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Article

Biological and Genomic Characterization of a Novel Jumbo Bacteriophage, vB_VhaM_pir03 with Broad Host Lytic Activity against Vibrio harveyi

1
Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Center for Marine Research, 71500 Heraklion, Crete, Greece
2
Department of Biology, University of Crete, 71003 Heraklion, Crete, Greece
*
Author to whom correspondence should be addressed.
Pathogens 2020, 9(12), 1051; https://doi.org/10.3390/pathogens9121051
Received: 18 October 2020 / Revised: 25 November 2020 / Accepted: 14 December 2020 / Published: 15 December 2020

Abstract

Vibrio harveyi is a Gram-negative marine bacterium that causes major disease outbreaks and economic losses in aquaculture. Phage therapy has been considered as a potential alternative to antibiotics however, candidate bacteriophages require comprehensive characterization for a safe and practical phage therapy. In this work, a lytic novel jumbo bacteriophage, vB_VhaM_pir03 belonging to the Myoviridae family was isolated and characterized against V. harveyi type strain DSM19623. It had broad host lytic activity against 31 antibiotic-resistant strains of V. harveyi, V. alginolyticus, V. campbellii and V. owensii. Adsorption time of vB_VhaM_pir03 was determined at 6 min while the latent-phase was at 40 min and burst-size at 75 pfu/mL. vB_VhaM_pir03 was able to lyse several host strains at multiplicity-of-infections (MOI) 0.1 to 10. The genome of vB_VhaM_pir03 consists of 286,284 base pairs with 334 predicted open reading frames (ORFs). No virulence, antibiotic resistance, integrase encoding genes and transducing potential were detected. Phylogenetic and phylogenomic analysis showed that vB_VhaM_pir03 is a novel bacteriophage displaying the highest similarity to another jumbo phage, vB_BONAISHI infecting Vibrio coralliilyticus. Experimental phage therapy trial using brine shrimp, Artemia salina infected with V. harveyi demonstrated that vB_VhaM_pir03 was able to significantly reduce mortality 24 h post infection when administered at MOI 0.1 which suggests that it can be an excellent candidate for phage therapy.
Keywords: jumbo phage; Vibrio harveyi; antibiotic resistance; phage therapy aquaculture jumbo phage; Vibrio harveyi; antibiotic resistance; phage therapy aquaculture

1. Introduction

The financial losses in aquaculture due to outbreaks of bacterial diseases are estimated to be in the range of billion US dollars globally. Disease outbreaks are among the most important threats for the economic sustainability of the aquaculture sector [1,2]. An important bacterial pathogen in aquaculture is Vibrio harveyi, which is a halophilic Gram-negative bacterium causing vibriosis disease in marine finfish, crustacean and molluscan species [3,4]. Vibrio harveyi is ubiquitous in the aquatic environment and can survive without a host. It is an opportunistic pathogen that will induce disease when the water temperature is optimal for its growth and at the same time its hosts are stressed [5]. Vibrio harveyi has also been increasingly reported in the Mediterranean aquaculture [6,7,8]. Intensification of aquaculture has been regularly considered as a major contributing factor to the outbreaks. In addition, the rising of the seawater temperature globally and climate change have also been associated with increasing Vibrio spp. detection in the environment [9,10,11,12]. Therefore, there is a high risk of more vibriosis outbreaks in the future. Antibiotics such as tetracyclines, fluoroquinolones and beta-lactamases have been extensively used and, in some instances, misused (or abused) in aquaculture as prophylactic or therapeutic means to control vibriosis [13]. As a consequence, resistant bacterial subpopulations develop rapidly in aquaculture through the exposure to subclinical dosages of antibiotic residues [14,15,16,17,18,19]. It is essential to reduce drastically the use of antibiotics in aquaculture or to be considered as a last resort option. To control bacterial diseases in aquaculture with reduced reliance on antibiotics, a working alternative is urgently needed. Bacteriophages or phages are ubiquitous viruses that exclusively infect bacteria. Recently, phage therapy has been revisited as a potential alternative to control bacterial diseases in aquaculture [20,21,22]. Phages are also the most abundant and diverse biological entities on earth therefore, finding phages that infect a specific strain of bacteria is relatively easy. Recent experimental phage therapy studies have also demonstrated positive results in controlling important bacterial fish pathogens such as Flavobacterium psychrophilum and Vibrio spp. [23,24,25,26,27]. However, phage therapy requires comprehensive knowledge of the applied bacteriophages therefore a full characterization of their biological and genomic attributes that includes their infectivity, lifestyle, stability, and possible virulence and antibiotic resistance encoding genes [28]. This study aimed to the isolation and characterization of a lytic phage against V. harveyi that could be used for phage therapy in aquaculture.

2. Results

2.1. Isolation and Morphology of vB_VhaM_pir03

vB_VhaM_pir03 was isolated from an environmental sample collected from the Port of Piraeus, Athens, Greece against Vibrio harveyi type strain DSM19623. A single plaque of vB_VhaM_pir03 was carefully isolated and purified through six times propagation. Throughout the propagation steps, vB_VhaM_pir03 showed a consistent plaque morphology. In the double layer agar plating assay, vB_VhaM_pir03 produced a pinhole-type plaque formation with a diameter of 0.27 ± 0.05 mm. We found that a comparison between the use of LB agar and diluted (LB/2) agar as the bottom layer for plating showed that a higher bacteriological nutrient composition reduced the visibility and plaque size of vB_VhaM_pir03 but not the actual count (data not shown). Transmission electron microscopy (TEM) showed that vB_VhaM_pir03 has a short neck, contractile tail and an icosahedral capsid (Figure 1) which indicated that its morphology is close to the phages of the Myoviridae family. Structural measurements of vB_VhaM_pir03 revealed relatively large virion dimensions.

2.2. Host Range and Efficiency of Plating (EOP) of vB_VhaM_pir03 against Multiple Antibiotic Resistant Strains

In the host range test (Table 1), vB_VhaM_pir03 was able to infect 31 out of 51 strains used. vB_VhaM_pir03 infected 21 of the 23 strains of V. harveyi, three of the seven strains of V. alginolyticus, the single strain of V. campbellii, both strains of V. owensii and four of the ten other unidentified presumptive Vibrio spp. There were no susceptible strains from V. parahaemolyticus, V. anguillarum and V. splendidus. EOP of vB_VhaM_pir03 was high for eight strains of V. harveyi, two strains of V. alginolyticus and two strains of unidentified Vibrio spp. and moderate for nine strains. For the antibiotic susceptibility tests (Table 1), all the phage susceptible strains were determined to be completely resistant against ampicillin. Eight strains were resistant against oxytetracycline, nine strains were resistant against oxalinic acid, nine strains were resistant against florfenicol, fifteen were resistant against sulfamethoxazole/trimethoprim and five strains were resistant against flumequine. Overall, vB_VhaM_pir03 was shown to be very effective against five multiple antibiotic-resistant strains and moderately effective against six multiple antibiotic-resistant strains.
Exposure to different temperatures (Figure 2a) showed that vB_VhaM_pir03 was stable up to 35 °C. Significant reduction (one-way ANOVA, p < 0.05) of its titer was observed between 40 to 45 °C while complete inactivation of vB_VhaM_pir03 was observed from 50 °C and above. When exposed to 0.001% benzalkonium chloride, BKC (Figure 2b), vB_VhaM_pir03 titer was significantly reduced (one-way ANOVA, p < 0.05) compared to the control. However, vB_VhaM_pir03 was complete inactivated when exposed to other organic solvents.

2.3. Adsorption Time and One-Step Growth of vB_VhaM_pir03

In the adsorption time assay (Figure 3a), it was estimated that the time required for 90% of the vB_VhaM_pir03 to irreversibly bind to bacterial host was 6 min. One step growth assay (Figure 3b) revealed that vB_VhaM_pir03 has a latent phase (period of between irreversible binding of vB_VhaM_pir03 to host cell until phage bursts) of 40 min. The rise phase (start of phage release from infected host until no more phages were released from its infected host) was estimated between 40 to 70 min. The plateau phase (period that indicated no more phages were released from the infected host cells) was reached at 70 min. In this assay, the burst size (number of new infective particles produced per each infected bacterial cell) of vB_VhaM_pir03 was 75 virions.

2.4. In Vitro Cell Lysis

In vitro lysis assay with DSM19623 (Figure 4) showed that vB_VhaM_pir03 was able to lyse the host bacterial population from MOI 0.1 to 10 after 18 h of incubation. Bacterial population infected at MOI 10 initially showed the lowest growth after 4 h of incubation but after 6 h the infected bacterial populations showed similar growth curves until 18 h of incubation to the other two MOIs used. Overall, vB_VhaM_pir03 still managed to decrease the bacterial population of DSM19623 by an approximate 40% at all MOIs compared to the uninfected population after 18 h of incubation. For in vitro lysis of vB_VhaM_pir03 with other strains, infected bacterial populations of Vh5, SerKid SA1.1, SA1.2 and SA4.1 (Supplementary Figure S1) showed similar growth patterns with DSM19623. The bacterial populations infected at MOI 10 initially showed the lowest growth however, all the infected bacterial populations eventually showed a similar growth curve pattern. For strains SNGR, VhP1 Liv, SA5.1, SA3.1, infections with vB_VhaM_pir03 with a MOI of at least 1 showed a discernible control of the host bacterial population growth. For strains Vh2, VhSerNFr, SA6.1, Vh No. 22, RG1, SA2.1, SA9.2, SA9.1, SA7.1, Art. 2, V1 and V2, infection of vB_VhaM_pir03 with MOI ≥1 was required to produce a similar outcome. However, vB_VhaM_pir03 was not effective in controlling bacterial population for strains VIB391, KS6, Barb A4/1.1, HCMR 1 Art.3, Rot. Vib. 5, Rot. 2 and Barb A4/1.2 even at MOI 10 despite those strains were susceptible in the plaque assay. The non-susceptibility of strains of Vh6 and VhKarx was also confirmed here. Finally, the bacterial population growth curves between infected and uninfected bacterial population for strains SA4.1 and Art. 2 converged when approaching 18 h of incubation.

2.5. Whole Genome Sequencing and Assembly

The sequenced genome of vB_VhaM_pir03 produced 41,500,540 clean reads with an average read length of 150 bp and 96.13% correct base calls. The GC content (%) was 43.6%. The per base calls scores produced good per sequence quality scores with a median of 36 for 150 bp reads. The per base sequence content and per sequence GC content showed that there was no bias in the proportion of each base position calls for four normal DNA bases or contamination during library preparation for vB_VhaM_pir03 sequencing. Finally, the per base N content result showed that no N substitutions were made which indicated that the sequencer had sufficient confidence to make base call. The genome of vB_VhaM_pir03 was assembled into a single contig with a minimum genome coverage of 5×. The total genome length of vB_VhaM_pir03 was 286,284 bp. A total of 99.91% of the raw reads were mapped back to the assembled genome resulting to an average coverage depth of 21,669×. In addition, the vB_VhaM_pir03 genome does not have any termini and was found to be terminally redundant and circularly permuted.

