New approach to use phage therapy against Aeromonas hydrophila-induced Motile Aeromonas Septicemia in Nile tilapia


El-Araby DA et al.

Infections with Aeromonas hydrophila is a progressive problem in aquaculture. The use of antibiotic such as Ciprofloxacine has contributed to the rapid and effective treatment of disease cause by this organism. However the Fast-paced increase of resistance to the Said antibiotics has posed problems and there is now a new approach to look for alternative method to control this bacterial pathogen. Phage therapy comes in as a new method to respond to these growing problems. This study demonstrated the promising action of isolated bacteriophage ΦZH1 and ΦZH2 for therapy against Motile Aeromonas Septicemia in Nile tilapia caused by Aeromonas hydrophila.
Egyptian aquaculture has developed rapidly in recent years, where there are many problems facing fish, one of them is bacterial infection for fish, which constitutes a huge menace for aquaculture farming, leading to disastrous economic loss and health risks for the consumer [1].
Aeromonas hydrophila a gram negative, rod shaped enterobacterium and distributed widely in aquatic environments [2]. It is one of the most important agents of the outbreaks in fresh water fish. The main problem involving the use of antibiotics against Aeromonas infections is the development of resistance by these bacteria [3].
A bacteriophage is a virus that infects bacteria and can either instantly kill a bacterial cell or integrate its DNA into the host bacterial chromosome [4]. If the phage DNA is integrated into the host, the phage can then stay within the bacteria causing no harm. This pathway is called the lysogenic cycle. On the other hand, the phage can also cause eventual lysis and death of the host after it reproduces inside the host and escapes with numerous progeny through the lytic cycle [5]. Phages are effective against multidrug resistant pathogenic bacteria because the mechanisms by which they induce bacteriolysis differ completely from those antibiotics. Moreover, phages have self-limitation, meaning that the number of phages remain in very low level after killing the target bacteria [6].
The role of bacteriophages in the environment has been the subject of intense investigation over the past several years [7]. The development of techniques to study natural viral populations in situ has progressed tremendously. Various aspects of bacteriophage ecology in nature-including abundance, role in microbial mortality and water column trophodynamics, viral decay rates, repair mechanisms and lysogeny are gradually being understood [8]. Much of research has focused on using phages to control diseases caused by a variety of human pathogenic bacteria including Salmonella [9], Listeria [10] and Campylobacter [11] species. In addition, the current attempts to apply phages in the control of human pathogens, aquatic animal pathogens have also been investigated as a target for phage therapy.
A number of phages have been isolated for potential use in phage therapy against important aquatic animal pathogens such as Aeromonas salmonicida in brook trout (Oncorhynchus fontinalis) [12], Vibrio harveyi in shrimp (Penaeus monodon) [13], Pseudomonas plecoglossicida in ayu (Plecoglossus altivelis) [14] and Lactococcus garvieae in yellowtail (Seriolaquin queradiata) [14].
The efficiency of an Aeromonas hydrophila bacteriophage isolated from ponds of Abbassa was compared to that of the antibiotic Ciproflixacine for the treatment of “Motile Aeromonas Septicemia” (MAS) in Oreochromis niloticus [15]. Hence, this study aims to isolate and identify of A. hydrophila of lytic phages and efficiency of phages to control A. hydrophila in aquria.
Material and Methods
Isolation of Aeromonas hydrophila bacteriophages
Bacteriophages were isolated from sewage samples by the specific enrichment method of Adams. The supernatant were filtered through a 0.45 µm pore size syringe filter and assayed for phage activity by double layer agar technique. The presence of phage in filtrate was detected by spot test and plaque assay methods as described by Eisenstark [16]. Phages were propagated and purified from single-plaque isolates according to Adams [17]. Plaques were distinguished by differences in plaque morphology, size and turbidity and were purified by successive single plaque isolation using the propagating host strain. Afterward, Phage suspensions of high titer lysates were prepared in two ways first Confluent plate lysates were prepared according to the method of Eisenstark [16].
Physical characterization of isolated bacteriophages
Effect of temperature on isolated phage: The effect of temperature on the viability of phage was studied by the method described by Clokie [18]. Phage suspension was incubated at 30, 40, 50, 60, 70, 80 and 90ºC in water bath for 10 min. Phages survival was determined Effect of irradiation by ultra violet light on the isolated bacteriophages: The effect of UV light on the viability of phages was studied by method described by Clokie [18]. UV sensitivity was determined by exposing 5 ml of phage lysate (4.8×10 11  pfu/ml and 5.0×10 11  pfu/ml) for phage ΦZH 1 and ΦZH 2 respectively (diluted 0.1 in saline solution) in an uncovered small Petri dish to UV-light at distance 20 cm from Cosmolux UVA, A1-11-40 W, PREHEAT- BIPIN, Mode In W-Germany, lamp was used as a UV source for the following times: 20, 40, 60, 80,100 and 120 minutes. Phages survival was determined by plaque assay technique [17].
