3.2.1. Bacterial strains
A total of 17 A. salmonicida strains of 3 different subspecies (subsp.
salmonicida, subsp. achromogenes and subsp. masoucida), 20 strains of motile Aeromonas spp. (10 of A. hydrophila, 5 of A. sobria and 5 of A. media) and 11 other strains of different species were used in this study (Table 3.1). The bacterial strains were cultured in tryptic soy broth (TSB) or sub-cultured on tryptic soy agar (TSA) at 20°C for all Aeromonas spp. and at 37°C for other bacterial species. All the strains were stored at -80°C with 10% glycerol until needed.
3.2.2. Phage isolation and host range determination
The conventional double-layered agar method described by Adams (3) was used for the phage isolation and enumeration of its plaque-forming units (PFUs). One of the previously confirmed antibiotic-resistant A. salmonicida subsp. salmonicida strains, AS01 (14), was used as a host bacterial strain for the phage isolation.
Plaque morphologies were observed after 18 to 24 h of incubation. Bacterial cultures in exponential phase were inoculated with environmental water samples of the final effluent from the sewage of the rainbow trout culture farms in Korea. The mixtures were incubated for 36 h at 20°C, centrifuged for 20 min at 10,000 × g and filtered through a 0.45-μm pore size membrane filter. Pure phage strain was obtained by three serial single-plaque isolations and designated as PAS-1. The
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filtered phage lysate was precipitated with 10% (w/v) polyethylene glycol (PEG) 8000 in 1 M NaCl at 4°C for 12 h and collected by centrifugation at 10,000 × g for 10 min at 4°C. The purified phage was prepared by CsCl step gradients ultra- centrifugation (gradient density: 1.15, 1.45, 1.50 and 1.70 g/ml; 250,000 × g; 22 h;
4°C), dialyzed in SM buffer (10 mM NaCl, 50 mM Tris [pH 7.5] and 10 mM MgSO4) and stored at 4°C until used.
The host range of the phage was determined by the double-layered agar method;
bacteria were inoculated with all the 37 Aeromonas spp. strains and then checked for the presence or absence of plaque formation. The plaque-forming ability of the phage against each A. salmonicida strain was measured as the efficiency of plating (EOP), which was measured against the standard of A. salmonicida subsp.
salmonicida AS01. To check the polyvalency of the phage, 10 strains of other bacterial species were tested against the phage PAS-1.
3.2.3. Electron microscopy
The purified phage sample was loaded onto a copper grid followed by negative staining with 2% uranyl acetate and drying. The morphology of the phage was observed using a Zeiss TEM EM902 (Zeiss) at an accelerating voltage of 80 kV.
Phage sizes were calculated by the means of at least 10 measurements.
3.2.4. One step growth
The 10 μl of purified phage suspension (9.3 × 109 PFU/ml) was added to 10 ml of inoculums of the host bacterial strain in early-exponential phase (OD600: 0.2) in TSB, absorbed for 5 min and then centrifuged at 10,000 × g for 1 min. After the
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supernatants were removed, the pellets containing the phages-infected bacterial cells were suspended in 20 ml of fresh TSB and incubated with shaking at 250 rpm and 20°C. Partial samples were taken at 10 min intervals for 120 min, and the titrations from the aliquots were immediately determined.
3.2.5. Thermo- and pH-stability
For the thermo-stability tests, the phage suspension was incubated in TSB (final phage concentration: 1.4 × 107 PFU/ml) at 4°C, 20°C, 40°C and 55°C for 60 min, and aliquots were taken at 30 and 60 min. For pH-stability tests, the phage suspension was inoculated in a series of tubes containing TSB (final phage concentration: 1.2 × 107 PFU/ml) with final pH of 3.0, 5.0, 7.0, 9.0 and 11.0 (adjusted with 1 M HCl or 1 M NaOH), incubated at 20°C, and then titered at 30 and 60 min.
3.2.6. Phage genome analysis
Purified phage genomic DNA (gDNA) was prepared as previously described (33), and it was subjected to nuclease treatment using DNase I (20 U/μl), RNase A (5 U/μl) and Mung bean nuclease (20 U/μl) (Takara) according to the manufacturer’s instructions. In addition, the size estimation and restriction analysis of phage gDNA were performed by pulsed-field gel electrophoresis as previously described (36), with some modifications. Briefly, 500 μl of phage suspension was mixed with 500 μl of 2% (wt/vol) NuSieve GTG agarose (FMC BioProducts), dispensed into plug molds and solidified. The plugs were punched out of the molds into a small volume of digestion buffer (500 mM EDTA, 10 mM Tris [pH 8.0], 1%
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SDS [w/v] and 1 mg/ml of proteinase K) and incubated at 50°C overnight. The digestion buffer was decanted, and the samples were washed three times using TE buffer and then digested with 10 U of SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI (New England Biolabs) for 1 h at 37°C, respectively. The plugs were washed three times using TE buffer, placed in wells of 1.2% Pulsed Field Certified agarose (Bio-Rad) in 0.5X TBE and overlaid with molten 0.5% NuSieve GTG agarose. The samples were electrophoresed using a CHEF-DR III System (Bio-Rad) at 6 V/cm with pulse ramps from 5 to 15 s for 16 h at 14°C in 0.5X TBE buffer. The phage genome sequencing was performed by Macrogen Inc. (Korea). Briefly, phage gDNA was sheared using a nebulizer (Invitrogen) and blunt-end repaired. DNA fragments of the desired size (2 to 3 kb) were blunt-end ligated into the pCR4 blunt-TOPO vector (Invitrogen) and introduced into E. coli DH10B. Partial genome sequences were obtained by sequencing with primer walking. The potential ORFs were predicted using GeneMark.hmm (17), and gene sequence similarity was investigated using the NCBI BLASTP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
3.2.7. Phage proteome analysis
Phage ghosts were prepared as previously described (15). Briefly, purified phage suspension (8.4 × 1011 PFU/ml) was re-concentrated by ultra-centrifugation at 100,000 × g at 4°C for 30 min. They were re-suspended in 10 M LiCl, heated to 46°C for 20 min and then ten-fold diluted with 50 mM Tris/HCl (pH 8.0) in 100 mM NaCl and 5 mM MgCl2, and treated with DNase I (20U/μl) (Takara) for 2 h at 37°C. Prepared phage ghosts were then analyzed by standard Tris-glycine sodium
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dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using SDS Ready-Gel (4 to 20% polyacrylamide gradient; Bio-Rad), and stained by PlusOne™ Silver Staining Kit, Protein (GE Healthcare). Protein bands were extracted from the gels, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the proteomics platform at the National Instrumentation Center for Environmental Management (NICEM) at Seoul National University. All MS/MS data were searched using ProteinPilotTM 3.0 Software (Applied Biosystems) against the GenBank non- redundant protein database.
