Abstract: Background and Objective: Bacterial fish diseases constitute a major problem in aquaculture, it was found in the environment and under stressors cause severe economic losses to fish. This work aimed to investigate the bacterial causes and suitable treatments of mass mortality in some cultured marine fish farms in Damietta governorate. Materials and Methods: The study was performed on 5 farms suffered from mass mortality. Total of 100 diseased fish (10 sea bass and 10 sea bream/farm) and 20 water samples were randomly collected from these farms. Bacteriological examinations were carried out followed by in vitro sensitivity tests. Treatment trial was performed using the most effective antibacterial agent on isolated bacteria. Results: From fish and water samples Pseudomonas spp., Aeromonas spp. and Vibrio spp. were isolated with the rat of (16, 10%), (22, 10%) and (28, 10%) respectively. These results were confirmed biochemically. Some virulence genes of isolated bacteria were detected using PCR; meanwhile, enrofloxacin reduced significantly the mortality rates in examined farms. Conclusion: It could be concluded that, Pseudomonas spp., Aeromonas spp. and Vibrio spp. are the main bacterial species causing mass mortality in marine fish farms. These bacteria were highly sensitive to enrofloxacin in vitro and in vivo.
INTRODUCTION
Fisheries represent an important sector in the Egyptian national income structure. Marine fishes are liable to many environmental stressors as chemicals, natural and biological invaders which induce immune suppression of fish. However, the bacterial invasion is the main immunosuppressive agent1.
In the long run, water resources will be the most limiting factor for aquaculture development in Egypt. Therefore, marine fisheries are the immediate alternative for water needed in aquaculture2. Fish diseases of bacterial origin have become one of the major agents of economic losses since the beginning of marine farming. Vibrio spp. are common inhabitants among the aquatic animals. Vibrio spp. may be the most devastating bacterial disease in cultured fish3. Vibrio spp. are widely distributed, Gram-negative bacteria that need sodium and chloride4-6. Pseudomonas spp. are Gram-negative bacteria that can develop resistance and virulence factors. P. aeruginosa produces two types of soluble pigments, a blue pigment, pyocyanin and a fluorescent pigment, pyoverdin7. Most Aeromonas spp. were catalase and oxidase-positive, motile, able to reduce nitrate to nitrite, sugars fermentative and gas may be produced8.
Although Aquaculture plays an important role in providing safe, reliable and low priced food, the threat of antimicrobial resistance is increased9,10 inducing treatment failure. Therefore, identification of antibiotic resistance gene in the virulent bacteria is highly important11,12. The outer membrane protein (oprL) of Pseudomonas spp. is virulent genes located on the chromosome, that enable it to play a great role in causing diseases13,14. Uncontrolled or sub-therapeutic use of antimicrobials may be responsible for the resistance to these chemotherapeutic agents. This explanation is further in harmony with the statement saying that increase use of antimicrobials increases the problem of drug-resistant strain15,16. Bla TEM gene is a B-Lactam resistance gene (e.g., ampicillin, amoxicillin), it is in plasmid pUC1917-20. It was found in many pathogenic bacteria12. Moreover, thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) genes were found in V. parahaemolyticus inducing outbreaks21.
Before starting any treatment, feed intake, daily losses and value of the fish should be measured against the cost of the treatment. Oral medications should preferably be given during the early stages of the bacterial diseases since fish in the late stages feed poorly. Antimicrobial therapy is frequently used to control bacterial fish diseases. Antimicrobial susceptibility testing is performed to select the most suitable antibiotics. It should be noted that improper use of antibiotics may led to the presence of resistant bacterial strains22,23.
The present research aimed to study the role of antibacterial drugs in controlling the bacteria involved in mass mortality in some cultured marine fish farms in Damietta governorate.
MATERIALS AND METHODS
Study area: The study was carried out at units of bacteriology and fish diseases Animal Health Research Institute, Kafr El-Sheikh branch, Egypt from December 2018-November 2019.