2.6. Genomic Features of vB_VhaM_pir03

The genome size of vB_VhaM_pir03 (286,284 bp) indicated that it is a jumbo phage (phages with total genome length more than 200,000 bp). The gene-coding potential of the global genome is 96.85% with 1.17 genes per kbp which suggests a dense genome arrangement. A total of 336 ORFs were identified with Rapid Annotation using Subsystem Technology (RASTk) server, 282 ORFs by Glimmer.hmm 2.0 and 286 ORFs by GeneMark. Comparison of the predicted ORFs showed that all ORFs called by Glimmer.hmm 2.0 and GeneMark were also called by RASTk. Manual inspection of each predicted ORF and gap between ORFs, and subsequent alignment in the NCBI nr database showed that 334 ORFS were present in vB_VhaM_pir03 genome. No tRNA was found in the genome. 303 ORFs used a start codon of ATG, 17 ORFs used GTG and 14 used TTG. A search on NCBI nr database showed that 119 ORFs (35.6%) had significant hits (expected value ≤10−3) with an average similarity of 55.8%. 71 ORFs (21.3%) were determined to have best hits with a jumbo Vibrio phage, vB_BONAISHI MH595538 which infects Vibrio coralliilyticus [29] while 20 ORFs (6.0%) had best hits with another four similar jumbo Vibrio phages; vB_VmeM-Yong MS31 MK308676.1, vB_VmeM-Yong MS32 MK308677.1, vB_VmeM-Yong XC31 MK308674.1 and vB_VmeM-Yong XC32 MK308675.1. In addition, protein structural homolog search for the predicted ORFs showed 26 hits in the Gene Ontology database, 35 hits with InterPro, 38 hits with the NCBI CDD and 61 hits with the HHPRED search tool. Overall, 137 (41.0%) ORFs were annotated based on amino acid sequence and protein structural homologies. No homologs of integrase, virulence or antibiotic-resistance encoding genes were found in vB_VhaM_pir03.

2.7. Genomic Arrangement and Functional Annotations of vB_VhaM_pir03

Generally, the genome of vB_VhaM_pir03 did not have any modular arrangement (Figure 5). However, genes encoding for head and tail proteins were arranged in subclusters while genes encoding for DNA replication and nucleotide metabolism proteins were scattered. Other genes that were functionally annotated are as in Table 2.

2.7.1. Phage Structural Proteins

Proteins required for phage assembly included baseplate protein (ORF 2), tail protein (ORF 4), membrane puncturing device (ORF 8), tail-tube (ORF 51), tail-sheath (ORF 52), capsid protein (ORF 143), internal head protein (ORF 148), major capsid protein (ORF 156), portal protein (ORF 167) and other virion structural proteins (ORFs 55, 139, 141, 142, 144, 150, 152, 165 and 166). The large terminase subunit involved for DNA packaging for tailed phages was identified at ORF 57. Interestingly, Proline-Alanine-Alanine-aRginine (PAAR) repeat proteins (ORF 10 and 11), a sharp conical structure for penetration of host cells [30] were also identified adjacent to the tail, baseplate protein and membrane puncturing device proteins. In addition, a prohead core protein protease which functions to facilitate the transition of a prohead or procapsid to a mature capsid [31] was identified at ORF 42.

2.7.2. DNA Replication, Repair, and Recombination

Proteins for DNA replication, recombination and repair were also identified; ribonuclease HI (ORF 16), RecA (ORF 18), HNH endonuclease (ORF 30, 107), homing endonuclease (ORF 35), DNA polymerases (ORF 101, 138, 272), SbcD nuclease (ORF 115), DNA helicases (ORF 125, 153, 198, 263), Holliday junction resolvase (ORF 164), HNH catalytic motif (ORF 185), UV-damage endonuclease (ORF 207), ribonuclease E/G (ORF 267), NAD-dependent DNA ligase LigA (ORF 275), ribonucleotide reductase (ORF 283, 286) and PIN protein (ORF 299). Glutaredoxin 2, a reducing agent for ribonucleotide reductase was identified at ORF 310. A DNA polymerase accessory protein for ATP hydrolase for DNA replication was also found at ORF 263.

2.7.3. Nucleotide Metabolism and Transcription

For nucleotide metabolism, enzymes such as putative nucleotidyl transferase (ORF 223), putative N-acetyltransferase (ORF 225), thymidylate kinase (ORF 281) and thymidylate synthase (ORF 323). DNA modification enzymes were also identified such as polynucleotide kinase (ORF193) and phosphagen kinase (ORF 262). For DNA transcription, multiple RNA polymerases (ORFs 20, 29, 34, 38, 106, 109, 122, 123, 133) and RNA binding proteins (ORF 64, 319) were identified. Two ORFs encoding transcription factor type II for site-specific DNA binding [32] were identified also identified (ORF 183, 184). Although no tRNAs were found in vB_VhaM_pir03 genome, a class 2b aminoacyl-tRNA synthetases whose function is to pair tRNA with their amino acids for accurate translation of the genetic code [33] was identified at ORF 134.

2.7.4. Miscellaneous Proteins

Several enzymes for lysis of host bacteria were also identified as glycoside hydrolase (ORF 28, 168, 182) which functions to degrade the host bacterial cell wall prior to phage burst. Additionally, two ORFs with no previous phage-associated descriptions were identified in vB_VhaM_pir03 genome which are ORF 200 with a structural homolog to palindromic amphipathic repeat coding elements (PARCEL) protein and ORF 64 with a structural homologs to Ro60-related proteins.

2.8. Genomic Synteny of vB_VhaM_pir03 with Other Similar Phages

Following whole genome alignment with the most similar phage genomes obtained from the NCBI nr database (Figure 6), vB_VhaM_pir03 was shown to have the highest degree of genomic synteny with vB_BONAISHI. Both phages also shared six collinear blocks with similar length. The longest shared collinear block had a sequence length almost 50,000 bp. Despite similar genomic arrangements, the shared collinear blocks showed very low DNA sequence similarities between them. vB_VhaM_pir03 also shared a single collinear block (<3000 bp) with Salmonella phage SKML 39. Alignment with another four similar jumbo phages; vB_VmeM-Yong_MS32; vB_VmeM-Yong_XC31; vB_VmeM-Yong_XC32 and vB_VmeM-Yong_MS31; showed shared collinear blocks of different length with no synteny and very low sequence similarities.

2.9. Phylogenetic Analysis

Wide genome proteomic tree analysis (Figure 7) showed that vB_VhaM_pir03 belong to the Myoviridae taxonomic family however, it was observed that the position of vB_VhaM_pir03 was in a subcluster within the Siphoviridae family. In addition, vB_VhaM_pir03 was also determined to infect a host from the Gammaproteobacteria class which includes Vibrionaceae family.
Phylogeny using large terminase subunits of jumbo phages (Figure 8) showed that vB_VhaM_pir03 have a recent common ancestor with vB_VmeM-Yong_MS32, vB_VmeM-Yong_XC31, vB_VmeM-Yong_XC32 and vB_VmeM-Yong_MS31 although with the bootstrap value (0.536) for this inference is below the acceptable threshold of 70% for bootstrapping [34]. However, vB_VhaM_pir03 has a high bootstrap support (0.996) with vB_BONAISHI. In addition, the branch length indicated that both phages share similar number of amino acid substitutions in their large terminase subunit since diverging from their common ancestor.

2.10. In Vivo Phage Therapy Trial with Artemia nauplii

At 24 h post infection, the survival of the Vibrio harveyi-infected Artemia was approximately 30% which was lower than the phage-treated groups (except for MOI 10) and comparable to the untreated control group (Figure 9). At 48 h post infection, survival of the phage-treated group was higher than the Vibrio harveyi-infected control group although this difference was not statistically significant. The delayed treatment resulted in similar survival to the Vibrio harveyi-infected control group both at 24 and 48 h post infection. Measurement of the presumptive Vibrio load at 24 and 48 h post infection showed no significant differences (two-way ANOVA, p < 0.05) between all treatments (data not shown). No colony forming units were observed in the control group at both times of measurement.

