Potentiality of Selected Commercial Antibiotics Challenged with Aeromonas sp

This study was intended to address, the effect of oxytetracycline, erythromycin, and streptomycin on the antimicrobial resistance of Aeromonas sp. isolated during the production process, from infected common carp (Cyprinous carpio). Antimicrobial resistance patterns were defined by determination of minimal inhibitory concentrations (MIC) of three antimicrobial drugs, and bacteria. Results indicated that In vitro bactericidal activity, oxytetracycline showed lowest number of bacterial colonies (1×10 c.f.u./ml) than erythromycin (3×10) and streptomycin (4.5×10) compared to the control (9×10). Oxytetracycline showed the highest maximum zone of inhibition (20 mm) than erythromycin (18 mm) and streptomycin (15 mm) in antibiotic susceptibility test. In infection inhibition assay, serum from oxytetracycline fed diets inhibited Aeromonas infection to common carp. The susceptibility of the fish fed antibiotics (50 μg/mg feed) and challenge with Aeromonas sp. (1×10) was examined for 30 days. There was high relative percent of survivility (RPS) in oxytetracycline was 73%. Very low RPS 63% and 47.37% was found in erythromycin and streptomycin against Aeromonas sp. infection. These results demonstrated that the oxytetracycline might be a potential for Aeromonas sp. infection to common carp (Cyprinous carpio).


Introduction
Fish production through aquaculture is increasing day by day. But the total production is still behind the demand. Disease is one of the major limiting factors to obtain the production target. Among various diseases bacterial disease in fish is a serious threat to aquaculture system in Bangladesh. Various types of disease such as ulcer type disease including epizootic ulcerative syndrome, bacterial hemorrhagic septicemia, tail and fin rot, bacterial gill rot, dropsy, columnaris disease, fungal disease and parasitic disease are important limiting factors for sustainable fish production [1]. Infections caused by motile aeromonads are probably the most common bacterial disease of freshwater fish [2]. Resistance of Aeromonas sp. to commonly used antibiotics is an emerging problem in the farmed fish. Therefore, the present work attempted to evaluate the potentiality of antibiotics to inhibition assay, bactericidal activity and susceptibility against Aeromonas sp. infection.

Fish and Management
Common carp, Cyprinous carpio (weight 270.7±10.4 g, N= 100) was obtained from fish farm located at Chachra, Jessore and were transported to the laboratory of Fisheries and Marine Bioscience (FMB), Jessore University of Science and Technology (JUST), on July to December 2013. The fish were immediately examined by their movement, eye condition and external lesion to find out their health status and acclimatized in the indoor aquarium (100 L) with recirculating aerated water. Continuous aeration was provided to maintain dissolved oxygen level at 7.5±0.5 mgl -1 and one-third of the aquarium water was exchanged daily and by siphoning the waste materials was removed. During the experimental period water temperature, pH and TDS (total dissolved solid) were 22±0.8°C, 5.94±0.21 and 434±0.29 mgl -1 respectively. Fishes were provided with normal feed (without herbal extract) at the rate of 5% of their body weight twice a day at 09:00 and 16:00 h for 3 days but at the first day of their arrival no feed was provided.

Aeromonas Hydrophila Isolation
A. hydrophila isolated from diseased common carp (Cyprinous carpio), was used in FMB laboratory for the study. Stocks were grown in brain heart infusion (BHI, Potentiality of Selected Commercial Antibiotics Challenged with Aeromonas sp. Hi-media, Indian) and nutrient broth for 24 hrs at 37 ºC and then kept in −20°C until use. The subculture was taken and centrifuged (4000 rpm for 15 min), after centrifugation the supernatant was discarded and the pellet was resuspended in sterile phosphate buffer saline (PBS). The culture was adjusted at 3.5×10 -7 colony forming units (CFU) ml -1 by 10 time's serial dilution and incubated at 37°C for 24 hours. The bacterium was confirmed by some biochemical test (Table  1).

Antibiotic Treatment Conditions
The study was carried out during a disease outbreak (caused by the bacteria Aeromonas hydrophila) occurring in a common carp, Cyprinous carpio lot. The fish were treated on decision of the veterinarian with antibiotics at 12 mg per kg of bodyweight per day during eight consecutive days. The trout were fed the antibiotics-supplemented food at a rate of 0.5% of body weight per day. At the onset of treatment, mean fish weight was approximately 270 g and water temperature was about 30 °C. This treatment was successful in curing the affected common carp lot. At the end of the study period, water temperature in the farm environment was about 25-30 °C.

Bleeding, Serum Separation and Bactericidal Activity Test
Blood from the randomly selected fish were drawn directly from the caudal vein with the help of a sterilized 1 ml hypodermal syringe containing EDTA (Ethylene-Diamine-Tetra-Acetic Acid) as an anticoagulant using 24 gauge needles. For serum separation blood was collected without anticoagulant in serological tubes and stored in a refrigerator overnight. The clot was then spun down at 4500 g for 10 min. The collected serum was stored in sterile serum tubes at −20 °C until used for assays. All the procedures were carried out in the sterilized condition. A. hydrophila was used as a model to examine the effectiveness of antibiotics to kill the bacterial infection. 100 µl of serum was added to 100 µl bacterial suspension and mixed before incubated for 1 hours at 37 0 C. A blank control was prepared by replacing serum with sterile PBS. The mixture was then diluted with sterile 0.05 M sodium phosphate buffer, PBS (pH 6.2) at a ratio of 1:10. The serum-bacterial mixture (100 µl) was plated onto the nutrient agar plates with incubation for 24 hours at 37 0 C before the number of colonies was counted.

