Isolation of a virulent Vibrio alginolyticus Bacteriophage and Its Application in Shrimp culture

Isolation of a virulent Vibrio alginolyticus Bacteriophage and Its Application in Shrimp culture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation of a virulent Vibrio alginolyticus Bacteriophage and Its Application in Shrimp culture Wenqi Wang, Xiansong Meng, Zhonghao Wu, Yubin Fu, Xiaoling Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4446997/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As an opportunist pathogen, Vibrio alginolyticus , causes disease outbreaks in marine farmed fish and invertebrates. Due to problems caused by the abuse of antibiotics, it is extremely important to develop green biocontrol methods for Vibrio diseases. Phage therapy is considered a safe and promising prevention and treatment method. Here, we report that a novel virulent bacteriophage VaPW, which infects V. alginolyticus , was isolated from seawater. The bacteriophage is morphologically similar to phages from Myoviridae family. It displayed good pH (7–9) and temperature (≤ 50°C) tolerance and had a narrow host range. Its genome consists of 34,637 bp with a GC content of 43.22%, while sequence analysis revealed the presence of 41 potential ORFs, of which 26 coding sequences showed homology to functionally characterized genes. The in vitro antibacterial experiment showed that phage VaPW could effectively inhibit the growth of V. alginolyticus ATCC 33787. Moreover, the shrimp survival test demonstrated that phage VaPW has the in vivo protection effect against V. alginolyticus infection. To determine whether the putative lysozyme and holin from phage VaPW have bactericidal activity, orf26 and orf27 were cloned for protein expression and analyses in E. coli . The data revealed that co-existence of holin and lysozyme exhibited a synergistic bactericidal effect on the lysis of target cells compared with single protein alone. These results suggest that phage VaPW and its proteins may provide good candidates to control recurrent diseases caused by V. alginolyticus in marine animals. Bacteriophage Isolation Shrimp culture growth inhibition Vibrio alginolyticus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The breeding technology of Penaeus vannamei has been mature since its introduction, and the annual output accounted for 80% of the total output of all kinds of shrimp. However, several Vibrio spp. , including Vibrio alginolyticus , are considered to be the most prevalent bacterial pathogens causing mortality of fish and invertebrates and considerable economic losses (Selvin and Lipton 2003; Austin et al. 2016; Grimes et al. 2020). Moreover, V. alginolyticus was reported as a human pathogen causing infections and food poisoning (Schmidt et al. 1979; Hernández-Robles et al. 2016). The widespread and frequent uses of antibiotics in aquaculture caused the emergence of antibiotic resistance and food safety problems. For example, antibiotics used for treating infectious diseases in aquaculture have resulted in the development of many antibiotic resistant strains of Vibrio spp. (Oh et al. 2011 ; Defoirdt et al. 2007 ). Phage therapy is a promising alternative to antibiotics. The main advantages of phage include its specificity and phage can achieve a dynamic balance with pathogenic bacteria without destroying the environmental balance. As a green and pollution-free new biological control technology, it has great application potential (O'Flaherty et al. 2009 ). Phages have been experimentally used to control important pathogens in aquacuture (Vinod et al. 2006; Kim et al. 2015 ; Srinivasan et al. 2017). Moreover, phage therapy has been also considered targeting V. alginolyticus (Kokkari et al. 2018 ; Le et al. 2020 ). Though phage therapy is ideal for the control of bacterial pathogens in aquaculture, it has not yet been fully exploited. In order to use this strategy safely, a full characterization of phage genomes is required to check the presence of potential genes related to virulence and antibiotic resistance and to confirm phages were obligate lytic. With recent advances in phage genome sequencing, bacteriophage-derived lytic enzymes and proteins are of interest to the scientific community as biocontrol and therapeutic agents against multidrug resistant bacteria (Chang et al. 2022 ; Lee et al. 2023 ; Liu et al. 2023 ). The bacterial lysis is achieved by phage-encoded lysozymes or endolysins that degrade the peptidoglycan (PG) layer present in the bacterial cell wall. The canonical lysis process is that lysozymes act on PG layer and trigger the lysis process with the help of a second phage-encoded membrane protein called holin, in a timely-controlled fashion (Young et al. 2013). Holins are phage-encoded proteins that control access of phage-encoded endolysins to the peptidoglycan through pores in the membrane. Several lysozymes/endolysins coded by V. alginolyticus had been reported, although the bactericidal activity of some genes has not been experimentally verified (Matamp et al. 2019; Li et al. 2019 ; Li et al. 2021 ). With more and more Vibrio phages being discovered, their genetic information will help to provide more comprehensive information for phage therapy. This study reports that a novel lytic phage VaPW was isolated from seawater and identified as members of the Myoviridae family by transmission electron microscopy (TEM). The characteristics and complete genome of VaPW were determined. Antibacterial ability of phage VaPW was verified in vitro and in vivo . Moreover, two putative genes, holin and lysozyme, were cloned and verified for their bactericidal effects. These results suggest that phage VaPW is a potential candidate to control diseases caused by V. alginolyticus in aquaculture. Materials and methods 2.1 Bacteriophage isolation To isolate bacteriophages, 50 mL sea water sample and 1–2 mL of overnight V. alginolyticus ATCC 33787 culture were added to 50 mL of double concentrated LB broth, shaking at 200 rpm overnight at 30°C. The broth mixture was then centrifuged at 10,000 ×g for 10 min and the supernatant was filtered through a 0.22 µm filter. The bacteriophage supernatant was diluted with a 10-fold gradient using SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH 7.5, 0.01% (w/v) gelatin). 100 microliters of each dilution were mixed with 1 mL of V. alginolyticus solution, and stood at 30 ℃ for 20 minutes. Then 9 mL of warm semi-solid top agar (culture medium with 0.6% agar) were added into the solution and immediately mix to prepare a double-layer plate. The double-layer agar plate was cultured at 30°C for 12 h, and then phage plaques were visualized. The isolation on double-layer agar plates was repeated at least three times to purify the phages. 2.2 Bacteriophage characterization The bacteriophages were morphologically characterized by electron microscopy. Purified phage (> 10 10 PFU/ml) were deposited onto carbon-coated copper grids and allowed to adsorb for 5 min. The grids were allowed to dry, stained with 2% phosphotungstic acid (pH 7.0) for 5 min, and washed with PBS. The morphology was examined under a Hitachi TEM System HC-1 at 80 kV. The host range of phage was also determined by the double-layer agar plate method as mentioned above. Specifically, 1 mL of each freshly grown potential host cultures (10 7 -10 8 cfu/ml) was mixed with 100 µL of phage and 3 ml of warm top agar, then poured on bottom agar plates. After incubation, plaque formation on the plates was observed to determine the sensitivity of the host bacteria to phage. Thermal stability was assessed by exposing aliquots of the phage at various temperatures (40, 50, 60, 70 and 80°C) for 30 minutes or 1 h. After incubation, samples were diluted with SM buffer, and titers were evaluated by the double-layer agar method in triplicates. For the determination of the stability of the phages at different pH values, phage lysates were added to SM buffer at different pH levels (pH 3–11), which was adjusted by using HCl or NaOH. After incubation of the mixtures at 30°C for 1 h, the titers were evaluated by the double-layer method. To determine the optimal multiplicity of infection (MOI), different titers of phage were co-cultured with V. alginolytics at MOI ranging from 0.001 to 100 for 10 h at 30 ℃ with shaking. The titer of the phage was then determined by the double-layer agar method. The optimal MOI was the MOI of the phage sample with the highest titer. 2.3 Genome sequencing and bioinformatic analysis Genomic DNA was extracted from the cell pellets with a Bacteria DNA Kit (OMEGA) according to the manufacturer’s instructions, and high qualified DNA sample (OD260/280 = 1.8 ~ 2.0, >6ug) is utilized to construct fragment library. Library construction and Illumina HiSeq sequencing were performed by Biozeron Biotechnology Co., Ltd. (Shanghai, China.). Paired-end libraries with insert sizes of ~ 400bp were prepared following Illumina’s standard genomic DNA library preparation procedure. The qualified Illumina pair-end library would be used for Illumina NovaSeq 6000 sequencing. ABySS (Jackman et al. 2017 ) was used to do genome assembly with multiple-Kmer parameters and got the optimal results of the assembly. The complete genome of phage was sequenced and assembled by Biozeron Biotechnology Company. Gene models were identified using GeneMark (Besemer et al. 2005). Then all gene models were blastp against non-redundant (NR in NCBI) database, SwissProt ( http://uniprot.org ), KEGG ( http://www.genome.jp/kegg/ ), and COG ( http://www.ncbi.nlm.nih.gov/COG ) to do functional annotation. In addition, tRNA were identified using the tRNAscan-SE (v1.23, http://lowelab.ucsc.edu/tRNAscan-SE ) and rRNA were determined using the RNAmmer (v1.2, http://www.cbs.dtu.dk/services/RNAmmer/ ). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 11 (Tamura et al. 2021 ). Phylogenetic trees were constructed in MEGA using the neighbor-joining algorithm. 2.4 Antibacterial effect in vitro V. alginolyticus ATCC 33787 was cultured to OD 600 0.1, and then phage (4×10 7 PFU) was added to the culture to achieve the optimal MOI (0.01). Host bacteria with chloramphenicol (finally 20 µg/mL) were used as positive controls, and host bacteria with PBS served as negative controls. The mixtures were shaken for 12 h at 30°C, and the optical density at 600 nm was monitored every 1 h. Each treatment was performed in triplicate. 2.5 Therapeutic efficacy of VaPW against V. alginolyticus infection in shrimp Phage therapy was performed using 10 randomly selected shrimp in a PE water tank with seawater for each experimental group. the V. alginolyticus was added into tank to a final concentration of 1.59 × 10 8 CFU/mL, with a total volume of 3 L. And then bacteriophage was added with MOI of 1, 0.01, and 0.0001, respectively. Three independent experiments were performed for each condition. A group adding PBS of the same volume was used as the negative control group. A control group containing only bacteriophages (~ 1.59 × 10 8 CFU/mL) was set up to test whether VaPW itself has toxic effects on shrimp health. Any dead shrimp was observed and recorded every 12 hours for a total of 7 days, and a survival curve was drawn. 2.6 Bactericidal activity of recombinant lysozyme and holin of VaPW The complete holin (ORF 26) gene, lysozame (ORF 27) gene, and holin-lysozyme (ORF 26–27) fragment were amplified using PrimSTAR Max DNA Polymerase (Takara, China). Plasmids and primers used in this study are listed in Table S1 & S2. The vector fragment was amplified using pSCT32 vector (Zhang et al. 2017 ) as template and then recombinated with the amplified lysozame, holin, and lysozame-holin gene fragments, respectively, using One Step Cloning Kit (Vazyme, China) according to the manufacturer’s instructions. The reaction mixtures were transformed into competent E. coli DH5α cells. The resulting pSCT-lysozyme, pSCT-holin, and pSCT-holin-lysozame plasmids were transferred into E. coli BL21 cells for expression. The BL21 cells containing the above-mentioned plasmids were cultured in LB medium with 50 µg/mL kanamycin until the OD 600 nm reached at about 1.0. The cultures were then supplemented with IPTG with the final concentration of 0.1 mM, and incubated for 8 hours at 30°C. The OD values of cultures were monitored and the expression of recombination proteins, which were tagged with a hexahistidine epitope tag, was analyzed by previous published SDS-PAGE and Western blotting methods (Zhang et al. 2018 ). 2.7 Statistical analysis The statistical study of the obtained results was performed with GraphPad Prism 7.0. Results are expressed as means ± the standard deviations (SD). One-way analysis of variance (ANOVA) was used to determine the statistical significance. Significant differences were considered present at *P < 0.05 and **P < 0.01. 2.8 Data availability The sequence data for the phage VaPW were deposited at GenBank under accession no. 2649232. Results 3.1 Isolation of V. alginolytics phage VaPW Phage VAPW was isolated from a seawater sample of a shrimp farm in Qingdao, Shandong, China. Phage VaPW produced small plaques plaques (1.08 ± 0.25 mm in diameter) on the lawn of host V. alginolyticus ATCC 33787 (Fig. 1 A). Phage morphology and size were determined by transmission electron microscopy (TEM) (Fig. 1 B). Phage VaPW has a regular hexagon head with a diameter of 90 nm, and about 310 nm long tail with has a contractile tail sheath and a central tube. The isolate was assigned to the Myoviridae family, order Caudovirales , based on morphology characteristics of the classification system of International Committee on Taxonomy of Viruses (ICTV). The host range of phage VaPW was determined by using 11 clinical or environmental strains. No sensitivity was observed in the other test genera or species shown in Table 1 , suggesting that phage VaPW had a narrow host range with specificity. Table 1 Lysis profile of phage VaPW Strain VaPW Reference or source V. parahaemolyticus ATCC17802 — Collection V. alginolyticus ATCC33787 + Collection V. alginolyticus YX — Collection V. alginolyticus ZJTCC — Chen, 2012 Vibrio anguillarum M3 — Mo, 2010 Vibrio vulnificus ATCC27562 — Collection Edwardsiella tarda EtSC — Mo, 2013 Vibrio harveyi ATCC 14126 — Collection Vibrio ichthyoenteri F1 — Collection Aeromonas hydrophila ATCC7966 — Collection Aeromonas salmonicida C4 — Kuang, 2022 3.2 Biological characteristics of phage Phage VaPW was propagated under various environments to evaluate stability. Survival of phage VaPW was stable during heat treatment from 40°C to 50°C lasting 30 min. The phage titer was reduced by approximately 40% at 60°C, and almost complete inactivation of the bacteriophage occurred at temperature ranges above 70°C (Fig. 2 A). In addition, the pH stability test showed that phage VaPW remains active at a pH range from 4 to 11 (Fig. 2 B). However, the phage was inactivated at pH 3. These findings show that phage VaPW is stable under wide ranges of temperatures and pHs. The effects of organic solvents (chloroform, diethyl ether), on the survival of the phage was determined. There was no significant difference in the titer between the untreated phages and the solvents-treated phages (Fig. 2 c), which indicates that phage VaPW was not affected by chloroform/diethyl ether treatment and it does not contain lipids. The V. alginolyticus strain, when infected by phage VaPW at a MOI of 0.01, generated the maximum phage titer of approximately 8.55 x 10 9 PFU/mL (Table 2 ), indicating that 0.01 was the optimal MOI for VaPW. Table 2 MOI of phage VaPW MOI Phage titer PFU Bacteria number CFU Phage titer PFU/mL 100 1×10 10 1×10 8 2.00×10 7 10 1×10 9 1×10 8 9.00×10 7 1 1×10 8 1×10 8 1.57×10 9 0.1 1×10 7 1×10 8 2.50×10 9 0.01 1×10 6 1×10 8 8.55×10 9 0.001 1×10 5 1×10 8 2.13×10 9 The effect of the phage on V. alginolytics ATCC 33787 in vitro was shown in Fig. 3 . Growth curves of V. alginolytics infected with phage VaPW at optimal MOI were measured by monitoring absorbance to evaluate the in vitro antibacterial efficacy. The negative control received PBS and positive control received chloramphenicol (CPL). Compared to the control group, the optical density of V. alginolyticus treated with phage started to show decreases after 2h infection, and the OD 600 decreased further and was significant (P < 0.05) compared to the control group. This result indicates that phage VaPW could effectively inhibits the growth of V. alginolyticus ATCC 33787, suggesting that it could be a candidate biocontrol agent for phage therapy against V. alginolyticus . 3.4 Genomic analysis of phage VaPW The genome of VaPW was sequenced and submitted to GenBank under the accession number 2649232. The complete genome of phage VaPW is 34,637 bp in length, with an average GC content of 43.22%. Forty-one putative open reading frames (ORFs) comprising 90.7% of the genome were subsequently predicted and 37 of them start with an ATG codon, except for gene 2, which start with TTG and gene 16、gene 27 and gene29, which starts with GTG. According to BLAST search, 38 of the 41 ORFs were identified to share similarities with previously reported genes in the database, of which 26 ORFs showed homology to functionally characterized genes. The classifications of the 26 coding sequences with functional annotations are being listed (Table S3), which includes 11 structural and packing protein genes, 10 DNA replication/regulation genes, and 5 other protein genes. No tRNA-encoding genes and no toxin genes were identified. The genome sequence comparison using the Blastn method revealed that the complete genome of this phage had a nucleotide similarity score of 59.13% with the known bacteriophage Vibrio phage Vp1B1MD-2 (NCBI Reference Sequence NC_055862.1). As a result, we concluded that VaPW is a new phage. To investigate the phylogeny of phage VaPW, two unrooted phylogenetic trees were constructed with amino acid sequences of ORF07 and ORF26, which are predicted to encode the capsid protein and lysozyme, respectively. Interestingly, we consistently found that phage VaPW seems to be most closely related to the Vibrio phage Vp1B1MD-2 on the phylogenetic trees (Fig. 4 ), which is classified as belonging to the Myoviridae family. 3.5 Therapeutic efficacy of phage VaPW against V. alginolyticus infection in shrimp To estimate the in vivo protection effect of phage VaPW against V. alginolyticus infection, a survival test was performed with shrimp. The survival rates of V. alginolyticus- infected shrimp in groups treated with phage at different MOIs were recorded for 7 days. As shown in Fig. 5 , the survival rate increased in a dose-dependent manner when V. alginolyticus -infected shrimp received phage treatment. The phage- groups at MOIs of 0.0001, which is much lower than the optimum MOI of 0.01, also showed a slight improvement (55%) in the survival rate. The phage-treated groups at MOIs of 0.01 and 1 showed much higher survival rates (65% and 70%, respectively). There was no die in control group without V. alginolyticus infection, indicating that the phage did not affect shrimp health. This result suggested that phage VaPW may be a candidate as an effective biocontrol agent for phage therapy against V. alginolyticus . 