3. Discussion

Phage therapy is a very promising alternative to antibiotics. While scientific publications about isolation of phages have increased in the last decade, there is only a small group of phages that have been applied for commercial use [35] and only one for aquaculture which is CUSTUS®YRS against Yersinia ruckeri [36]. One of the main reasons for the lack of commercialized phages is insufficient characterization which is a prerequisite for phage therapy [37]. These characterizations are necessary to identify and reduce risks associated with phage therapy which in turn, will support progress towards regulatory approval [28,38]. Therefore, these considerations were the highest priorities when performing characterization of bacteriophages in this study.
In the present study, we have isolated and characterized a novel jumbo bacteriophage, vB_VhaM_pir03 with broad host lytic activity against Vibrio harveyi type strain DSM19623 and analyzed its therapeutic potential for aquaculture. Transmission electron microscopy revealed that vB_VhaM_pir03 is related to the Myoviridae family based on the presence of an icosahedral head and long contractile tail [39]. In addition, vB_VhaM_pir03 also had relatively large structural dimensions compared to other Myoviruses infecting Vibrio spp. [40,41]. The main factor for large structural dimensions is still undetermined but it has been posited that a ratcheting mechanism is present for large phages especially jumbo phages for the accommodation of large genome sizes and potential acquisition of more genes [42]. In relation to plaque formation, it has also been suggested previously that large structural dimensions of phages have contributed to small plaque formations as observed for vB_VhaM_pir03 due to reduced diffusion capacity of the large virion particles [29].
Several factors affect the reproductivity and stability of phages. In our study, the significant reduction of vB_VhaM_pir03 titers was first observed at 40 °C which was lower than that of previously described Vibrio phages [24,25,43,44]. Poullain et al. [45] have shown that heat treatment caused damage to tailed phages such as detachment of head and tail, empty capsids, and aggregation of tails. However, since the vB_VhaM_pir03 thermal tolerance was lower than previous reports, we suggest that this phage was more sensitive due to its large genome. Previous reports [46,47] have shown that phages with large genomes have high internal capsid pressure due to dense genome packaging. These phages were demonstrated to eject their genomic material at 37 °C by the internal capsid pressures. Nevertheless, the thermal stability of vB_VhaM_pir03 do not hinder its potential direct application in aquaculture since rearing temperatures of aquatic organisms do not reach 37 °C. vB_VhaM_pir03 was also found to be completely inactivated by chloroform in the present study therefore, chloroform was not used in all assays conducted subsequently. Tailed dsDNA phages do not contain lipid membrane [48] and are not sensitive to chloroform. A recent study [49] however has reported that several tailed dsDNA phages including members of Myoviridae and Podoviridae families showed reduction in phage titer after exposure to chloroform. In addition, vB_VhaM_pir03 was also completely inactivated after exposure to organic solvents in this study except for benzalkonium chloride, BKC. This suggests that vB_VhaM_pir03 applications can be controlled for contaminations and unintentional transfers such as observed in inactivation of Lactobacillus phages during milk productions [50].
vB_VhaM_pir03 showed a rapid adsorption time to its host compared to previously reported Vibrio and jumbo phages [24,25,51,52,53]. This indicated the efficiency of vB_VhaM_pir03 to locate and irreversibly bind to the host receptors under controlled conditions. Although the main factor determining adsorption time is the rate of phage–host encounters [54], application of rigorous aeration in an aquaculture system may increase the rate of phage–host encounters by reduction of phage and host sedimentations [55]. In one-step growth assay, vB_VhaM_pir03 displayed a short latent period and high burst size similar to previously reported jumbo phages [44,56,57]. A short latent period would result in low burst sizes of phages due to limited time for viral reproduction cycles [54]. Nonetheless, vB_VhaM_pir03 was shown in this study to have an efficient viral replication mechanism which allowed high virion productions in short latent periods. As a result, a minimal concentration of vB_VhaM_pir03 can be considered in therapeutic applications in aquaculture due to its rapid adsorption time and high multiplication rate. Finally, high multiplication rate of phages has been suggested as an evolutionary trade off with low phage survivability under stressful conditions [46] which may provide an insight to the low thermal stability of vB_VhaM_pir03.
In small phages (<200,000 bp), the arrangement of core genes that encode for head, tail, DNA replication and nucleotide metabolism proteins are conserved in a modular order for maintenance of functions throughout its replication cycles [58,59] however, we found the arrangement of the core genes in the vB_VhaM_pir03 genome was generally scattered and formed subclusters as previously described by Yuan and Gao [60]. Interestingly, we also found genes in vB_VhaM_pir03 that encode multisubunit RNA polymerases (RNAPs). RNAPs are found in jumbo phages and function to trigger early DNA transcription during infection without requiring the host’s DNA machinery [61]. Furthermore, we also found genes which encode proteins that have no definitive described functions in phages. A very large gene, ORF 200 with a nucleotide length of 19,254 bp contained a protein domain termed PARCEL (Palindromic Amphipathic Repeat Coding Elements) which has not been described in phages up to date. These repeats have dyad symmetry and variable hydrophilic and conserved hydrophobic regions and have been found in bacteria, eukaryotes [62] and recently in giant viruses [63]. PARCEL protein is related to mobile elements and has been considered as products of horizontal gene transfer [64], however the GC content of ORF 200 is not different to the GC content of the phage genome, which contradicts this likelihood. It has also been suggested that PARCEL protein facilitate diversification of bacterial surface protein [62] and also play a role in the coevolution of viruses with their hosts [63]. In addition, we also found a Ro60 related protein which has been described to have Y RNAs bound to its structure. The exact functions of this protein are unknown, but several phages have been reported to have Yr1A, a protein module within Y RNA that can mimic the structure of tRNA [65]. This supports the suggestion on the diverse genetic resources contained within jumbo phage genomes as a result of the large number of gene acquisition [66].
Phages have been associated with risks of horizontal gene transfer [20] therefore, it is imperative that any phage considered for therapy are investigated for temperateness and transduction potential. In our present study, we did not find any integrase, virulence or antibiotic resistance encoding genes in vB_VhaM_pir03 genome. Hence, this phage has an exclusive lytic lifestyle and does not risk antibiotic resistance and virulence gene transfers for applications. The vB_VhaM_pir03 genome is also absent of any termini, is circularly permuted and terminally redundant which suggest a headful packaging mechanism [67]. Based on the reads mapped to the vB_VhaM_pir03 genome, the potential host sequence was ≤0.09% which indicated that vB_VhaM_pir03 does not exhibit any transduction potential [68]. From whole genome sequence homolog search, a novel jumbo phage, vB_BONAISHI which infects Vibrio coralliilyticus, was determined to be the only similar phage to vB_VhaM_pir03 with an average ORF similarity of 52.3%. Analysis of genomic synteny in this study revealed that both phages shared similar genomic arrangements but with low nucleotide sequence similarities. In addition, phylogenetic analysis using large terminase subunits of vB_VhaM_pir03 and other described jumbo phages produced strong bootstrap support to the evolutionary relationship between vB_VhaM_pir03 and vB_BONAISHI. This suggested that both phages may have diverged from a common ancestor but have since undergone multiple nucleotide substitution events [61]. Since, vB_BONAISHI was previously described as a singleton phage in the jumbo phage phylogenetic tree [29], the phylogenetic and phylogenomic analyses indicated that vB_VhaM_pir03 is a novel phage.
vB_VhaM_pir03 was shown to have a broad host lytic activity against different Vibrio species within the Harveyi clade [69,70]. To our knowledge, only one other Vibrio phage, KVP40 was reported to infect multiple Vibrio species [71]. The ability of phages to infect different strains and species of bacteria has been suggested as an adaptation tool for survival [72]. For jumbo phages, their diversity of gene functions was suggested to be responsible for their broad host lytic activity [73,74]. In our study, the broad host lytic activity of vB_VhaM_pir03 can be considered analogous to broad-spectrum antibiotics [75] therefore in aquaculture, pathogenic Vibrio species are ubiquitous and diverse [76,77] thus a broad host phage such as vB_VhaM_pir03 would be advantageous in controlling the Vibrio population. It has been previously reported that there is an inverse relationship between bacterial antibiotic and phage resistance due to the high biological cost to maintain each resistance mechanism [78]. In this study, the susceptible host strains to vB_VhaM_pir03 were determined to be multiple antibiotic resistant bacteria. Therefore, the use vB_VhaM_pir03 would be a suitable approach against antibiotic resistant bacteria either as an antibiotic alternative or as a co-therapeutant.
In vitro lysis is typically carried out as an intermediate step to large scale applications. In this step, the therapeutic effects of phages at different MOIs and the host resistance development are measured concurrently against time [38]. In the present study, several strains including the host were determined to be susceptible to vB_VhaM_pir03 suggesting that when applied in vivo, pharmacokinetics and pharmacodynamics of the phage therapy are the major factors in the treatment efficacy [79]. Based on the in vitro lysis assays, host bacterial growth which were inhibited by vB_VhaM_pir03 at MOIs 10 and below would be ideal for immersion type delivery method since the concentration of phage required for application in an aquaculture setting would be practical. However, for hosts needing higher concentrations of vB_VhaM_pir03, further investigations are required to determine if site-specific delivery methods such as oral or intraperitoneal injection can provide effective therapeutic effect. Nonetheless, several host bacterial strains used in the in vitro lysis showed continuous growth even after inoculation of vB_VhaM_pir03. This suggested the development of resistant bacterial subpopulation due to selective pressure [80]. This supports further investigations into co-administration of vB_VhaM_pir03 with another phage as a cocktail in which each phage utilizes different infection mechanisms to overcome bacterial phage resistance mechanisms and avoid competitive phage infection [53]. In addition, the continuous bacterial growth even after inoculation of vB_VhaM_pir03 during the in vitro lysis assays may also suggest the development of a phage carrier state population in which the phage and host exist in equilibrium without any lysogenic activity or viral replication. In a phage carrier state, the phage may reside in its host cell without any lysogenic conversion, as a possible means of protection against environmental factors and avoidance of bacterial phage resistance mechanisms [81]. While the development of phage carrier state during vB_VhaM_pir03 infections was not examined, previous studies have reported the existence of phage carrier states resulting in reduction of virulence of Pseudomonas aeruginosa in biofilms and new colonization of Campylobacter jejunii in chickens [82,83].
In the in vivo trial with Artemia nauplii, we found that a single dosage of vB_VhaM_pir03 was effective in increasing survival of Artemia nauplii infected with Vibrio harveyi strain Vh5 at 24 h post infection even at MOI 0.1. This result was showed that vB_VhaM_pir03 performed slightly better than a previously reported phage therapy trial with Artemia spp. in which the survival was measured at 50% [26] However, it was observed that vB_VhaM_pir03 was unable to provide protection to the Artemia nauplii at 48 h post infection. Nonetheless, Artemia nauplii population still showed a higher percentage of survival to the untreated population which suggested a residual effect of protection. Despite the short protective period provided by vB_VhaM_pir03, a single dosage of vB_VhaM_pir03 can also be considered as a potential live feed disinfectant since Artemia nauplii are fed to fish within 24 h post hatch. For the infected Artemia nauplii that received a delayed treatment, vB_VhaM_pir03 was unable to provide protection which suggested that the damage caused by vibriosis was irreversible similarly to the reported results of Diaz et al. [84]. Phages are conventionally used in therapeutic purposes [27], however, for pathogens which cause irreversible damage, consideration may be given to use of phages as prophylaxis such as suggested by Silva et al. and Zaczek et al. [85,86].

4. Conclusions and Future Directions

In this study, we have provided a comprehensive biological and genomic characterization of vB_VhaM_pir03 as a candidate for phage therapy against Vibrio harveyi. The biological characterization of vB_VhaM_pir03 showed that it can rapidly locate and adsorb to a host and produce a high burst size within a short latent phase. Further characterization showed that vB_VhaM_pir03 has a broad lytic activity against thirty-one multiple antibiotic resistant strains of species belonging to the Harveyi clade. This is a unique ability of vB_VhaM_pir03 which has only been reported in only one other Vibrio phage, KVP40. Genomic analysis revealed a wealth of diverse gene functions that contributed to the efficacy of vB_VhaM_pir03. Furthermore, we also suggest that vB_VhaM_pir03 is not a temperate phage, does not harbor virulence or antibiotic resistance genes and does not exhibit transduction potential. Evaluation of the performance vB_VhaM_pir03 in vitro showed that it can inhibit several host bacterial growths at low MOI which supports its application in phage therapy. Finally, in the in vivo trial, vB_VhaM_pir03 was able to provide some protection to Artemia nauplii against vibriosis at 24 h post infection at all MOIs. Further characterization of its genome to understand its underlying mechanism is suggested to be carried out. In addition, we also suggest that large scale phage therapy trial with vB_VhaM_pir03 which includes investigations into different types of delivery methods. Finally, we would also like to emphasize that phage characterizations should be comprehensive to ensure a safe and practical phage therapy. This is very important towards the progress of phage therapy from the regulatory perspective.

5. Materials and Methods

5.1. Bacterial Strains Used in This Study

Thirty-one strains of Vibrio harveyi, V. alginolyticus, V. owensii, V. anguillarum, V. campbellii, V. parahaemolyticus and other Vibrio spp. (Table 3) used in this study were obtained from the bacterial collection of the Laboratory of Aquaculture Microbiology, Institute of Marine Biology, Biotechnology and Aquaculture (IMBBC), Hellenic Center for Marine Research (HCMR) in Heraklion, Crete. The bacterial strains were previously identified either through their NCBI or ENA accession numbers for the type strains, biochemical test (BIOLOG GENiii) and PCR method (16 s rRNA and toxR amplifications). In addition, unidentified Vibrio spp. isolated from the live feeds of HCMR were also used. All the bacterial strains were maintained in microbeads (MicroBank) at −80 °C and were grown in Lysogeny Broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 1L deionized water, 0.75 g/L MgSO4, 1.5 g/L KCl, 0.73 g/L CaCl2) at 25 °C when used.

5.2. Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was carried out for the bacterial strains used in this study according to standard disk diffusion test [87]. Bacterial suspension of the 31 selected strains were diluted to obtain a 0.7 absorbance read at OD600. The diluted bacterial suspensions were then plated on Mueller-Hinton agar (Difco, Detroit, MI, USA) with 2% NaCl. Antimicrobial susceptibility disks (ThermoFisher Scientific, Waltham, MA, USA) (Table 4) were placed on the agar plates and incubated at 25 °C (optimal temperature for the bacteria used) for 24 h. The recorded diameters were interpreted as susceptible, medium, or resistant according to the Clinical Laboratory Standards Institute (CLSI) guidelines CLSI M45-A2 [88] and CLSI M100-S25 [89] as in Table 4.

