Characterization of a novel Vibrio parahaemolyticus host-phage pair and antibacterial effect against the host

doi:10.21203/rs.3.rs-342426/v1

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Characterization of a novel Vibrio parahaemolyticus host-phage pair and antibacterial effect against the host

https://doi.org/10.21203/rs.3.rs-342426/v1

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Vibrio parahaemolyticus (V.parahaemolyticus) is widely recognized pathogenic Vibrio species duo to numerous outbreaks and its wide occurrence in the marine environment. This study investigates the characteristics of a novel V.parahaemolyticus strain BTXS2 and its associated phage VB_VpP_BT-1011 isolated from the Bohai Coast (Tianjin, China). The strain BTXS2 is a short coryneform bacterium with terminal flagellum, and has an excellent ability to utilize and metabolize organic matter due to its unique carbon source utilization and enzyme activity. The host grows well in media between pH 5.0–9.0 and salinities of simulated freshwater, estuary and seawater (NaCl 0.5%-3%). Genome sequencing of BTSX2 found multiple antibiotic resistance genes and virulence genes that endanger human health. The phage VB_VpP_BT-1011 that infects BTSX2, is a 43841 bp double-stranded DNA virus of Myoviridae family with a latent time of 30 min and burst size of 24 PFU/cell. The phage tolerates a broad range of environmental conditions (salinity 0%-3% NaCl, pH 5.0–9.0, temperature 4^oC-37^oC) as that of the host. The host range tests showed that the phage only infected and inhibited the isolated BTXS2. This study is an investigation of a novel V.parahaemolyticus host–phage pair and the antibacterial effect on V.parahaemolyticus, providing novel insights into marine microbial ecology.

Architecture, Design and Planning

Virology

Marine and Freshwater Ecology

Civil Engineering

Environmental Engineering

Vibrio parahaemolyticus

lytic bacteriophage

antibacterial effect

antibiotic resistance

pathogen

V.parahaemolyticus exists in estuarine waters and marine environments around the world, and V.parahaemolyticus infection in humans causes open wound infections and life-threatening sepsis[1]. V.parahaemolyticus is also common in seafood products such as fish, shrimp and shellfish[2]. Ingestion of seafood contaminated with V.parahaemolyticus was reported to cause vibriosis, a gastrointestinal disease leading to diarrhea, abdominal cramps, vomiting, fever, headache, etc.[3]. Ever since the first outbreak of vibriosis in Japan in 1950[4], outbreaks have been reported all over the world, such as France[5], Spain [6, 7], Sweden and Finland[8].

The global increase of infectious diseases caused by V.parahaemolyticus warns us that the prevention and control of this bacteria is imminent. The current clinical treatment of related diseases is treatment with antibiotic[9]. However, antibiotics increases the production and spread of antibiotic-resistant strains (AMR) and resistance genes, further endangering public health[10, 11]. Antibiotic resistance of V.parahaemolyticus has been reported in the United States, China, Italy and other countries [9, 12-15], covering multiple types of antibiotics such as ampicillin, cefazolin, streptomycin, gentamicin, kanamycin, tetracycline, chloramphenicol, polymyxin and so on[9, 16, 17]. It indicates that better treatment and prevention strategies are urgently needed due to increasing resistance of V.parahaemolyticus and the lack of new antibiotics[18, 19]. Since bacteriophages can recognize and infect host bacterial cells in various environments, and are highly specific and can only proliferate in live bacteria, researchers began to use phages to control V.parahaemolyticus [20, 21]. For example, some new types of V.parahaemolyticus bacteriophages were isolated and found to have antibacterial effects[22, 23]. Li, et al. [24] isolated and sequenced a V.parahaemolyticus phage VPP1 and studied the antibacterial activity of the endolysin derived from VPP1. These studies suggest that we can utilize the phage to control, inhibit and block the infection and the spread of resistant V.parahaemolyticus.

In this study, a new strain of V.parahaemolyticus and its specific bacteriophage were isolated in the coastal water of the Bohai Sea. A series of research on the morphological structure, biological characteristics, genetic information and functions were launched. Co-cultivation of host and specific bacteriolytic phage was implemented to study the antibacterial effect of the bacteriophage on its host, so as to provide a new medium and choice for the future treatment of V.parahaemolyticus with phage instead of antibiotics. This research provides a theoretical basis for the use of bacteriophages to maintain the ecological health of estuaries, to ensure the development of marine economy such as fisheries and aquaculture, and the clinical treatment of V.parahaemolyticus infections.

2.1 Sample collection

Ten-liter surface seawater sample was collected from Bohai Coast in Beitang, Tianjin and transported to the laboratory within 2 h at low temperature in October 2019. Total dissolved solids (TDS,14.01 ppt), pH (8.1), and temperature (20.3^oC) were measured using a handheld pH meter (pH808, SMART SENSOR, China) and a TDS measure instrument (AR8011, SMART SENSOR, China), respectively. The water sample was stored at 4^oC in darkness for no more than 1 h before the experiment.