3.5 Bactericidal activity of recombinant lysozyme and holin of VaPW Bacteriophage proteins have been applied to the prevention and control of food-borne pathogens recently. In phage VaPW, BLASTX showed that the hypothetical product of orf27 has identity values (41– 99%) with a series of lysozymes from a wide variety of sources. Orf26 is located upstream of orf27 and also encodes a putative phage holin. A conserved phage_holin superfamily domain (Fig. S3) and a conserved bacteriophage Lyz-like superfamily domain (Fig. S4) were found in Orf27 and Orf26 protein, respectively. To determine whether the lysozyme and holin genes from phage VaPW have bactericidal activity, Orf26 and orf27 were cloned for protein expression and analyses. The E. coli carrying the plasmids pSCT32-hol or pSCT32-lys were cultured to OD 600 1.0, and then induced by IPTG. Growth curves showed that growth of E. coli expressing recombinant holin was slightly inhibited when comparing to the control groups within 3 h (Fig. 6 A). On the other hand, the expression of lysozyme exhibited significant inhibition on growth of E.coli within 2 h based on the growth curves. The WB experimental data showed that holin was stably expressed in cells after induction, while the expression level of lysozyme continued to decrease over time (Fig. 7B). These results indicated that the lysozyme from phage VaPW have better bactericidal ability than holin on growth inhibition of E. coli . The growth of pSCT32-lys E. coli and was gradually recovered to OD 600 1.0 after 5 h, indicating that the effect on growth inhibition of the lysozyme is valid (Fig. 6 B). To verify a synergistic effect on the lysis of target cells, the holin and lyzosyme were cloned together from the phage VaPW genome and co-expressed in E. coli , and then bactericidal activity was determined. The data revealed that co-existence of holine and lyzosyme in E. coli pSCT3-hol-lys exhibited the stronger bactericidal activity than single protein alone ( E. coli pSCT3-hol or E. coli pSCT3-lys), as showed in the growth curve (Fig. 6 B). Discussion Nowadays, phage therapy has been suggested as an alternative to antibiotic administration in both human and veterinary medicine due to the developing problem of antibiotic resistance (O'Flaherty et al. 2009 ; Kortright et al. 2019 ; Hatfull et al. 2022 ). In aquaculture, the use of bacteriophages and their derived enzymes to combat resistant bacterial infections can help to control bacterial populations in delicate environments, whereas antibiotic administration would alter the natural microflora of the environment and the intestinal microflora of cultured organisms (Nachimuthu, et al, 2021 ). In this study, a novel lytic phage VaPW that targets V. alginolyticus ATCC 33787 was isolated and characterized. The results presented here showed that phage VaPW is efficient in reducing the growth of the host bacteria during in vitro testing at a low MOI (0.01). Moreover, phage VaPW stability test under various temperature and pH conditions showed that phage is extremely stable remaining active at 50°C and under a wide range of pHs, which suggested that its antimicrobial activity could be retained during storage and production operations. Phage VaPW has a very narrow lytic rang being able to infect only its host V. alginolyticus ATCC 33787. However, we cannot rule out its effect on other sensitive V. alginolyticus strains. Further research would include collecting more clinic, environmental isolates and other standard strains of V. alginolyticus and other Vibrio spp. to test the infectivity of VaPW. In addition to the in vitro antibacterial test of phage VaPW against V. alginolyticus (Fig. 3 ), an in vivo shrimp survival test with V. alginolyticus infected shrimp revealed that phage VaPW protected shrimp from the virulence of V. alginolyticus . In this study, bacteriophages were directly applied to the culture pond before shrimp were infected with V. alginolyticus . The aim of the operation is to reduce the Vibrio load in the culture environment, thereby reducing the risk of bacterial disease outbreaks. In the shrimp protection experiment, we used a higher concentration of bacteria (final 1.59 × 10 8 CFU/mL) to observe the biocontrol effect in a short time. When the phage MOI (0.0001) is 100 times lower than the optimal MOI (0.01), phage VaPW can still effectively improve the survival rate of shrimp after infection (Fig. 5 ), indicating that it has a certain disease prevention effect evern if the phage concentration is low. When the MOI was 0.01, its antibacterial effect was similar to that of MOI = 1 group, indicating that the phage (MOI above 0.01) could prevent the infection of V. alginolyticus in P. vannamei . After thoroughly understanding the genetic information of bacteriophages, it thus becomes possible to engineer phages with enhanced therapeutic properties, safety features and host-range (Strathdee et al. 2023 ). The genome of VaPW was sequenced and analyzed to gain a further understanding of this phage. Its genome is 34,637 bp, similar to other Vibrio phages, such as Vibrio phage Vp1B1MD-2 with a maximal nucleotide similarity score of 59.13%. Since RNA polymerase was not annotated in phage VaPW genome, three representative phage conserved proteins, capsid protein, lysozyme and endonuclease, were used for phylogenetic tree analysis. The phylogenetic analysis revealed that phage VaPW clustered closely with Vibrio phage Vp1B1MD-2 on the phylogenetic tree based on the capsid protein and lysozyme analysis (Fig. 4 ). Interestingly, additional endonuclease sequence analysis revealed that orf6 of phages VaPW seems to be most closely related to a HNH nuclease of a Myoviridae sp. phage (GenBank: DAN88581.1), but not nuclease of phage Vp1B1MD-2 (Fig. S1). Phage Vp1B1MD-2 also belongs to the Myoviridae phage family. These results showed that there are a wide variety of phages with strong diversity. More phage genomes are needed to fill the gap in the database. Currently, VaPW was confirmed to be a novel phage belonging to the Myoviridae phage family, which was consistent with the results of TEM observation. The genomic analysis did not reveal the presence of virulence or antibiotic resistance genes in VaPW, implying that VaPW might be suitable for phage therapy. A phage used as a potential biocontrol agent should preferably be a virulent phage with no side effects on marine animal health. However, as more than one-third of the predicted ORFs (15 of the 41 ORFs) in VaPW are hypothetical proteins with uncharacterized function, it is not known whether this phage carries potentially hazardous genes. Fortunately, the safety of VaPW has been evaluated as non-toxic to shrimp when tested in vivo (Fig. 5 ). The main mechanism of bacteriophage lysis of the host has been thoroughly studied, mainly through their encoded lytic proteins (lysozyme and horin) synthesized in the host (Young et al. 2000 ; Wang et al. 2000 ). It is generally believed that (an oligomeric membrane lesion is formed firstly by the holin, allowing escape of endolysin. Then, lysozyme penetrate the pores to hydrolyze peptidoglycans and lyse the host. Holins control the length of the infective cycle for lytic phages and achieve lysis at an optimal time. Besides, lytic single-stranded RNA and DNA phages accomplish lysis by producing a single lysis protein without muralytic activity (Young et al. 2000 ). Phage and their lysins have already been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections (O'Flaherty S et al., 2009 ). In this study, genome sequence analysis of phage VaPW revealed that it has an holin-encoding gene ( orf 26 ) and endolysin-encoding gene ( orf 27 ) (Table S2). Two genes were cloned and induced for protein expression in E. coli . The experimental results showed that the expression of either protein could inhibit the growth of E. coli , though lysozyme (Orf 27) had a stronger antibacterial effect. These data suggested that holin might not be essential for the action of endolysin in the phage VaPW. Some researches also demonstrated that the bactericidal activity of endolysins of phage are not dependent on holins in the lysis system for export (Xu et al. 2004 ; Wang et al. 2000 ; Li et al. 2021 ). After 5 hours of induction, the inhibited E. coli by lysozyme expression began to slowly recover and grew, which might probably be because that the host cell detected the toxicity of overexpressed lysozmy and the host defense mechanism reduced the expression level of the recombinant protein. Interestingly, while two lytic proteins were expressed simultaneously, a highly significant host cell lysis phenomenon was observed, which indicating a synergistic effect between two proteins. Hence, we speculate that in phage VaPW lysis system, holin helps to some extent improve the efficiency of lysozyme to break through the cell membrane and degrade the cell wall. A similar example was also reported in another V. alginolyticus phage HH109 (Li et al. 2021 ). Although the synergetic effect needs to be further elucidated in the other bacterial species, our results demonstrated that the holin and lysozyme from phage VaPW could also be used as new potential biocontrol agents against V. alginolyticus . Taken together, we isolated a novel V. alginolyticus phage VaPW from seawater sample and identified its biological characteristics. Its antibacterial effect was evaluated by the in vitro and in vivo shrimp infection test. The complete genome was sequenced and analyzed. Two lytic protein genes, holin and lysozyme, were cloned and verified their bactericidal activity in E.coli . In conclusion, our work demonstrated the phage VaPW and its proteins could be used as new type of biocontrol agents to be exploited against V. alginolyticus in aquaculture. Declarations Conflict of Interest: The authors declare that they have no conflict of interest. Author Contribution SC and WW conceived the experiments, WW, XM, ZW,YF and XL conducted the experiments, WW, XM and ZW analyzed the results. All authors assisted in writing the manuscript, discussed the results and reviewed the manuscript. Acknowledgments This work was financially supported by the Natural Science Foundation of Shandong Province (ZR2020MC197), the National Natural Science Foundation of China (32273183 & 31372564), Technical system of shrimp and crab industry in Shandong Province (SDAIT-11), “First class fishery discipline” program in Shandong Province, and a special talent program “One Thing One Decision (Yishi Yiyi)” Program in Shandong Province, China. References AustinB, Austin DA (2016) Bacterial Fish Pathogens, 6th ed.; Springer: Chichester, UK, ISBN 978-3-319-32673-3 Besemer J, Borodovsky M (2005) GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. 