A Novel Vibriophage Vb Vcas HC Containing Lysogeny-Related Gene Has Strong Lytic Ability Against Pathogenic Bacteria

Virologica Sinica www.virosin.org https://doi.org/10.1007/s12250-020-00271-w www.springer.com/12250 (0123456789().,-volV)(0123456789().,-volV) RESEARCH ARTICLE A Novel Vibriophage vB_VcaS_HC Containing Lysogeny-Related Gene Has Strong Lytic Ability against Pathogenic Bacteria 1,2 1,2 1,2 1 3 3 Chengcheng Li • Zengmeng Wang • Jiulong Zhao • Long Wang • Guosi Xie • Jie Huang • Yongyu Zhang1,2 Received: 2 March 2020 / Accepted: 8 June 2020 Ó Wuhan Institute of Virology, CAS 2020 Abstract To avoid the negative effects of antibiotics, using phage to prevent animal disease becomes a promising method in aquaculture. Here, a lytic phage provisionally named vB_VcaS_HC that can infect the pathogen (i.e., Vibrio campbellii 18) of prawn was isolated. The phage has an isometric head and a non-contractile tail. During phage infection, the induced host mortality in 5.5 h reached ca. 96%, with a latent period of 1.5 h and a burst size of 172 PFU/cell. It has an 81,566 bp circular dsDNA genome containing 121 open reading frames (ORFs), and ca. 71% of the ORFs are functionally unknown. Comparative genomic and phylogenetic analysis revealed that it is a novel phage belonging to Delepquintavirus, Siphoviridae, Caudovirales. In the phage genome, besides the ordinary genes related to structure assembly and DNA metabolism, there are 10 auxiliary metabolic genes. For the first time, the pyruvate phosphate dikinase (PPDK) gene was found in phages whose product is a key rate-limiting enzyme involving Embden-Meyerhof-Parnas (EMP) reaction. Interestingly, although the phage has a strong bactericidal activity and contains a potential lysogeny related gene, i.e., the recombinase (RecA) gene, we did not find the phage turned into a lysogenic state. Meanwhile, the phage genome does not contain any bacterial virulence gene or antimicrobial resistance gene. This study represents the first comprehensive characterization of a lytic V. campbellii phage and indicates that it is a promising candidate for the treatment of V. campbellii infections. Keywords Vibrio campbellii Á Phage Á Lysogeny Á RecA Á Auxiliary metabolic gene Introduction example, the emergence of a large number of drug-resistant bacteria has made the diseases of aquaculture animal more In recent decades, the prevention and treatment of bacterial difficult to be controlled. There is an urgent need to find diseases have relied heavily on antibiotics. However, the alternatives to antibiotics (Defoirdt et al. 2011). The viru- negative effects of antibiotics are gradually emerging. For ses that infect bacteria, i.e., phages, are the most abundant microorganisms in marine environments (Suttle 2005; Liang et al. 2019) and one of the most important factors Electronic supplementary material The online version of this article leading to bacterial death (Wang et al. 2019). In recent (https://doi.org/10.1007/s12250-020-00271-w) contains supplementary material, which is available to authorized users. years, the use of phage to remove pathogenic bacteria in aquaculture has been proven to be a very promising disease & Yongyu Zhang prevention strategy (Hoshiba et al. 2010; Jun et al. 2014; [email protected] Nishikawa et al. 2008). 1 Key Laboratory of Biofuels, Shandong Provincial Key Vibrios are ubiquitous in the marine and estuarine Laboratory of Energy Genetics, Qingdao Institute of environments, accounting for more than 10% of the total Bioenergy and Bioprocess Technology, Chinese Academy of aquatic culturable bacteria (Lin et al. 2018;Liet al. 2019; Sciences, Qingdao 266101, China Yang et al. 2019). Many members of vibrios are patho- 2 University of Chinese Academy of Sciences, Beijing 100049, genic bacteria that can infect aquatic animals and induce China foodborne diseases (Drake et al. 2007). Among them, 3 Yellow Sea Fisheries Research Institute, Chinese Academy Vibrio campbellii is an opportunistic pathogen widely of Fishery Sciences, Qingdao 266071, China 123 Virologica Sinica distributed in aquaculture seawater. It can cause serious overnight incubation at 28 °C, single plaque was picked diseases of prawns, finfish, and mollusks, such as bacterial out and purified at least five times by the double-layer agar septicemia, luminescent vibriosis or red-leg disease, etc. method. (Defoirdt et al. 2007) and lead to huge losses in marine aquaculture (Halet et al. 2007). Observation of Phage Morphology To our knowledge, only seven lytic phages infecting by Transmission Electron Microscope V. campbellii have been isolated so far, but there is no comprehensive identification in both physiological and Phage particles were prepared according to the conven- genetic levels. Among them, the genomes of five lytic tional negative staining method (Liang et al. 2016). Briefly, phages OPB45, OPB48, OPB54, OPB58 and OPB66 iso- phage stock suspension was placed on