Bacterial strains and growth conditions
Two epidemic strains of high virulence isolated from diseased crucian carp, A. hydrophila NJ-35 (Genebank: CP006870.1) from Nanjing, Jiangsu in 2010 and XY-16 (GenBank of gyrB gene: JX025797.1) from Xinyi, Jiangsu in 2009 [52, 53], were used for phage isolation. An additional 203 Aeromonas strains, including 73 A.hydrophila, 85 A.veronii, 12 A. caviae, 1 A. bestiarum, 12 A. sobria, 10 A. media, 3 A. salmonicida, 3 A. jandaei and 4 A. aquariorum were used for host range analysis. All bacterial strains were routinely cultured in Luria Bertani broth (LB) (Difco/Becton Dickinson) at 28 °C with shaking at 180 rpm. All Aeromonas strains used in this study are listed in Table 3.
Phage isolation, purification and propagation
A. hydrophila strains NJ-35 and XY-16 were used as indicator host for phages. Ten water samples were collected from ponds, sewage and rivers in different areas of Nanjing. The samples were centrifuged at 4000 g for 30 min and the supernatants were filtered through 0.22-µm membrane filters. A 10 mL of the supernatant was added into 20 mL of 3 ´ LB liquid media, and 1mL cultures of NJ-35 or XY-16 (late logarithmic phase) were used to inoculate the mixture. After adding 5 M CaCl2 to a final concentration of 0.1 mM, the phages were enriched by culturing for 14 h at 28 °C. A 3 mL of the culture was added to a 5 mL LB media, followed by the addition of chloroform with a final concentration of 3%, vigorously shaken for 2 min. After static layering, 1 mL of the supernatant was filtered through a 0.22 μm filter. To confirm the presence of the lytic phage in the fltrate, the double-layer agar method [54] was performed using the filtrate. After incubating at 28 °C for 8 h, a single plaque was picked up with a sterile pipette tip into LB media with the addition of host bacteria. After proliferation, the phages were purified several times using the double-layer agar method.
Host range
The host range of the harvested phages was determined using a spot assay. Aeromonas strains were spread evenly on the LB solid plate. Five microliters of the phage cultures of 108 PFU/mL were dropped onto the overlaid top agar. After cultured for 12 h at 28 °C, the presence or absence of a lysis zone was observed.
TEM analysis
The phages were cultured at 28 °C for 8-10 h using the double-layer plates. A plate with plenty of plaques whose edges were faintly visible was added with 2 mL of sterilized ddH2O. The plate was shaken horizontally for 5 minutes to fully wash off the phages. Suspensions in the plate were centrifuged at 6000 g for 5 min. For TEM analysis, 15 μL of the phage supernatant was spotted on a copper grid covered with a Formva film. After 3 min, the suspension was removed by filter paper and baked it under incandescent light for 5-10 s. The phages were negatively stained with 2% uranyl acetate for 1 min. Excess dye was removed by the filter paper and the grid was dried under an incandescent lamp. The morphology of the phage was imaged by transmission electron microscopy (H-7650,Hitachi, Japan) operated at 80 kV. Phage dimensions were calculated by measuring the dimensions of five independent phages.
Determination of optimal MOI
The optimal MOI is the ratio of the number of phages to that of host bacteria present in a defined space that is best for phage proliferation to obtain maximum titers. The host A. hydrophila strains grown to the log phase were washed three times with PBS and adjusted to corresponding densities of 105, 106, 107 and 108 CFU/mL, respectively. The phages and bacteria were mixed with MOIs of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001, respectively. After incubation for 2 h at 28 °C, the phage titers were measured by the double-layer agar method.
One-step growth curve
According to the optimal MOI, phage solutions were inoculated into 10 mL host bacteria in logarithmic phase (1×108 CFU/mL). The phages were allowed to absorb for 15 min and then centrifuged at 13,000 g for 5 min. After discarding the supernatants, the phage-infected bacterial pellets were resuspended in 10 mL of preheated LB media and incubated at 28 °C with shaking at 180 rpm. At a interval of 5 min, 100-μL aliquots were taken until 115 min and phage titers were immediately determined by the double-layer agar method.
pH and thermal stability assays
For pH stability tests, 100 μL phage suspension (1.0 × 107 PFU/mL) was used to inoculate 900 μL physiological saline adjusted to pH values of 3~12 with NaOH or HCl. The mixtures were incubated at 28°C for 2 h and aliquots were taken to measure the titers of phages at different pH values. For thermal stability tests, 2 mL phage suspension (1.0 × 107 PFU/mL) was incubated at 30 °C, 40 °C, 50 °C, and 60 °C. At a interval of 20 min, 100-μL aliquots were collected until 100 min. Survived phages were counted and the survival rates were calculated by the PFU at each time point divided by that at the primary PFU. All tests were performed in triplicate.