2.2 Isolation, purification and 16S rRNA gene identification of host bacteria

The isolation method of the strain is modified on the basis of the method by Rodela, et al. [25]. One liter seawater was filtered through a 0.22 µm filter membrane (Millipore), and the bacteria trapped on the filter membrane were resuspended with sterile phosphate buffer saline (PBS). The bacterial resuspension was streaked on the Vibrio chromogenic medium and TCBS plate (Qingdao Hopebio) and cultured at 37^oC for 24 h. The round blue-green colonies were selected and inoculated in 3% NaCl LB broth (30 g/L NaCl, 10 g/L Tryptone, 5 g/L Yeast extract) and cultivated overnight at 37^oC. Streak-plate isolations on TCBS agar were performed and repeated five times by using inoculum from the culture broth that showed growth to ensure strain purity. The monoclonal bacteria were named as BTXS2.

The host bacteria BTXS2 was cultured overnight in 3% NaCl LB at 37^oC. The bacterial cells were collected after centrifuging at 4000 rpm (Eppendorf centrifuge 5804R) for 10 min. The DNA was extracted from cell pellets with TIANamp Bacteria DNA Kit (TIANGEN). Primers 27F: AGTTTGATCMTGGCTCAG, 1492R: GGTTACCTTGTTACGACTT) targeting 16S rDNA genes were used for PCR amplifications. The PCR amplification products were purified using TIANquickMidi Purification Kit (TIANGEN). Samples were sent to the Allwegene Technologies (Beijing, China) for Sanger sequencing. The sequencing results were aligned in the NCBI database.

2.3 Bacteria physiology, biochemistry and antibiotic Resistance

The carbon source utilization of host bacteria was analyzed by Biolog. V.parahaemolyticus BTXS2 was incubated onto BIOLOG GENIII plates (Biolog, Microbiologics) to determine the carbon source utilization profiles and the physiological versatility as previously described in detail by Sabet, et al. [26]. The API ZYM Kit (bioMerieux) was used to determine the enzymatic characteristics. Antibiotic sensitivity determination was carried out with K-B paper dispersion (Beijing Tiantan Pharmaceutical Biotechnology Development Company) as described previously with some modifications [27]. Briefly, the concentration of the bacterial solution was adjusted to 0.5McF (1.5×108 CFU/ml). The bacterial suspension was soaked with a sterile cotton swab and spread evenly on the medium plate at an inclination angle of about 30°. The following 13 types of antibiotic sensitive paper were used in this study (Table 1). The antibiotic sensitive paper was stuck on the coated plate with a distance no less than 25 mm. Then the plate was incubated at 30°C for 18 h to observe the results.

According to the inferred resistance gene sequence from the sequencing results of BTXS2 strain, primers for PCR experiment verification were designed. The primer sequences are shown in Table S1.

2.4 Physicochemical tolerance experiments of BTXS2

The BTXS2 was characterized by determining its tolerance to different salinity (NaCl) and pH. The 96-well plates were used to incubated 200 μL of bacterial cells (about 10⁵CFU/mL) in triplicate. At each time point, the absorbance value of each culture medium was measured automatically at 600 nm (OD600) with a microplate reader (SpectraMax M5, Molecular Devices).

For pH experiments, BTXS2 was inoculated in 3% NaCl LB broth at different pH. The initial pH of the broth medium was from 5.0 to 9.0 and adjusted by using HCl and NaOH. The cultures were divided into sterilized 96 well plates and incubated at 37^oC for 13 h.

For salinity experiments, BTXS2 was inoculated in LB broth with NaCl concentrations of 0.5%, 1.8%, 2.6%, and 3% respectively and the pH was adjusted to 7.0. The cultures were divided into sterilized 96 well plates and incubated at 37^oC for 13 h.

2.5 Screening and purification of phage

The phage was isolated from 2 L water centrifuged at 10000 × g for 5 min to pellet the larger organisms and debris. The supernatant was filtered by 0.22 µm filter membrane (EMD Millipore, Billerica, MA, USA). The filtrate was centrifuged at 4000 rpm (Eppendorf centrifuge 5804R) for 10 min with an ultrafiltration centrifuge tube (Millipore Amicon Ultra-15, 30 kDa) to concentrate the phage. The retentate was eluted with 2 mL SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris HCl, pH 7.5). The phage stock was mixed with 500 μL exponential phase host cells and incubated at 37^oC for 24 h to perform a top agar overlay [28]. The representative plaques were resuspended in 1 mL SM buffer. This sample was serially diluted and mixed with host cells again to do another round of top agar overlay and plaque isolation. The process was performed many times to ensure the purity of the phage stock. The phage stock was mixed with an equal volume of 20% sterile glycerol and stored in a freezer at -80°C. The phage, designated as VB_VpP_BT-1011, that infected the BTXS2 host were selected for characterization in this study.

2.6 Phage one-step growth curve

The one-step growth experiment was conducted as described by previous with some modifications [29]. The one-step growth curve was used to observe the growth characteristics of the phage. Exponential-phase host cells were inoculated with the phage at MOI 0.1 (The ratio of the number of phages to host bacteria is equal to 0.1.) and incubated with shaking (160 rpm) at 37^oC for 10 min for adsorption. Cells were then washed three times using 5 mL fresh LB culture with 3% NaCl to remove unabsorbed phage and resuspended in 30 mL fresh LB culture with 3% NaCl. The experiment was carried out in triplicate. Samples were collected every 10 min for the first 30 minutes, every 30 min for 30-120 minutes, and every 60 min for 120-360 minutes to perform a plaque count (PFU/ml).