5.3. Isolation and Purification of Bacteriophages

Water samples were collected from three locations: (a) the Port of Piraeus, Athens, (b) the Karavolas beach, Heraklion, Crete and (c) a fish tank in the broodstock section of HCMR in Heraklion. 250 mL of the collected water samples were then enriched with 25 mL of concentrated LB and 2.5 mL of the host strain, Vibrio harveyi type strain DSM19623 liquid culture. The enriched water sample were incubated at 25 °C with a shaking speed of 70 rpm for 24 h. Following filtration through a 0.22 µm sterile filter (GVS Life Sciences, Sanford, ME, USA), 10 µL of each sample were spotted on bacterial lawns of the host strain. Following 24 h incubation at 25 ℃, the clearest plaque formations were collected. Serial propagations for phage purification were then made for the collected plaques against its host by double agar layer method according to Clokie et al. [90]. A single plaque was carefully collected, serially diluted, and propagated again in host bacterial lawn. This step was repeated at least five times for a phage to be considered purified. Phages that showed drastic decrease or loss in plaque formation during purification steps were discarded. One of the purified phages was selected for further characterization and was named vB_VhaM_pir03.

5.4. Transmission Electron Microscopy

For transmission electron microscopy, aliquot of vB_VhaM_pir03 suspension with a titer of ~1010 PFU/mL was prepared and negatively stained with 4% w/v uranyl acetate (pH 7.2). The phage was observed using a JEOL transmission electron microscopy operated at 80 kV at the Electron Microscopy Laboratory in the University of Crete. From the obtained digital micrographs, structural dimensions of individual phages were measured with ImageJ software version 1.52t [91] for capsid width, capsid length, tail length, baseplate length and baseplate width.

5.5. Host Range Test

For determination of host range for the purified phage, fresh cultures of bacterial strains used in this study (Table 1) were grown in LB at an approximate concentration of 107 CFU/mL and were then mixed with top molten LB agar (0.75% agar) and poured on bottom LB/2 agar (5 g/L tryptone, 2.5 g/L yeast extract, 10 g/L NaCl, 1L deionized water, 0.75 g/L MgSO4, 1.5 g/L KCl, 0.73 g/L CaCl2) which only contained half of the tryptone and yeast content from the LB agar. After solidification of top agar, 10 µL of vB_VhaM_pir03 were spotted on the host lawn. The phage titer was determined after the agar plates were incubated at 25 °C for 24 h.

5.6. Efficiency of Plating (EOP)

Efficiency of plating (EOP) was performed in this study according to [90]. The phage was serially diluted to ~100, 10−1, 10−2, 10−3, 10−4 and 10−5 and spotted on the bacterial lawns of the 31 susceptible strains. The phage titer was determined after the agar plates were incubated at 25 °C for 24 h. The EOP was calculated as a percentage of the number of plaque-forming units formed on a bacterial strain against the number of plaque-forming units formed on the host DSM19623. EOP more than 10 was categorized as high, EOP between 9.9 and 0.5 was considered medium while EOP less than 0.5 was considered low.

5.7. Stability of Phage in Different Temperatures and Organic Solvents

Phage thermal stability was measured by exposing the phage aliquots at ~107 PFU/mL to different temperatures (25, 30, 35, 40, 45, 50 and 55 °C) for 1 h before being rested at 25 °C for 10 min. The aliquots were then serially diluted and spotted on host bacterial lawn. The phage titer was determined after the agar plates were incubated at 25 °C for 24 h. vB_VhaM_pir03 stored at 4 °C for 24 h was used as a control.
The sensitivity of vB_VhaM_pir03 to chloroform was determined by exposing ~107 PFU/mL of the phage aliquots to 10% chloroform at 4 °C for 1 h while the stability of the vB_VhaM_pir03 against commonly used disinfectants in aquaculture was measured by exposing ~107 PFU/mL of vB_VhaM_pir03 to 0.001% benzalkonium chloride, BKC; 3% hydrogen peroxide, H2O2; 1% sodium hypochlorite, NaOCl; 70% ethanol, EtOH and; 1% formaldehyde, CH2O at 25 °C for 1 h. vB_VhaM_pir03 incubated at 25 °C for 1 h were used as control. Each treatment was serially diluted and spotted on host bacterial lawn. The phage titer was determined after the agar plates were incubated at 25 °C for 24 h. All assays were done with triplicates.

5.8. Adsorption Time and One-Step Growth

Adsorption time and one-step growth of vB_VhaM_pir03 was determined according to Kutter [92] with some modifications. Briefly, host culture in exponential phase (~108 CFU/mL) was infected with vB_VhaM_pir03 at multiplicity of infection (MOI) 0.01. Aliquots of the infected culture were then collected and transferred to chilled Eppendorf tubes at 0, 2, 4, 6, 10, 15, 20, 30 min, and kept in ice. The aliquots were centrifuged at 13,000 rpm for 3 min and supernatants were collected and serially diluted. The serial dilutions were then spotted on the host bacterial lawn on LB/2 agar plates. The phage titer was determined after the agar plates were incubated at 25 °C for 24 h.
For one-step growth, 1 mL of host culture in exponential phase (~108 CFU/mL) was centrifuged at 13,000 rpm for 3 min. The supernatant was then discarded and the pellet was washed and resuspended in 1 mL of SM buffer (5.8 g/L NaCl, 2 g/L MgSO4, 50 mL 1M Tris-Cl pH 7.5 and 2% gelatin, 1 L deionized H2O). This step was then repeated twice before the pellet was finally resuspended in 1 mL of LB. The fresh host culture was then infected with vB_VhaM_pir03 at MOI 0.01. After incubation for 10 min at 25 °C, the infected DSM19623 culture was then transferred to LB with the final volume of 30 mL. Afterwards, 1 mL aliquots were then collected from the infected host culture and immediately transferred to chilled Eppendorf tubes. The aliquots were then centrifuged for 13,000 rpm for 3 min. Subsequently, the supernatants were collected and serially diluted. The serial dilutions were then spotted on the host bacterial lawn on LB/2 agar plates. This step was repeated at 10 min intervals. The phage titer was determined after the agar plates were incubated at 25 °C for 24 h.

5.9. In Vitro Cell Lysis

The in vitro cell lysis of vB_VhaM_pir03 against DSM19623 was carried out by loading 180 µL of fresh host bacterial culture in each well of sterile 96-well plates. The plates were then read at OD600 using TECAN microplate reader (Infinite PRO 200) at 25 °C with orbital shaking. A total of 20 µL of vB_VhaM_pir03 was then added at MOIs 0.1, 1 and 10 when host culture was at exponential phase (~108 CFU/mL). Phages added to LB without host bacteria served as control. The assay was also carried out for the remaining 30 susceptible hosts of vB_VhaM_pir03. The growth curves of the cultures were then measured every 10 min for 18 h. All assays were done in triplicates.

5.10. DNA Extraction and Purification

The DNA extraction of vB_VhaM_pir03 was carried out using the phenol-chloroform method according to Higuera et al. [51]. The extracted DNA was visualized for quality on 1% agarose gel electrophoresis at 80 kV for 1 h with a 50 kbp ladder. Milli-Q® Reference Water (Merck KGaA, Darmstadt, Germany) was used as a negative control. The extracted DNA of vB_VhaM_pir03was then stored in −20 °C.

5.11. Genomic Analysis

The whole genome of vB_VhaM_pir03 was sequenced, assembled, and annotated previously as described in Misol et al. [93]. The genome sequence of phage vB_VhaM_pir03 is available in GenBank under accession number MT811961. The associated BioProject, SRA, and BioSample accession numbers are PRJNA665717, SRR12712979, and SAMN16261552, respectively. QUAST v4.6.3 [94] and BBMap v38.88 [95] were used to map the reads back to the assembled genome while PhageTerm was used to predict phage termini [68] through the Galaxy server [96]. Proteins of vB_VhaM_pir03 were automatically annotated in Blast2GO Suite [97] using (i) NCBI Basic Local Alignment Search Tool (BLAST) [98] adjusted at non-redundant (nr) protein database, (ii) Gene Ontology [99] and (iii) InterPro [100]. Predicted proteins of vB_VhaM_pir03 were also manually annotated with NCBI Conserved Domain Database (NCBI CDD) [101] and HHpred tool [102]. All ORF predictions and annotations were manually inspected. Integrase, virulence and antibiotic resistance-encoding genes in vB_VhaM_pir03 were searched using INTEGRALL Database webserver [103], Virulence Factor Database (VFDB) [104], VirulenceFinder and ResFinder webservers [105]. The host DSM19623 genome was analyzed for prophage-like sequences using Prophinder [106] and PHAge Search Tool Enhanced Release (PHASTER) [107]. For protein structural homologies, only probabilities above 90% were accepted for manual protein function assignment of the vB_VhaM_pir03 predicted ORFs. All hits in existing databases with expected value above 10−3. The genome of vB_VhaM_pir03 with annotated predicted ORFs was then visualized in a circular representation with Geneious software (Geneious v9.1, Biomatters, Auckland, http://www.geneious.com).

5.12. Genome Alignment and Phylogenetic Analysis of vB_VhaM_pir03

The whole proteome of vB_VhaM_pir03 was searched for similarity to other phages using the NCBI BLASTP nr protein database. The phage genomes with significant similarities were then downloaded and aligned with vB_VhaM_pir03 using the progressiveMauve: Multiple Genome Alignment [108] for analysis of the genomic synteny. The viral taxonomic family of vB_VhaM_pir03 and its host taxonomic group were determined using ViPTree: the viral proteomic tree server [99]. A six-frame translation proteome of vB_VhaM_pir03 was generated and compared to other six-frame translations of dSDNA phages in the NCBI database [109]. Phylogeny and molecular evolutionary analyses with other jumbo phages were conducted with Molecular Evolutionary Genetics Analysis (MEGA X) webserver [110]. Forty-nine large terminase subunits of described jumbo phages were downloaded from NCBI database and were aligned with the large terminase subunit of vB_VhaM_pir03 using MUSCLE algorithm [111]. Gaps in the amino acid sequence alignments were trimmed. A maximum likelihood phylogenetic tree was constructed using JTT matrix-based model [112] with bootstrap test = 1000. The tree was visualized using Interactive Tree of Life web server [113].