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Protein J 42(5):463–476 Luo P, Yun L, Li Y et al (2018) Complete genomic sequence of the Vibrio alginolyticus bacteriophage Vp670 and characterization of the lysis-related genes, cwlQ and holA. BMC Genom 19:741 Matamp N, Bhat SG (2019) Phage Endolysins as Potential Antimicrobials against Multidrug Resistant Vibrio alginolyticus and Vibrio parahaemolyticus : Current Status of Research and Challenges Ahead. Microorganisms 7(3):84 Mo Z, Guo D, Mao Y et al (2010) Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease. Chin J Oceanol Limnol 28(1):55–61 Mo Z, Li J, Li G et al (2013) Phenotypic characterization, virulence, and immunogenicity of Edwardsiella tarda LSE40 aroA mutant. Appl Microbiol Biotechnol 97:6325–6335 Nachimuthu R, Royam MM, Manohar P et al (2021) Application of bacteriophages and endolysins in aquaculture as a biocontrol measure. Biol Control 160:104678 O'Flaherty S, Ross RP, Coffey A (2009) Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol Rev 33(4):801–819 Oh EG, Son KT, Yu H et al (2011) Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007. J Food Prot 74(3):380–386 Srinivasan P, Ramasamy P (2017) Morphological characterization and biocontrol effects of Vibrio vulnificus phages against Vibriosis in the shrimp aquaculture environment. Microb Pathog 111:472–480 Strathdee SA, Hatfull GF, Mutalik VK (2023) Schooley RT. Phage therapy: From biological mechanisms to future directions. Cell 186(1):17–31 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38(7):3022–3027 Wang IN, Smith DL, Young R (2000) HOLINS: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54:799–782 Xu M, Struck DK, Deaton J et al (2004) A signal-arrest-release sequence mediates export and control of the phage P1 endolysin, Proc. Natl Acad Sci USA 101:6415–6420 Young I, Wang I, Roof WD (2000) Phages will out: strategies of host cell lysis. Trends Microbiol 8(3):120–128 Young R (2013) Phage lysis: Do we have the whole story yet? Curr Opin Microbiol 16:790–797 Zhang YJ, Chen G, Lin H et al (2017) Development of a regulatable expression system for the functional study of Vibrio vulnificus essential genes. Antonie Van Leeuwenhoek 110(4):607–614 Zhang YJ, Lin H, Wang P et al (2018) Subcellular Localization of lipoproteins of Vibrio vulnificus by the identification of outer membrane vesicles components. Antonie van Leeuwenhoek . 2018, 111(11):1985–1997 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4446997","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":304767245,"identity":"0930a78e-0766-4205-b5ae-c480a624db4e","order_by":0,"name":"Wenqi Wang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenqi","middleName":"","lastName":"Wang","suffix":""},{"id":304767246,"identity":"099d7298-02e7-4ca8-86bb-6c98302d5ded","order_by":1,"name":"Xiansong Meng","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiansong","middleName":"","lastName":"Meng","suffix":""},{"id":304767247,"identity":"abef5da1-b57f-4781-b156-b565e4f96a8c","order_by":2,"name":"Zhonghao Wu","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhonghao","middleName":"","lastName":"Wu","suffix":""},{"id":304767248,"identity":"46dc2a2d-9a37-47a9-a5bb-ecef360d00dc","order_by":3,"name":"Yubin Fu","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yubin","middleName":"","lastName":"Fu","suffix":""},{"id":304767249,"identity":"e160a368-9119-44bd-a77c-7c476471312e","order_by":4,"name":"Xiaoling Li","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Li","suffix":""},{"id":304767250,"identity":"35fddb86-d098-4020-b877-209c98341e87","order_by":5,"name":"Shiyong Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACCcYGMG3AwMD4ACKUQLwWZgMitUBpoHI2CaK0yM9ubpP4uKNW3py991jFm5rDDPzsOQYMP3fg1mJw52Cb5Mwzxw139pxLuznn2GEGyZ43Boy9Z/BokUhsk+ZtO5ZgcCPH7DYP22EGIMOAmbENj8NmwLTcf2NWzPPvMIM9IS0MN8BaaoC28Jgx87YBbZEgoMXgRmKz5cy2A4YbzuQYS87tS+eROPOs4GAvXoelP7zxsa1O3uD4GcMPb75Zy/G3J2988BOfwxgYWIDRcRjC5AEjBoYDeDUAI/0DA0MdXMsoGAWjYBSMAgwAAGfVU6Dn6tR4AAAAAElFTkSuQmCC","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shiyong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-05-20 06:24:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4446997/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4446997/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57498945,"identity":"fe4248d8-856b-4c4d-aa7d-fa430b7866b0","added_by":"auto","created_at":"2024-05-31 13:24:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":636254,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of phage VaPW. Phage plaques (A) in 2216E media and electron micrograph (B) as revealed by transmission electron microscopy.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/8f1cf6dc0a46d78241853dcc.jpeg"},{"id":57498948,"identity":"a549b59b-86e3-48ab-b507-78f6a77c01ed","added_by":"auto","created_at":"2024-05-31 13:24:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":274755,"visible":true,"origin":"","legend":"\u003cp\u003eBiological characteristics of phage VaPW. (A) Thermal stability of phages treated with different temperatures (40, 50, 60, 70 and 80°C). (B) pH stability of phages treated with different pH 2 to 11 for 2 h. (C) Effects of chloroform and ether on phages. The data represent the means and SD (n = 3).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/f1f0a3ac1c37b3608e4f5eac.jpeg"},{"id":57498943,"identity":"f942673e-d849-4941-a972-3e1fa32f4e6a","added_by":"auto","created_at":"2024-05-31 13:24:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11325,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves displaying the antibacterial effect of phage VaPW on \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787 growth at MOIs of 0.01. The negative control was host bacteria with PBS; the positive control included host bacteria and chloramphenicol (CPL). (n = 4, mean ± SD)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/5e04660017911cc069e231ed.png"},{"id":57498944,"identity":"8a8a1baa-eea1-4cf9-90e8-1378f041034c","added_by":"auto","created_at":"2024-05-31 13:24:40","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":976220,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis based on the amino acid sequence of capsid protein (A) and lysozyme (B). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA11. Phage VaPW is highlighted by a box.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/9c712efbca71862a16b5700a.jpeg"},{"id":57499496,"identity":"472322a6-938c-4159-b2c2-7c3de0fa0c91","added_by":"auto","created_at":"2024-05-31 13:32:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e survival test using phage VaPW against \u003cem\u003eV. alginolyticus\u003c/em\u003e infection in shrimp at different multiplication of infection (MOI) of 0, 0.001, 0.01, 1. n = 3.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/f8357493216dc3b9e3560e4b.png"},{"id":57498947,"identity":"722bf642-1c99-42d2-bd77-bb70fd86f53c","added_by":"auto","created_at":"2024-05-31 13:24:41","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":651177,"visible":true,"origin":"","legend":"\u003cp\u003eBactericidal effect of recombinant lysozyme and holin of VaPW on \u003cem\u003eE. coli \u003c/em\u003egrowth. Various plasmids (pET32-hol, pET32-lys, pET32-hoA-lysin) were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21. IPTG was used to induce protein expression, and PBS was used as control. Values, at the OD\u003csub\u003e600\u003c/sub\u003e, are represented by the mean ± standard deviation. A: growth curves of E. coli carrying different plasmids. B: Expression of recombinant lysozyme and holin of VaPW in IPTG-induced \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/6672a3c30bbe3553a9d1b3ad.jpeg"},{"id":78674567,"identity":"4d472945-c754-44f7-8f47-9cac4f893180","added_by":"auto","created_at":"2025-03-17 13:23:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3137980,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/54727b28-2962-4bd0-8b64-c441c2c0de79.pdf"},{"id":57498942,"identity":"662e9edd-a5c1-43ca-9172-ee30f5bfa21e","added_by":"auto","created_at":"2024-05-31 13:24:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":103242,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.docx","url":"https://assets-eu.researchsquare.com/files/rs-4446997/v1/156fa453dbefa6d1e2d88fa9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation of a virulent Vibrio alginolyticus Bacteriophage and Its Application in Shrimp culture","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe breeding technology of \u003cem\u003ePenaeus vannamei\u003c/em\u003e has been mature since its introduction, and the annual output accounted for 80% of the total output of all kinds of shrimp. However, several \u003cem\u003eVibrio spp.