2.7 Environmental Tolerance of VB_VpP_BT-1011 Phage

1 mol/L Na₂HPO₄ and NaH₂PO₄ stock solution were prepared and mixed to form phosphate buffers with pH values of 5.0, 6.0, 7.0, 8.0 and 9.0. VB_VpP_BT-1011 phage stock solution was added to phosphate buffers with different pH respectively and incubated at 37^oC for 24 h. Artificial seawater with NaCl concentrations of 0%, 1.8%, 2.6%, and 3% were prepared to simulate freshwater, estuary, offshore and Bohai seawater environments. VB_VpP_BT-1011 phage stock solution was added to different salinities and incubated at 37^oC for 24 h. A certain amount of VB_VpP_BT-1011 phage solution was added to simulated fresh water and sea water, and placed at typical temperatures of 4°C, 11°C, 25°C and 37°C representing different seasons respectively for 24 h. Double-layer plates were made to count the number of phages at the beginning and end of the experiment. And the survival rate of phage was calculated with formula 1.

SR= N₂₄/N₀ (1)

where SR means the survival rate of the phage after 24 h incubation;

N₀ means the number of the phage at the beginning of the experiment;

N₂₄ means the number of the phage at the end of the experiment.

2.8 Phage decay curve

Artificial seawater with 0% and 3% of NaCl concentrations were prepared and adjusted to pH 7.0. VB_VpP_BT-1011 phage stock solution was added and incubated at 11^oC. The survival rate of the phage at the beginning, the first week, the third week, the fifth week, and the seventh week was calculated.

2.9 Antibacterial effect of bacteriophages on host BTXS2

The VB_VpP_BT-1011 phage was mixed with the BTXS2 host at different MOIs of 10^-6-10³ respectively and incubated at 37°C [23]. The same concentration BTXS2 host culture without inoculating VB_VpP_BT-1011 phage was served as positive control. The optical density (OD600) of the culture was measured at 15 min intervals on a microplate reader (SpectraMax M5, Molecular Devices). The experiment was performed in eight copies to ensure the reliability of the experimental results.

2.10 Host range test

The host range of phage VB_VpP_BT-1011 was tested against 24 strains of bacteria (8 strains of V.parahaemolyticus, 4 strains of Vibrio fluvialis, 3 strains of Vibrio cholerae, 3 strains of Vibrio alginolyticus, 2 strains of Vibrio vulnificus, 1 strain of Staphylococcus aureus, 1 strain of Coccus, 1 strain of Escherichia coli and 1 strain of Pseudomonas aeruginosa) (Table 2) by using the spot inoculation method [30]. 5 μL phage stock solution was spotted onto the plate with different bacterial lawn and incubated at 37^oC for 24 h. After incubation, the appearance of plaques was examined.

2.11 The morphology structure of bacteria and phage

BTXS2 cells were Gram stained. Briefly, the bacteria liquid cultivated to the exponential growth phase was dropped onto a clean glass slide and dried naturally to fix the bacteria on the glass slide. The ammonium oxalate crystal violet staining solution dropwise was added on the bacterial smear to dye for 60 s, after that excess dye solution was washed off. Iodine solution dropwise was added to the bacterial smear to mordant for 60 s followed with a flushing process. In order to remove all water droplets, the ethanol dropwise was added to the bacterial smear to decolorize 30~45 s. The safranin staining solution dropwise was added on the bacterial smear followed with counterstain 60～90 s and flushing process. After drying, Gram-stained hosts were imaged using an BX51 inverted scope at 100x (Olympus, Center Valley, PA, USA) and C4742-95 digital camera (Hamamatsu, Bridgewater, NJ, USA).

The BTXS2 grown to exponential growth phase was centrifuged at 3000 rpm for 10 min and washed with PBS solution. This process was repeated three times. The BTXS2 was fixed at 4^oC in darkness for 24 h with 2.5% glutaraldehyde. The BTXS2 was then serially treated with ethanol (30% for 15 min, 50% for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min, 95% for 15 min, and twice with 100% for 15 min each time). The dehydrated BTXS2 was sputter coated with gold and visualized by using optical microscope and scanning electron microscope SEM (Quanta200, FEI).

Phage VB_VpP_BT-1011 and BTSX2 host were mixed and poured on a double-layer plate in advance and then the plaques were selected and resuspended in the PBS solution followed by a centrifugation at low speed to remove solid impurities such as agar. The supernatant was added onto the surface of a copper grid and adsorbed for 15 min. The phages were negatively stained with 2% phosphotungstic acid (2% w/v, pH 7.2) in darkness for 10 min and observed with a transmission electron microscope (TEM) (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The size of the phage was estimated from the electron micrograph.

