Investigation of gcat gene and antibiotic resistance pattern of Aeromonas hydrophila isolated from hemorrhagic septicemia’s cases in fish farms

Article history: Received April 5, 2020 Accepted May 14, 2020 Available online March 15, 2021 The significance of Aeromonas hydrophila concerning hemorrhagic septicemia in aquaculture farms production in Duhok province, Iraq was investigated. Antibiotic-resistant profiles of isolates were also investigated with 8 antibiotics. Bacterial isolates were identified by using morphological and biochemical tests and confirmed molecularly by amplification of gcat gene. Out of 25 examined fish, only 19 fish were harbored A. hydrophila. Twenty-four A. hydrophila strains were isolates from 100 organ samples. Ninety-six percentages of the isolates were resistant to each of the imipenem and gentamicin, followed by doxycycline 92%, ciprofloxacin and trimethoprimsulfamethoxazole 88%, norfloxacin 58% and ceftriaxone 33%. None were resistant to levofloxacin. Eighty-eight percentages were multiple antibiotics resistant. The high isolation rate of A. hydrophila in our study indicates that this species was the major cause of the outbreak in hemorrhagic septicemia’s cases in our area affecting carp farms and the high rate of resistance should be considered as these isolate can serve as a resistance source for human being during food series and make great challenge for their therapeutic opportunity.


Introduction
The largest and most dangerous form of diseases affecting fish production is the bacterial infections, which account for 80% of fish mortality (1). In turn, this would adversely affect aquaculture (2). Aeromonas hydrophila is considered the primary cause of septicemia disease, including carp, tilapia, perch, catfish, salmon, and many other freshwaters and marine species (3). The marked clinical signs that seen on the abdominal wall and at the base of fins are congestion and hemorrhage as well as scale erosion at all body surfaces. While the prominent post mortem lesions are highly congested internal organs, ascitic fluid build-up in the abdominal cavity and swollen kidney and spleen (2). In addition, a variety of human illnesses have involved by A. hydrophila, it causes a broad spectrum of infections (gastroenteritis, septicemia, meningitis, endocarditis) in humans, often in immune-compromised hosts (4). A. hydrophila is easy to grow on standard laboratory enteric media and produces β-hemolysis on blood agar with grey, flat, round and shiny colonies about 2.0-3.0 mm in diameter, ability to grow on MacConkey agar with non-lactose fermentation and they are oxidase-positive (5,6). Different reports have revealed that A. hydrophila is widely antibiotic-resistant.
Considerations, including the indiscriminate overuse of antibiotics in fisheries are responsible for this. This contributes to high selection pressure in bacteria to develop resistance through a variety of mechanisms such as genetic mutation and horizontal gene transfer. This is a public and marine health hazard (7).
Little information is available on the fact that is it A. hydrophila regarded as the primary cause of hemorrhagic septicemia in fish as well as antibiotic resistance rate of A. hydrophila from clinical cases of fish suffering from hemorrhagic lesions in all around the world. The present study was intended to assess the accurate rate of A. hydrophila incidence as a causal agent of septicemias in different fish farms in Duhok city and also to assess their resistant rate against different antibiotics that most commonly used for therapeutic purposes against this bacterium in fish farms and human being.

Sample collection
The sample collection process and the choosing of sample types, was carried out according to Aboyadak, (2) and Hassan et al. (8). Samples were taken from 5 carp fish farms (Agricultural 1, 2 and 3, Mosul dam and one pond in Xhanke Village) with symptoms of septicemia, which include skin hemorrhage with hemorrhagic skin ulcers and de-pigmented zones in the skin, and necropsy for internal organ lesions including extreme congestion and internal organ hemorrhage, enlarged liver and gall bladder. Each dead fish was picked in a separate sterile labeled plastic bag and transported to the laboratory in icebox. From each fish, four different samples were taken from heart blood, liver, kidney tissues, and hemorrhagic or ulcerative area on the skin. The sampling from each part of the fish lesion was carried out under strict sterile conditions to prevent contamination with normal flora, in which bacteria were isolated with a sterile loop from each organ of each fish. Four samples from each fish organ lesions were pooled to make one sample for testing. From 25 collected fish, a total of 100 organ lesion samples were taken.

Isolation and identification of Aeromonas hydrophila by phenotypic methods
Each loop sample at the collection time was directly put into tubes containing 10 ml of brain heart infusion broth (HiMedia, India) or tryptic soy broth (Lab M, UK) and incubated at 37°C for 20-24 hrs., as a pre-enrichment step (8). One to two loopful of pre-enrichment broth was inoculated on to blood agar supplemented with 7% sheep blood and incubated as previous. Beta-haemolytic 2-3 mm colonies were directly sub-cultured onto MacConkey agar and incubated as previously. Pale (non lactose fermenter) colonies of Aeromonas hydrophila were presumptively identified by Grams stain, indole production test, oxidase test, catalase test and urease test (5,6). Colonies from positive samples were directly preserved at -20°C as stock culture in tubes containing brain heart infusion broth with 25% glycerol (9). The final confirmation was carried out by PCR amplification of gcat (glycerophospholipidcholesterolacyltransferase) gene which is common for Aeromonas hydrophila (10).

