Antibiotic susceptibility and pathogenicity of Aeromonas hydrophila isolated from red hybrid tilapia (Oreochromis niloticus×Oreochromis mossambicus) in Malaysia

Background and Aim: Aeromonas hydrophila is a major cause of bacterial infections affecting a wide range of warm water fishes worldwide. In Malaysia, A. hydrophila isolations from diseased fishes were previously reported; however, with limited information. The present study investigates the antibiotic susceptibility and pathogenicity of A. hydrophila isolated from farmed red hybrid tilapia (Oreochromis spp.) in Malaysia. Materials and Methods: A. hydrophila was biochemically identified and subjected to antibiotic susceptibility tests. The isolate was then intraperitoneally injected into red hybrid tilapia, and the mortality, clinicopathological changes, and LD50 were determined up to 240 h post-infection (hpi). Results: The isolate demonstrated multiple antibiotic resistances (MAR) toward amikacin, ampicillin, cefotaxime, amoxicillin, trimethoprim-sulfamethoxazole, erythromycin, and streptomycin, with a MAR index of 0.5. The experimental infection of A. hydrophila at 105 CFU/mL in the red hybrid tilapia resulted in 100% mortality at 240 hpi. The LD50 was determined at 1.1×104 CFU/mL. Infected fish demonstrated occasional erratic swimming patterns, localized hemorrhages and depigmentation on the body and operculum areas, fin erosion, enlargement of the gall bladder, and hemorrhage in internal organs. Microscopic observation of infected fish revealed brain congestion, tubular necrosis, and glomerular shrinkage in the kidneys, necrosis of hepatocytes, and congestion of blood vessels in the liver. Conclusion: The high virulence of A. hydrophila to the red hybrid tilapia emphasizes the importance of active, on-going monitoring of its prevalence in Malaysian tilapia farming.


Introduction
Tilapia (Oreochromis spp.) is among the major commercially important freshwater fish intensively farmed worldwide. World tilapia production has exceeded 5 million tonnes, generating incomes of approximately USD 9.8 billion in 2015, and has been increasing annually [1]. In Malaysia, the production of tilapia in 2017 was approximately 31,400 tonnes with a wholesale value of RM 286 million, which indicates the significance of tilapia farming in the country [2]. Although tilapia culture is steadily growing, the sector frequently suffers from disease outbreaks that represent a major obstacle to its growth.
Aeromonas hydrophila is recognized as a significant pathogen which can have a devastating impact on the fish farming industry [3]; it can be potentially ubiquitous in both marine and freshwater environments, and under favorable conditions, it has emerged as an opportunistic pathogen. Infection by A. hydrophila is usually characterized by hemorrhagic ulcers on the skin and fin erosion [3]. It has been previously reported in various freshwater fish including channel catfish (Ictalurus punctatus), Siberian sturgeon (Acipenser baerii), Russian sturgeon (Acipenser gueldenstaedtii), banded knifefish (Gymnotus omarorum), and Nile tilapia (Oreochromis niloticus) [4][5][6].
In this study, we determined the antibiotic susceptibility and pathogenicity of A. hydrophila isolated from red hybrid tilapia in Malaysia.

Ethical approval
The handling of fish and all experimental procedures, in this study, was performed in accordance with the methods approved by the Institutional Animal Care and Use Committee, Universiti Putra Malaysia (AUP No.: R006/2016).

Study period and location
The study was carried out for a period of 3 months from March until June 2019 at the Department of Biology, Faculty of Science, Universiti Putra Malaysia, Selangor, Malaysia.

Bacterial strain
The A. hydrophila isolate used in this study was obtained from the Aquatic Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Malaysia. The isolate had been previously isolated from the spleen of diseased red hybrid tilapia and identified as A. hydrophila using biochemical and molecular methods [12]. Glycerol stock (−80°C) of A. hydrophila was streaked on Tryptic Soy Agar (TSA) (Merck, Darmstadt, Germany) and incubated at 30°C for 24 h. A pure colony of the isolate was subjected to Gram staining, catalase testing, oxidase testing, and the use of an API 20NE test kit (bioMérieux, Marcy l'Etoile, France) for species confirmation.
Following the positive confirmation of A. hydrophila, the isolate was then intraperitoneally (IP) injected into three red hybrid tilapias (1 mL at 10 5 CFU/mL of A. hydrophila per fish) for re-virulent purposes. The bacteria were re-isolated from moribund fish, cultured onto TSA, incubated at 30°C for 24 h, and reconfirmed as A. hydrophila.

Experimental infection study
For the pathogenicity study, ten colonies of 24 h cultures of A. hydrophila were inoculated into 100 mL of Tryptic Soy Broth (TSB) (Merck) and incubated at 30°C for 24 h. A ten-fold serial dilution was made with sterile TSB, and the standard spread-plate technique was used for bacterial enumeration.
One hundred eighty red hybrid tilapia (length: 9±2 cm) were purchased from local suppliers and kept in a tank (1500 L) for 1 week for acclimatization. No sign of disease or mortality was observed during the acclimatization period. Five fish were randomly sampled, and their gills and body surfaces were examined microscopically for the presence of parasites. Swabs were taken from internal organs and tested for bacterial presence. The samples were determined to be free from parasite and bacterial infection. Before a challenge test, the fish were distributed into five treatment groups and one control group of 10 fish. The experiment was conducted in triplicate. The fish in each of the five treatment groups were IP injected with 1 mL of A. hydrophila at concentrations increasing from 10 1 to 10 5 CFU/mL. The fish in the control group were injected with sterile TSB. Within 240 h post-infection (hpi), the clinical signs of gross lesions and mortality patterns were recorded, and the LD 50 value was calculated [16]. Swabs were taken from the organs of the diseased red hybrid tilapia for isolation and identification of bacteria.