2.12 Bacteria and phage DNA extraction

The purified and preserved BTXS2 bacteria were cultured to the exponential phase in advance and centrifuged at 8000 rpm (Eppendorf centrifuge 5804R) for 5 min. Then the bacterial genome was extracted using the TIANamp Bacteria DNAKit (TIANGEN). The extracted genome was gel electrophoresed to observe the purity of the DNA band.

Bacteriophage VB_VpP_BT-1011 was added to BTXS2 in exponential phase, amplified and cultured for 24 h. 2% (v/v) chloroform was added to lyse the bacteria. Then the lysis solution was centrifuged at 8000 rpm for 10 min to remove bacterial debris. After being filtered through a 0.22μm membrane, the filtrate was added to a 30 kDa ultrafiltration tube and concentrated by ultrafiltration. DNase I and RNase A were added to the concentrated solution to a final concentration of 1 μg/mL and the mixture was incubated at 37^oC for 30 min, and then incubated at 80°C for 15 min to inactivate DNase I and RNase A. The phage DNA was extracted with DNA Viral Genome Extraction Kit (Solarbio), and the purity and integrity of the band were checked by gel electrophoresis.

2.13 Sequencing and assembly of bacterial and phage

Bacteria and phage gene library construction and library inspection were conducted with the SMRT bell TM Template Kit (version 1.0) to construct a 10K SMRT Bell library. The DNA samples qualified by electrophoresis were broken into target fragments and the DNA damage repair and end repair were performed. The constructed library was quantified by Qubit concentration, and the insert size was detected by Agilent 2100. Finally, sequencing was performed on the PacBio platform. After filtering out low-quality sequences, SMRT Linkv5.0.1 software (https://www.pacb.com/support/software-downloads/) was used to perform genome assembly on reads to obtain preliminary assembly results that reflect the basic situation of the sample genome.

The complete genome sequence was annotated using Subsystem Technology (RAST, http://rast.nmpdr.org) and GeneMarkS (Version 4.17） (http://topaz.gatech.edu/GeneMark/). All predicted open reading frames (ORFs) in BTXS2 and VB_VpP-BT-1011 were both verified using the online BLASTP (http://www.ncbi.nlm.nih.gov/BLAST). The putative transfer RNA (tRNA)-encoding genes were searched using tRNA scan-SE（Version 1.3.1）（http://lowelab.ucsc.edu/tRNAscan-SE）. The bacteria gene function were further annotated by the database such as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) and Cluster of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/). The bacteria gene island was predicted by IslandPath-DIOMB software (Version 0.2) (http://www.pathogenomics.sfu.ca/islandpath/). The complete genome sequence of BTXS2 host and the phage VB_VpP_BT-1011 have been deposited in the GenBank database under the accession number CP063525, CP063525, CP063527 and MW009675, respectively.

2.14 Statistical analysis and phylogenetic trees

Each experiment was independently performed at least thrice. Data were expressed as the mean ± SD and analyzed with the SPSS version 25.0 statistical software. Significant differences were assessed using the Student-Newman-Keuls test (S-N-K test). P < 0.05 was considered to be statistically significant. The pre-selected bacterial 16S rDNA and phage whole genome were aligned by ClustalW in Mega 5. After deleting non-aligned bases, the Neighbor Joining method was used to construct the phylogenetic trees with 1000 bootstraps.

3.1 Characterization of bacteria

3.1.1 Morphological structure of bacteria

Strain BTXS2 presented as typical large green single colony with smooth surface and flat edges on Vibrio chromogenic medium (Figure 1A); The morphology of the bacteria was observed using an optical microscope after Gram staining, and the results showed BTXS2 was a Gram-negative bacterium with a short rod-like shape (Figure 1B); After observing under SEM, the BTXS2 was short rod-shaped (0.5-0.8×1.4-2.0 μm) with a terminal flagellum (Figure 1C and D). The shape of the bacteria was similar to that of V.parahaemolyticus reported by references [27, 31], but no flagellum was observed in their results. It may be due to the glutaraldehyde fixation and centrifugation process that damaged the flagella and caused it to fall off. According to the morphological observation, it was preliminarily identified as V.parahaemolyticus.

3.1.2 Physiology, Biochemistry and Antibiotic resistance of BTSX2

The carbon source utilization and enzymatic results of the bacteria were compared with Bergey's Manual [32], and the result showed that BTSX2 was indeed a strain of V.parahaemolyticus. It can use 44 of 71 types of carbon source including eleven sugars in the BIOLOG plate (Table S2). BTSX2 has a great advantage in competition with other bacterial species due to its ability to use a broad range of carbon sources. The strain BTXS2 can utilize lactose and inositol but cannot utilize L-aspartic acid, which is not consistent with the result reported by Heinemeyer [33]. It indicates the specificity of the BTXS2 in carbon source utilization, which may affect and determine the survival and distribution of the BTXS2. The enzymatic reaction of BTXS2 showed positive results for alkaline phosphatase, esterase (C4), lipid esterase (C8), lipase (C14), and naphthol-AS-BI-phosphohydrolase, indicating that it has a high metabolic diversity (Table S3). The excellent physiological, biochemical and metabolic capabilities of BTXS2 provide guarantee for its widespread in estuaries and oceans.