\u003c/em\u003e, including \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, are considered to be the most prevalent bacterial pathogens causing mortality of fish and invertebrates and considerable economic losses (Selvin and Lipton 2003; Austin et al. 2016; Grimes et al. 2020). Moreover, \u003cem\u003eV. alginolyticus\u003c/em\u003e was reported as a human pathogen causing infections and food poisoning (Schmidt et al. 1979; Hern\u0026aacute;ndez-Robles et al. 2016). The widespread and frequent uses of antibiotics in aquaculture caused the emergence of antibiotic resistance and food safety problems. For example, antibiotics used for treating infectious diseases in aquaculture have resulted in the development of many antibiotic resistant strains of \u003cem\u003eVibrio spp.\u003c/em\u003e (Oh et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Defoirdt et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhage therapy is a promising alternative to antibiotics. The main advantages of phage include its specificity and phage can achieve a dynamic balance with pathogenic bacteria without destroying the environmental balance. As a green and pollution-free new biological control technology, it has great application potential (O'Flaherty et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Phages have been experimentally used to control important pathogens in aquacuture (Vinod et al. 2006; Kim et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Srinivasan et al. 2017). Moreover, phage therapy has been also considered targeting \u003cem\u003eV. alginolyticus\u003c/em\u003e (Kokkari et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Le et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThough phage therapy is ideal for the control of bacterial pathogens in aquaculture, it has not yet been fully exploited. In order to use this strategy safely, a full characterization of phage genomes is required to check the presence of potential genes related to virulence and antibiotic resistance and to confirm phages were obligate lytic. With recent advances in phage genome sequencing, bacteriophage-derived lytic enzymes and proteins are of interest to the scientific community as biocontrol and therapeutic agents against multidrug resistant bacteria (Chang et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The bacterial lysis is achieved by phage-encoded lysozymes or endolysins that degrade the peptidoglycan (PG) layer present in the bacterial cell wall. The canonical lysis process is that lysozymes act on PG layer and trigger the lysis process with the help of a second phage-encoded membrane protein called holin, in a timely-controlled fashion (Young et al. 2013). Holins are phage-encoded proteins that control access of phage-encoded endolysins to the peptidoglycan through pores in the membrane. Several lysozymes/endolysins coded by \u003cem\u003eV. alginolyticus\u003c/em\u003e had been reported, although the bactericidal activity of some genes has not been experimentally verified (Matamp et al. 2019; Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). With more and more \u003cem\u003eVibrio\u003c/em\u003e phages being discovered, their genetic information will help to provide more comprehensive information for phage therapy.\u003c/p\u003e \u003cp\u003eThis study reports that a novel lytic phage VaPW was isolated from seawater and identified as members of the \u003cem\u003eMyoviridae\u003c/em\u003e family by transmission electron microscopy (TEM). The characteristics and complete genome of VaPW were determined. Antibacterial ability of phage VaPW was verified \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Moreover, two putative genes, holin and lysozyme, were cloned and verified for their bactericidal effects. These results suggest that phage VaPW is a potential candidate to control diseases caused by \u003cem\u003eV. alginolyticus\u003c/em\u003e in aquaculture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e2.1 Bacteriophage isolation\u003c/p\u003e \u003cp\u003eTo isolate bacteriophages, 50 mL sea water sample and 1\u0026ndash;2 mL of overnight \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787 culture were added to 50 mL of double concentrated LB broth, shaking at 200 rpm overnight at 30\u0026deg;C. The broth mixture was then centrifuged at 10,000 \u0026times;g for 10 min and the supernatant was filtered through a 0.22 \u0026micro;m filter. The bacteriophage supernatant was diluted with a 10-fold gradient using SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH 7.5, 0.01% (w/v) gelatin). 100 microliters of each dilution were mixed with 1 mL of \u003cem\u003eV. alginolyticus\u003c/em\u003e solution, and stood at 30 ℃ for 20 minutes. Then 9 mL of warm semi-solid top agar (culture medium with 0.6% agar) were added into the solution and immediately mix to prepare a double-layer plate. The double-layer agar plate was cultured at 30\u0026deg;C for 12 h, and then phage plaques were visualized. The isolation on double-layer agar plates was repeated at least three times to purify the phages.\u003c/p\u003e \u003cp\u003e2.2 Bacteriophage characterization\u003c/p\u003e \u003cp\u003eThe bacteriophages were morphologically characterized by electron microscopy. Purified phage (\u0026gt;\u0026thinsp;10\u003csup\u003e10\u003c/sup\u003e PFU/ml) were deposited onto carbon-coated copper grids and allowed to adsorb for 5 min. The grids were allowed to dry, stained with 2% phosphotungstic acid (pH 7.0) for 5 min, and washed with PBS. The morphology was examined under a Hitachi TEM System HC-1 at 80 kV.\u003c/p\u003e \u003cp\u003eThe host range of phage was also determined by the double-layer agar plate method as mentioned above. Specifically, 1 mL of each freshly grown potential host cultures (10\u003csup\u003e7\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e cfu/ml) was mixed with 100 \u0026micro;L of phage and 3 ml of warm top agar, then poured on bottom agar plates. After incubation, plaque formation on the plates was observed to determine the sensitivity of the host bacteria to phage.\u003c/p\u003e \u003cp\u003eThermal stability was assessed by exposing aliquots of the phage at various temperatures (40, 50, 60, 70 and 80\u0026deg;C) for 30 minutes or 1 h. After incubation, samples were diluted with SM buffer, and titers were evaluated by the double-layer agar method in triplicates.\u003c/p\u003e \u003cp\u003eFor the determination of the stability of the phages at different pH values, phage lysates were added to SM buffer at different pH levels (pH 3\u0026ndash;11), which was adjusted by using HCl or NaOH. After incubation of the mixtures at 30\u0026deg;C for 1 h, the titers were evaluated by the double-layer method.\u003c/p\u003e \u003cp\u003eTo determine the optimal multiplicity of infection (MOI), different titers of phage were co-cultured with \u003cem\u003eV. alginolytics\u003c/em\u003e at MOI ranging from 0.001 to 100 for 10 h at 30 ℃ with shaking. The titer of the phage was then determined by the double-layer agar method. The optimal MOI was the MOI of the phage sample with the highest titer.\u003c/p\u003e \u003cp\u003e2.3 Genome sequencing and bioinformatic analysis\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from the cell pellets with a Bacteria DNA Kit (OMEGA) according to the manufacturer\u0026rsquo;s instructions, and high qualified DNA sample (OD260/280\u0026thinsp;=\u0026thinsp;1.8\u0026thinsp;~\u0026thinsp;2.0, \u0026gt;6ug) is utilized to construct fragment library. Library construction and Illumina HiSeq sequencing were performed by Biozeron Biotechnology Co., Ltd. (Shanghai, China.). Paired-end libraries with insert sizes of ~\u0026thinsp;400bp were prepared following Illumina\u0026rsquo;s standard genomic DNA library preparation procedure. The qualified Illumina pair-end library would be used for Illumina NovaSeq 6000 sequencing.\u003c/p\u003e \u003cp\u003eABySS (Jackman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was used to do genome assembly with multiple-Kmer parameters and got the optimal results of the assembly. The complete genome of phage was sequenced and assembled by Biozeron Biotechnology Company. Gene models were identified using GeneMark (Besemer et al. 2005). Then all gene models were blastp against non-redundant (NR in NCBI) database, SwissProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://uniprot.org\u003c/span\u003e\u003cspan address=\"http://uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and COG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/COG\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/COG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to do functional annotation. In addition, tRNA were identified using the tRNAscan-SE (v1.23, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lowelab.ucsc.edu/tRNAscan-SE\u003c/span\u003e\u003cspan address=\"http://lowelab.ucsc.edu/tRNAscan-SE\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and rRNA were determined using the RNAmmer (v1.2, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/RNAmmer/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/RNAmmer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhylogenetic and molecular evolutionary analyses were conducted using MEGA version 11 (Tamura et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Phylogenetic trees were constructed in MEGA using the neighbor-joining algorithm.\u003c/p\u003e \u003cp\u003e2.4 Antibacterial effect \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787 was cultured to OD\u003csub\u003e600\u003c/sub\u003e 0.1, and then phage (4\u0026times;10\u003csup\u003e7\u003c/sup\u003e PFU) was added to the culture to achieve the optimal MOI (0.01). Host bacteria with chloramphenicol (finally 20 \u0026micro;g/mL) were used as positive controls, and host bacteria with PBS served as negative controls. The mixtures were shaken for 12 h at 30\u0026deg;C, and the optical density at 600 nm was monitored every 1 h. Each treatment was performed in triplicate.