Antibiotic Susceptibility Test
Antibiotic susceptibility tests were carried out against A. hydrophila with three antibiotics by disc diffusion method [3]. Antibiotics disc of oxytetacyclene (30µg), erithromycine (30µg) and streptomycin (30µg) were used for the study [4]. NA and BHI agar was poured into the plates and allowed to solidify. The standard inoculum (10 6 cfu/ml) was swabbed over the surface of the media using sterile cotton swab to ensure the confluent growth. The plates with incubation for 24 hours at 37 0 C before the number of colonies were counted. All the experiments were carried out in triplicate.

Agglutination Titer Assay
The test for agglutination titer was realized individually for bacterium Aeromonas sp. according to Yildirim, et al. 2003 [5]. The bacterial cell suspension was centrifuged at 300 × g for 15 min and supernatant was discarded. The resulting pellets were washed twice with 0.85% phosphate buffer saline (PBS) solution and pellets were resunpended in PBS to an optical density of 0.8 at 540 nm. Starting with a dilution of 1:10 (10-µl serum and 90 µl PBS), two-fold serial serum dilutions were made in 96-well round bottom microtiter plates by adding 50 µl of diluted serum into the reaming wells plated with 50 µl of PBS. Thereafter, 50 µl of bacterial cell suspension was added to each well; thus, the initial serum dilution was 1:20. The plates were covered with plastic film and incubated at room temperature for 2 hours at 4 0 C for 24 hours. The agglutination end point was estimated as the last serum dilution where cell agglutination was visible after incubation. Agglutination titers were reported as log 10 of the reciprocal of the highest serum dilution showing visible agglutination as compared to the positive control.

Assessment of Aeromonas infection inhibition
Aeromonas infection inhibition assay was carried out as follows: Aeromonas cells were grown in 96-well plates at 37°C until the density reached to 1×10 6 cfu/ml. The cells were washed gently with PBS and then incubated for 4 hours Immunology and Infectious Diseases 3(2): 11-15, 2015 13 at 37°C with 100 μl serum from oxytetracycline fed diets against the target bacterium diluted at 1:50, 1:100 or 1:200 with maintenance medium. After incubation, the cells were washed twice with PBS and then infected with Aeromonas sp. at LD 50 at 37°C for 1 hour. After bacterial adsorption, the unattached bacteria were removed by washing with PBS and 100 μl maintenance media were added. The infection was allowed for 5 days at 37°C and the cytopathic effect (CPE) was monitored by photography microscope, serum (1:50 diluted) was used as negative control. The experiment was performed twice in duplicate.

Challenge Test
To evaluate the effects of antibiotics in the diet on fish disease resistance, Aeromonas sp. was used as the pathogenic agent. The susceptibility of the fish fed with different antibiotics and challenged with Aeromonas sp. (1×10 -4 c.f.u./ml) was examined for 30 days. The relative percent survival (RPS) was calculated by following formula [6]:

Statistical Analysis
All the data are expressed as mean±SE. Statistical analysis of data involved one way analysis of variance (ANOVA) followed by the comparison of means following Stat Professional Software and SPSS windows 15.0 version. The level of significance was expressed as p-value at 0.05 levels. The lowest number of bacterial colonies indicated the efficiency of antibiotics to kill the pathogen. With oxytetracycline the lowest number of colonies i.e. 1×10 -4 on media with the 30 µg/ml dose with highly significant differences to the control, 9×10 -4 . The 30 µg/ml dose recorded a highest number of colonies; 3×10 -4 and 4.5×10 -4 for erythromycin and streptomycin respectively in Figure 1.

Antibiotic Susceptibility Test
In the drug susceptibility assays, Aeromonas sp. was susceptible against oxytetracycline (20 mm), erythromycin (18 mm) and streptomycin (15 mm). In the antibiotic susceptibility test, oxytetracycline exhibited maximum activity than that of erythromycin and streptomycin in Figure  2.

Bacterial Agglutination Test
The agglutination antibody titers were quantified following the standard micro-agglutination procedure using serial two fold dilution of each antibiotic pool. Using highest antibiotic dilution showed visible agglutination as compared to the positive control. Negative agglutinations were found usually lowest antibiotic dilution. with Aeromonas sp., less than 25% of cells showed obvious CPE at the second day after infection, and more than 50% CPE occurred at the fifth day (Table 3). By contrast, the CPE didn't appear until the fifth day in the presence of 1:50 diluted post-immune sera, but the CPE was gradually aggravated by pre-incubation with lower concentrations of post-immune sera at 1:100 or 1:200 dilutions (Table 3).