The antibiotic sensitivity tests showed that the BTXS2 was resistant to penicillin and vancomycin, but sensitive to other antibiotics tested in the experiment (Table 1), indicating that the V.parahaemolyticus BTXS2 isolated from the environment is resistant to multiple antibiotics. It should be noted that the BTXS2 has acquired resistance to the last-line antibiotic vancomycin, which is rarely reported [34]. And it may increase the cost of treatment of antibiotic-resistant V.parahaemolyticus based on the fact that vancomycin is often used to clinical treatment of antibiotic-resistant bacteria.

3.1.3 The effect of pH and salinity on bacterial growth

As shown in Figure 2A, different initial pH values had no obvious effect on the growth of V.parahaemolyticus BTXS2 except for pH 5.0. In the exponential growth phase, the growth rates at pH 6.0 and 7.0 were almost the same, followed by that at pH 8.0 and pH 9.0. And in the stationary phase, the numbers of BTXS2 could reach an equal value at different pH. The BTXS2 entered the exponential growth phase later at pH 5.0 compared with that at other pH, but it reached the stable phase almost at the same time as pH 9.0. The growth properties of BTXS2 at different pH were inconsistent with the strains isolated by Yaashikaa, et al. [27] and Soto-Rodriguez, et al. [35] which grew best at pH 9.0 and pH 5.0 respectively. The BTXS2 isolated from Bohai Coast had good stable performance in the natural water environment, enhancing the possibility of its spread in different water bodies.

The growth of BTXS2 in 1.8%, 2.6% and 3% NaCl LB culture had no statistically significant difference (Figure 2B). At the end of exponential growth, the growth of bacteria in 1.8% NaCl LB was slightly faster than 2.6% and 3%, and reached the stable phase slightly earlier. The growth of V.parahaemolyticus in 0.5% NaCl LB culture was relatively slow, and began to grow rapidly after 230 min, which was consistent with the results reported by Soto-Rodriguez, et al. [35]. It shows that the BTXS2 could grow well at salinities of estuary, offshore and seas. The wide tolerance of the BTXS2 may enhance the survival and spread within an ecological niche [36].

3.1.4 Phylogeny analysis

After comparing the 16S rRNA sequence of BTXS2 with the NCBI nucleotide database, nine sequences belonging to V.parahaemolyticus were selected and compared with the 16S rRNA sequence to finally form phylogenetic tree (Figure 3). The results showed that the conservative sequence of BTXS2 was not significantly different from other V.parahaemolyticus. The strain of V.parahaemolyticus similar to BTXS2 widely distributed all over the world such as America, China, Japan and so on, and they are widely distributed in fishponds, shrimp ponds and sediments [37]. The 16S rRNA sequence of BTXS2 isolated from Tianjin estuary was the most homologous to ATCC 17802 that was a standard strain of BTXS2, and it was clustered under the same branch as FROC, indicating that they had a strong genetic relationship. Based on their pathogenicity, it shows that BTXS2 poses a security threat to humans all over the world.

3.1.5 Genome analysis of BTXS2

After sequencing and assembly optimization, the genome sequence was submitted to NCBI. The DNA size was 5068431 bp, which included two chromosomes and a plasmid. The G+C content was 45.32%, 45.65 and 67.99%, respectively. A total of 6182 genes were predicted to be the coding genes of the newly sequenced genome. Using RepeatMasker (Version 4.0.5) software to predict scattered repetitive sequences, a total of 384 scattered repetitive sequences and 138 tandem repetitive sequences were obtained. Sequencing analysis revealed that BTXS2 contained 131 tRNAs, 37 rRNAs and 15 sRNAs. The distribution of 11 gene islands is shown in the Figure S1. The islands contain virulence genes encoding putative type III secretion systems, type VI secretion systems and superoxide dismutase, as well as streptomycin resistance genes (Table S4). The annotation with the GO database revealed that BTXS2 contained functional genes related to virus structure and reproduction (Table S5), indicating that there were phage-related genes on the genome. Compared with the PHI database, it was found that the BTXS2 had a variety of pathogenic genes that threatened human health, such as causing human food poisoning, fever, diarrhea, gastritis, and other diseases. And there were also pathogenic genes that cause skin infections and sepsis in Danio rerio and Penaeus monodon (Table S6 and Figure S2 and S3). The BTXS2 may pose a great threat and negative impacts on human health and fish in the estuaries and coasts.

The antibiotic resistance genome were found in the gene sequence, and the result of PCR showed that the BTXS2 contained antibiotic resistance genes for β-lactamases (Figure S4), which commonly presented in various V.parahaemolyticus isolated from other studies[9, 16, 38]. A resistance gene related to the efflux pump mechanism was found in the PCR results of BTXS2, which might play an important role in antibiotic resistance. The antibiotic resistance gene for quinolones was also founded in BTXS2, but it is interesting that it did not show resistance in the antibiotic susceptibility test, which may be due to the fact that the gene is not expressed in the BTXS2. Gene alas, an alanyl-tRNA synthetase conferring resistance to novobiocin, was first founded in the V.parahaemolyticus genome, which was not reported in other V.parahaemolyticus, and it would bring greater challenges to the treatment of V.parahaemolyticus.