\u003c/p\u003e \u003cp\u003e2.5 Therapeutic efficacy of VaPW against \u003cem\u003eV. alginolyticus\u003c/em\u003e infection in shrimp\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003ePhage therapy was performed using 10 randomly selected shrimp in a PE water tank with seawater for each experimental group. the \u003cem\u003eV. alginolyticus\u003c/em\u003e was added into tank to a final concentration of 1.59 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, with a total volume of 3 L. And then bacteriophage was added with MOI of 1, 0.01, and 0.0001, respectively. Three independent experiments were performed for each condition. A group adding PBS of the same volume was used as the negative control group. A control group containing only bacteriophages (~\u0026thinsp;1.59 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was set up to test whether VaPW itself has toxic effects on shrimp health. Any dead shrimp was observed and recorded every 12 hours for a total of 7 days, and a survival curve was drawn.\u003c/p\u003e \u003cp\u003e2.6 Bactericidal activity of recombinant lysozyme and holin of VaPW\u003c/p\u003e \u003cp\u003eThe complete holin (ORF 26) gene, lysozame (ORF 27) gene, and holin-lysozyme (ORF 26\u0026ndash;27) fragment were amplified using PrimSTAR Max DNA Polymerase (Takara, China). Plasmids and primers used in this study are listed in Table S1 \u0026amp; S2. The vector fragment was amplified using pSCT32 vector (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) as template and then recombinated with the amplified lysozame, holin, and lysozame-holin gene fragments, respectively, using One Step Cloning Kit (Vazyme, China) according to the manufacturer\u0026rsquo;s instructions. The reaction mixtures were transformed into competent \u003cem\u003eE. coli\u003c/em\u003e DH5α cells. The resulting pSCT-lysozyme, pSCT-holin, and pSCT-holin-lysozame plasmids were transferred into \u003cem\u003eE. coli\u003c/em\u003e BL21 cells for expression.\u003c/p\u003e \u003cp\u003eThe BL21 cells containing the above-mentioned plasmids were cultured in LB medium with 50 \u0026micro;g/mL kanamycin until the OD\u003csub\u003e600\u003c/sub\u003e nm reached at about 1.0. The cultures were then supplemented with IPTG with the final concentration of 0.1 mM, and incubated for 8 hours at 30\u0026deg;C. The OD values of cultures were monitored and the expression of recombination proteins, which were tagged with a hexahistidine epitope tag, was analyzed by previous published SDS-PAGE and Western blotting methods (Zhang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2.7 Statistical analysis\u003c/p\u003e \u003cp\u003eThe statistical study of the obtained results was performed with GraphPad Prism 7.0. Results are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard deviations (SD). One-way analysis of variance (ANOVA) was used to determine the statistical significance. Significant differences were considered present at *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\n\u003cp\u003e2.8 Data availability\u003c/p\u003e\n\u003cp\u003eThe sequence data for the phage VaPW were deposited at GenBank under accession no. 2649232.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e3.1 Isolation of \u003cem\u003eV. alginolytics\u003c/em\u003e phage VaPW\u003c/p\u003e\n\u003cp\u003ePhage VAPW was isolated from a seawater sample of a shrimp farm in Qingdao, Shandong, China. Phage VaPW produced small plaques plaques (1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mm in diameter) on the lawn of host \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Phage morphology and size were determined by transmission electron microscopy (TEM) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Phage VaPW has a regular hexagon head with a diameter of 90 nm, and about 310 nm long tail with has a contractile tail sheath and a central tube. The isolate was assigned to the \u003cem\u003eMyoviridae\u003c/em\u003e family, order \u003cem\u003eCaudovirales\u003c/em\u003e, based on morphology characteristics of the classification system of International Committee on Taxonomy of Viruses (ICTV).\u003c/p\u003e\n\u003cp\u003eThe host range of phage VaPW was determined by using 11 clinical or environmental strains. No sensitivity was observed in the other test genera or species shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, suggesting that phage VaPW had a narrow host range with specificity.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eLysis profile of phage\u003c/strong\u003e VaPW\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVaPW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference or source\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eV. parahaemolyticus\u003c/em\u003e ATCC17802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC33787\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e YX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eV. alginolyticus\u003c/em\u003e ZJTCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChen, 2012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eVibrio anguillarum\u003c/em\u003e M3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMo, 2010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eVibrio vulnificus\u003c/em\u003e ATCC27562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEdwardsiella tarda\u003c/em\u003e EtSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMo, 2013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eVibrio harveyi\u003c/em\u003e ATCC 14126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eVibrio ichthyoenteri\u003c/em\u003e F1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAeromonas hydrophila\u003c/em\u003e ATCC7966\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAeromonas salmonicida C4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKuang, 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e3.2 Biological characteristics of phage\u003c/p\u003e\n\u003cp\u003ePhage VaPW was propagated under various environments to evaluate stability. Survival of phage VaPW was stable during heat treatment from 40\u0026deg;C to 50\u0026deg;C lasting 30 min. The phage titer was reduced by approximately 40% at 60\u0026deg;C, and almost complete inactivation of the bacteriophage occurred at temperature ranges above 70\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, the pH stability test showed that phage VaPW remains active at a pH range from 4 to 11 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, the phage was inactivated at pH 3. These findings show that phage VaPW is stable under wide ranges of temperatures and pHs.\u003c/p\u003e\n\u003cp\u003eThe effects of organic solvents (chloroform, diethyl ether), on the survival of the phage was determined. There was no significant difference in the titer between the untreated phages and the solvents-treated phages (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), which indicates that phage VaPW was not affected by chloroform/diethyl ether treatment and it does not contain lipids.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eV. alginolyticus\u003c/em\u003e strain, when infected by phage VaPW at a MOI of 0.01, generated the maximum phage titer of approximately 8.55 x 10\u003csup\u003e9\u003c/sup\u003e PFU/mL (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that 0.01 was the optimal MOI for VaPW.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eMOI of phage\u003c/strong\u003e VaPW\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMOI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhage titer PFU\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBacteria number CFU\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhage titer PFU/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.00\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.00\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.57\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.50\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.55\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.13\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe effect of the phage on \u003cem\u003eV. alginolytics\u003c/em\u003e ATCC 33787 \u003cem\u003ein vitro\u003c/em\u003e was shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Growth curves of \u003cem\u003eV. alginolytics\u003c/em\u003e infected with phage VaPW at optimal MOI were measured by monitoring absorbance to evaluate the \u003cem\u003ein vitro\u003c/em\u003e antibacterial efficacy. The negative control received PBS and positive control received chloramphenicol (CPL). Compared to the control group, the optical density of \u003cem\u003eV. alginolyticus\u003c/em\u003e treated with phage started to show decreases after 2h infection, and the OD\u003csub\u003e600\u003c/sub\u003e decreased further and was significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the control group. This result indicates that phage VaPW could effectively inhibits the growth of \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787, suggesting that it could be a candidate biocontrol agent for phage therapy against \u003cem\u003eV. alginolyticus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e3.4 Genomic analysis of phage VaPW\u003c/p\u003e\n\u003cp\u003eThe genome of VaPW was sequenced and submitted to GenBank under the accession number 2649232. The complete genome of phage VaPW is 34,637 bp in length, with an average GC content of 43.22%. Forty-one putative open reading frames (ORFs) comprising 90.7% of the genome were subsequently predicted and 37 of them start with an ATG codon, except for gene 2, which start with TTG and gene 16、gene 27 and gene29, which starts with GTG. According to BLAST search, 38 of the 41 ORFs were identified to share similarities with previously reported genes in the database, of which 26 ORFs showed homology to functionally characterized genes. The classifications of the 26 coding sequences with functional annotations are being listed (Table S3), which includes 11 structural and packing protein genes, 10 DNA replication/regulation genes, and 5 other protein genes. No tRNA-encoding genes and no toxin genes were identified.