or in 2216E broth (Difco, Detroit, MI, USA), and incubated at 28 ˚C [21].Soft top agar containing 0.5% (w/w) agar in 2216E broth was used for phage plaque confirmation, and 2216E agar plates containing 1.8% (w/w) agar were used for bacterial growth.V. splendidus HS0 was stored at −80 ˚C in 20% (v/v) glycerol.

Phage isolation and purification
V. splendidus phage was isolated from A. japonicus breeding pond silt in Dalian, China.The propagation method was modified from Park [22].Briefly, 5 g of a A. japonicus breeding pond silt sample was mixed with 15 mL phosphate buffer solution in a 50 mL centrifuge tube and incubated with shaking at 180 rpm for 1 h at room temperature.Then, the sewage sample was centrifuged at 6,000 × g for 10 min, and the supernatants were filtered with a 0.22 μm filter membrane.After filtration, 10 mL of each filtrate was inoculated onto log phase-grown V. splendidus in 40 mL of 2216E culture broth and incubated for 48 h.Then, the culture was centrifuged at 6 000 × g for 10 min, and the supernatant was filtered with a 0.22 μm filter membrane.The filtrate was serially diluted 10 times, mixed with 5 mL molten 0.5% 2216E soft agar containing V. splendidus HS0 (2 × 10 8 cfu/mL), and immediately added to a 2216E plate.The growth of overnight cultures and plaque formation were observed after 24 h.A single phage plaque was selected for phage purification, and the process was repeated three times.

Growth curve and adsorption rate of the isolated phage
To measure the adsorption rate, the co-culture of phage vB_VspM_VS1 (1 × 10 8 pfu/mL) and logarithmic phase V. splendidus HS0 were mixed at an multiple of infection (MOI) of 1 and cultured at 28 ˚C; the phage titer was measured after 0, 5, 10, 15, 20, 25 and 30 min.The onestep growth curve of phage vB_VspM_VS1 was carried out as follows.Briefly, 10 mL of exponential phase V. splendidus HS0 culture was harvested by centrifugation (5,000 g, 4 min, 28 ˚C), and the pellet was resuspended in 20 mL of fresh 2216E to obtain an OD 600 of 1.0.Next, 20 mL of phage vB_VspM_VS1 was added to reach an MOI of 1 and allowed to adsorb for 10 min at 28 ˚C.The mixture was centrifuged at 5,000 × g for 4 min at 28 ˚C, and the pellet was resuspended in 10 mL of fresh 2216E.Samples were taken every 10 min for 120 min, after which the supernatants were plated on 2216E agar to determine the phage titer.

TEM and antibacterial analyses of the isolated phage
The morphology of the phage vB_VspM_VS1 particles was analyzed using transmission electron microscopy (TEM) mainly with the steps described below.Dilutions of the phage vB_VspM_VS1 stock (approximately 6×10 9 pfu/mL) were deposited on carbon film and stained with 2% uranyl acetate.Phage vB_VspM_VS1 samples were observed using a Philips EM 300 electron microscope operated at an acceleration voltage of 120 kV at Ningbo University (Ningbo, China).Phage vB_VspM_VS1 was identified and classified according to the International Committee on Taxonomy of Viruses.Antibacterial assessment was performed by placing 6 mL diluted phage vB_VspM_VS1 (1 × 10 8 pfu/mL) and 6 mL exponential phase V. splendidus HS0 culture (1 × 10 9 cfu/mL) at an MOI of 0.1 in 150 mL 2216E culture, followed by incubation at 28 ˚C for 12 h; for the control, no phage was included.The absorbance was measured at a wavelength of 600 nm with a microplate reader.Phage concentration was determined on double layer 2216E agar at every 2 h.The experiment was repeated three times.

Sequencing and analysis
Purified phage vB_VspM_VS1 was concentrated through a 10 kDa filter and treated with DNase and RNase at 37 ˚C for 1 h.A Takara Minibest Viral RNA/DNA Extraction Kit (Cat#9766) was used to extract phage vB_VspM_VS1 genomic DNA.Sequencing of the phage vB_VspM_VS1 genomic DNA was carried out using the Illumina HiSeq platform (Sangon Biotech, China) and assembled using SPAdes and FastQC assembler software.NCBI BLAST was used to compare sequences from multiple databases and open reading frames (ORFs), including TrEMBL, KOG, COG, CDD, NT, PFAM, SwissProt and NR, to obtain functional annotation information for the phage vB_VspM_VS1 gene protein sequences.The database VFDB (http://www.mgc.ac.cn/VFs/main.htm)was used to detect the virulence factors in the phage vB_VspM_VS1.The database ARG-ANNOT (http://backup.mediterranee-infection. com/article.php?laref=282&titre=arg-annot) was used to detect antimicrobial resistance genes in the phage vB_VspM_VS1.The genome similarity between phages was determined by BLASTN analysis via NCBI.The phylogenetic analysis of the phage vB_VspM_VS1 and related bacterial phage major capsid proteins amino acid sequences was performed using Molecular Evolutionary Genetics Analysis (MEGA7.0)software using the neighbor-joining method.The nodal reliability of the trees was assessed with bootstrapping using 500 pseudo replicates.