copper grids for lated from cockle samples are not sequenced (Sangseedum about 30 min and then stained with 2% uranyl acetate for et al. 2017), and the physiological characteristics of the 3 min. The negatively stained phage was examined by other two phages VHS1 and SIO-2 are unknown (Baudoux H-7650 transmission electron microscope at an accelerat- et al. 2012; Khemayan et al. 2012). In addition, two tem- ing voltage of 80 kV. perate phages that can infect V. campbellii have been isolated, but they are not suitable for the treatment of Detection of Phage Lipids pathogens in aquaculture (Lorenz et al. 2016). Recently, a pathogenic bacterium V. campbellii 18 was To investigate the presence of lipid in vB_VcaS_HC, the isolated from diseased Kuruma prawn. Meanwhile, a lytic phages were incubated with 0%, 2%, or 20% (v/v) chlo- phage, which is capable of infecting V. campbellii 18, was roform with vigorously shaking for 1 min and then held at also obtained. In order to evaluate the application potential room temperature for 30 min. After centrifugation at of this phage in the treatment of V. campbellii infection in 6000 9 g for 5 min, the phages remaining in the super- aquaculture, its physiological and genomic characteristics, natant were dropped onto V. campbellii 18 plate. Whether potential metabolic gene functions and biological safety the phage capsid contains lipid was judged according to the were analyzed comprehensively. Our study provides new emergence of plaques (Zhang and Jiao 2009). insights into the species diversity of Vibrio phages and their application potential in treating aquaculture One-Step Growth Curve pathogens. For the one-step growth curve, the previously described method was used with minor modifications (Yang et al. Materials and Methods 2017). Briefly, V. campbellii 18 was infected with the phage at a multiplicity of infection (MOI) of 0.01 and then Phage Isolation incubated at 28 °C for 20 min. The unabsorbed phage particles were removed by centrifugation at 6000 9 g for The V. campbellii 18 strain was kindly provided by Yellow 10 min and the pellets were resuspended in 1 mL of RO Sea Fisheries Research Institute, Chinese Academy of medium. This process was repeated twice. Then 50 lLof Fishery Sciences, which is a pathogen isolated from dis- the resuspended culture were transferred into 50 mL of RO eased Kuruma prawn. It was cultured from a glycerol stock medium and incubated at 28 °C with continuous shaking. in RO medium (yeast extract 1 g, tryptone 1 g, sodium Samples were collected at 15 min intervals (up to 5.5 h), acetate 1 g, artificial seawater 1 L, pH 7.8–8.0) at 28 °C then immediately diluted, and plated for phage titration with shaking at 200 rpm for 12 h. The phage named with the double-agar plaque assay. Three replicates of the vB_VcaS_HC was isolated using standard virus enrich- experiments were carried out in this study. The burst size ment and double-layer agar method (Zhang and Jiao 2009). was calculated as the ratio of the final titer of liberated In detail, the samples for phage isolation were collected phages to the initial count of infected bacterial cells. from the surface coastal seawater in Qingdao (36°17042.5600 N, 120°39011.1900 E) and filtered through Bactericidal Activity 0.22 lm sterile microfilter. Then 10 mL filtrate was added to 50 mL V. campbellii 18 culture and incubated at 28 °C In vitro lysis assays were performed as described previ- for 7 days in a shaker. Samples were taken from the mixed ously with some modification (Yuan et al. 2019). In brief, culture at an interval of 24 h. Each sample was filtered with phage suspensions were added to the cultures of V. 0.22 lm sterile microfilter and then mixed with V. camp- campbellii 18 (OD600 = 0.3–0.4) at an MOI of 0.01. Then bellii 18 using the double-layer agar method. After samples were taken from the mixture every 15 min for 5.5 h and serially diluted (tenfold each) with cold RO 123 C. Li et al.: Characterization of a phage against Vibrio campbellii medium. The number of bacteria in each sample was method. Whether the phage can replicate independently quantified with the agar plate method. All assays were can be inferred from the number of plaques on the plate. repeated in triplicates. The bacterial mortality caused by phage infection was calculated by comparing the number Phage DNA Isolation, Genome Sequencing of bacteria in the phage infection group and the control and Analysis group without the addition of phages. The phage DNA was extracted according to the method Host Range Analysis described previously (Zhang and Jiao 2009). Briefly, the CsCl-purified phages were treated with DNase I (2 ng/L) Spot test (Zhang and Jiao 2009) was carried out on 14 and RNase A (2 ng/L) at room temperature for 2 h to Vibrio sp. strains listed in Table 1 to determine the host reduce the host contamination. Then proteinase K range of vB_VcaS_HC.

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