5.13. In Vivo Phage Therapy Trial Using Artemia nauplii

Six different treatments were investigated to assess the efficacy of vB_VhaM_pir03 in controlling pathogenic Vibrio harveyi strain VH5 in brine shrimp, Artemia salina nauplii. The first two treatments were control groups: a negative control containing Artemia nauplii only and a positive control of Artemia nauplii with V. harveyi. The other three treatments were single doses of vB_VhaM_pir03 at MOI 0.1, 1 and 10. The final treatment was a group that received a delayed single dose of vB_VhaM_pir03 at MOI 10 at 24 h post infection. The V. harveyi strain Vh5 was determined earlier to be the most pathogenic to Artemia nauplii (data not shown). Newly hatched Artemia nauplii were obtained from the live feed section of IMBBC, HCMR and were disinfected according to the protocol by Gomez-Gil et al. [114]. The Artemia nauplii were then washed three times with autoclaved and filtered borehole water (T: 25 °C). Afterwards, 50 Artemia nauplii were transferred to each well in Thermo Scientific™ Nunc™ Cell-Culture 6-well plates (Thermo Fisher Scientific 168 Third Avenue, Waltham, MA, USA) with 10 mL autoclaved and filtered borehole water. Aliquots from the washed Artemia nauplii were spotted on a bacterial lawn to observe presence of sodium hypochlorite residues. All treatments except for the negative control were inoculated with Vh5 at ~10−5 CFU/mL. At 2 h post infection, the single dose vB_VhaM_pir03 treatments were inoculated. For the delayed treatment, inoculation of vB_VhaM_pir03 was only carried out at 24 h post infection. The Artemia nauplii were fed with autoclaved Aeromonas hydrophila at 10 cells per individuals daily [115]. The sterility of the autoclaved A. hydrophila was tested earlier by transferring 10 µL from its suspension to LB and streaking on a LB agar. Individual counts of the Artemia nauplii were carried out by visual counting using a NIKON SMZ-800 Stereomicroscope (Nikon Instruments Inc., 1300 Walt Whitman Road Melville, NY 11747-3064, USA) at 24 and 48 h post infection to determine survival percentage. 100 µL of water from each treatment were taken and serially diluted before spotted on TCBS agar at 24 and 48 h post infection to determine the total Vibrio load. All treatments were done in triplicates at 25 °C.

5.14. Statistical Analysis

One-way ANOVA was performed for the thermal stability and effects of organic solvents assays. Two-way ANOVA was performed for calculation the survival of Artemia nauplii (factor A: hours post infection, factor B: treatments) and total Vibrio load (factor A: hours post infection, factor B: treatments). Tukey’s HSD post hoc test [116] was used as a multiple comparison tool after ANOVA was performed. Standard error of the mean was displayed for n = 3. All statistical analyses were carried out with PAST: Paleontological Statistics Software Package for Education and Data Analysis version 4.03 [117].

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-0817/9/12/1051/s1, Figure S1: in-vitro lysis of vB_VhaM_pir03 against 31 Vibrio spp. strains at multiplicity of infections (MOI) 0.1, 1 and 10 for 18 h. Bacterial growth was indicated by the absorbance (OD600) value. SE bars were shown for the mean of n = 3.

Author Contributions

Conceptualization and methodology, G.N.M.J., P.K. and C.K.; formal analysis, G.N.M.J.; investigation, G.N.M.J.; resources, P.K.; data curation, G.N.M.J.; writing—original draft preparation, G.N.M.J.; writing—review and editing, G.N.M.J., P.K. and C.K.; visualization, G.N.M.J.; supervision, P.K.; funding acquisition, P.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by the project ROBUST-Prevention of vibriosis caused by Vibrio harveyi with innovative tools, MIS5045915, Operational Programme Competitiveness, Entrepreneurship and Innovation 2014–2020, General Secretariat of Research and Technology.

Conflicts of Interest

The author declares that there is no conflict of interest.