\u003c/p\u003e\n\u003cp\u003eThe genome sequence comparison using the Blastn method revealed that the complete genome of this phage had a nucleotide similarity score of 59.13% with the known bacteriophage \u003cem\u003eVibrio\u003c/em\u003e phage Vp1B1MD-2 (NCBI Reference Sequence NC_055862.1). As a result, we concluded that VaPW is a new phage. To investigate the phylogeny of phage VaPW, two unrooted phylogenetic trees were constructed with amino acid sequences of ORF07 and ORF26, which are predicted to encode the capsid protein and lysozyme, respectively. Interestingly, we consistently found that phage VaPW seems to be most closely related to the \u003cem\u003eVibrio\u003c/em\u003e phage Vp1B1MD-2 on the phylogenetic trees (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), which is classified as belonging to the \u003cem\u003eMyoviridae\u003c/em\u003e family.\u003c/p\u003e\n\u003cp\u003e3.5 Therapeutic efficacy of phage VaPW against \u003cem\u003eV. alginolyticus\u003c/em\u003e infection in shrimp\u003c/p\u003e\n\u003cp\u003eTo estimate the \u003cem\u003ein vivo\u003c/em\u003e protection effect of phage VaPW against \u003cem\u003eV. alginolyticus\u003c/em\u003e infection, a survival test was performed with shrimp. The survival rates of \u003cem\u003eV. alginolyticus-\u003c/em\u003einfected shrimp in groups treated with phage at different MOIs were recorded for 7 days. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the survival rate increased in a dose-dependent manner when \u003cem\u003eV. alginolyticus\u003c/em\u003e-infected shrimp received phage treatment. The phage- groups at MOIs of 0.0001, which is much lower than the optimum MOI of 0.01, also showed a slight improvement (55%) in the survival rate. The phage-treated groups at MOIs of 0.01 and 1 showed much higher survival rates (65% and 70%, respectively). There was no die in control group without \u003cem\u003eV. alginolyticus\u003c/em\u003e infection, indicating that the phage did not affect shrimp health. This result suggested that phage VaPW may be a candidate as an effective biocontrol agent for phage therapy against \u003cem\u003eV. alginolyticus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e3.5 Bactericidal activity of recombinant lysozyme and holin of VaPW\u003c/p\u003e\n\u003cp\u003eBacteriophage proteins have been applied to the prevention and control of food-borne pathogens recently. In phage VaPW, BLASTX showed that the hypothetical product of \u003cem\u003eorf27\u003c/em\u003e has identity values (41\u0026ndash; 99%) with a series of lysozymes from a wide variety of sources. \u003cem\u003eOrf26\u003c/em\u003e is located upstream of \u003cem\u003eorf27\u003c/em\u003e and also encodes a putative phage holin. A conserved phage_holin superfamily domain (Fig. S3) and a conserved bacteriophage Lyz-like superfamily domain (Fig. S4) were found in Orf27 and Orf26 protein, respectively.\u003c/p\u003e\n\u003cp\u003eTo determine whether the lysozyme and holin genes from phage VaPW have bactericidal activity, \u003cem\u003eOrf26\u003c/em\u003e and \u003cem\u003eorf27\u003c/em\u003e were cloned for protein expression and analyses. The \u003cem\u003eE. coli\u003c/em\u003e carrying the plasmids pSCT32-hol or pSCT32-lys were cultured to OD\u003csub\u003e600\u003c/sub\u003e 1.0, and then induced by IPTG. Growth curves showed that growth of \u003cem\u003eE. coli\u003c/em\u003e expressing recombinant holin was slightly inhibited when comparing to the control groups within 3 h (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). On the other hand, the expression of lysozyme exhibited significant inhibition on growth of \u003cem\u003eE.coli\u003c/em\u003e within 2 h based on the growth curves. The WB experimental data showed that holin was stably expressed in cells after induction, while the expression level of lysozyme continued to decrease over time (Fig. 7B). These results indicated that the lysozyme from phage VaPW have better bactericidal ability than holin on growth inhibition of \u003cem\u003eE. coli\u003c/em\u003e. The growth of pSCT32-lys \u003cem\u003eE. coli\u003c/em\u003e and was gradually recovered to OD\u003csub\u003e600\u003c/sub\u003e 1.0 after 5 h, indicating that the effect on growth inhibition of the lysozyme is valid (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eTo verify a synergistic effect on the lysis of target cells, the holin and lyzosyme were cloned together from the phage VaPW genome and co-expressed in \u003cem\u003eE. coli\u003c/em\u003e, and then bactericidal activity was determined. The data revealed that co-existence of holine and lyzosyme in \u003cem\u003eE. coli\u003c/em\u003e pSCT3-hol-lys exhibited the stronger bactericidal activity than single protein alone (\u003cem\u003eE. coli\u003c/em\u003e pSCT3-hol or \u003cem\u003eE. coli\u003c/em\u003e pSCT3-lys), as showed in the growth curve (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNowadays, phage therapy has been suggested as an alternative to antibiotic administration in both human and veterinary medicine due to the developing problem of antibiotic resistance (O'Flaherty et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kortright et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hatfull et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In aquaculture, the use of bacteriophages and their derived enzymes to combat resistant bacterial infections can help to control bacterial populations in delicate environments, whereas antibiotic administration would alter the natural microflora of the environment and the intestinal microflora of cultured organisms (Nachimuthu, et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, a novel lytic phage VaPW that targets \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787 was isolated and characterized. The results presented here showed that phage VaPW is efficient in reducing the growth of the host bacteria during \u003cem\u003ein vitro\u003c/em\u003e testing at a low MOI (0.01). Moreover, phage VaPW stability test under various temperature and pH conditions showed that phage is extremely stable remaining active at 50\u0026deg;C and under a wide range of pHs, which suggested that its antimicrobial activity could be retained during storage and production operations. Phage VaPW has a very narrow lytic rang being able to infect only its host \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787. However, we cannot rule out its effect on other sensitive \u003cem\u003eV. alginolyticus\u003c/em\u003e strains. Further research would include collecting more clinic, environmental isolates and other standard strains of \u003cem\u003eV. alginolyticus\u003c/em\u003e and other \u003cem\u003eVibrio spp.\u003c/em\u003e to test the infectivity of VaPW.\u003c/p\u003e \u003cp\u003eIn addition to the \u003cem\u003ein vitro\u003c/em\u003e antibacterial test of phage VaPW against \u003cem\u003eV. alginolyticus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), an \u003cem\u003ein vivo\u003c/em\u003e shrimp survival test with \u003cem\u003eV. alginolyticus\u003c/em\u003e infected shrimp revealed that phage VaPW protected shrimp from the virulence of \u003cem\u003eV. alginolyticus\u003c/em\u003e. In this study, bacteriophages were directly applied to the culture pond before shrimp were infected with \u003cem\u003eV. alginolyticus\u003c/em\u003e. The aim of the operation is to reduce the \u003cem\u003eVibrio\u003c/em\u003e load in the culture environment, thereby reducing the risk of bacterial disease outbreaks. In the shrimp protection experiment, we used a higher concentration of bacteria (final 1.59 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL) to observe the biocontrol effect in a short time. When the phage MOI (0.0001) is 100 times lower than the optimal MOI (0.01), phage VaPW can still effectively improve the survival rate of shrimp after infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating that it has a certain disease prevention effect evern if the phage concentration is low. When the MOI was 0.01, its antibacterial effect was similar to that of MOI\u0026thinsp;=\u0026thinsp;1 group, indicating that the phage (MOI above 0.01) could prevent the infection of \u003cem\u003eV. alginolyticus\u003c/em\u003e in \u003cem\u003eP. vannamei\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAfter thoroughly understanding the genetic information of bacteriophages, it thus becomes possible to engineer phages with enhanced therapeutic properties, safety features and host-range (Strathdee et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The genome of VaPW was sequenced and analyzed to gain a further understanding of this phage. Its genome is 34,637 bp, similar to other \u003cem\u003eVibrio\u003c/em\u003e phages, such as \u003cem\u003eVibrio\u003c/em\u003e phage Vp1B1MD-2 with a maximal nucleotide similarity score of 59.13%. Since RNA polymerase was not annotated in phage VaPW genome, three representative phage conserved proteins, capsid protein, lysozyme and endonuclease, were used for phylogenetic tree analysis. The phylogenetic analysis revealed that phage VaPW clustered closely with \u003cem\u003eVibrio\u003c/em\u003e phage Vp1B1MD-2 on the phylogenetic tree based on the capsid protein and lysozyme analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, additional endonuclease sequence analysis revealed that \u003cem\u003eorf6\u003c/em\u003e of phages VaPW seems to be most closely related to a HNH nuclease of a \u003cem\u003eMyoviridae sp.