Fish examination and samples collection
Fish samples: A total of 100 diseased fish were collected from 5 different marine fish farms situated in Damietta, Egypt and suffered from mass mortality (sea bass 100-120 g and sea bream 80-100 g). Fish were carefully examined for symptoms of diseases with a special focus towards the lesions as pale gills, exophthalmia, abdominal distension and skin lesions as blisters, ulcers and hemorrhages. These live fish have transported in battery aerated tanks to the lab for examination.
Water samples: Total 20 water samples from the 5 fish farms (10 samples from the water inlet and 10 samples from farm water) were collected in a sterilized glass bottle. A total 30 mL of water samples were centrifuged at 5000 rpm for 5 min. A total 1 mL of the sediment was incubated into a test tube containing 9 mL of trypticase soya broth at 30°C/24 hrs for bacteriological examination22.
Clinical and post mortem examination: Naturally infected marine fish were carefully examined in ponds of the farm for swimming, feeding and any abnormal sings on the body3. Also, any post mortem lesions were recorded (Fig. 1a-b).
Bacteriological examinations isolation: In complete aseptic conditions, bacteriological isolation was carried out from spleen, liver, kidney, brain and skin lesions of infected fish and inoculated into Tryptic soy broth with NaCl 2% at 30°C for 24-48 hrs then cultured into general bacteriological media (saline Nutrient agar and Tryptic soy agar with NaCl 2%) and incubated24 at 30°C for 48 hrs. The colonies were streaked on specific medium as Rimler’s-Shotts medium (R.S. medium), Pseudomonas selective agar base, Aeromonas selective agar base with ampicillin supplement and TCBS agar then incubated at 30°C for 24 hrs. Semisolid nutrient agar was used for measuring the motility.
| Fig. 1(a-b): | Naturally infected sea bass |
(a) congestion in the liver and darkening of the skin and (b) Congestion in gallbladder, gills and paleness in liver |
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Identification
Phenotypic characterization and Biochemical identification: The identification of the isolates was performed according to Bergey25. Smears of suspected bacterial colonies were prepared, stained (gram stain) for microscopic examination. For phenotypic characterization of isolated bacteria (Biochemical identification), VITEK2 COMPACT SYSTEM (BIOMERIUX, FRANCE) were used.
Polymerase chain reaction: DNA extraction: DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH)
Oligonucleotide Primer: Primers from Metabion (Germany) was used. This is shown in Table 1.
PCR amplification: Amplification was carried out through the Applied biosystem 2720 thermal cycler.
Analysis of the PCR products: 1% agarose gel (Applichem, Germany, GmbH) in 1xTBE buffer was used for gel electrophoresis. About 40 μL of the products were loaded in the gel holes. Gelpilot100 bp (Qiagen, Germany, GmbH) and GeneRuler 100 bp ladder (Fermentas, Thermo, Germany) were used for measuring the fragments. Photographing the gel was performed utilizing the documentation system (Alpha Innotech, Biometra). The data were analyzed using computer software.
| Table 1: | Target genes, primers sequences, applicant sizes and cycling conditions |
Antibiogram: Antibiogram (sensitivity test) was performed using several antibiotics for the detection of the most effective one for the treatment of diseased fish farms24,29.
Treatments trials: Enrofloxacin (the most effective in vitro antibiotics against isolated bacteria) was used at a level of 50 mg kg–1 for 7 days in each bacterial infected farm30. One mL blood sample per fish was collected from 20 diseased fish of each farm one day before starting treatment and one day after the end of treatment. Blood was used to obtain serum for some biochemical studies (ALT, AST31, urea32 and creatinine33).
Statistical analysis: The obtained results were analyzed using SAS.34.
RESULTS
Bacteriological examination: Table 2 shows bacteria isolated from marine fish farm suffered from mass mortality and situated in Damietta, Egypt. It was found that Pseudomonas spp., Aeromonas spp. and Vibrio spp. were isolated from all farms.