V. splendidus phage vB_VspM_VS1 tail fibers structure domain
The protein crystal structures of the vB_VspM_VS1 tail fiber genes ORF42 (a), ORF61 (b), ORF64 (c) and ORF82 (d) of the V. splendidus phage were compared in the Protein Data Bank (PDB) database.a, b, c, and d of V. splendidus phage vB_VspM_VS1 tail fiber gene domains were predicted in the SMART (http://smart.embl.de)database.

Determination of lytic capacity of phage vB_VspM_VS1 at different MOI
Four hundred microliter of the overnight culture was sub-cultured in 100 mL fresh 2216E liquid medium, added phage in culture according to the MOI = 0.1, 1 and 10.Sampling the culture of every 1 h (0-20 h) and use spectrophotometer to determination optical density (OD 600 ).The effect of phage lytic capacity was examined in culture of V. splendidus by measuring the OD 600 every hour at various MOI from 0.1 to 10.At the same time, Phages titers in the filtrates were determined by titration on the double-layer agar (0-12 h).

Assessment of the effects of phage vB_VspM_VS1 on biofilm formation
To begin with, a 24-well cell slide was placed into a 12-well plate.The seed solution of V. splendidus was inoculated into 200 mL of 2216E culture at a concentration of 4‰. 1 mL of V. splendidus culture was inoculated into a 12-well plate.Phage vB_VspM_VS1 was added, with no addition used as a control group (the phage vB_VspM_VS1 was added at an MOI = 1), the cultures were incubated at 28 ˚C for 48 h and detected at 14, 24 and 48 h.Next, the recovered culture was washed three times with PBS buffer and fixed for 15 min with 99% methanol.Then, the methanol was discarded, and the V. splendidus cells were dried.A solution of 2% crystal violet was added and incubated with the cells for 15 min.The OD 570 value was detected after rinses with water, and the results were obtained through epifluorescence microscopy.Three repeated tests were performed.The experiment was repeated three times.

Characteristics and morphology of the isolated phage
Virulent V. splendidus phage vB_VspM_VS1 was isolated from a A. japonicus breeding pond silt in Dalian, China.The plaques of phage vB_VspM_VS1 were 3 mm in diameter after overnight incubation at 28 ˚C (Fig 1a).Negative staining of purified V. splendidus phage vB_VspM_VS1 was observed with an electron microscope.TEM showed that the phage vB_VspM_VS1 particles possessed an icosahedral head with a diameter of 143 ± 5 nm and a short tail with a length of 34 ± 3 nm (Fig 1b).The morphology and genomic data of phage vB_VspM_VS1 indicated that it belongs to the family Straboviridae [23].A one-step growth curve of phage vB_VspM_VS1 was obtained by inoculation of V. splendidus HS0 at an MOI of 1 at 28 ˚C (Fig 1c).The latent period of phage vB_VspM_VS1 was 65 min, and the titers of phage vB_VspM_VS1 reached peaks very quickly in 2 h.In addition, the burst size of phage vB_VspM_VS1 was approximately 140 PFU/cell.After standing for 15 min at 28 ˚C, nearly 96% of the phage particles were adsorbed to the host bacterium V. splendidus HS0.After incubation for 30 min, almost all phages were adsorbed to the host bacterium V. splendidus HS0 (Fig 1d ).