Biological Characteristics
Host range
The host ranges of the phages were determined by spot tests [16]. The adsorption experiments and single step growth curve of phages were carried out as described by Adams [17].
Effect of different MOI on bacterial growth
Multiplicity of infection (MOI) was defined as the ratio of virus particles to potential host cells and prepared according to Birge [19]. Morphological characteristics (Electron microscopy) High stock titer of phages (4.8×10 11  pfu/ml, 5.0×10 11  pfu/ml ) were negatively stained with 2% (w/v) aqueous uranyl acetate (pH 4.0) on a carbon-coated grid and examined by transmission electron microscopy JEOL( JEM-1400cx) at an accelerating voltage of 80 KV.
Effect of bacteriophages on mortality of Nile tilapia by Aeromonas hydrophila infections
Nile tilapia (O. niloticus) (weight range: 25-40 g) were obtained from ponds of Fish Research Center of Abbassa, Abo- Hammad, Sharkia. “Where these fishes were transferred alive to Microbiological laboratory, Faculty of Veterinary Medicine, Zagazig University. All fishes were kept in tanks (40 cm×70 cm×60 cm) with approximately 45 l de-chlorinated tap water. Acclimatized for 1 week prior to the experiment and fed with organic feeds. Four aquarium were used for the experiment and each aquarium contained 5 fishes. The aquaria were maintained at 28 ± 1°C with a pH of 7. Bacterial inoculum of A.hydrophila was prepared using a 24 hrs old culture of A. hydrophila inoculated in TSB. The inoculum was subjected to to 10 -7 , 10 -8 and 10 -9 dilutions. These were transferred to falcon tubes and were centrifuged for 30 min at 3,000×g. After centrifugation, the supernatant was removed and 5 mL of normal saline solution (0.9%) was added. The lethal dose (LD) of A. hydrophila was determined by intraperitoneally injecting the fishes with 0.5 mL of doses [20]. The dose sufficient to cause death among the fishes within 72-96 h was taken as the optimum LD100 (lethal dose causing 100% mortality). Clinical signs of MAS such as skin lesions, hyperemia, rotting of caudal and dorsal fins, and hyperemia in fin bases were observed prior to the experimental treatment [21].
The mortality in the tanks was monitored after 15 days for each challenge. The concentration of bacteria and phage in the water tanks was monitored by inoculating the corresponding dilution in TSA plates to detect the bacteria, and using the double-layer agar plaque assay to determine the phage concentration [17].
Two groups only were injected with A. hydrophila. Group 1, which contain water only. Group 2, which contain water and fishes served as the negative control. Group 3, which was used as positive control contain water and fishes were injected with A. hydrophila but was not treated with bacteriophages. Group 4, was also injected with A. hydrophila and was treated with bacteriophage. Administration of bacteriophage (Group 4) was done 24 hrs after injection of A.hydrophila.
Morphology of plaques and phage(s)
Result in Table 1 and Figures 1 and 2 showed that two phenotypic plaques were appeared. One of them remarked as ΦZH 1  which measured 3.0 mm in diameter with turbid center (LTC), while other remarked as ΦZH 2 which measured 4.0 mm in diameter with clear center (LCC).
Morphology of isolated plaques under electron microscope
Five successive transfer plaques with clear area and center were selected to prepare height titer phage stocks (4.8×10 11  pfu/ml) and (5.0×10 11  pfu/ml). Each phage stock viewed under electron microscope after staining by (1%) potassium phosphotungstate at pH 6.4. Results in Table 1 and Figures 3-5 showed that, two phages were appeared. Icosahedral heads of these phages (φZH1 and φZH 2 ) measured 100 and 50 nm respectively. Also these phages had very short non-contractile tail measured 30 and 7 nm respectively. Phages (φZH1 and φZH 2 ) adsorbed on cell wall and not have receptor on flagella (Figure 6). On the basis of phage morphology, the phages φZH1 and φZH 2 belongs to the family Podoviridae [22].
Table 1:  Morphology of plaques and isolated phages under electronmicroscope
after negatively staining.
Figure 1:  Plaques morphology of A.hydrophils phage ΦZH 1  with turbid center
on the TSA Plate.