Histopathological analysis
The livers, brains, kidneys, and eyes of moribund fish were collected and preserved in 10% buffered formalin for histopathological analysis. Briefly, the organs were processed using a tissue processor (Leica TP 1020, Leica, Germany), embedded in paraffin, sectioned at 4 µm thick (Leica Jung Multicut 2045, Germany), and stained with Harris's hematoxylin and eosin. The sections were then examined under a light microscope (Nikon Eclipse 50i, Japan) and analyzed using Nikon NIS-Element D 3.2 Image Analysis software (Nikon Instruments Inc., USA).

Results
Following identification using API 20NE, the result showed 99.9% similarity with A. hydrophila. The A. hydrophila isolate in this study formed yellowish, opaque colonies on TSA agar, produced beta-hemolysis on horse blood agar, and were motile, oxidase-and catalase-positive, and Gram-negative short rods (Table-1). The isolate tested positive for arginine dihydrolase and β-galactosidase, production Available at www.veterinaryworld.org/Vol.13/October-2020/17.pdf of nitrate and indole, and hydrolysis of β-glucosidase and gelatin. It also assimilated arabinose, capric acid, glucose, malate, maltose, mannitol, mannose, N-acetylglucosamine, and potassium gluconate.
Following IP injection, infected tilapia exhibited occasional erratic swimming patterns; hemorrhagic foci and depigmentation on body and operculum areas and Available at www.veterinaryworld.org/Vol.13/October-2020/17.pdf tail erosion were also observed (Figure-2). Internally, infected fish demonstrated enlarged gall bladders and hemorrhage of internal organs. A. hydrophila was successfully isolated from the freshly dead fish. Microscopic observation of infected red hybrid tilapia brain tissue revealed mild-to-moderate congestion (Figure-3). In addition, mild-to-moderate tubular necrosis and glomerular shrinkage in the kidneys and mild-to-moderate necrosis of hepatocytes and congestion of blood vessels in the livers of infected red hybrid tilapia were observed.

Discussion
This study reports the antibiotic susceptibility and pathogenicity of A. hydrophila isolated from diseased red hybrid tilapia in Malaysia. A. hydrophila has been responsible for significant losses in the tilapia aquaculture industry worldwide. In Malaysia, its presence has been reported in the aquatic environment, retail, and cultured fish [7][8][9][10]; however, the pathogenicity of local A. hydrophila isolate has never been investigated.
The morphological and biochemical characteristics of A. hydrophila observed in this study are consistent with previous reports [17], except for its inability to ferment glucose and assimilate adipic acid. The isolate demonstrated multiple resistances toward the tested antibiotics including amikacin, ampicillin, cefotaxime, amoxicillin, trimethoprim-sulfamethoxazole, erythromycin, and streptomycin with a MAR index of 0.5. The resistance of A. hydrophila toward the penicillin group has been previously reported in Thailand [18], India [19], and Pakistan [17], probably due to the prolonged and excessive use of these antibiotics. Significantly, in this study, the quinolone group of antibiotics successfully inhibited the growth of A. hydrophila; hence, it can be utilized as a treatment in tilapia farms. However, with the concerns of newly emerging MAR pathogens, the responsible use of antibiotics in fish farming should be adopted, and alternative treatments to combat A. hydrophila infection should be applied.
The pathogenicity study revealed that red hybrid tilapia was susceptible to A. hydrophila infection following IP injection. The previous experimental studies on A. hydrophila infection in various hosts showed cumulative mortality ranging from 60% to 100%, depending on the challenge doses and route of infection [20][21][22][23], which indicates a wide range of fish host susceptible to this pathogen. In this study, the LD 50 was determined at 1.1×10 4 CFU/mL, suggesting a low tolerance of red hybrid tilapia to A. hydrophila infection. In previous studies on the experimental infection of A. hydrophila using IP routes, the LD 50 were recorded at 4.1×10 8 CFU/mL for snakehead fish (Channa striata) [23] and 4.53×10 6 CFU/mL for gourami (Osphronemus goramy) [22].
In the present study, the observed clinical signs were similar to those observed in affected catfish and Nile tilapia in natural outbreaks of A. hydrophila infection [10,24] but with the lower severity. Other gross lesions such as exophthalmia and the presence of ascites caused by A. hydrophila infection in fish were not observed in this study [10,24]. Highly pathogenic strains may cause fish to die quickly without showing apparent signs of disease, as observed in this study where mortality started early at 6 hpi. In addition, other factors, such as the type and age of the host and the time of exposure, can also influence the severity of the clinical symptoms [5,6]. This study revealed that the kidneys of infected red hybrid tilapia showed tubular necrosis and glomerular shrinkage that was similar to previous reports [10,25]. Necrosis of the hepatocytes and congestion of blood vessels in the livers of infected red hybrid tilapia have   also been observed in infected golden mahseer (Tor putitora) [26] and channel catfish [25].

Conclusion
This study demonstrates the susceptibility of red hybrid tilapia to A. hydrophila infection. Apart from its threat to cultured fish, the MAR of A. hydrophila isolates may also pose a health threat to humans. With respect to the potential pathogenicity of A. hydrophila to tilapia, more attention should be given to the diagnostics of the disease in farms and the effective control of this pathogen through the use of appropriate and environment-friendly therapeutic measures.