Inhibition assay of growth and biofilm formation
A. hydrophila strains were cultured in LB medium to logarithmic period and then normalized to 1×106 CFU/mL. The suspensions were mixed with equal volume of phages with the MOI of 0.01, 0.1, 0, 1, 10, and 100, respectively. Two hundred microliters of the mixtures were added into each well of the 96-well plates. Each treatment was performed in eight replicates. Fresh LB medium served as a blank control. Then the plates were incubated at 28 °C for 24h without shaking. The biomass of bacteia was evaluated by measuring OD450 using a micro-plate reader (Tecan, Switzerland). Next, the culture supernatants were discarded and the plates were washed three times with sterile PBS to remove all planktonic cells. Biofilms formed in each well were fixed with 200 μL of 99% (vol/vol) methanol for 15 min. After drying, biofilms were stained with 1% crystal violet for 10 min. Then the wells were washed with distilled water to remove unbound dye. Crystal violet was dissolved in 200 μL of 95% ethanol for 10 min and the absorbance was measured at 595 nm (OD595).
Biofilm clearance assay
A. hydrophila strains were cultured in LB medium to logarithmic period and then normalized to OD600 of 1.0. The suspensions were inoculated to LB meida (200 μL per well) in 96-well plates at a ratio of 1:1000. The plates were incubated at 28 °C for 24 h without shaking. After removing all planktonic cells, each well were treated with 200 μL phage dilution of 1×108 PFU/mL with sodium chloride-magnesium sulfate (SM) buffer (100 mM NaCl, 50 mM Tris pH 7.5, and 10 mM MgSO4) for 6 h, 12 h, and 24 h. Biofilms treated with SM buffer served as the control. The microplates were then washed twice with PBS and the biofilms left were stained by crystal violet method described above.
Phage therapy assay in mice model
The animal experiment was performed in accordance with the animal welfare standards, complied with the guidelines of the Experimental Animal Welfare Ethics Committee, Chinese Association for Laboratory Animal Sciences, and was approved by the Ethical Committee for Animal Experiments of Nanjing Agricultural University, China (approval number: SYXK(Su) 2017-0007). Six-week-old female ICR mice (body weight, 18 ± 2 g) were purchased from the Experimental Animal Center of Yangzhou University and raised in the Experimental Animal Center of College of Veterinary Medicine, Nanjin Agricultural University under specific-pathogen-free (SPF) conditions. The dose of A. hydrophila used in this study was chosen on the basis of a preliminary study that the mice were expected to show obvious clinical signs but no death. A. hydrophila NJ-35 and XY-16 grown to log phase were washed three times with PBS and adjusted to CFU/mL, respectively. For phage therapy assay, a total of 120 mice were randomly divided into three groups and housed in cages (5 mice per cage). Feed and water were allowed ad libitum. To evaluate treatment efficiency of phages in A. hydrophila NJ-35 infection, 40 mice were intraperitoneally inoculated with 100 μL of bacterial suspension of A. hydrophila NJ-35; after 30 min, the infection group of 10 mice were then intraperitoneally injected with 100 μL of sterile PBS, while each 10 out of the other 30 mice in the treatment group were injected with 100 μL of phages N21, W3 or G65 at the MOI of 1. The blank control group of 30 mice were intraperitoneally injected with 100 μL of sterile PBS, followed by injecting 100 μL of each of the phage suspensions. Similarly, 50 mice were used to determine the therapy effect of phages Y71 and Y81 on A. hydrophila XY-16 infection. After 6 h and 24 h, five mice in each treatment were euthanized by CO2, respectively. Hearts, livers, spleens, lungs and kidneys were aseptically removed from the mice, weighed, added to 1 mL of sterilized PBS, homogenized using a tissue homogenizer (Bioprep-24, Allsheng, Hangzhou, China), and then 10-fold serially diluted in PBS. Bacterial loads in tissues were determined by plating the dilutions on LB plates. The results are expressed as the numbers of CFU/g tissue.
Statistical analyses
Data were collected and analyzed using GraphPad Prism version 5 software. The bacterial loads in the phage-treatment and the control groups were analyzed using t-test. Error bars presented in the figures represent standard deviations of the means of three replicate experiments. A P-value < 0.05 was considered as a significant difference.