Abstract: Background and Objective: Antibiotics have been used to treat Aeromonas hydrophila infections in fish farming. However, their extensive uses can cause many negative effects including the development of drug-resistant bacterial strains. The main objective of this study was to find an alternative to antibiotics to inhibit A. hydrophila both in vitro and in vivo. Materials and Methods: A bacteriophage infecting A. hydrophila was isolated from a fish a pond water sample. It was classified based on its genome type studied by enzymatic digestion and morphology investigated by transmission electron microscopy. Its ability to control experimental A. hydrophila infection in tilapia (Oreochromis niloticus) was examined by feeding tilapia with fish diets supplemented with different titers of the bacteriophage. Results: A bacteriophage specific to Aeromonas hydrophila UR1 designated PAh4 was isolated and classified as a member of the family Myoviridae. When tilapia experimentally infected with A. hydrophila at the median lethal dose (3.16×105 CFU per fish) were fed the fish diets supplemented with the bacteriophage PAh4 at doses ranging from 105-108 PFU g–1 of diet, the diets could reduce the mortality rate of infected tilapia in a dose-dependent manner. Conclusion: The bacteriophage PAh4 can be used as an alternative to antibiotics to control A. hydrophila infection in tilapia.
INTRODUCTION
Aeromonas hydrophila is a rod-shaped, gram-negative, motile, facultative anaerobic bacterium. It is known to infect fish, reptiles and amphibians and human1. Aeromonas hydrophila causes disease in fish known as Motile Aeromonas Septicemia (MAS), Haemorrhagic Septicemia, Ulcer Disease, or Red-Sore Disease. The disease is characterized by the presence of small surface lesions leading to sloughing off the scales, hemorrhaging in the gills and anus, ulcers, exophthalmia, dropsy, the presence of ascitic fluid in the peritoneal cavity and swelling of the kidney and liver2. The occurrence of the disease relates to stress conditions of fish which can be arisen when fish are mishandled, overcrowded, transported under poor conditions, grown in poor water quality and reared with poor nutritional status. The disease primarily affects freshwater fish such as channel catfish (Ictalurus punctatus) and tilapia (Oreochromis niloticus), several species of bass including striped bass (Morone saxatilis) and largemouth bass (Micropterus salmoides) and many species of tropical and ornamental fish3. In Thailand, A. hydrophila infection is one of the major causes of economical damage to fish producers, especially tilapia farmers.
The most common approach for the treatment of A. hydrophila infection in fish farming is the use of chemotherapeutic agents, especially antibiotics. Many of them are used to control fish disease including oxytetracycline, chloramphenicol, furanace, florfenicol, oxolinic acid, piromidic acid, thiamphenicol, sulphonamide, nitrofuran derivatives and pyridine carboxylic acids4,5. However, the use of antibiotics in aquaculture has many negative impacts. It can result in the development of drug-resistant bacteria and therefore to reduce the efficacy of the drugs. The accumulation of antibiotics in the environment and the fish can cause potential risks to consumers and the environment6,7. Due to the adverse effects of antibiotics, the search for a safe and environmentally friendly strategy to control the fish disease caused by A. hydrophila infection has become a major issue of study for many research groups.
Bacteriophages (or phages) are viruses infecting bacteria. They have very narrow target spectra and some phages may be active against only a specific strain8. This high degree of specificity allows phages to be used against targeted microorganisms in a mixed population without disturbing the microbial ecosystem. Based on their characteristics, phages are interesting choices to replace antibiotics for treating A. hydrophila infection in fish. There have been many reports describing phages of fish pathogenic bacteria suggested that phages can be useful for controlling bacterial infections in fish9-11.
In this study, a phage infecting A. hydrophila was isolated and characterized. The isolated phage was examined for the ability to control experimental A. hydrophila infection in tilapia (Oreochromis niloticus) which is one of the most cultured fish species in Thailand with a high incidence of A. hydrophila infection.
MATERIALS AND METHODS
Study area: The study was carried out at the Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Thailand from January, 2018-December, 2019.
