Observation of Inherently Contaminated Oncorhynchus mykiss Walbaum, 1792 by Aeromonas veronii with MALDİ-TOF and Culture Methods and Specification of Antibiotic Sensitivity Profiles of Agent in a Commercial Farms

Aquaculture is one of the world’s quickest developing food sectors to supply the growing nutritional requirements worldwide. Rainbow trout (Oncorhynchus mykiss) is a significant species in aquaculture with its production of approximately 848 million tons in the world of aquaculture in 2018 (FAO, 2020). This species is the most frequently farmed freshwater fish in Turkey and its production was 125 thousand tons in 2019 (TUİK, 2020). Cultured fish are weak to various bacterial diseases that often economically cause high prices for fish farmers (Pasqualetti et al., 2021). Many opportunistic pathogens are present in the aquatic environment of fish farms. Aeromonas spp. naturally inhabit the aquatic environment such as freshwater, coastal water and sewage of which many are regarded pathogenic for aquatic animals (Han et al., 2021). These bacteria were previously reported as the causative agent of bacterial hemorrhagic septicemia, motile aeromonad septicemia and epizootic ulcerative syndrome in many marine and freshwater fish species (Liu et al., 2016). Aeromonas veronii is a dangerous opportunistic bacterium in terrestrial and aquatic milieus (Chen et al., 2019). Presently, an increasing number of A. veronii infections causing epidemic diseases in diverse fish species have been described (Hoai et al., 2019; Raj et al., 2019). This pathogen in aquaculture, can infect a variety of aquatic animals, including freshwater goldfish, Nile tilapia,  Chinese Longsnout catfish and catfish (Wang et al., 2021) A. veronii is a Gram-negative bacterial pathogen and the clinical manifestation of contaminated fish mostly includes internal organ hemorrhage and skin ulcers (Tekedar et al., 2019). Aeromonas species affect many fish species, thus the interest in understanding the role of the pathogen increases in fish farms (Guzman-Murillo et al., 2000). This study aimed to define the potential pathogen, A. veronii, in diseased rainbow trout that may explain the high mortalities observed in the Southeastern Anatolia Region commercial farms. To this end, diseased fish were investigated from fish farms in varied zones of the Southeastern Anatolia Region.

In January 2021, we visited to thirty commercial fish farms of the Southeastern Anatolia Region (Diyarbakır, Batman, Şanlıurfa, Adıyaman). We randomly collected 40 live fish that average live weight of 200-250 g per farm and macroscopically examined them to define the disease prevalence. External clinical consultations regarded hemorrhage patches, open wounds, detached scales and dark discoloration as disease signs. Therefore the contaminated fish were determined with evident symptoms including hemorrhages below the pectoral fin and anal fin petechial hemorrhages in the skin surface, skin ulceration, intestinal fluid accumulation and liver hemorrhages (Fig 1).  The liver and kidney, tissues were used for bacterial isolation. Blood Agar (BA) and McConkey Agar (McA) were employed for bacteria isolation for 24 h at 28^oC and the dominant uniform bacterial colonies were purified by streaking three times onto the BA and McA plates (Fig 2). Then, the causative agents according to Gram staining and culture growing profiles were identified with MALDI-TOF mass spectrometry by Maldi Biotyper (Bruker, U.S.A) (Fig 3). Antibiotic susceptibility testing was performed using the Kirby - Bauer Disc Diffusion method, Mueller - Hinton medium and Bauer et al., (1966) and was evaluated according to the procedures reported by the Clinical and Laboratory Standards Institute (CLSI, 2004), Ruangpan and Tendencia (Ruangpan, Tendencia 2004) and Becton Dickinson and Company (BD, 2011).