3.2 Characterization of phage

3.2.1 VB_VpP_BT-1011 morphology

Phage VB_VpP_BT-1011 could form a single-character plaque with a diameter of about 3 mm on double-layer plates (Figure 4A). After staining, the results of TEM showed that the phage was an isometric eicosahedral structure with a total length of 54 ± 1 nm, a tail length of 23 ± 0.5 nm, and a head length of 31 ± 0.5 nm (Figure 4B). VB_VpP_BT-1011 belonged to Mycoviridae family. Compared with the V.parahaemolyticus myophage found in other studies, VB_VpP_BT-1011 was smaller in size and had longer tail fibers[24, 39, 40]. The process of phage adsorption (Figure 4C) and cell membrane damage (Figure 4D) were observed.

3.2.2 Biological characteristics of VB_VpP_BT-1011

The latent time and burst size of the phage were determined by the one-step growth experiment. The one-step growth curve was divided into three stages, including the incubation period, the rising period and the stable period (Figure 5A). The one-step growth curve showed that the latent time of VB_VpP_BT-1011 was 30 min, and the rising period was 60 min. The burst size was 24 PFU/cell. Compared with the V.parahaemolyticus bacteriophage isolated by Matamp and Bhat [39] and Yang, et al. [41], VB_VpP_BT-1011 had a little longer latent time and a smaller burst size, but compared with four out of five phages isolated by Yu, et al. [42], they had a shorter latent time. The different latent time may be related to the specific recognition and assembly mechanism between host and phage. Moreover, mathematical modeling studies have demonstrated that both host quantity and quality are involved in determining the latent time [43, 44].

In the simulated water environment of different salinity, VB_VpP_BT-1011 could survive stably, and its survival rate after 24 h was higher than 84%. In addition, there was no significant difference in the survival rate under different salinity conditions (Figure 5B). VB_VpP_BT-1011 reached the highest survival rate (94.15%) at salinity of 1.8%. The survival rate under low-salinity conditions and high-salinity conditions were almost the same, indicating that the phage had good adaptability under the salinity conditions of natural water bodies.

The survival rate of VB_VpP_BT-1011 phage decreased after incubation in a simulated water environment in which pH values were ranging from 5.0 to 9.0 for 24 h, but there was no statistically significant difference in the survival rate under different pH (Figure 5C). After 24 h, the highest survival rate reached 83.45% at pH 7.0.

In the simulated freshwater environment with salinity of 0%, there was no significant difference in the effects of different temperatures on the survival rate of VB_VpP_BT-1011 (Figure 5D). And it reached the highest survival rate (94.54%) at 11^oC. In the simulated seawater environment with salinity of 3%, there was no significant difference in the effect of the activity of VB_VpP_BT-1011 phage with the temperature ranging from 4^oC to 37^oC. After 24 h, it also reached the highest survival rate (96.28%) at 11^oC. The conclusion can be drawn that the phage VB_VpP_BT-1011 could keep activity well throughout the year in freshwater and seawater. In the absence of the host, the bacteriophage VB_VpP_BT-1011 could exist stably in simulated freshwater and seawater. The results of the experiment that lasted for seven weeks showed that the survival rate of bacteriophages in simulated freshwater and seawater dropped by about 40% (Figure 5E). However, the survival rate of bacteriophages in the simulated seawater decreased more slowly than in the simulated freshwater environment, indicating that the bacteriophages have better adaptability to environments with higher NaCl concentrations.

Salinity, pH, and temperature are important factors affecting the survival and activity of bacteriophages [45]. In this study, VB_VpP_BT-1011 can survive stably under these environmental conditions, which shows its strong viability and the possibility of the phage's distribution and spread in various places. Thus, VB_VpP_BT-1011 may have the potential impact on the microbial community structure and ecological health in the natural environment since that bacteriophages occupy an important niche in the water environment [46].

3.2.3 Host Range

The results of spot incubation test showed that the phage could only infect BTXS2 (Table 2), indicating that phage VB_VpP_BT-1011 was highly specific to BTXS2, which was similar to phages in other studies [47, 48]. The specificity may be related to the phage-host interaction mechanism [49, 50]. In order to understand the host range of the phage better, more bacteria need to be tested. At the same time, it can be seen that the high specificity makes the use of this phage to treat V.parahaemolyticus more promising without affecting other microorganisms.

3.2.4 Phage genome analysis

The dsDNA genome size of the phage VB_VpP_BT-1011 was 43841 bp. The total G+C content was 42.77%. Three encoded tRNAs (starting aminoacyl-tRNA, arginine-carrying tRNA, and glycine tRNA) were found and a total of 72 coding genes were identified. A total of 75 CDSs were predicted using RAST, of which 15 were known phage functional genes, and the remaining 60 CDSs were predicted hypothetical proteins. A total of 268 ORFs were found using the ORF Finder online tool, and only 82 ORFs were found to have homology with the amino acid sequence of known proteins using the SMARTBLAST tool. The remaining 124 ORFs were speculative proteins and 62 ORFs with unknown functions, which might be due to the novelty of VB_VpP_BT-1011 and the lack of database information on environmental phages.