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Figure 1. Electron micrograph of vB_VhaM_pir03: (a) virion morphology and dimensions—CW, capsid width; CL, capsid length; TL, tail length; BPL, baseplate length; and BPW, baseplate width; (b) uncontracted tail; (c) contracted tail sheath; (d) tail tube exposed; (e) absence of genetic material in the capsid.
Figure 1. Electron micrograph of vB_VhaM_pir03: (a) virion morphology and dimensions—CW, capsid width; CL, capsid length; TL, tail length; BPL, baseplate length; and BPW, baseplate width; (b) uncontracted tail; (c) contracted tail sheath; (d) tail tube exposed; (e) absence of genetic material in the capsid.
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Figure 2. (a) Effect of different temperatures on the stability of vB_VhaM_pir03. Incubation at 4 °C was used as control. (b) Effect of organic solvents to the stability of vB_VhaM_pir03. Incubation with LB was used as control. SE bars were shown for the mean of n = 3. Statistical significance indicated by different superscript letters was determined at p < 0.05.
Figure 2. (a) Effect of different temperatures on the stability of vB_VhaM_pir03. Incubation at 4 °C was used as control. (b) Effect of organic solvents to the stability of vB_VhaM_pir03. Incubation with LB was used as control. SE bars were shown for the mean of n = 3. Statistical significance indicated by different superscript letters was determined at p < 0.05.
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Figure 3. (a) Adsorption rate of vB_VhaM_pir03 measured against V. harveyi type strain DSM19623 at multiplicity of infection (MOI) 0.01. (b) One-step growth of vB_VhaM_pir03 measured against V. harveyi type strain DSM19623 at multiplicity of infection (MOI) 0.01. SE bars were shown for the mean of n = 3.
Figure 3. (a) Adsorption rate of vB_VhaM_pir03 measured against V. harveyi type strain DSM19623 at multiplicity of infection (MOI) 0.01. (b) One-step growth of vB_VhaM_pir03 measured against V. harveyi type strain DSM19623 at multiplicity of infection (MOI) 0.01. SE bars were shown for the mean of n = 3.
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Figure 4. In vitro lysis of vB_VhaM_pir03 against V. harveyi type strain DSM19623 at multiplicity of infections (MOI) 0.1, 1 and 10 for 18 h. Bacterial growth indicated by the absorbance (OD600) read. SE bars were shown for the mean of n = 3.
Figure 4. In vitro lysis of vB_VhaM_pir03 against V. harveyi type strain DSM19623 at multiplicity of infections (MOI) 0.1, 1 and 10 for 18 h. Bacterial growth indicated by the absorbance (OD600) read. SE bars were shown for the mean of n = 3.
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Figure 5. Circular representation of the vB_VhaM_pir03 genome in which the genome GC skew was shown as the inner blue circle. The predicted open reading frames (ORFs) were labelled at the outer circle. The color of the ORFs refer to annotated biochemical function; phage assembly proteins (red), DNA polymerases (green), RNA polymerases (yellow); DNA-directed RNA polymerases (blue), endolysins (grey). Other ORF colors refer to specific biochemical functions.
Figure 5. Circular representation of the vB_VhaM_pir03 genome in which the genome GC skew was shown as the inner blue circle. The predicted open reading frames (ORFs) were labelled at the outer circle. The color of the ORFs refer to annotated biochemical function; phage assembly proteins (red), DNA polymerases (green), RNA polymerases (yellow); DNA-directed RNA polymerases (blue), endolysins (grey). Other ORF colors refer to specific biochemical functions.
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Figure 6. Whole genome alignment with progressive MAUVE of vB_VhaM_pir03 with similar phages. From top is vB_VhaM_pir03, Salmonella phage SKML-39, Vibrio phages; vB_BONAISHI, vB_VmeM-Yong_MS31, vB_VmeM-Yong_MS32, vB_VmeM-Yong_XC31 and vB_VmeM-Yong_XC32. The colored collinear blocks indicate homologous regions between genome sequences while the height of the similarity profile in the collinear blocks indicate average level of conservation in the regions of the genome sequence. Inverted blocks indicate homologous regions that align in the complement orientation.
Figure 6. Whole genome alignment with progressive MAUVE of vB_VhaM_pir03 with similar phages. From top is vB_VhaM_pir03, Salmonella phage SKML-39, Vibrio phages; vB_BONAISHI, vB_VmeM-Yong_MS31, vB_VmeM-Yong_MS32, vB_VmeM-Yong_XC31 and vB_VmeM-Yong_XC32. The colored collinear blocks indicate homologous regions between genome sequences while the height of the similarity profile in the collinear blocks indicate average level of conservation in the regions of the genome sequence. Inverted blocks indicate homologous regions that align in the complement orientation.
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Figure 7. Determination of taxa and host group for vB_VhaM_pir03 by a proteomic tree using VIPTree. vB_VhaM_pir03 was determined to belong to Myoviridae family and infect Gammaproteobacteria group (red star and line). vB_VhaM_pir03 (asterisk) proteome was compared with 2688 dsDNA phages proteomes. The branch length scale was calculated as log values. The inner and outer ring indicate the taxonomic virus family and host group, respectively.
Figure 7. Determination of taxa and host group for vB_VhaM_pir03 by a proteomic tree using VIPTree. vB_VhaM_pir03 was determined to belong to Myoviridae family and infect Gammaproteobacteria group (red star and line). vB_VhaM_pir03 (asterisk) proteome was compared with 2688 dsDNA phages proteomes. The branch length scale was calculated as log values. The inner and outer ring indicate the taxonomic virus family and host group, respectively.
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Figure 8. Phylogenetic tree of vB_VhaM_pir03 with other jumbo phages. The large terminase subunits of jumbo phages were downloaded from NCBI database and aligned using MUSCLE and a maximum likelihood (bootstrap = 1000) phylogenetic tree was constructed using MEGA X. The tree was visualized using Interactive Tree of Life (ITOL). The bootstrap support value was denoted in each branch.
Figure 8. Phylogenetic tree of vB_VhaM_pir03 with other jumbo phages. The large terminase subunits of jumbo phages were downloaded from NCBI database and aligned using MUSCLE and a maximum likelihood (bootstrap = 1000) phylogenetic tree was constructed using MEGA X. The tree was visualized using Interactive Tree of Life (ITOL). The bootstrap support value was denoted in each branch.
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Figure 9. Survival of Artemia nauplii infected with Vh5 strain in an experimental phage therapy trial. Artemia nauplii infected with Vh5 were inoculated with vB_VhaM_pir03 with different multiplicities of infection (MOI) at 2 h and at 24 h of post-infection. Survival was measured at 24 and 48 h post infection. SE were shown for the mean of n = 3. Statistical differences between treatments and time post-infection were indicated by the superscript letter(s) and *** respectively (p < 0.05).
Figure 9. Survival of Artemia nauplii infected with Vh5 strain in an experimental phage therapy trial. Artemia nauplii infected with Vh5 were inoculated with vB_VhaM_pir03 with different multiplicities of infection (MOI) at 2 h and at 24 h of post-infection. Survival was measured at 24 and 48 h post infection. SE were shown for the mean of n = 3. Statistical differences between treatments and time post-infection were indicated by the superscript letter(s) and *** respectively (p < 0.05).
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Table 1. Host range and efficiency of plating of vB_VhaM_pir03 against selected Vibrio spp. On the right is the sensitivity of these strains against antibiotics used in aquaculture.
Table 1. Host range and efficiency of plating of vB_VhaM_pir03 against selected Vibrio spp. On the right is the sensitivity of these strains against antibiotics used in aquaculture.
Efficiency of Plating of vB_VhaM_pir03Antibiotic Susceptibility Testing
Species/StrainHost RangeEOPZone of Inhibition Diameter (mm)
10010−110−210−310−410−5(%)AMPTEOTOAFFCSXTUB
Vibrio harveyi
*DSM19623+++++++++++++++HighRSIRSSS
SNGR++++++++++++++++HighRSRISRS
KS6++++++++-LowRSRRRRS
Vh2+++++++++++-MediumRSSRSIS
Vh5+++++++++++++++HighRSISSSS
VhSernFr+++++++++++++++HighRSSRIIS
VhP1Liv+++++++++++++++HighRSISSSS
Vhp1Spl++++---Low
VhKarx++-----NFRSIRSSS
RG1++++++++--MediumRSRSSSS
Barb A4/1.1------NFRSIISSS
SerKid+++++++++++++++MediumRSIISSS
SerKid2+++++++++++++++High
SerSd+++++++++++High
SA 5.1+++++++++++++MediumRSIISRS
SA 6.1++++++--LowRSIIRRR
SA 9.2+++++++++MediumRSRISRI
SA 1.2++++++--LowRSIRRRR
SA 7.1+++++++++--LowRSSIIRS
SA 3.1+++++++++++++MediumRSIRSRS
SA 4.1++++++--LowRSIIRRR
SA 2.1+++++--LowRSRRSSS
Vh No. 22+++++++--LowRSIIISI
Vh6------NF RSIISIS
Vibrio alginolyticus
V1+++++++++++++++HighRSIISSS
V2+++++++++++-LowRSIISSS
HCMR 1 Art. 3+++++++++-HighRSSISRS
Vibrio campbellii
VIB391+++++++++++--MediumRSIISSS
Vibrio owensii
SA 1.1++++++++++MediumRSIRRRR
SA 9.1+++++--LowRSRIRRS
Other Vibrio spp.
Art. 2+++++++++++++HighRSRIRRR
Rot. 2+++++----LowRSRSRRI
Barb A4/1.2+++++++++++-HighRSIISSS
Rot. Vib. 5+++++++++++--MediumRSSIRRS
Abbreviations: EOP, efficiency of plating; AST, antibiotic susceptibility testing; AMP, ampicillin; TE, tetracycline; OT, oxytetracycline; OA, oxalinic acid; FFC, florfenicol; SXT, sulfamethoxazole/trimethoprim; UB, flumequine. Efficiency of plating: NF, no formation; +++, single large clearing zone; ++, ≥30 small plaques; +, <30 small plaques; NF, no formation; High, EOP > 10.0%; medium, 0.5% < EOP < 9.9%; low, EOP < 0.5%. *DSM19623 is used as the reference strain for efficiency of plating (EOP) calculations. Antibiotic susceptibility testing: R, resistant; I, intermediate; S, susceptible2.3. Stability of vB_VhaM_pir03 in different temperatures and organic solvents.
Table 2. Summary table of vB_VhaM_pir03 ORFs that were annotated with relevant information based on significant amino acid sequence and protein structural homologies (E-value ≤ 10−3).
Table 2. Summary table of vB_VhaM_pir03 ORFs that were annotated with relevant information based on significant amino acid sequence and protein structural homologies (E-value ≤ 10−3).
Predicted FunctionsStartEndLengthDirectionNCBI BLASTP Best HitE-ValueSimilarity Score (%)Gene OnthologyInterProNCBI CDD Best HitE-ValueHHPRED Best HitE-ValueProbability (%)
ORF 2Baseplate structural protein324262683027ReverseVibrio phage BONAISHI AXH71039.11.05 × 10−1542.48 cl33689|long tail fiber, proximal subunit5.51 × 10−31S2E_B|Bacteriophage T41.20 × 10−598.20
ORF 3Hypothetical protein62787180903ReverseVibrio phage BONAISHI AXH71040.12.0 × 10−4057.70
ORF 4Putative tail protein71928181990ReverseVibrio phage BONAISHI AXH71041.15.39 × 10−7256.00