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Rainbow trout inherently contaminated with A. veronii mostly display subacute death in fish farm conditions. After 3-4 days of contamination, fish start to swim sluggishly leading to high mortality. Most of the infected fish exhibit big regions of body surface ulceration (Fig 1). After dissecting the fish with intestinal inflammation, internal organ hemorrhages were frequently observed (Fig 1). Additionally, some diseased fish were determined with ischemia, liver and kidney, hemorrhage and spleen darkening (Fig 1). The predominant disease symptoms include skin ulceration, skin surface, hemorrhages the and pelvic fin and anal fin. This study aimed to identify the potential pathogen Aeromonas veronii in fish farms of the Southeastern Anatolia Region. We visited commercial fish farms, covering different regions of the Southeastern Anatolia Region and observed A. veronii infection from 0% to 100%. Clinical darkening of color, irregular swimming tendency, stagnation, decreased feed consumption, formation of exophthalmos and respiratory distress were observed in rainbow trout taken from the farms. The macroscopic examination, revealed that the most common lesion was petechial hemorrhages and ulcers formed on the skin in different body parts. Other macroscopic findings on the skin include skin darkening increased mucus structure and shedding of scales, erosion and large-scale fluid-filled ulcer formations up to the deep muscle layers. The present study investigated a total of 1,200 samples and detected A. veronii in the liver, kidneys, tissues of the fish collected from 28 of the 40 farms. Fish deaths were found in some of the visited farms. Antimicrobial sensitivity testing indicated that the isolated strain A. veronii was sensible to enrofloxacine (5 µg) but was resistant to florfenicol, neomycin, amoxicillin, oxytetracycline and erythromycine. Therefore, we could select sensitive antibiotics for disease prevention (Table 1). Infectious epidemic diseases that are caused by aeromonads are one of the very important problems in trout farms that impact the aquaculture sector economy (Austin and Austin, 2012). The characteristic habitats for these bacteria include freshwater. Aeromonas spp. are significant pathogens that are considerably found in aquatic environments, causing infection in injured fish or individuals under stress conditions (Janda and Abbott, 2010). A. hydrophila has been considered the very detrimental pathogens for the enviroment of aquatic animals; however, A. veronii has increasingly affected the fishes in the last years (Chen et al., 2019). A. veronii is a dangerous pathogen of human beings and aquatic animals and maybe frequently isolated from infected aquatic environments and aquatic animals (Weiss, 2019). A. veronii has been verified as a dangerous pathogen of ulcerative disease in loach (Zhu et al., 2016), Nile tilapia (Raj et al., 2019), Gibel carp (Chen et al., 2019), guppy (Lazado and Zilberg, 2018) and catfish (Hoai et al., 2019). Symptoms with fin rot and petechial hemorrhage on body surface were shown in the crucian carp (Carassius auratus gibelio) (Chen et al., 2019), goldfish (Carassius auratus) (Shameena et al., 2019), guppy (Poecilia reticulata) (Lazado and Zilberg, 2018), zebrafish Danio rerio and Nile tilapia (Song et al., 2018), which were also observed in diseased fish in our study (Fig 1). The present study, examined A. veronii from various fish farms and was isolated from liver and kidney tissues of fish (Chen et al., 2019; Raj et al., 2019). A. veronii has a diversity of hosts and may live in habitat and in aquatic environments and aquatic animals, which might harm the aquaculture in the future (Wu et al., 2019). Most fish species can be contaminated by A. veronii, including tilapia, rainbow trout, loach, European sea bass and a variety of catfish. These fish are cultured with variable production quantities in different countries (Han et al., 2021). Thus, that the opportunist pathogen has a chance of host specificity and likely spread, which may induce extreme future problems for aquaculture. Bacterial resistance to antibiotics influences the environment and the health of humans and animals (Hoai et al., 2019). Antibiotic resistant bacteria in aquacultures may be also human opportunistic pathogens and some fish pathogens. Thus, it has a particularly critical threat to public health. Resistant bacteria can be transferred to humans with infected fish or by direct contact with aquaculture ecosystems, e.g., farm fish workers, fish scoops, hand fishing (Laukova et al., 2018).  Previous study results revealed that A. veronii was resistant to antibacterial drugs, including ampicillin, amoxicillin and oxacillin (Zhixiu et al., 2016). These fast-spreading pathogens can then be transferred to the environment and eventually become contagious to humans (Romero et al., 2012). Previous studies have indicated that A. veronii exposes high-level resistance to a diversity of antibiotics including kanamycin, neomycin, tetracycline, sulfamethoxazole, deoxycycline, penbritin, midecamycin, amikacin, tetracycline, azithromycin, tobramycin, cefadine, gentamycin and amoxicillin (Hoai et al., 2019). The current study verified some of these conclusions and indicated that A. veronii species were further resistant to neomycin, amoxicillin, oxytetracycline and erythromycin. A. veronii exhibited antimicrobial resistance to six antibiotics; however, the pathogen could be regulated by the implementation of antibiotics such as enrofloxacine and florfenicol. Fishes that are successfully healed should be emphasized owing to the prescribed antibiotic treatment, thus no further mortality was reported in the commercial farm.

The investigations made in the current study remind us that the occurrence of multiple antimicrobial resistant pathogens and their pathogenicity in fishes and aquaculture environments is unavoidable and timely follow-up coupled with sensible antibiotic use is required for sustainable commercial farming.

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