The sequence of the phage was compared with the antibiotic resistance gene database, and the results showed that the phage did not have any antibiotic resistance gene. The bacteriophage VB_VpP_BT-1011 had the same modular genome structure as most dsDNA bacteriophages. RAST database was used to annotate the sequencing results, we found the phage recombinant protein NinG, DNA replication helicase protein DnaC/DnaI, recombinant functional protein, single-stranded DNA binding protein, phage-related endonuclease protein, phage terminal large subunit, phage major capsid protein and minor capsid protein genes (Figure 6A). The genes encoding these proteins play an important role in the structure of bacteriophage and the function of infection, recombination, and replication.

By comparing the genome of VB_VpP_BT-1011 with the genome of NCBI, ten similar phage genomes were selected to establish phylogenetic tree. The results showed that this phage had no significant similarity with other Vibrio phages in the NCBI database，but had a certain genetic relationship with the published Vibrio phages (Figure 6B). The phylogenetic tree analysis results based on the comparison of the whole genome showed that the phage was most similar to the Vibrio_phage_V039C genome. VB_VpP_BT-1011 and Vibrio_phage_V039C were in the same branch, and the relative distance was closer. In the course of evolution, horizontal gene transfer may lead to the widespread exchange of phage shared genes [51]. By comparison, it was found that both VB_VpP_BT-1011 and Vibrio_phage_V039C possessed large terminal subunits, phage capsids, single-stranded DNA binding proteins and DNA helicase genes. But they are significantly different from each other. Vibrio_phage_V039C contained a tape measure protein gene, HigA family antitoxin and Rha family regulatory protein, while the phage VB_VpP_BT-1011 isolated in this study contained a phage base plate protein, phage recombinant protein and VgrG protein. The difference in the types of genes leads to the difference in structure and function of each other, which will further affect their living habits and interaction with their hosts.

3.3 Inhibition of VB_VpP_BT-1011 on BTXS2

The result showed that the phage had antibacterial effect on V.parahaemolyticus BTXS2. When the MOIs were 0.00001-0.001, the higher the MOI was, the more obvious antibacterial effects would have on the host bacteria, which showed that MOI was an important factor affecting the antibacterial of phages. When the MOI was 0.01, the concentration of host bacteria increased first, but with the expansion of phage, the concentration of host bacteria began to decrease after 150 min. When the host bacteria were suppressed to a low concentration, a new resistant subgroup was selected and proliferated, resulting in the increase of bacterial concentration in the later period. When the MOIs were 0.1-1000, the growth of the host bacteria BTXS2 was inhibited, but the concentration of the host bacteria increased after 350 min, which might be related to bacterial resistance because bacteria could resist phage infection through a variety of mechanisms, including spontaneous mutation, restriction endonuclease modification system and adaptive immunity of CRISPR-Cas system [49]. Spontaneous mutation is an important mechanism for phage resistance and phage-host co-evolution [52] that may confer resistance to phage by changing the structure of the receptor on the surface of the bacteria [53], and it also determines the specificity of the phage[54]. Due to the selection of the anti-phage subgroup of BTXS2, they can regrow after the lysis of the bacterial population initially induced by the VB_VpP_BT-1011 [55]. In summary, it can be seen from the Figure 7 that the antibacterial effect of VB_VpP_BT-1011 on the BTXS2 was properly obvious, indicating that it has a strong antibacterial effect on the host. the antibacterial effect varied with different MOIs, but high MOI can effectively inhibit the growth of host bacteria, which provides possible theoretical data for the future use of bacteriophages in food and clinical applications to inhibit and kill pathogenic bacteria. This study provides foundation for further research on the relationship between V.parahaemolyticus related phages, and further research is needed on the diversity of bacteriophages and the mechanism of the antibacterial effect on bacteria especially pathogenic bacteria.

In this study, we isolated new strain of V.parahaemolyticus BTXS2 and specific phage VB_VpP_BT-1011 from the water of Bohai Coast. The BTXS2 has specific carbon source utilization and oxidase types that are different from other V.parahaemolyticus, and has potential multiple antibiotic resistance and pathogenicity. The bacteriophage VB_VpP_BT-1011 that infected BTSX2 belongs to the Myoviridae family and has no obvious similarity with the V.parahaemolyticus phage isolated from other studies. BTXS2 and VB_VpP_BT-1011 isolated in this study can stably exist and has good adaptability in simulated freshwater, estuaries, offshore and deep-sea pH, NaCl concentration and seasonal temperatures. The genomic analysis of the phage shows that the phage has abundant and important genetic genes including genes closely related to phage infection, recognition and replication. The co-culture of phage and host shows that the phage has obvious control effect on the host, and the control effect is different at different MOIs.

This study provides foundation for further research on the relationship between V.parahaemolyticus related phages, and further research is needed on the diversity of bacteriophages and the mechanism of the biological control effect on bacteria especially pathogenic bacteria.