ORF 6Hypothetical protein831595261212ForwardVibrio phage vB_VmeM-Yong XC31 QAX96157.13.0 × 10−3047.20
ORF 7ABC-type ATPase956812,2762709ReverseVibrio phage vB_VmeM-Yong XC31 QAX96150.11.76 × 10−10950.20 cd00267|ATP-binding cassette transporter nucleotide-binding domain1.30 × 10−76S6V_D|Escherichia coli4.40 × 10−2399.96
ORF 8Membrane-puncturing device12,11312,937825ForwardVibrio phage BONAISHI AXH70744.12.51 × 10−6661.50 6ORJ_A|Pseudomonas virus phiKZ4.60 × 10−89100.00
ORF 9Hypothetical protein12,94713,567621ForwardVibrio phage BONAISHI AXH70745.11.43 × 10−1056.31
ORF 10PAAR-repeat containing protein13,56013,859300ForwardVibrio phage BONAISHI AXH70746.11.30 × 10−2169.40 IPR008727cd14737|proline-alanine-alanine-arginine (PAAR) domain1.46 × 10−294KU0_D|Bacteriophage T42.70 × 10−1199.40
ORF 11PAAR-motif protein13,71813,891174ForwardVibrio phage 03O._10N.2646.F8] AUR83144.19.00 × 10−36.64
ORF 12Hypothetical protein13,90114,512612ReverseVibrio phage vB_VmeM-Yong XC31 QAX96156.14.76 × 10−3154.80
ORF 13Hypothetical protein14,523158,1811296ReverseVibrio phage vB_VmeM-Yong XC31 QAX96155.18.43 × 10−6853.60
ORF 16Ribonuclease HI (EC 3.1.26.4) CDS16,66718,0281362ForwardVibrio phage BONAISHI AXH70751.11.35 × 10−5948.00F: GO:0016787 IPR036397cd09278|RNase HI family found mainly in prokaryotes1.40 × 10−364MH8_A|Moloney murine leukemia virus2.90 × 10−999.07
ORF 17Hypothetical protein18,08118,920840ReverseVibrio phage BONAISHI AXH70752.18.97 × 10−2150.70
ORF 18UvsX protein18,75720,4841728ForwardVibrio phage BONAISHI AXH70753.13.12 × 10−17167.50 3IO5_B|Bacteriophage T4.4.60 × 10−2699.95
ORF 20DNA-directed RNA polymerase20,72621,496771ForwardVibrio phage BONAISHI AXH70756.12.85 × 10−6166.70 2A6H_M|Thermus thermophilus3.80 × 10−3299.98
ORF 21Hypothetical protein21,48921,896408ForwardVibrio phage BONAISHI AXH70757.17.61 × 10−751.18
ORF 22Hypothetical protein21,88922,572684ForwardVibrio phage BONAISHI AXH70758.13.23 × 10−2349.10
ORF 25Hypothetical protein23,63824,297660ForwardVibrio phage BONAISHI AXH70761.12.49 × 10−4258.30
ORF 26Hypothetical protein24,39525,4891095ForwardVibrio phage BONAISHI AXH70762.11.87 × 10−940.40
ORF 27Hypothetical protein25,52927,5532025ReverseVibrio phage BONAISHI AXH70763.14.37 × 10−16259.50
ORF 28Glycoside hydrolase 27,59633,3555760ReverseVibrio phage BONAISHI AXH70764.16.29 × 10−8152.30 pfam01551|Peptidase family M231.40 × 10−354RNZ_A|Helicobacter pylori4.70 × 10−1599.57
ORF 29DNA-directed RNA polymerase subunit alpha33,45834,9601503ForwardVibrio phage BONAISHI AXH70765.11.65 × 10−17374.10 5ZX3_D|Mycobacterium tuberculosis2.00 × 10−49100.00
ORF 30HNH endonuclease 35,01935,972954ForwardChryseobacterium gleum] WP_002984461.15.39 × 10−1357.30F: GO:0004519 IPR010896 pfam13392|HNH endonuclease1.45 × 10−101U3E_M|Bacillus phage SPO14.90 × 10−2999.97
ORF 33Hypothetical protein36,32236,495174ForwardVibrio phage vB_VmeM-Yong XC31 QAX96130.16.44 × 10−1070.90
ORF 34RNA-polymerase beta subunit 36,50437,8891386ForwardVibrio phage BONAISHI AXH70766.17.28 × 10−7155.40
ORF 35Homing endonuclease 37,93939,0271089ForwardVibrio phage vB_VmeM-Yong XC32 QAX96446.12.5 × 10−10161.50 3R3P_A|Bacillus phage 0305phi8-361.70 × 10−397.46
ORF 38DNA-directed RNA polymerase subunit alpha39,43742,0012565ForwardVibrio phage vB_VmeM-Yong XC31 QAX96126.10.0 × 10064.00F: GO:0003899SSF64484 6J9E_C|Xanthomonas oryzae (strain PXO99A)1.50 × 10−77100.00
ORF 40Hypothetical protein42,27843,7741497ForwardVibrio phage BONAISHI AXH70769.14.42 × 10−3846.31
ORF 41Hypothetical protein43,83044,558729ForwardVibrio phage BONAISHI AXH70770.11.11 × 10−6571.50
ORF 42Prohead core protein protease44,56745,388822ForwardVibrio phage vB_VmeM-Yong XC31 QAX96122.11.91 × 10−3750.40 5JBL_E|Bacteriophage T42.20 × 10−2599.93
ORF 51Tail-tube protein 48,26749,133867ReverseVibrio phage vB_VmeM-Yong XC31 QAX96106.14.32 × 10−6159.70 5IV5_DE|Bacteriophage T45.80 × 10−296.67
ORF 52Tail sheath protein 49,18851,2182031ReverseVibrio phage BONAISHI AXH70778.16.24 × 10−16259.70 3SPE_A|Pseudomonas phage phiKZ2.40 × 10−69100.00
ORF 54Hypothetical protein51,43752,378942ForwardVibrio phage vB_VmeM-Yong XC31 QAX96104.18.14 × 10−1948.10
ORF 55Virion structural protein52,34154,9922652ForwardVibrio phage BONAISHI AXH70780.11.42 × 10−10148.90
ORF 56Hypothetical protein55,00456,6651662ForwardVibrio phage BONAISHI AXH70781.11.49 × 10−12058.20
ORF 57Phage DNA helicase or terminase, large subunit 56,71158,9002190ForwardVibrio phage BONAISHI AXH70782.10.0 × 10069.50 3CPE_A|Bacteriophage T42.60 × 10−34100.00
ORF 58Hypothetical protein58,94360,1591217ReverseVibrio phage BONAISHI AXH70784.13.34 × 10−11065.90
ORF 60Hypothetical protein60,31461,015702ForwardVibrio phage BONAISHI AXH70786.11.02 × 10−848.60
ORF 62Hypothetical protein61,41562,4461032ForwardVibrio phage BONAISHI AXH70788.12.60 × 10−4660.50
ORF 64RNA-binding protein 62,62564,1751551ReverseVibrio phage BONAISHI AXH70790.16.79 × 10−544.30F: GO:0003723 IPR037214 2NVO_A|Deinococcus radiodurans1.60 × 10−62100.00
ORF 90Hypothetical protein69,56370,327765ReverseVibrio phage BONAISHI AXH70799.15.98 × 10−3758.00
ORF 99Hypothetical protein74,16974,585417ForwardVibrio phage BONAISHI AXH70809.12.55 × 10−1051.20
ORF 101DNA polymerase 74,76776,9382172ForwardVibrio phage BONAISHI AXH70810.10.0 × 10064.80F: GO:0000166IPR006134 cl33389|DNA polymerase type-B family4.62 × 10−33QEX_A|Bacteriophage RB692.60 × 10−2899.97
ORF 102Hypothetical protein76,98378,0471065ReverseVibrio phage BONAISHI AXH70811.11.42 × 10−3652.10
ORF 105Hypothetical protein78,38680,3231938ForwardVibrio phage BONAISHI AXH70812.17.30 × 10−6847.20
ORF 106DNA-directed RNA polymerase80,41681,7711356ForwardVibrio phage vB_VmeM-Yong XC31 QAX96059.18.5 × 10−13067.60F: GO:0003677 SSF64484 cl37096|DNA-directed RNA polymerase, beta subunit1.24 × 10−36RFL_A|Vaccinia virus GLV-1h681.30 × 10−36100.00
ORF 107HNH endonuclease 81,59882,6801083ForwardPseudomonas sp. JY-Q WP_064614171.11.0 × 10−945.21
ORF 109RNA polymerase beta prime subunit 82,88883,265378ForwardVibrio phage BONAISHI AXH70813.16.86 × 10−853.10
ORF 112Hypothetical protein84,23984,934606ForwardVibrio phage vB_VmeM-Yong XC31 QAX96047.12.32 × 10−1749.00
ORF 113Hypothetical protein 84,96885,369402ForwardVibrio phage BONAISHI AXH70815.15.83 × 10−2054.50
ORF 114Hypothetical protein 85,37185,847477ForwardVibrio phage BONAISHI AXH70816.13.08 × 10−1859.20
ORF 115Putative nuclease SbcD subunit D85,75986,9611203ForwardVibrio phage BONAISHI AXH70817.11.88 × 10−7456.10F: GO:0016787 IPR029052 cl33866|DNA repair exonuclease SbcCD nuclease subunit1.92 × 10−96S6V_B|Escherichia coli4.30 × 10−30100.00
ORF 116Hypothetical protein 86,95887,755798ForwardVibrio phage vB_VmeM-Yong XC31 QAX96043.16.97 × 10−4455.00
ORF 117Hypothetical protein 87,81888,516699ForwardVibrio phage BONAISHI AXH70819.17.16 × 10−3555.40
ORF 118Hypothetical protein 88,69290,1551464ForwardVibrio phage vB_VmeM-Yong XC31 QAX96041.12.29 × 10−6852.10
ORF 119Hypothetical protein 90,17091,6271458ForwardVibrio phage BONAISHI AXH70821.11.49 × 10−6750.00
ORF 120Hypothetical protein91,67193,5871917ForwardVibrio phage BONAISHI AXH70822.17.01 × 10−2749.00
ORF 122RNA polymerase beta subunit93,89896,1052208ForwardVibrio phage BONAISHI AXH70824.11.04 × 10−15255.90F: GO:0003677IPR007120cl37028|DNA-directed RNA polymerase, beta subunit.2.01 × 10−86PST_I|Escherichia coli1.00 × 1079100.00
ORF 123RNA polymerase beta subunit 96,11698,0801965ForwardVibrio phage vB_VmeM-Yong XC31 QAX96036.10.00 × 10060.40F: GO:0003899 6PST_J|Escherichia coli1.30 × 10−35100.00
ORF 125ATP-dependent DNA helicase uvsW98,30599,6931389ForwardVibrio phage vB_VmeM-Yong XC31 QAX96032.11.91 × 10−15368.70 cl34083|Superfamily II DNA or RNA helicase 1.64 × 10−122OCA_A| Bacteriophage T49.20 × 10−32100.00
ORF 127ATP-dependent Clp protease proteolytic subunit 100,236100,730495ForwardVibrio phage BONAISHI AXH70828.19.08 × 10−2654.00F: GO:0004252IPR001907 cl23717|Crotonase/Enoyl-Coenzyme A (CoA) hydratase superfamily5.81 × 10−252FZS_H|Escherichia coli1.20 × 10−2299.93
ORF 129Hypothetical protein101,203102,6721470ReverseVibrio phage BONAISHI AXH70830.11.17 × 10−1444.60
ORF 132Hypothetical protein103,126103,947822ForwardVibrio phage BONAISHI AXH70832.17.46 × 10−4155.60
ORF 133RNA polymerase beta prime subunit 103,982105,2111230ForwardVibrio phage BONAISHI AXH70833.12.45 × 10−12366.00F: GO:0003899 cl32391|DNA-directed RNA polymerase subunit beta1.46 × 10−86PST_J|Escherichia coli7.20 × 10−46100.00
ORF 134Putative replication protein A family105,237105,911675 cl09930|Replication protein A, class 2b aminoacyl-tRNA synthetases
ORF 137Hypothetical protein106,600107,9461347ForwardVibrio phage BONAISHI AXH70836.15.35 × 10−545.90
ORF 138DNA polymerase 107,997109,7241728ReverseVibrio phage BONAISHI AXH70837.10.00 × 10074.50F: GO:0003676 IPR036397 smart00486|DNA polymerase type-B family5.325 × 10−83QEX_A|Bacteriophage RB696.40 × 10−44100.00
ORF 139Virion structural protein 109,804111,0661263ForwardVibrio phage BONAISHI AXH70838.11.04 × 10−3648.90F: GO:0004222 6AIT_C|Escherichia coli (strain K12)3.30 × 10−699.05
ORF 140Hypothetical protein111,070113,0461977ForwardVibrio phage BONAISHI AXH70839.14.21 × 10−942.80
ORF 141Virion structural protein 113,085115,9312847ReverseVibrio phage BONAISHI AXH70840.11.42 × 10−17856.70
ORF 142Virion structural protein 115,924116,9701047ReverseVibrio phage BONAISHI AXH70841.11.32 × 10−9560.60
ORF 143Capsid protein 117,030118,1361107ForwardVibrio phage BONAISHI AXH70842.11.63 × 10−6858.60
ORF 144Virion structural protein 118,147119,028882ForwardVibrio phage BONAISHI AXH70843.17.55 × 10−2450.00
ORF 146Hypothetical protein119,612120,9131302ForwardVibrio phage BONAISHI AXH70845.12.53 × 10−643.20
ORF 148Putative internal head protein122,313123,3771245Forward cl20461|phiKZ-like phage internal head proteins1.32 × 10−3.
ORF 149Hypothetical protein 123,449125,0531605ForwardVibrio phage BONAISHI AXH70848.12.17 × 10−10856.10
ORF 150Virion structural protein 125,053126,2701218ForwardVibrio phage BONAISHI AXH70849.11.52 × 10−6555.20
ORF 152Virion structural protein 126,970128,3341365ForwardVibrio phage BONAISHI AXH70851.11.52 × 10−4650.80
ORF 153DNA helicase 128,374129,9211548ReverseVibrio phage BONAISHI AXH70852.12.72 × 10−17770.10F: GO:0003678 6BBM_A|Escherichia coli O1111.50 × 10−36100.00
ORF 156Major capsid protein 130,604132,7692166ForwardVibrio phage BONAISHI AXH70854.13.91 × 10−14763.10
ORF 159Hypothetical protein 134,018135,5891572ForwardVibrio phage BONAISHI AXH70856.19.35 × 10−14161.50
ORF 162DUF723 domain-containing protein 138,774139,013240ForwardVibrio mediterranei WP_096444327.15.15 × 10−1261.90F: GO:0004519
ORF 163Endonuclease 139,080139,541462ForwardVibrio phage 1.225.O._10N.261.48.B7 AUR96455.12.54 × 10−746.00 6SEI_A|Thielavia terrestris1.80 × 10−497.77
ORF 164Holliday junction resolvase 139,578140,171594ReverseVibrio phage BONAISHI AXH70859.18.65 × 10−4162.90P: GO:0009987IPR036397cl21482|Crossover junction endodeoxyribonuclease RuvC and similar proteins1.05 × 10−36LW3_B|Escherichia coli4.70 × 10−2299.91