Phenotypic and biochemical characteristics of isolated bacteria
Pseudomonas species: Table 4 shows the Phenotypic and biochemical characteristics of Pseudomonas spp. Morphological and biochemical characteristics confirmed that Pseudomonas is a rod-shaped motile gram-negative bacterium, oxidative and nitrate reduction positive. The colony color of Pseudomonas is yellow (P. fluorescens colony is Yellowish green)
Vibro species: Table 5 shows the Phenotypic and biochemical characteristics of Vibrio spp. It was observed that Vibro is a rod-shaped motile gram-negative bacterium. It is oxidative and catalase-positive. It grows on TCBS (thiosulfate-citrate-bile salts-sucrose agar) producing a yellow colony.
Aeromonas hydrophila: Table 6 shows the Phenotypic and biochemical characteristics of Aeromonas hydrophila. It was found that A. hydrophila is a rod-shaped motile gram-negative bacterium. It is catalase-positive and hydrolyzes starch and gelatin.
Analysis of the PCR products: Figure 2 showed that isolated V. parahaemolyticus and V. alginolyticus were positive for the presence of trh and tdh virulence genes respectively, meanwhile, isolated P. aeruginosa was positive for oprl virulence gene and the examined P. fluorescens were positive for bla TEM virulence as shown in Fig. 3.
| Table 2: | Bacteria isolated from of diseased fish farms |
Bacteriological examinations revealed isolation of Pseudomonas spp., Aeromonas spp. and Vibrio spp. from the examined fish and water samples with rate of (16, 10%), (22, 10%) and (28, 10%), respectively. The isolates were Pseudomonas spp. (P. aeruginosa, P. fluorescens, P. putida and P. alcaligenes), Aeromonas spp. (A. sobria, A. hydrophila and A. caviae) and Vibrio spp. (V. parahaemolyticus, V. alginolyticus, V. vulnificus and V. harveyi) | |
| Table 3: | Bacteriological examination of water and fish samples |
It grows on TCBS (thiosulfate-citrate-bile salts-sucrose agar) producing a yellow colony.
Aeromonas hydrophila: Table 6 shows the Phenotypic and biochemical characteristics of Aeromonas hydrophila. It was found that A. hydrophila is a rod-shaped motile gram-negative bacterium. It is catalase-positive and hydrolyzes starch and gelatin.
| Table 4: | Phenotypic and biochemical characteristics of Pseudomonas species |
| -ve : Negative; +ve : Positive, MR-VP: Methyl red -Voges proskauer test | |
| Fig. 2: | The examined isolates of Vibrio parahaemolyticus and Vibrio alginolyticu |
Lane L: 100 bp ladder as a molecular size DNA marker, Pos: control positive, Neg: control negative, Lane 1-3: Positive virulence gene (tdh) for Vibrio parahaemolyticus, Lane 1-3: positive virulence gene (trh) for Vibrio Alginolyticus |
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| Table 5: | Phenotypic and biochemical characteristics of V. harveyi and V. alginolyticus |
-ve : Negative; +ve : Positive, TCBS: Thiosulfate-Citrate-Bile salts-Sucrose agar, MR: Methyl red test, VP: voges proskauer test | |
| Table 6: | Phenotypic and biochemical characteristics of Aeromonas hydrophila |
-ve : Negative; +ve : Positive, MR: Methyl red test, VP: Voges proskauer test | |
| Table 7: | Agar disc diffusion test results showing the sensitivity of isolated bacteria to different antibiotics |
S: Sensitive (more than 50 and less than 75% of isolates were susceptible to the antimicrobial agents), MS: Moderately susceptible (50% of the isolates were susceptible to the antimicrobial agents), HS: Highly sensitive (75% or more of isolates were susceptible to the antimicrobial agents), R: Resistant (more than 50 and less than 75% of isolates were resistant to the antimicrobial agents), HR: Highly resistant (more than 75% of isolates were resistant to the antimicrobial agents) | |
| Fig. 3: | The examined isolates of P. aeruginosa and P. fluorescens |
Lane L: 100 bp ladder as a molecular size DNA marker, Pos.: control positive, Neg.: Control negative, Lane 1-3: Positive virulence gene (oprl) for P. aeruginosa, Lane 1-3: Positive virulence gene (blaTEM) for P. fluorescens |
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| Table 8: | Effect of enrofloxacin administration (50 mg kg–1 feed) for 7 days on mortalities of infected farms |
| Table 9: | Effect of enrofloxacin on AST, ALT, urea and creatinine levels of diseased fish |
Aspartate aminotransferase, ALT: Alanine aminotransferase, Means within the same column of different superscript digits are significantly different at (p<0.05) |
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Analysis of the PCR products: Figure 2 showed that isolated V. parahaemolyticus and V. alginolyticus were positive for the presence of trh and tdh virulence genes respectively, meanwhile, isolated P. aeruginosa was positive for oprl virulence gene and the examined P. fluorescens were positive for bla TEM virulence as shown in Fig. 3.