Bacterial strains and culture conditions: Eleven strains of Aeromonas hydrophila were used in this study. Ten of them designates as UR1-UR10, were isolated from kidneys of diseased tilapia collected from 6 different fish farms in Ubon Ratchathani province, Thailand. The other strain of A. hydrophila, A. hydrophila ATCC 700183, was obtained from the American Type Culture Collection (ATCC). The other twelve fish pathogenic bacteria listed in Table 1 were used to determine the phage host range. Trypticase Soy Broth (TSB) and Trypticase Soy Agar (TSA) were used for culturing the bacterial strains. Bacterial stock cultures were stored as frozen cultures at -80°C in TSB containing 20% glycerol (v/v).
| Table 1: | Host range of phage PAh4 |
| aATCC: American type culture collection, b+: Lytic activity against bacterial host, -: No lytic activity against bacterial host | |
Phage isolation and enrichment: In this experiment, A. hydrophila UR1 was used as a bacterial host for phage isolation. Phage was isolated from water collected from ponds where the diseased fish used in this study were obtained. Phage isolation and enrichment were performed as previously described12.
Phage detection and host range: Phage enriched samples were initially tested for the presence of phage by using the spot-on-lawn method12. Four milliliter of soft TBA (0.4% agar) was inoculated with 100 μL of a log phase culture of A. hydrophila UR1, mixed gently and poured onto a TSA plate. Ten microliter of each phage enriched sample was spotted on the solidified soft agar. The plate was incubated at 30°C for 24 hrs before checking for a clear zone at the position on which the phage enriched sample was spotted. A clear zone in the plate, resulting from the lysis of host cells, indicated the presence of phage. The spot-on-lawn assay was also used to determine the phage host range against all fish pathogenic bacteria listed in Table 1.
Phage titer determination: The phage enriched sample producing a clear zone against A. hydrophila UR1 was subjected to phage titer determination by using plaque assay12. The phage titer was expressed as plaque-forming unit per milliliter (PFU mL–1).
Phage purification: The phage of interest was purified from a phage enriched sample by using the following protocol described previously12. The resulting purified phage was called phage suspension.
Analysis of phage morphology and phage genome: Phage morphology was studied by transmission electron microscopy as mentioned in our previous report12. Phage genome was analyzed by enzymatic digestion with S1 nuclease, RNase A and PvuI12.
Fish preparation: Tilapias of mixed sexes were obtained from Nong Khon Farm (Ubon Ratchathani, Thailand). They were maintained in 500 L plastic containers at 30°C, subjected to a 12 hrs light/12 hrs dark cycle and fed a commercial fish diet (Thai Spring Fish Co. Ltd., Rayong, Thailand) for 2 weeks before experiments. To verify that the fish were free of bacterial infection, they were randomly sampled and their livers and kidneys were aseptically streaked on TSA and incubated at 30°C for 24 hrs.
All experiments with the fish were conducted in 45 L aquaria at 30°C. Fish weighing 10±1 g were stocked in the aquaria (10 fish per aquarium) 24 hrs before the experiments. The commercial tilapia diet was supplied twice daily at the rate of 5% of fish body weight per day.
Examination of Pathogenicity of A. hydrophila: The pathogenicity of A. hydrophila UR1 was examined using the method previously described by Phumkhachorn and Rattanachaikunsopon13. The median lethal dose (LD50) was calculated by the method of Reed-Muench14 using the following Eq.:
log LD50 = [α log b]+c
where, α is the mortality rate >50%-50%/mortality rate >50%-mortality rate <50%, b is the dilution rate (10–1) and c is the log of minimum dilution rate in which the mortality rate was >50%.
Fish diets preparation: Fish diets supplemented with a phage suspension and oxytetracycline were prepared. Diets 1-5 were prepared by mixing a phage suspension with the commercial fish diet. The final concentrations of the phage in Diet 1, 2, 3 and 4 were 105, 106, 107 and 108 PFU g–1 of diet, respectively. Diet 5 was the fish diet supplemented with oxytetracycline at the concentration of 0.5% (w/w). The control diet (Diet 6) was prepared using the same process as the other fish diets except for no added phage or oxytetracycline.