DNA extraction and detection of gcat gene by PCR amplification
DNA samples were extracted by thermal extraction method according to Taha and Yassin (11). From stock culture 100 µl was inoculated onto MacConkey agar and incubated as previous. From MacConkey agar 2-3 pure (similar morphology) colonies were chosen and mixed with 200 µl of sterile double distilled water in a 1.5 ml tube. For at least 15 s, the mixture was vortexed and directly heated at 95ºC for 10 min; the samples then cooled directly by ice, the cooled suspension was centrifuged. One hundred fifty µl supernatant was used as a template DNA for PCR. The purity and concentration of extracted DNA were examined using a nano drop (Thermo Scientific, USA) (12). The extracted DNA samples were stored under freezing (-20) until used as DNA templates for PCR analysis (13). To identify the A. hydrophila, gcat gene was amplified as potential markers of detection (10). The gcat-PCR amplification was made in a total volume of 25 µl containing 12.5 µl of hot start premix (Genedirex, Taiwan), 1 µl of each of reverse and forward primer (F:5'-CTCCTGGAATCCCAAGTATCAG-3' and R: 5'-GGCAGGTTGAACAGCAGTATCT-3'), which amplify a 237 bp fragment (concentration: 10 pmol), 2 µl of sample DNA (50-150ng/µl), the remainder was filled with 8.5 µl nuclease-free water (Qiagen, Germany). The process of amplification was performed in thermocycler (GeneAmp ® PCR System 9700, Singapore, Applied Biosystems) according to Latif-Eugenín et al. (14). PCR program was 95°C for 3 min followed by 35 amplification cycles of denaturation, annealing and elongation, were 94ºC for 1 min, 56°C for 1 min, and 72ºC for 1 min, respectively and final elongation of 72ºC for 5 min. Amplification of PCR products was confirmed in 1.5% agarose gel prepared with 1× Trisacetate-EDTA (TAE) buffer and stained by red safe DNA staining solution (GeNetBio, Korea). Successful gcat gene amplification is considered when bands in agarose gel at the anticipated size of 237 bp seen.

Antibiotic susceptibility test
The identified isolates by PCR assay were tested against 8 antibiotics as described by Stratev and Odeyemi (7) and Li et al. (15), using the disk diffusion method on Mueller-Hinton agar (Lab M, UK).The concentration of each antibiotic per disc (Bio-analyze, Turkey) was as follow: doxycycline 30 μg, trimethoprim-sulfamethoxazole 1.25 and 23.75 μg, gentamicin (10 μg), ciprofloxacin 5 μg, ceftriaxone 30 μg, imipenem 10 μg, levofloxacin 5 μg and norfloxacin 10 μg. The protocol and interpretations of results breakpinots were carried out according to clinical and laboratory standards institute (CLSI) (16). Isolates were marked as either susceptible or resistant isolates that were intermediately susceptible to specific antibiotic were classified as resistant. Any isolate that was resistant to three or more antibiotics (≥ 3) was known as multiple antibiotics resistance (MAR).

Phenotypic methods for the detection of Aeromonas hydrophila
The cultural characteristics of all isolates of Aeromonas hydrophila on blood agar were found as β-hemolytic, with grey, flat, round and shiny colonies about 2-3 mm in diameter. On MacConkey agar, their colonies were showed as pale non lactose fermenter. With Grams stain all isolates were showed as medium-sized, straight, Gram-negative rods under oil immersion objective lens (Figure 1). While the results of biochemical tests, all isolates were indole positive characteristic red ring above fluid media in tryptophan broth, oxidase test positive (the color was changed of cotton swab with a loopful of bacterial culture to dark purple within 10 seconds after adding of 1% solution of N, N-dimethyl-pphenylenediamine hydrochloride, catalase test positive bubble formation after adding of one drop of hydrogen peroxide on a loopful of bacterial culture on sterile microscopic slide and urease test negativity the color of Christensen's urea agar contained 40% urea solution was changed to yellowish as a result of acidic byproduct from glucose utilization (Figure 2).