3.2.8. Selection of a virulent A. salmonicida strain and lysis test
To prepare host bacterial strain for experimental therapeutic applications of PAS-1, the presence of the type III secretion system (TTSS) gene ascV was screened by PCR in all 17 A. salmonicida strains used in this study. A pair of primers, ascVF (5’-CAG CTC GCT ATA GCT CCC CT-3’) and ascVR (5’-GCC CTC TAT CTC GAT CTC GG-3’), were designed based on the ascV gene of A.
salmonicida A449 plasmid 5 [GenBank accession number: NC_009350], and PCR was carried out with using a GeneAmp PCR system 2720 (Applied Biosystems) with the following steps: a predenaturation at 95°C for 3 min, followed by amplification for 30 cycles at 95°C for 30 s, 52°C for 60 s and 72°C for 90 s and a final extension at 72°C for 7 min. The amplified 399 bp of PCR products were sequenced for final confirmations of the ascV gene in A. salmonicida.
Based on the bacterial screening result, the purified phage and A. salmonicida subsp. salmonicida AS05 strain in early-exponential phase (OD600: 0.06, 1.3 × 107
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CFU/ml) were co-cultured in 10 ml of fresh TSB at several doses of multiplicity of infection (MOI): 0, 0.01,1, 100 and 10,000. The preparations were incubated with shaking at 250 rpm and 20°C. Bacteria inoculated into TSB without phage (MOI:
0) were used as a control. The absorbance dose (OD600) was determined 0, 3, 6, 12, 24 and 48 h after inoculation, respectively.
3.2.9. Fish experiments
All the animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University. Experiments were performed using 3- to 4-month-old triploid juvenile rainbow trout (avg. body length: 13.1 cm; avg. body weight: 17.1 g) purchased from a private culture farm in Korea. Prior to the experiment, liver, kidney and spleen of the fish were randomly sampled and screened for A. salmonicida infection by PCR assay (4), and the fish were acclimatized at 14-15°C for 1 week.
All the fish were euthanized or anaesthetized by Ethyl 3-aminobenzoate methanesulfonate (Tricaine methanesulfonate; Sigma-aldrich) before or after the experiments.
In advance of the therapeutic applications, the fate of PAS-1 in fish kidneys and the neutralizing activity of the phage-administrated fish serums against the phage were preferentially investigated. The fish were treated with 0.1 ml of the purified phage suspension by intra-muscular (IM) administration (3.0 × 107 PFU/fish), and the fish kidneys (0.1 g) and aquarium waters were randomly sampled at 0, 6, 24, 48, 72, 96, 120, 144, 200, 240 and 360 h post-administration (pa), respectively.
Additionally, blood samples of the phage-administrated fish were obtained at 10,
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15, 20, 25 and 30 days pa by caudal vein-puncture. The serum was collected by centrifugation (1,500 × g, 30 min), and de-complementation was performed by heat treatment for 30 min at 45°C as recommended by Sakai (31). Twenty microliters of prepared serum samples was mixed with an equal volume of purified phage suspension (1.9 × 107 PFU/ml) and incubated at 20°C for 2 h. The 0 h samples, which were collected before phage administration, were used as a control in all the experimental groups, and the phage PFUs were counted by the double-layered agar method.
The protective effects of PAS-1 against A. salmonicida infection were evaluated as below. The fish (20 fish in each experimental group) with identical conditions (as previously described) were challenged with 0.1 ml of the bacterial suspension in phosphate-buffered saline (PBS) containing fresh culture of A. salmonicida AS05 by the IM method (2.5 × 102 CFU/fish). Following bacterial challenge, the fish were immediately given 0.1 ml of the purified phage suspension by the IM method (2.4 × 106 PFU/fish). The fish given SM buffer without the phage were used as control. The fish were monitored for 14 days at 20°C, and bacteria from kidneys of dead fish were re-isolated and confirmed by PCR assay (4).
3.2.10. Statistical analysis
Statistically significant differences in all the experiments were determined using the student’s t-test. A P value of less than 0.05 was accepted as statistically significant. The SPSS statistical software package version 13.0 (SPSS, Inc., Chicago, IL) was used for all statistical analyses.
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The nucleotide sequence data of the RNA polymerase, DNA polymerase, large subunit terminase, tail fiber, muramidase, head portal protein and one hypothetical protein gene in the phage PAS-1 were deposited in the GenBank database under the accession numbers JF342689, JF342683, JF342686, JF342690, JF342687, JF342688 and JF342684, respectively.