Fish feeding experiment: To study the effect of phage supplemented fish diets on A. hydrophila UR1 infection in vivo, the following experiment was conducted. Groups of 10 uninfected fish were fed Diets 1, 2, 3, 4, 5 and 6 separately for 10 days. On the eleventh day, the fish were infected with A. hydrophila UR1 by intraperitoneal injection at a dose causing 50% mortality (LD50). The fish continued to be fed the assigned diets for 10 days. Mortality was observed daily from the day of bacterial injection which was considered as day zero. Dead fish were removed from the aquaria daily and their livers and kidneys were subjected to bacterial isolation on TSA to examine the presence of A. hydrophila. Bacterial isolation was also performed with livers and kidneys of surviving fish to confirm that they were free of A. hydrophila infection. The experiment was conducted in five replicates.
RESULTS
Phage detection and characteristics of plaques: From the initial screening of pond water samples for a phage infecting Aeromonas hydrophila UR1 using spot-on-lawn assay, one sample gave the positive result with a clear inhibition zone indicating that the detected phage was a lytic phage. The phage was designated PAh4. When the phage was subjected to the plaque assay, it formed small, clear round plaques (about 1.5 mm in diameter) on the lawn of A. hydrophila UR1.
Phage host range: Spot-on-lawn assay was used to examine the ability of the phage PAh4 to infect various strains of fish pathogenic bacteria listed in Table 1. All of the strains of A. hydrophila used in this study were lysed by the phage. However, other strains of Aeromonas and the rest of the fish pathogenic bacteria were not susceptible to the phage.
Phage morphology: The ultrastructure of phage PAh4 examined by transmission electron microscopy revealed that it was a tailed phage (Fig. 1). The phage had an isometric head (about 102 nm in diameter) and a short contractile tail (about 246 nm long and 18 nm wide) with a base plate at the end of the tail.
Analysis of phage genome: The phage PAh4’s genome was tested for its sensitivity to several enzymes digesting nucleic acid and checked by agarose gel electrophoresis. It was found that the genome was digested by PvuI but not by S1 nuclease and RNase A (Fig. 2). Sizes of the bands resulting from digesting the genome with PvuI were approximately 10, 5.5, 3.5, 2.5 and 1.2 kb.
Examination of pathogenicity of A. hydrophila UR1: Pathogenicity of A. hydrophila UR1 for tilapia is shown in Table 2. All of the dead fish died within 5 days after bacterial injections and the pathogen was found in their livers and kidneys. In Table 2, mortality rates obtained from injecting tilapia with A. hydrophila UR1 at the doses of 105 and 106 CFU per fish were 40 and 60%, respectively. The median lethal dose (LD50) of A. hydrophila UR1 for tilapia calculated from these results was 105.5 CFU per fish or 3.16×105 CFU per fish.
Fish feeding experiment: Before testing the effects of fish diets supplemented with the phage PAH4 and oxytetracycline on tilapia infected with A. hydrophila, all of the diets (Diet 1-6) were fed separately on uninfected fish twice a day for 10 days.
| Fig. 1: | Transmission electron micrograph of phage PAh4 showing its head (H) and tail (T) |
| Bar = 50 nm | |
| Fig. 2: | Analysis of genome extracted from phage PAh4 using agarose gel electrophoresis. Lane 1: DNA standard (1 kb ladder, New England Labs), Lane 2: Cut with PvuI, Lane 3: Cut with S1 nuclease, Lane 4: Cut with RNase A and Lane 5: Uncut |
It was found that the diets had no adverse effect on the fish based on mortality, appearance, feeding response and behavioral alterations of the fished which were observed daily. When A. hydrophila infected tilapia fed diets supplemented with the phage (Diet 1-4), reduction in mortality of the fish was observed in a dose-dependent manner (Fig. 3).
| Table 2: | Mortality of tilapia intraperitoneally injected with different dilutions of A. hydrophila UR1 suspension |
| aResults from all replicates (replicate 1-5) | |
| Fig. 3: | Effect of fish diets supplemented with phage PAh4 at doses of 105 (Diet 1), 106 (Diet 2), 107 (Diet 3), 108 (Diet 4) PFU g–1 of diet and oxytetracycline (Diet 5) on the mortality rate of A. hydrophila UR1 infected tilapia compared with the control fish diet (Diet 6) |
Moreover, the mortality of the infected fish treated with Diet 4 (containing the phage at the concentration of 108 CFU g–1 of diet) was not different from that treated with Diet 5 (containing 0.5% (w/w) of oxytetracycline).