\u003c/em\u003e phage (GenBank: DAN88581.1), but not nuclease of phage Vp1B1MD-2 (Fig. S1). Phage Vp1B1MD-2 also belongs to the \u003cem\u003eMyoviridae\u003c/em\u003e phage family. These results showed that there are a wide variety of phages with strong diversity. More phage genomes are needed to fill the gap in the database. Currently, VaPW was confirmed to be a novel phage belonging to the \u003cem\u003eMyoviridae\u003c/em\u003e phage family, which was consistent with the results of TEM observation. The genomic analysis did not reveal the presence of virulence or antibiotic resistance genes in VaPW, implying that VaPW might be suitable for phage therapy. A phage used as a potential biocontrol agent should preferably be a virulent phage with no side effects on marine animal health. However, as more than one-third of the predicted ORFs (15 of the 41 ORFs) in VaPW are hypothetical proteins with uncharacterized function, it is not known whether this phage carries potentially hazardous genes. Fortunately, the safety of VaPW has been evaluated as non-toxic to shrimp when tested in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe main mechanism of bacteriophage lysis of the host has been thoroughly studied, mainly through their encoded lytic proteins (lysozyme and horin) synthesized in the host (Young et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). It is generally believed that (an oligomeric membrane lesion is formed firstly by the holin, allowing escape of endolysin. Then, lysozyme penetrate the pores to hydrolyze peptidoglycans and lyse the host. Holins control the length of the infective cycle for lytic phages and achieve lysis at an optimal time. Besides, lytic single-stranded RNA and DNA phages accomplish lysis by producing a single lysis protein without muralytic activity (Young et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Phage and their lysins have already been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections (O'Flaherty S et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this study, genome sequence analysis of phage VaPW revealed that it has an holin-encoding gene (\u003cem\u003eorf 26\u003c/em\u003e) and endolysin-encoding gene (\u003cem\u003eorf 27\u003c/em\u003e) (Table S2). Two genes were cloned and induced for protein expression in \u003cem\u003eE. coli\u003c/em\u003e. The experimental results showed that the expression of either protein could inhibit the growth of \u003cem\u003eE. coli\u003c/em\u003e, though lysozyme (Orf 27) had a stronger antibacterial effect. These data suggested that holin might not be essential for the action of endolysin in the phage VaPW. Some researches also demonstrated that the bactericidal activity of endolysins of phage are not dependent on holins in the lysis system for export (Xu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After 5 hours of induction, the inhibited \u003cem\u003eE. coli\u003c/em\u003e by lysozyme expression began to slowly recover and grew, which might probably be because that the host cell detected the toxicity of overexpressed lysozmy and the host defense mechanism reduced the expression level of the recombinant protein. Interestingly, while two lytic proteins were expressed simultaneously, a highly significant host cell lysis phenomenon was observed, which indicating a synergistic effect between two proteins. Hence, we speculate that in phage VaPW lysis system, holin helps to some extent improve the efficiency of lysozyme to break through the cell membrane and degrade the cell wall. A similar example was also reported in another \u003cem\u003eV. alginolyticus\u003c/em\u003e phage HH109 (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although the synergetic effect needs to be further elucidated in the other bacterial species, our results demonstrated that the holin and lysozyme from phage VaPW could also be used as new potential biocontrol agents against \u003cem\u003eV. alginolyticus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTaken together, we isolated a novel \u003cem\u003eV. alginolyticus\u003c/em\u003e phage VaPW from seawater sample and identified its biological characteristics. Its antibacterial effect was evaluated by the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e shrimp infection test. The complete genome was sequenced and analyzed. Two lytic protein genes, holin and lysozyme, were cloned and verified their bactericidal activity in \u003cem\u003eE.coli\u003c/em\u003e. In conclusion, our work demonstrated the phage VaPW and its proteins could be used as new type of biocontrol agents to be exploited against \u003cem\u003eV. alginolyticus\u003c/em\u003e in aquaculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSC and WW conceived the experiments, WW, XM, ZW,YF and XL conducted the experiments, WW, XM and ZW analyzed the results. All authors assisted in writing the manuscript, discussed the results and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the Natural Science Foundation of Shandong Province (ZR2020MC197), the National Natural Science Foundation of China (32273183 \u0026amp; 31372564), Technical system of shrimp and crab industry in Shandong Province (SDAIT-11), \u0026ldquo;First class fishery discipline\u0026rdquo; program in Shandong Province, and a special talent program \u0026ldquo;One Thing One Decision (Yishi Yiyi)\u0026rdquo; Program in Shandong Province, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAustinB, Austin DA (2016) Bacterial Fish Pathogens, 6th ed.; Springer: Chichester, UK, ISBN 978-3-319-32673-3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBesemer J, Borodovsky M (2005) GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. 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Antonie Van Leeuwenhoek 110(4):607\u0026ndash;614\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YJ, Lin H, Wang P et al (2018) Subcellular Localization of lipoproteins of \u003cem\u003eVibrio vulnificus\u003c/em\u003e by the identification of outer membrane vesicles components. \u003cem\u003eAntonie van Leeuwenhoek\u003c/em\u003e. 2018, 111(11):1985\u0026ndash;1997\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacteriophage, Isolation, Shrimp culture, growth inhibition, Vibrio alginolyticus","lastPublishedDoi":"10.21203/rs.3.rs-4446997/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4446997/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs an opportunist pathogen, \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, causes disease outbreaks in marine farmed fish and invertebrates. Due to problems caused by the abuse of antibiotics, it is extremely important to develop green biocontrol methods for \u003cem\u003eVibrio\u003c/em\u003e diseases. Phage therapy is considered a safe and promising prevention and treatment method. Here, we report that a novel virulent bacteriophage VaPW, which infects \u003cem\u003eV. alginolyticus\u003c/em\u003e, was isolated from seawater. The bacteriophage is morphologically similar to phages from \u003cem\u003eMyoviridae\u003c/em\u003e family. It displayed good pH (7\u0026ndash;9) and temperature (\u0026le;\u0026thinsp;50\u0026deg;C) tolerance and had a narrow host range. Its genome consists of 34,637 bp with a GC content of 43.22%, while sequence analysis revealed the presence of 41 potential ORFs, of which 26 coding sequences showed homology to functionally characterized genes. The \u003cem\u003ein vitro\u003c/em\u003e antibacterial experiment showed that phage VaPW could effectively inhibit the growth of \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 33787. Moreover, the shrimp survival test demonstrated that phage VaPW has the \u003cem\u003ein vivo\u003c/em\u003e protection effect against \u003cem\u003eV. alginolyticus\u003c/em\u003e infection. To determine whether the putative lysozyme and holin from phage VaPW have bactericidal activity, \u003cem\u003eorf26\u003c/em\u003e and \u003cem\u003eorf27\u003c/em\u003e were cloned for protein expression and analyses in \u003cem\u003eE. coli\u003c/em\u003e. The data revealed that co-existence of holin and lysozyme exhibited a synergistic bactericidal effect on the lysis of target cells compared with single protein alone. These results suggest that phage VaPW and its proteins may provide good candidates to control recurrent diseases caused by V. \u003cem\u003ealginolyticus\u003c/em\u003e in marine animals.\u003c/p\u003e","manuscriptTitle":"Isolation of a virulent Vibrio alginolyticus Bacteriophage and Its Application in Shrimp culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-31 13:24:36","doi":"10.21203/rs.3.rs-4446997/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2f132207-b7a1-4a56-a1b0-4d0a81cd5305","owner":[],"postedDate":"May 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T13:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-31 13:24:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4446997","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4446997","identity":"rs-4446997","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}