Acknowledgments:

Thanks to the support from the National Key R&D Program of China (grant number 2018YFD0800104), Special Fund of China (grant number AWS18J004), the Natural Science Foundation of Tianjin, China (grant number 17JCZDJC39100 and 19JCYBJC23800) and the National Natural Science Foundation of China (grant number 51808468). And thanks to Dan Huang for her help in analyzing the genetic information and function of phage. And we thank Kun He, Yanping Yang and Chunchun Zhang for their assistance in conducting host growth and phage experiments.

Funding

This research was funded by National Key R&D Program of China (grant number 2018YFD0800104), Special Fund of China (grant number AWS18J004), the Natural Science Foundation of Tianjin, China (grant number 17JCZDJC39100 and 19JCYBJC23800) and the National Natural Science Foundation of China (grant number 51808468).

Conflicts of interest/Competing interests

The authors declare no conflicts or competing interests.

Availability of data and material

The nucleic acid sequence information of V.parahaemolyticus BTX2 can be viewed in the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/?term=BTXS2); And The nucleic acid sequence information of the phage can be viewed at https://www.ncbi.nlm.nih.gov/nuccore/MW009675.1.

Code availability

Not applicable.

Authors' contributions

Conceptualization, Chao Gao, Xiaobo Yang, Chen Zhao, Chenyu Li, Shang Wang, Xi Zhang, Bin Xue, Zhiqiang Shen, Jingfeng Wang, Lingli Li, Pingfeng Yu, Zhiguang Niu and Zhigang Qiu; Data curation, Chao Gao, Xiaobo Yang, Zhiguang Niu and Zhigang Qiu; Formal analysis, Chao Gao, Xiaobo Yang, Chen Zhao, Shang Wang, Zhuosong Cao, Hongrui Zhou, Yutong Yang, Zhiguang Niu and Zhigang Qiu; Funding acquisition, Shang Wang, Jingfeng Wang, Lingli Li and Zhigang Qiu; Investigation, Chao Gao, Xiaobo Yang, Chenyu Li, Shang Wang, Xi Zhang, Bin Xue, Zhiqiang Shen, Jingfeng Wang, Zhiguang Niu and Zhigang Qiu; Methodology, Chao Gao, Xiaobo Yang, Chen Zhao, Shang Wang, Xi Zhang, Bin Xue, Zhiqiang Shen, Jingfeng Wang, Pingfeng Yu, Zhiguang Niu and Zhigang Qiu; Project administration, Jingfeng Wang, Zhiguang Niu and Zhigang Qiu; Resources, Jingfeng Wang and Zhigang Qiu; Validation, Chao Gao, Xiaobo Yang, Chen Zhao, Shang Wang, Zhuosong Cao, Hongrui Zhou, Yutong Yang, Zhiguang Niu and Zhigang Qiu; Writing – original draft, Chao Gao, Xiaobo Yang and Zhigang Qiu; Writing – review & editing, Chao Gao, Xiaobo Yang and Zhigang Qiu. All authors have read and agreed to the published version of the manuscript.

Ethics approval

This research complies with all ethical guidelines, and relevant departments and institutions have approved the research.

Consent to participate

All prevailing local, national and international regulations and conventions, and normal scientific ethical practices, have been respected.

Consent for publication

All contributing authors agree to give consent to publication, if accepted.

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Table 1. BTXS2 antibiotic sensitivity test

Antibiotic	Dosage	BTXS2
Chloramphenicol	30 μg	-
Streptomycin	10 μg	-
Ofloxacin	5 μg	-
Penicillin	10 IU	+
Tetracycline	30 μg	-
Vancomycin	30 μg	+
Erythromycin Cefotaxime Cefuroxime sodium Cefepime Polymyxin B Cefoperazone/Sulbactam 2:1 Fosfomycin	30 μg 30 μg 30 μg 30 μg 300 units 105 μg 50 μg	- - - - - - -
H₂O（Negative control）	30 μL	+
Note: +, grow; -, not grow

Table 2. The result of host range test

Host	Result	Host	Result
V.parahaemolyticus BTXS2	+	Vibrio fluvialis 1.1608	-
V.parahaemolyticus 1.1997	-	Vibrio fluvialis 1.1611	-
V.parahaemolyticus ATCC17802	-	Vibrio cholerae 860072	-
V.parahaemolyticus 20502	-	Vibrio cholerae 860172	-
V.parahaemolyticus 20516	-	Vibrio cholerae 860084	-
V.parahaemolyticus 20572	-	Vibrio alginolyticus ATCC17749	-
V.parahaemolyticus (isolated from CDC)	-	Vibrio alginolyticus TJF-15	-
V.parahaemolyticus 1.1615	-	Vibrio alginolyticus CICC21611	-
Vibrio vulnificus CICC10383	-	Staphylococcus aureus	-
Vibrio vulnificus ATCC27562	-	Streptococcus PCF10	-
Vibrio fluvialis 17002	-	Pseudomonas aeruginosa	-
Vibrio fluvialis 1.1609	-	Escherichia coli 1655	-
Note: +, positive; -, negative.

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Characterization of a novel Vibrio parahaemolyticus host-phage pair and antibacterial effect against the host

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