Characterization of the genome
To discern the novelty of vB_VspM_VS1 and its safety for biocontrol applications in the aquaculture industry, we characterized the phage genome.Phage vB_VspM_VS1 genomic DNA is a double-stranded with 248,270 base pairs in length.The G + C content of vB_VspM_VS1 was 42.5% (Fig 2).We identified 389 protein-coding genes [open reading frames (ORFs)] in the phage vB_VspM_VS1 genome, and 116 ORFs were annotated with specific functional genes (Table 1).The complete genomic sequences of phage vB_VspM_VS1 were deposited into the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/nuccore)(GenBank accession number OK905446).Functionally annotated ORFs are shown in Table 1.Homologs of any integrases, virulence genes, transposases, and antibiotic resistance genes was absent from the genome; thus, we hypothesize that phage vB_VspM_VS1 would not form lysogens.Moreover, the phage vB_VspM_VS1 genome showed no homology with any published antimicrobial resistance genes (ARGs) or phage virulence factors.Additionally, related sequences were mostly found in Vibrio phage nt-1 (HQ317393.2).Furthermore, we analysed the genome

Tail fiber gene domain and crystal structure information
Additionally, there were 4 tail fiber genes in the positive and negative strands of the phage vB_VspM_VS1 genome.The specific locations of phage vB_VspM_VS1 a, b, c and d tail fiber genes were 55021-58761 bp (ORF42), 87870-89291 bp (ORF61), 93768-94724 (ORF64) bp and 134694-137990 bp (ORF82), respectively.Protein domain analysis of the phage tail fiber proteins showed that tail fiber gene a likely generates a contractile tail, tail fiber gene b likely generates a short tail, tail fiber gene c likely generates a proximal long caudal fiber, and tail fiber gene d likely generates a long tail fiber.Prediction of the vB_VspM_VS1 tail fiber gene domains using the SMART (http://smart.embl.de)database showed that the tail fiber gene a, b, c-1, c-

Phylogenetic analysis
The amino acid sequences of V. splendidus and related bacterial phage major capsid proteins were obtained via the NCBI nucleotide database and compared (Vibrio phage nt-1, HQ317393.2;Vibrio phage V09, MT135026.1;Vibrio phage VH1_2019, MN794232.1;Vibrio phage V07, MT135025.1;Vibrio phage phi-ST2, KT919973.1,etc).Genome sequences from 17 other Vibrio group phages were obtained from the NCBI in GenBank format.A phylogenetic analysis of the major capsid proteins of all 18 phages (including   vB_VspM_VS1) was conducted using the neighbor-joining method in MEGA7 and Clus-talW (Fig 4).ClustalW was used to align the sequences, and MEGA7 was used to construct a neighbor-joining tree with 500 bootstrap replicates.This tree showed that phage vB_VspM_VS1 is highly homologous to the Vibrio phages nt-1, phi-ST2 and VH7D, which all belong to evolutionary Group I of a subclass of the Schizotequatrovirus, which share a common evolutionary branch (Fig 4).

Antibacterial analyses of the isolated phage
Co-culture of phage vB_VspM_VS1 and V. splendidus showed increased eradication compared to V. splendidus HS0 culture according to the results of the microplate reader OD 600 .
After culturing for 12 h, the OD 600 value of V. splendidus HS0 was 2.15 at an inoculate rate of 4‰.There were significant differences seen in the co-culture of phage vB_VspM_VS1 and V. splendidus HS0 based on statistical data analysis software version 17.0 (ANOVA) (IBM, Chicago, USA).The OD 600 value was significantly reduced to 0.023, and the bacteriostatic effect of phage vB_VspM_VS1 on V. splendidus HS0 culture was 93.5 times that of the control culture (  (OD 600nm < 0.01) in 12 h at an MOI of 10, whereas the OD 600nm started to increase after 3 h at MOI = 0.1 and 1.

Anti-biofilm activity of phage vB_VspM_VS1
Crystal violet staining experiments showed that V. splendidus had strong biofilm formation ability.To evaluate the ability of vB_VspM_VS1 to inhibit biofilm formation, the OD 570 values of V. splendidus were measured at 14, 24 and 48 h, and it was found that the most biofilm was formed at 48 h.The effect of vB_VspM_VS1 (MOI = 1) on biofilm formation by V. splendidus was assessed using 96-well plates.The results showed that the OD 570 values of the vB_VspM_VS1-treated group (MOI = 1) decreased significantly to 0.04 ± 0.01 compared with that of the control group (0.48 ± 0.08) at 24 h (Fig 8).This result suggested that vB_VspM_VS1 could prevent biofilm formation.This analysis demonstrated that phage vB_VspM_VS1 had a greater bactericidal effect (Figs 7 and 8).