DISCUSSION
Recently, the use of antibiotics to control diseases in fish aquaculture has raised a major concern on their safety to the environment, farmers and consumers15. Phage therapy for the fish disease has come to our attention because bacteriophages have several characteristics suitable to replace antibiotics. Bacteriophages are generally present everywhere including foods and water we consumed. Since they infect only a specific host, the use of them to control pathogenic bacteria in food animals does not disturb normal flora normally residing in animals and consumers. Phage therapy has been successfully used to control diseases in a wide range of animals including mice16, cattle, poultry, pigs17 and fish18.
The pathogenic bacteria used in this study were A. hydrophila isolated from tilapia suffered by Motile Aeromonas Septicemia cultured in 6 different fish farms in Ubon Ratchathani Provinces, Thailand. Aeromonas hydrophila UR1 was selected to be a representative of all isolated pathogenic bacteria to be used as a major host throughout this study. Its pathogenicity was confirmed by the determination of its median lethal dose (LD50) for tilapia which was 3.16×105 CFU per fish. Because bacteriophages and their hosts are generally present in the same environment, we decided to use water from ponds where all A. hydrophila used in this study were isolated as samples for searching a phage specific to the bacteria. By using A. hydrophila UR1 as a host, the phage PAh4 was isolated and found to be able to infect the host bacterium.
The phage PAh4 had a narrow host range. It specifically infected A. hydrophila. Aeromonas in different species and the bacteria in different genus used in this study were not susceptible to the phage. Phages with narrow host ranges might be more suitable as biocontrol agents than those with broad host ranges because they are likely to cause less harm to normal flora. Although phages with narrow host range sometimes cause limitations in their use, this problem can be overcome by using cocktails or combinations of several phages. For example, Mateus et al.19 reported the success of using phage cocktails (containing two or three phages of VP-1, VP-2 and VP-3 phages) to control fish pathogen Vibrio parahaemolyticus.
Information on the phage genome and morphology are necessary for phage classification. The sensitivity of phage PAh4’s genome to PvuI (but not to S1 nuclease and RNase A) suggested that its genome was double-stranded DNA. Transmission electron microscopy revealed that the phage PAh4 was a tailed phage with an isometric head and a noncontractile tail. According to the International Committee on Taxonomy of Viruses20, tailed phages with double-stranded DNA are classified in the Caudovirales order. This order contains three families including the Myoviridae (with long, contractile tail), the Siphoviridae (with long, noncontractile tail) and the Podoviridae (with short tail). Based on its nucleic acid and morphological characteristics, the phage PAh4 was tentatively classified as a member of the Myoviridae family. Phages specific to fish pathogenic bacteria previously reported did not only exist in the Myoviridae family but also in the other two families of the Caudovirales order10,21,22.
In vivo experiments were conducted by feeding tilapia with fish diets supplemented with phage at doses ranging from 105-108 PFU per fish. The phage supplemented fish diets were shown to be able to reduce the mortality rate of tilapia experimentally infected with A. hydrophila in a dose-dependent manner. Besides, all of the tested fish diets had no adverse effect on the fish. No difference was found in mortalities between the fish treated with phage at the dose of 108 PFU per fish and those treated with oxytetracycline. The results suggest that there is therapeutic potential to phage PAh4. It could be used to replace antibiotics to control the disease of tilapia. Experiments to study the use of phage PAh4 supplemented fish diets in tilapia farm against natural A. hydrophila infections is underway in order to develop a control treatment for the disease in aquaculture of tilapia.
CONCLUSION
It can be concluded that there is therapeutic potential to phage PAh4. It could be used to replace antibiotics to control the disease of tilapia. Experiments to study the use of phage PAh4 supplemented fish diets in tilapia farm against natural A. hydrophila infections are underway to develop a control treatment for the disease in aquaculture of tilapia.
SIGNIFICANCE STATEMENT
This study discovers a new phage designated as PAh4 that can be beneficial for control infection of Aeromonas hydrophila in aquaculture. This study will help the researcher to develop environmentally-friendly aquaculture by using the phage as an alternative to antibiotics that many researchers were not able to accomplish. Thus, antibiotic-free aquaculture is proven to be possible.