ORF 165Virion structural protein 140,171141,001831ReverseVibrio phage BONAISHI AXH70860.12.26 × 10−6960.40
ORF 166Virion structural protein 141,013143,0702058ReverseVibrio phage BONAISHI AXH70861.11.93 × 10−12556.50
ORF 167Putative portal protein 143,175145,9402766ForwardVibrio phage BONAISHI AXH70862.13.34 × 10−13255.60 cl27451|Hypothetical protein3.55 × 10−63JA7_I|Bacteriophage T42.60 × 10−598.45
ORF 168Putative hydrolase 145,952146,905954ForwardVibrio phage BONAISHI AXH70863.19.39 × 10−3150.20C: GO:0016020 4F55_A|Bacillus cereus1.10 × 10−2499.92
ORF 177Hypothetical protein150,443151,204762ForwardVibrio phage vB_VhaS-a ANO57550.11.74 × 10−3057.50
ORF 178Hypothetical protein151,281151,952672ForwardVibrio phage vB_VhaS-a ANO57549.19.91 × 10−4061.50
ORF 182Glycohydrolase 154,582155,496915ForwardVibrio phage BONAISHI AXH70879.12.61 × 10−6254.81F: GO:0016787 IPR002477
ORF 183Transcription factor: type II DNA-Binding155,609156,363855 1WTU_A|Bacillus phage SPO1 6.60 × 10−397.04
ORF 184Transcription factor: type II DNA-Binding156,379156,868510 1WTU_A|Bacillus phage SPO16.60 × 10−397.04
ORF 185Hypothetical protein156,896157,618723ForwardVibrio phage BONAISHI AXH70881.16.55 × 10−1247.83
ORF 193Putative DNA repair exonuclease 161,416162,201786ForwardVibrio phage pVa-21 AQT28114.11.92 × 10−2053.60F: GO:0004527 IPR036412 5UJ0_A|Bacteriophage T47.70 × 10−1199.26
ORF 194Nucleotide binding protein 162,149162,760612ForwardVibrio phage BONAISHI AXH70900.19.48 × 10−1654.40 cl17018|Fanconi anemia ID complex proteins FANCI and FANCD27.57 × 10−33GH1_B|Vibrio cholerae O1 biovar El Tor str. N169615.20 × 10−798.66
ORF 196Hypothetical protein163,750164,421672ForwardVibrio phage vB_VmeM-Yong XC31 QAX96244.12.14 × 10−10081.60
ORF 197Hypothetical protein164,476164,808333ForwardVibrio phage vB_VmeM-Yong XC31 QAX96240.1|1.00 × 10−338.89
ORF 198DEAD-like helicase 164,871166,8411971ForwardVibrio phage vB_VmeM-Yong XC31 QAX96243.11.70 × 10−16660.90P:GO:0000733 IPR014001 cd18793|C-terminal helicase domain of the SNF family helicases.1.80 × 10−162OCA_A|Bacteriophage T41.10 × 10−2999.98
ORF 199EAR-like protein166,948168,5911647 IPR009039
ORF 200Putative Palindromic Amphipathic Repeat Coding Elements (PARCEL)168,504187,86719,254 IPR011889pfam03382|Mycoplasma protein of unknown function, DUF2851.29 × 10−44
ORF 202DNA-packaging protein: hydrolase188,001188,966966ForwardPseudomonas virus phiKZ NP_803591.15.69 × 10−2053.10C: GO:0016020 2O0J_A|Bacteriophage T41.80 × 10−2499.92
ORF 207UV-endonuclease 191,809192,780972ForwardVibrio phage 1.084.O._10N.261.49.F5 AUR86431.16.23 × 10−6755.90F: GO:0004519 IPR004601cl23721|AP endonuclease family 22.84 × 10−483TC3_B|Sulfolobus acidocaldarius1.20 × 10−2699.96
ORF 209Dihydrofolate reductase (EC 1.5.1.3) CDS193,421194,152732ForwardMeiothermus ruber HFG20084.12.1 × 10−2259.30P: GO:0008152IPR001796cd00209|Dihydrofolate reductase (DHFR)9.07 × 10−391JUV_A|Bacteriophage T41.50 × 10−2099.87
ORF 223Putative nucleotidyl transferase202,749203,570822ForwardYersinia phage phiR1-37 YP_004934311.13.51 × 10−1351.80F: GO:0016740IPR043519cl35051|elongation factor Tu3.27 × 10−42FCL_A|Thermotoga maritima6.70 × 10−1299.44
ORF 225Putative N-acetyltransferase204,006204,428423 IPR016181 5Z6N_A| Escherichia coli (strain K12)
ORF 229Putative HTH-type transcription I regulator MqsA205,857506,309453 3GA8_A|Escherichia coli K-12
ORF 230Hypothetical protein VPIG_00040 206,325206,852528ForwardVibrio phage PWH3a-P1 YP_007675900.15.86 × 10−956.40
ORF 231Hypothetical protein SAMN05421742_1266 206,849207,121453ForwardRoseospirillum parvum
SDH93001.1|
1.94 × 10−554.70
ORF 243Putative glycosylhydrolase214,545215,8221278 IPR013320
ORF 245Hypothetical protein BCS93_11070217,217218,5391323ReverseVibrio breoganii PMP10208.18.63 × 10−1359.50 3ZYP_A|Hypocrea jecorina6.00 × 10−397.33
ORF 247Hypothetical protein218,552219,9491398ReverseVibrio breoganii WP_133150968.11.32 × 10−1042.50 3ZYP_A|Hypocrea jecorina8.80 × 10−497.72
ORF 248Hypothetical protein NVP1169O_83221,333222,6821350ReverseVibrio phage 1.169.O._10N.261.52.B1 AUR92111.15.68 × 10−1040.40
ORF 249Hypothetical protein BDU10_8600222,682223,677996ReverseBurkholderia sp. CF145 OYD65949.19.15 × 10−1553.10
ORF 250Polynucleotide kinase 223,801224,589789ForwardAeromonas virus Aeh1 NP_943967.11.30 × 10−1137.40 cl40282|HAD domain in Swiss Army Knife RNA repair proteins.1.12 × 10−85UJ0_A|Bacteriophage T41.80 × 10−498.02
ORF 256SH3 protein228,286228,981695 cl17036|Src Homology 3 domain superfamily6.19 × 10−3
ORF 262Phosphagen kinase232,254232,973720Forward cl02823|Phosphagen (guanidino) kinases
ORF 263 DNA polymerase accessory protein 44: AAA+, ATP hydrolase233,047234,3901344ForwardSalmonella enterica EAZ2022740.12.56 × 10−3050.70F: GO:0000166IPR003593 3U61_D|Bacteriophage T4}9.00 × 10−1299.45
ORF 267Ribonuclease E/G236,230236,958729 cl29166|Ribonuclease E/G family1.32 × 10−3
ORF 272DNA polymerase II large subunit239,038239,373336 cl36419|DNA-directed DNA polymerase II large subunit3.90 × 10−3
ORF 275NAD-dependent DNA ligase LigA 240,880242,8171938ForwardSalinivibrio sp. ES.052 WP_074213176.12.87 × 10−13157.20P: GO:0006259 IPR001357 cl35633|NAD-dependent DNA ligase LigA; Validated0.00 × 1005TT5_A|Escherichia coli K121.50 × 10−121100.00
ORF 278GTP cyclohydrolase II 244,297244,800504ForwardVibrio phage PWH3a-P1 YP_007676007.12.19 × 10−3061.60
ORF 281Thymiylate kinase 245,594246,316723ForwardFirmicutes bacterium CAG:582 CDB28696.13.93 × 10−3454.80P: GO:0006796.IPR039430 cl17190|Nucleoside/nucleotide kinase (NK).1.10 × 10−263LV8_A|Vibrio cholerae O1 biovar El Tor2.50 × 10−2299.92
ORF 283Ribonucleotide reductase of class Ia (aerobic), alpha subunit 246,875249,1632289ForwardRodentibacter pneumotropicus WP_077664105.10.00 × 10071.30F: GO:0000166IPR005144 cl32350|ribonucleoside-diphosphate reductase subunit alpha.0.00 × 1002XAP_A|Escherichia coli5.10 × 10−23100.00
ORF 286Ribonucleotide reductase of class Ia (aerobic), beta subunit 250,213251,3311119ForwardSulfurivirga caldicuralii WP_074201546.18.00 × 10−13969.40F: GO:0004748 cl00264|Ferritin-like superfamily of diiron-containing four-helix-bundle proteins0.00 × 1001MXR_B|Escherichia coli1.90 × 10−56100.00
ORF 287BspA family leucine-rich repeat surface protein 251,408255,0913684ForwardHelicobacter bizzozeronii WP_158656920.11.25 × 10−3646.00C: GO:0016020 IPR005046cl37689|Mycoplasma protein of unknown function, DUF2852.51 × 10−27
ORF 299PIN terminus261,891262,838978 IPR002716cl28905|PIN (PilT N terminus) domain: Superfamily2.33 × 10−62HWY_A|Homo sapiens4.10 × 10−396.35
ORF 308Hypothetical protein 266,214266,954741ForwardCellulomonas aerilata WP_146903668.12.66 × 10−441.20 6HIY_DS|Trypanosoma brucei brucei5.50 × 10−1199.13
ORF 310Glutaredoxin267,350268,000651 cl35908|glutaredoxin 22.14 × 10−3
ORF 316Asp/Glu/Hydantoin racemase270,693271,8771185 cl00518|Asp/Glu/Hydantoin racemase5.48 × 10−3
ORF 319RNA-binding protein274,073274,750678ForwardVibrio phage BONAISHI AXH70995.14.95 × 10−2051.30F: GO:0016787IPR036397cl10012|DnaQ-like (or DEDD) 3′-5′ exonuclease domain superfamily1.01 × 10−136N6A_A|Vibrio cholerae1.60 × 10−1599.70
ORF 323Thymidylate synthase (EC 2.1.1.45) 276,131277,057927ForwardVibrio phage 2.275.O._10N.286.54.E11 AUS02985.13.26 × 10−7662.60P: GO:0008152 IPR023451cl19097|Thymidylate synthase and pyrimidine hydroxy methylase. 1TIS_A|Bacteriophage T4
ORF 326Nucleoside Triphosphate Pyrophosphohydrolase277,926278,654729ForwardVibrio phage vB_VmeM-Yong XC31 QAX96185.14.77 × 10−1058.40 cl16941|Nucleoside Triphosphate Pyrophosphohydrolase (EC 3.6.8) MazG-like domain superfamily4.07 × 10−32YF4_B|Deinococcus radiodurans8.80 × 10−2099.83
ORF 329Hypothetical protein279,832280,650819Forward IPR006530
ORF 330Hypothetical protein 280,703281,116414ReverseVibrio phage BONAISHI AXH71034.11.91 × 10−1354.40
ORF 331Hypothetical protein yiiX281,272281,703432ReverseVibrio phage vB_VmeM-Yong XC31 QAX96165.12.49 × 10−2755.60 IPR038765cl21534|NlpC/P60 family.4.98 × 10−32IF6_A|Escherichia coli4.10 × 10−2199.87
ORF 332Hypothetical protein281,713282,711999ReverseVibrio phage BONAISHI AXH71036.11.57 × 10−4147.40
Table 3. List of bacterial strains used in this study with their methods of identification and location of isolation.
Table 3. List of bacterial strains used in this study with their methods of identification and location of isolation.
Bacterial StrainsMethod of IdentificationLocation
Vibrio harveyi
DSM19623ENA Accession No: BAOD01000001USA
SNGRBIOLOG GENiiiGreece
KS6BIOLOG GENiiiGreece
Vh2BIOLOG GENiii, toxR (+)Greece
Vh5BIOLOG GENiii, toxR (+)Greece
VhSernFrBIOLOG GENiii, toxR (+)Greece
VhP1 LivtoxR (+)Greece
VhP1 SpltoxR (+)Greece
VhKarxBIOLOG GENiii, toxR (+)Greece
RG1toxR (+)Greece
Barb A4/1.1BIOLOG GENiii, toxR (+)Greece
SerKidBIOLOG GENiiiGreece
SerKid2BIOLOG GENiiiGreece
SerSdBIOLOG GENiiiGreece
SA 5.116S rRNAKSA
SA 6.116S rRNAKSA
SA 9.216S rRNAKSA
SA 1.216S rRNAKSA
SA 7.116S rRNAKSA
SA 3.116S rRNAKSA
SA 4.116S rRNAKSA
SA 2.116S rRNAKSA
Epith. DBIOLOG GENiiiGreece
Vh No. 22BIOLOG GENiiiGreece
Vh6BIOLOG GENiiiGreece
Vibrio alginolyticus
V1BIOLOG GENiiiGreece
V2BIOLOG GENiiiGreece
HCMR 1 Art. 3Clinical strainGreece
DSM 2171ENA Accession No.: AB372523Japan
Valg HCMRBIOLOG GENiiiGreece
Skironis-2BIOLOG GENiiiGreece
NS A6BIOLOG GENiiiGreece
Rot. Vib. 5BIOLOG GENiiiGreece
Vibrio anguillarum
90-11-286Clinical strainDenmark
VIB 44Clinical strainItaly
VIB 64Clinical strainSpain
VIB 243Clinical strainUSA
Vibrio campbellii
VIB391NCBI RefSeq No: GCF_002078065.1Thailand
Vibrio owensii
SA 1.116S rRNAKSA
SA 9.116S rRNAKSA
Vibrio parahaemolyticus
VPINHBIOLOG GENiiiGreece
Vibrio splendidus
Barb A4/2BIOLOG GENiiiGreece
VaAnClinical strainGreece
Other Vibrio spp.
Art. 1TCBSGreece
Art. 2TCBSGreece
Rot. 2toxR (+)Greece
Barb A4/1.2TCBSGreece
Rot. Vib. 1TCBSGreece
Rot. Vib. 2TCBSGreece
Rot. Vib. 3TCBSGreece
Rot. Vib. 4TCBSGreece
Rot. Vib. 6TCBSGreece
Abbreviations: TCBS, thiosulfate-citrate-bile salts; ENA, European Nucleotide Archive; NCBI, National Center of Biotechnology Information; KSA, Kingdom of Saudi Arabia.
Table 4. Disk diffusion interpretive criteria for antibiotic susceptibility testing.
Table 4. Disk diffusion interpretive criteria for antibiotic susceptibility testing.
Antimicrobial AgentDisk Diffusion (μg)Zone Diameter (mm) Interpretive Criteria
SIR
Ampicillin10≥1714–16≤13
Tetracycline30≥1512–14≤11
Sulphamethoxazole/trimethoprim25≥1611–15≤10
Oxytetracycline a30≥2717–26≤16
Florfenicol b30≥1813–17≤12
Oxalinic acid c2≥1914–18≤13
Flumequine c30≥1914–18≤13
Abbreviations: S, susceptible; I, medium; R, resistant. a based on oxytetracycline breakpoint established by Uhland and Higgins (2006). b based on analogues in [88] clinical breakpoints for Vibrio spp. including Vibrio cholerae. c based on analogues in [89] clinical breakpoints for Vibrio spp. including Vibrio cholerae.
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