Figure 2:  Plaques morphology of A.hydrophils phage ΦZH 2  with clear center
on the TSA Plate.
Host range
The host range of isolated phages (ΦZH 1 and ΦZH 2 ) was determined against isolates of Aeromonas bacteria and 4 strains of non-Aeromonas bacteria. Results in Table 2 revealed that the isolated phages ΦZH 1 and ΦZH 2 was very specific to infect Aeromonas and does not have the ability to infect any isolates of non-Aeromonas bacteria.
Effect of thermal inactivation
The infectivity of both phages was highly sensitive to temperature above 40°C. Whereas both phages lost its infectivity by percentage reached to 88% and 50% for ΦZH 1 and ΦZH 2  respectively (Figure 7).
Effect of irradiation by ultraviolet light on the isolated phages
The exposure of 10 11  pfu/ml purified phages suspensions to UV irradiation (at high 20 cm) for different periods of time (0-120 min) illustrated by Figure 8 from these results, isolated phages (ΦZH 1 and ΦZH 2 ) are resistant to UV irradiation. Whereas the infectivity of this phage still active after exposure to UV (40w) for 120 min. ΦZH 1 lost 50% of its infectivity after exposure to UV irradiation for 100 min While, phage ΦZH 2 reached to this percentage after exposure to UV for 80 min.
Figure 3:  Electron micrograph of A. hydrophila phage ΦMH 1 . The purified bacteriophage preparation was negatively stained with (1%) potassium phosphotungstate (pH 6.4).
Figure 4:  Electron micrograph of A. hydrophila phage aggregation of ΦMH 1 & ΦMH 2 . The purified bacteriophage preparation was negatively stained with (1%) potassium phosphotungstate (pH 6.4).
Figure 5:  Electron micrograph of A. hydrophila phage ΦMH 2 . The purified bacteriophage preparation was negatively stained with (1%) potassium phosphotungstate (pH 6.4).
Figure 6:  Electron micrograph of A. hydrophila both phage ΦMH 1 & ΦMH 2 adsorbed on cell wall (A) and not have receptor on A. hydrophila flagella (B).
Table 2:  Host range of A.hydrophila phages ΦZH 1  and ΦZH 2 .
Figure 7:  Effect of temperature on phage ΦZH 1 and ΦZH 2 . Phages was incubated at different temperature for 5 min and the phage infectivity was investigated.
Figure 8:  Effect of time exposure to UV irradiation on ΦZH1 1 and ΦZH 2 phages.
Adsorption rates
The two phages in current studies ΦZH1 1 and ΦZH 2  exhepted to different rates. The maximum adsorption and percentage of adsorption are presented in Table 3. Adsorption rate were fast since the maximum phages adsorbed reached 51% and 66.8% of phages ΦZH1 1 and ΦZH 2 were adsorbed after 20 and 30 min respectively the adsorption constant (K) were (2.7×10 -13 ml/min) and (2.2×10 -13 ml/min) for ΦZH1 1 and ΦZH 2 respectively as determined by the formula K=2.3/ (B) t × log (Po/P), where Po= phage assay at zero time, P= phage not adsorbed at time t min, (B) = concentration of bacteria as number of cells/ml and K=velocity constant expressed as ml/min. Adsorption rate constant for both phages were similar.
One-step growth experiment
One step growth curve (Figure 9) shows that the latent period was about 20 min, the rise period was 60 min, and the mean burst size was about 113 and 114 pfu per infected cell for ΦZH 1  and ΦZH 2  respectively.
Effect of different concentration of phages on growth of A. Hydrophila
Each phage was used at MOI = 10, 1 and 0.1 over a time from 0-24 hrs. Data in Table 4 showed that, the highest reduction in bacterial count was observed when phage ΦZH 1  and ΦZH 2  added separately to A.hydrophila at M.O.I = 10 and incubated at 37°C for 12 hrs. On other hand, addition of phage ΦZH 1  or ΦZH 2 to A.hydrophila at M.O.I less than 10 (1.0 or 0.1) not gave efficient in reduction of bacterial growth.
The challenges to motile Aeromonas Septicemia (MAS) causing bacteria by both ΦZH 1  and ΦZH 2
Result in Table 5 showed that the addition of A. hydrophila to fishes in aquaria containing Nile water increases the mortality compared to it control. 2 nd aquaria mortality of this treatment reached 68%. Addition of phage to such treatment reduced the mortality to 18% with reduction efficiency reached above 50%. Also phage reduced total count of bacteria from 4.8×10 13  cfu/ml to 7.3×10 7 cfu/ml. on other hand, phages titer in fourth treatments increased above the initial titer of phages it changed from 8.1×10 9 to 8.1×10 13  pfu/ml.
