Isolation of A. hydrophila phages
Three phages designated as N21, W3 and G65 with A. hydrophila NJ-35 as an indicator host, and two phages Y71 and Y81 with A. hydrophila XY-16 as an indicator host, were isolated from fish ponds and polluted rivers in Nanjing. Clear plaques appeared after 12 h incubation at 28 °C. As shown in Fig. 1A, plaques of all the five phages were morphologically similar with diameters of 1 mm to 3 mm, and transparent in the middle. The plaque edges of phages W3, Y71 and Y81 were clear with no halo, while those of phages N21 and G65 were blurred with haloes.
Phage morphology
Purified phages were examined by transmission electron microscopy (TEM) and classifed based on the criteria proposed by Ackermann [23]. TEM observation revealed that all the phages (Fig. 1B) had tails and thus belonged to the order Caudovirales. Phages N21, W3 and G65 possessed a morphology typical of the Myoviridae family, displaying an icosahedral head with the diameter of (62.6 ± 1.9) nm, (64.9 ± 3.2) nm and (58.8 ± 4.1) nm, respectively, a contractile tail with the length of (153.1 ± 6.2) nm, (154.1 ± 1.4) nm and (152.3 ± 9.8) nm with six long fibers, respectively, and collar and base plate structures. Phages Y71 and Y81 morphologically belonged to the Podoviridae family, possessing an icosahedral head of (62.8 ± 1.1) nm and (54.8 ± 2.9) nm in diameter, respectively, and a short tail of (22.0 ± 0.2) nm, (20.0 ± 2.2) nm in length, respectively.
Host ranges
The lytic spectrum of five phages was determined against nine Aeromonas species of a total of 205 isolates, including 75 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 (Table 1), representative of major Aeromonas species pathogenic in fish. As shown in Table 2, it was found that 22.67% (n=17), 21.33% (n=16), 21.33% (n=16), 20% (n=15 ) and 22.67% (n=17) of 75 A. hydrophila isolates were susceptible to phages N21, W3, G65, Y71 and Y81. Additionally, all of the phages could infect one strain of A. caviae; phages N21, W3 and G65 also showed infectivity to A. veronii of 1, 3, and 3 strains, respectively; and phage W3 was able to infect the only A. bestiarum isolate tested in this study, showing broad infectivity against phylogenetically distant species in Aeromonadaceae.
Multiplicity of infection (MOI)
A. hydrophila cultures of exponential growth phase were infected with different amount of phages as designed. The phage titers were measured after incubation for 2 h. The results indicated that the optimal MOIs of phage isolates N21, W3, G65, Y71 and Y81 were 0.01, 1, 0.001, 0.1 and 0.001, respectively, which gave the highest production of phage progeny (Table 3).
Latent times and phage burst sizes
Single step growth experiment was performed to determine the latent time and phage burst size. As shown in Fig. 2, the latent periods of all five phages were found to be about 15 min, and the burst sizes of phages N21, W3, G65, Y71 and Y81 were 316 PFU, 160 PFU, 210 PFU, 200 PFU and 220 PFU per infected host cell, respectively.
pH and thermal stability
The pH and thermal stabilities of phages were estimated by determining the changes in survival based on the number of plaque-forming units (PFU). As shown in Fig. 3, growth of phage N21 showed no obvious change after 2 h incubation at pH5.0-11.0, but 75.26% recovery at pH4.0. The survival of phage W3 could maintain relatively stable at pH4.0-10.0; very few phages could recovery at pH3.0 or pH11.0. Phage Y81 displayed similar pH stability to phage W3. More than 75% phage G65 could survive at pH4.0-11.0. Phage Y71 showed relatively stable between pH5.0-10.0. The data suggested that the phages can remain active under a wide range of pH conditions but sensitive to strong acid or alkali.
Thermal stability of the isolated phages was assayed at pH7.0. All of the five phages maintained almost 100% infectivity after cultured at 4 °C or 30 °C for one day (data not shown). Fig. 4 showed that all the phages remained relatively stable at 30°C and 40°C, but sensitive to higher temperatures. No more than 50% phages remained alive after a 40-minute incubation at 50°C. At 60°C, no more than 1% of phages W3, G65, and Y81 survived for 20 minutes, and phages N21 and Y71 for 40 minutes.
Bacteriolytic activity in vitro
The bacteriolytic activities of five phages were evaluated using A. hydrophila strains NJ-35 and XY-16 at different doses of MOIs. As shown in Fig. 5, the absorbance of NJ-35 and XY-16 cultured without phages increased continuously within 24 hours, whereas the absorbance of the cultures with the phages increased gradually during the first 2 h, then decreased remarkably in a MOI-dependent manner (2-6 h), and at 6 h, dropped to the minimum at all different MOIs. From 6 h to 24 h, the absorbance of the cultures with phage G65 or Y81 remained stable at its lowest level. However, notably, the absorbance of the cultures began to rise remarkably from 12 h after treatment by phage N21, W3, or Y71, indicating a possibility of the presence of phage-resistant bacteria.
Effect of phages on bacterial biofilm formation
The capability of the phages to prevent biofilm formation of A. hydrophila NJ-35 and XY-16 was detected at 24 h after coculture using crystal violet method. As shown in Fig. 6, compared to A. hydrophila cultured alone, the bacteria cocultured with phages showed a considerably decreased biofilm formation at each MOI, and the decrease was MOI-dependent. At a MOI of 1.0, all the phages exhibited a significantly inhibited effect on the biofilm formation of their corresponding stains (P<0.05 or P<0.001). Interestingly, similar to what was observed in bacteriolytic activity, phages G65 and Y81 had stronger abilities to prevent biofilm formation than the other three phages. Very few biofilm could be detected when the A. hydrophila was incubated with G65 or Y81 even at a small MOI of 0.01.
Biofilm clearance ability
Decrease of mature biofilm formed by A. hydrophila strain NJ-35 or XY-16 treated with phages with a titer of 1.0 × 108 PFU/mL was determined. As shown in Fig. 7, phage treatment caused an obvious reduction in biofilm biomass of A. hydrophila in a time-dependent manner, as compared with untreated controls, except for the treatment with phage Y71 which resulted in a slight decrease with no statistical difference. Phages G65, W3 and N21 showed a relatively stronger biofilm clearance efficiency after treating for 24 h, with the clearance rates of 75.12%, 73.35% and 67.08%, respectively, whereas phages Y81 and Y71 only exhibited biofilm removal of 34.26% and 12.47%, respectively.
Phage therapy
To corroborate whether the phages can prevent the proliferation of A. hydrophila in vivo, we performed an infection and therapy assay in mice. Mice infected with A. hydrophila were sacrificed at 6 h and 24 h post-treatment with phages, and hearts, livers, spleens, lungs and kidneys were collected for determination of bacterial loads. As shown in Fig. 8, phages N21 and Y81 treatment lead to significant decrease of the bacterial loads in all tested tissues at 6 h compared to the non-treated controls. After treating for 24 h, the bacterial loads in all tissues of phage-treated groups showed significant decrease compared to the non-treated controls. The results indicated that the phages can act as scavengers to eliminate pathogens in vivo.