Challenge Test
The highest survivility was 75 % and 65% in fish fed with 50 µg/mg of oxytetracycline and erythromycin for 30 days against Aeromonas sp. The lowest survivility of 5% was observed in fish fed with 0% dose diet. The survivility decreased to 50% with 50 µg/mg of streptomycin dose diet (Fig. 3). The lowest survivility of 5% was noted in the control while 50% survivility was observed for streptomycin 50 µg/mg dose applications (Fig. 3). The highest RPS; 73% was found for oxytetracycline, 63% and 47.37% for erythromycin and streptomycin respectively.

Discussion
The motile Aeromonas group is responsible for severe fish diseases, such as hemorrhagic septicemia [7,8,9] and ulcerative disease of fish [10]. The worldwide distribution of this group is a consequence of its great adaptability to different aquatic environments [11]. Infections by Aeromonas had been observed in various freshwater fish species and eventually in marine fish, reptile, bovine, and man [7,12]. MIC has been recorded for several Aeromonas hydrophila strains around the world [13,14,]. In the present study Aeromonas sp. was isolated from hemorrhagic septicemia and ulcerative disease affected common carp.
In the present study, Studied on pure cultures of Aeromonas sp. were isolated from the skin, kidney, heart, and liver of the common carp. The isolates were found to be sensitive to and oxytetracycline, erythromycin and streptomycin. Oxytetracycline showed the better result than the erythromycin and streptomycin. Pure cultures of Aeromonas hydrophila were isolated from the skin, kidney, heart, and liver of the gold fish. All of the isolates were found to be susceptible to oxytetracycline, neomycin, streptomycin and resistant to ampicilin, amphotericin, bacitrain, cephalexin and moderately sensitive to Erythromycin, Kanamycin, [15] which support the present study. The isolates of Aeromonas hydrophilia were tested for their susceptibility to florephenikol, flumequine, rithromycin olaqindox and oxitetracycline [16], which support the present study. Antibiotic susceptibility assay with commercial antibiotics and oxytetracycline showed the better result than erythromycin and streptomycin [4], which supports the present study.
In the present study bacterial agglutination was performed and oxytetracycline showed the better result than the erythromycin and streptomycin at the same dose in 96 wells plate. In the present study assessment of MIC was also performed and result showed that, 50 µg/ml for oxytetracycline and 55 µg/ml, 60 µg/ml for erythromycin and streptomycin respectively. Miranda and Zemelman, (2002) studied on bacterial resistance to oxytetracycline in Chilean salmon farming by spread plate method and agar plate dilution method. They recommended that 30 to 100 µg/ml oxytetracycline is helpful for bacterial inhabitation [17], which supports the present study. Singh et al., (2009) isolated twelve isolates of oxytetracycline resistant bacteria were mainly dominated in adult fishes. Their selected strains exhibited high levels of oxytetracycline resistance with minimum inhibitory concentrations (MICs) ranging from 50 to 600μg/ml [18], which also support the present study. Guz and Kozinska, (2004), isolated twenty one Aeromonas pathogen from carp (Cyprinus carpio) and tested susceptibility to 22 antimicrobial agents by disc division method. They resulted that, 100% were resistant to ampicillin and penicillin, and sensitive to trimethoprim-sulphamides, oxolinic acid, flumequine, chloramphenicol, norfloxacin, linkomycin, pefloxacin. Most isolates were resistant to cephalothin (57%) and erythromycin (52%) [19]. Haque et al., (2014) observed the effectiveness of antibiotic, oxytetracycline reducing the bacterial load in rohu fish under artificial culture condition in the laboratory. They used live fish method for experiment and resulted that 2 gm/kg oxytetracycline in diet, twice daily is potential antibiotic to reduce bacterial load in fish and can be used commercially for maintaining the fish health in aquarium condition [20], dose did not support the present study because fishes may be treated prior to study time and bacteria gained resistancy against minimum dose or dose may be varied species to species. In the present study challenge test was performed with oxytetracycline, erythromycin, streptomycin and control against Aeromnas sp. affected fish. Oxytetracycline treated fish showed highest RPS value than the other two antibiotics and resulted that antibiotics have the potentiality to cure the fishes, which support various workers in this field.
Aeromonas sp. is very common bacterial pathogen which affects fish species frequently and can cause severe disease. At the end of present study it can be concluded that, Aeromonas sp. was responsible for hemorrhagic septicemia and ulcerative disease of fish. The Aeromonas sp. was found to be sensitive to oxytetracycline, erythromycin and streptomycin.

Conclusions
In vitro bactericidal activity, oxytetracycline showed lowest number of bacterial colonies (1×10 -4 c.f.u./ml) and highest maximum zone of inhibition (20 mm) in antibiotic susceptibility test. In infection inhibition assay, serum from oxytetracycline fed diets inhibited Aeromonas infection to common carp, which suggested that the serum from oxytetracycline fed diets played an important role in the infection of Aeromonas sp. to common carps. There was high relative percent of survivility (RPS); 73% in oxytetracycline against Aeromonas sp. infection, so the oxytetracycline than other antibiotic tested in this study might be a potential for Aeromonas sp. infection to common carp (Cyprinous carpio).