Fish diseases are major problem for fish farming industry and among those bacterial infections are considered to be a major cause of mortality in fish [23]. These awaited drawbacks enforced the fish pathologists to seek for other alternatives; the use of natural immunostimulants in fish culture for the prevention of diseases was a promising new development and could solve the problems of massive antibiotic use. Natural immunostimulants were biocompatible, biodegradable and safe for both the environment and human health. Moreover, they possess an added nutritional value [24].
Table 3:  Adsorption rate of A.hydrophila phages ΦZH 1  and ΦZH 2 in TS broth at 37ºC MOI >1.0
Figure 9:  One step growth curve of A. Hydrophila phages ΦMH in TSB at 37ºC. Initial titer of ΦZH 1 =4.8×10 11 (pfu/ml) and Initial titer of ΦZH 2 =5.0×10 11 (pfu/ml) and MOI<1.0.
Table 4:  Effect of incubation of several different multiplicity of infection (M.O.I) on growth curve of the host A.hydrophila.

Table 5:  The challenges to Motile Aeromonas Septicemia (MAS) causing bacteria by both ΦZH 1  and ΦZH 2 .
Aeromonas hydrophila was described as the dominant infectious agent of ‘fish-bacterial-septicemia’ in freshwater cultured finfish all over the world [25]. A hydrophila was also associated with EUS, which was a major problem in different countries [26]. The observed clinical signs in the examined fish suffering from motile Aeromonas Septicemia (MAS) were previously reported by Samal [25]. They reported that septicemia, ascitis, erosion, ulceration, detachment of scale, exophthalmia and muscular necrosis were the most predominant clinical signs of MAS in Nile tilapia.
In this study, the results showed that two bacteriophages ΦZH 1 and ΦZH 2 possess infection on its specific host A.hydrophila, isolated from Nile water. According to the electron micrograph; the two phages were characterized as podoviruses. The dimensions of the isolated podoviruses were similar or semi-similar to each other and also resembled those which were previously isolated for A.hydrophila [27]. A hydrophila phages ΦZH1 and ΦZH2 infected some Ahydrophila strains, but none of other genera or species tested. These results are in accordance with those obtained by Mitchell Sc [28].
Temperature is a crucial factor for bacteriophage survivability [29]. The results in showed that ΦZH 1 and ΦZH 2 phages were thermos table, between a temperature ranges of 30-60°C where it still remained active after 10 min exposure at 60°C. Interestingly, ΦZH 1 and ΦZH 2 phages survived at 37°C, with no significant loss in phage particle number, which is a very important parameter for phages considered for therapeutic application.
Phages examined here were tolerant to UV irradiation with distinct rate of inactivation where ΦZH 1 and ΦZH 2 lost 50% of their infectivity after exposure time reach to 100 and 80 min respectively. These result are in accordance with those obtain by Ramanandan [29]. This finding indicated that phages are suitable to use in field experiment where their infectivity not affected by UV in solar reached to water in aquarium.
The data obtained in one- step growth experiment were comparable with data presented by Cheng [30]. These authors conducted a similar growth experiment with Aeromonas species. It as reported by those authors that the latent periods of phage DH1 were 90 min which was much longer than Aeromonas hydrophila phages Aeh1 and Aeh2 [28] on other hand in our studies the latent period of phages ΦZH1 and ΦZH2 were 20 min. the average burst size of phage DH1 were about 125 PFU/cell, which was also bigger than Aeh1 and Aeh2 [28], but the latent period of phages ΦZH1 and ΦZH2 were 113 and 114 pfu per infected cell. The isolated phages (ΦZH 1 and ΦZH 2 ) administered via injection was found to be effective in treating fish infected with Aeromonas hydrophila shown through the significant decrease in number of A.hydrophila found in the water of treated fish. Where our results showed that the addition of phages ΦZH 1 and ΦZH 2 (M.O.I=2.1) to Nile water in aquaria. Which was inoculated by A.hydrophila (3.37×10 9 CFU/ml) reduced the percentage of mortality from 68% to 18% after treatment for 15 days. Also total number of bacteria in polluted aquarium changed from 4.18×10 13  cfu/ml to 7.5×10 7  Cfu/ml after three days of treatments. The efficiency of isolated phages reduction of A.hydrophila in Nile water more than that founded by Donn Cruz-Papa [15].  


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Article made possible through the contribution of El-Araby DA et al.

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