Discussion
The V. splendidus HS0 strain used in this study was isolated from A. japonicus suffering from skin ulceration syndrome at indoor farms at the Jinzhou Hatchery; unfortunately, it was resistant to ampicillin.The emergence of multidrug-resistant (MDR) strains urgently requires new measures to inhibit pathogens.With the overuse of antibiotics, multidrug-resistant bacteria and superbacteria have emerged and have led to public health problems [21].The isolated V. splendidus phage vB_VspM_VS1 was identified as an ideal substitute for antibiotics due to its strong lytic activity.We have conducted two experiments on phage lytic activity against V. splendidus.When the MOI of phage vB_VspM_VS1 to V. splendidus was 0.01, phage V. splendidus is one of the important pathogens to aquatic animal in mariculture.It has broad susceptible hosts, including shellfish, A. japonicusins [25], which has caused very large economic losses to the aquaculture industry.With the increase prevalence of bacteria antibiotic resistance in aquaculture, phage can be used to control bacterial disease as antibiotics are becoming an increasingly substandard option for disease control in aquaculture.Li et al. found that phage freeze-dried powder can significantly improve the survival rate of A. japonicusins seedlings that attributed to phage can obviously inhibit the growth of pathogenic V. splendidus [23].This work tested the ability of the phage vB_VspM_VS1 to control V. splendidus.Phage vB_VspM_VS1 shown strong lytic activity against V. splendidus suggesting that the activity of the phage was specifific for V. splendidus.At the same time, this study annotated the phage genome in detail, which provides research materials for the development of genetic engineering technologies such as phage lyase and hydrolase.The isolated V. splendidus phage vB_VspM_VS1 belongs to the family Myoviridae, and its genome size is 248270 bp.TEM revealed the phage vB_VspM_VS1 virions to have an icosahedral head of 119 × 143 ± 5 nm and a tail of 34 × 53 ± 5 nm in length (Fig 1b).In comparison, the V. splendidus Myoviridae phage PVS-1 virions have an icosahedral head of 45 ± 2 nm and a tail of 74 ± 1 nm in length, and phage PVS-2 virions have an icosahedral head of 95 ± 3 nm and a tail of 42 ± 2 nm in length.V. splendidus Siphoviridae phage PVS-3 virions have an icosahedral head of 47 ± 2 nm and a tail of 152 ± 2 nm in length [11,23].
The structure and molecular specificity of phage tail fibers affects the adsorption, host range, gene expression and the development of cell fate of host bacteria [26].The genomic characteristics of V. splendidus phage vB_VspM_VS1 revealed that vB_VspM_VS1 encodes 4 tail fiber genes in the positive and negative strands of the phage vB_VspM_VS1 genome.Phage tail fibers play an important role in infecting host bacteria.Kells et al. reported that T4 phage tail fibers irreversibly bind to the lipopolysaccharide core region of E. coli and are responsible for initial phage attachment [17].Miernikiewicz et al. found that gp12, a phage T4 tail protein had the ability to bind to lipopolysaccharide on the bacterial surface [27].Through natural evolution, structural modeling and site-directed mutagenesis, Yehl et al. determined that the HRDR region of the tail fiber of the T3 phage determines its host range [28].
In this study, a new V. splendidus phage, vB_VspM_VS1, was isolated and characterized.Through the comparison of key proteins of the major capsid and the large terminal subunit of phage vB_VspM_VS1, there no similar phages were found in the Blastn database.In addition, genome identity between phage vB_VspM_VS1 and nt-1 (HQ317393.2) was 84.18%.Here, we reported the genome characterization of a new V. splendidus phage.Thus, phage vB_VspM_VS1 has potential application value as an antimicrobial agent in the aquaculture industry.

Fig 2 .
Fig 2. Line map of the vB_VspM_VS1 genome.In the vB_VspM_VS1 genome track, the arrows represent the ORFs and point in the direction of transcription.The colour intensity corresponds to G+C skew level.https://doi.org/10.1371/journal.pone.0289895.g002

Fig 6 )
. The phage titer increased substantially within 4 h (Fig 5).We observed phage vB_VspM_VS1 propagation in the V. splendidus HS0 culture at different MOIs of 0.1, 1 and 10, whereas the phage production was ~2-fold lower in the MOI = 10 culture than in the MOI = 0.1 culture (Fig5).The growth curves of V. splendidus HS0 indicated that the phage vB_VspM_VS1 had a better inhibitory effect at an MOI = 10, 1 and 0.1 (Fig6).The combination of phage vB_VspM_VS1 and V. splendidus HS0 resulted in maximum lysis of the host strain at different MOIs (Fig6).The growth of V. splendidus HS0 was apparently inhibited

Fig 4 .Fig 5 .Fig 6 .
Fig 4. Neighbor-joining tree showing the relationship of various phages from the NCBI database to vB_VspM_VS1.The tree was drawn based on the major capsid amino acid protein of each phage.Bootstrap values were set for 500 repetitions.Phage vB_VspM_VS1 and nt-1 have very close evolutionary distance.Scale bar means distance ruler.https://doi.org/10.1371/journal.pone.0289895.g004