Genotypic method for the detection of Aeromonas hydrophila
According to the PCR amplification of gcat gene, all identified Aeromonas hydrophila isolates by both morphological and biochemical tests, were showed a band size of about 237 bp on agarose gel after staining with red safe DNA staining solution and after electrophoresis process ( Figure 3). These were indicated that all isolates were A. hdrophila.  Out of 25 carp examined, only 19 fish were found to be infected with A. hydrophila. While out of 100 collected organ samples (25 livers, 25 kidneys, 25 hearts, and 25 skins) from a total of 25 examined carps, only 24 strains of A. hydrophila were identified. These were in accordance with phenotypic methods (Figure 1 and 2) and genotypic methods (PCR amplification of gcat gene) (Figure 3). The liver was the predominated organ to have A. hydrophila (9 isolates) followed by kidney and heart each of (6 isolates) and skin of about (3 isolates) (Table1). Regarding the geographical region, fishes from Khanke area and agricultural pond 3 were showed a higher isolation rate (100%) followed by Mosul dam (80%), Agricultural 2 (75%), and Agricultural 1 (43%) ( Table 1).

Discussion
Aeromonas hydrophila is a significant opportunistic pathogen in the aquatic environment of freshwater where the most critical stressors include rough handling, crowding, malnutrition, heavy free ammonia (NH3) and elevated nitrites (NO2) are present (1,8). Adding to these, in muddy water with heavy organic content, A hydrophila is abundant (17). Any changes in water parameters can lead to immune stress situations and this will predispose fish from being infected with A. hydrophila, and encourage opportunist bacterial infections (1,18). The high isolation rate of A. hydrophila seen in this study can all be due to these factors. However, in this study, not all fish showing clinical signs of septicemia were harbored A. hydrophila, this mostly due to that a large number of Aeromonas spp other than hydrophila are responsible for hemorrhagic septicemia in fish. There is strong evidence that many Aeromonas spp as a cause of diseases have been found in aquatic environments (wild and farmed fresh water and/or marine species) (19). The causes of epizootic ulceration and hemorrhagic septicemia in fishes have become well established with Aeromonas veronii (8).
In another study which was carried in China, proposed that motile Aeromonas spp. other than A. hydrophila were also found in moribund fish with hemorrhagic septicemia (17).
According to the clinical and laboratory standards institute CLSI (16) isolates that are initially susceptible or intermediately resistant to one antibiotic may become resistant after initiation of therapy. Therefore, in this study any isolate that was intermediately resistant to specific antibiotic was classified as resistant isolate to antibiotics. In this study, all the isolates showed a high degree of resistance to the tested antibiotics with a significant resistance to both imipenem and gentamicin. However, Sreedharan et al. (20), reported a total sensitivity of Aeromonas spp., to imipenem and gentamicin. As well as, Al-Dabbagh (21) in Mosul, Iraq, also found that A. hydrophila isolated from milk samples in bovine mastitis was susceptible to gentamicin. On the other hand, Stratev and Odeyemi (7) reported that 50% of A. hydrophila isolated from Tilapia were resistant to imipenem. This finding most likely reflects the presence of selective pressures in these ecosystems mainly because antimicrobial agents are used in both aquatic and human clinical prevention and treatment strategies (15,22). However, imipenem has never been used neither therapeutically nor prophylactically in an aquatic environment but the resistant in these fields is most likely created due to human wastewaters (hospital wastewaters) or rivers containing imipenem resistant genes (carbapenems) or antibiotic residues with subsequent contamination to the aquatic environment which in turn lead to the development of selective pressure of resistant in these environments (23,24). In a study carried out to explore the existence of antibiotic resistance in hospitals wastewater collected in Mumbai, India, identified new resistance genes and novel carbapenemases including NDM, VIM, IMP, KPC, and OXA-48 (25). In comparison, all isolates were levofloxacin susceptible in this study and this is consistent with Li et al. (15). This indicates that this antibiotic is not yet used in aquatic environments as a therapeutic or prophylactic purpose.
An alarming level of MAR in this work was observed especially in isolates from Agricultural 2 and 3. This indicates that in these farms different antibiotic types were used as a prophylactic purpose to reduce the probability of the occurrence of infectious diseases or to accelerate aquaculture growth (24,26). The presence of bacteria in overcrowding microbial ecosystems favorable for the transfer of genetic materials such as plasmid, transposons, and integrons carrying multiple antimicrobial-resistant genes is usually regarded a reason for MAR development (27) or that isolates carried genes codes for efflux pumps to multiple antibiotics, resulting to the higher rates of MAR (28). The presence of MAR in A. hydrophila has also been reported by others (7,22,29).

Conclusion
A high isolation rate of A. hydrophila found in this study, means that this is a common organism associated with disease outbreaks in aquaculture in the Dohuk province, Iraq. This indicates that A. hydrophila is a primary invader in farmed fish in our area. The high levels of antibiotic resistance recorded in this study should be carefully considered as these isolates can serve as a human source of infection during food series and pose a major challenge for the therapeutic possibility.