Antibiogram: Table 7 explains the effect of different antibiotics on isolated bacteria in vitro. The results showed that the most effective antibiotic on isolated bacteria was enrofloxacin, meanwhile, Pseudomonas spp. were resistant to many antibacterial agents (Table 7).
Treatments trials: Table 8 shows the effects of dietary enrofloxacin supplementation (50 mg kg–1 feed) for 7 days on the mortality rate in examined farms. It was noticed that enrofloxacin reduced significantly the mortality rate. Furthermore, data in Table 9 revealed that enrofloxacin administration induced a significant reduction of serum AST, ALT, urea and creatinine.
DISCUSSION
Fish is an important animal protein source in Egypt. It is important to control fish diseases to avoid high economic losses35. The study was performed on 5 farms that suffered from mass mortality. One hundred diseased fish (20 per farm) were randomly collected from these farms. Bacteriological examinations were carried out on the diseased fish. The recorded P.M. lesions were hemorrhages on the liver and darkness of skin as in (Fig. 1a-b). Bacteriological examinations of diseased fish revealed isolation of Pseudomonas spp., Aeromonas spp. and Vibrio spp. from the examined fish and water samples with the rat of (16, 10%), (22, 10%) and (28, 10%) respectively as in Table 3, these results were confirmed with biochemical tests. The isolates were Pseudomonas spp. (P. aeruginosa, P. fluorescens, P. putida and P. alcaligenes),
Aeromonas spp. (A. sobria, A. hydrophila and A. caviae) and Vibrio spp. (V. parahaemolyticus, V. alginolyticus V. vulnificus and V. harvey). Bacteriological examinations of ration revealed no isolation of any previous bacteria. These results agree with that recorded by Tison et al.35, Zorrilla et al.36 and Moustafa et al.37 but in low prevalence. Phenotypic and biochemical characteristics of isolated Pseudomonas, Vibrio and Aeromonas species were nearly similar to that recorded by Moustafa et al.37.
The usage of molecular and conventional methods together for Vibrio species identification is necessary38. Further identification for Vibrio species were done for detection of tdh and trh virulence genes, as in Fig. 2, the pathogenicity of Vibrio species was detected by the presence of tdh (B hemolytic nature) and tdh which is cytotoxic to many types of cells39. As shown in Fig. 2 the examined isolates of V. alginolyticus and V. parahaemolyticus were positive for the presence of both trh and tdh virulence genes respectively. Immunological, biological and physicochemical characteristics of trh were found to be similar to those of tdh: 84% sequence identity40,41. Both gens are responsible for many outbreaks21. The outer membrane protein (oprL) of Pseudomonas spp. plays an important role of antibiotic resistance through efflux systems42. As result of mass use of antibiotics agents many pathogenic bacteria acquired virulence genes which made them more pathogenic. As shown in Fig. 3 the examined P. aeruginosa were positive for oprl virulence gene and the examined P. fluorescens were positive for bla TEM virulence gene. These genes and other virulence genes enable them to play a great role in causing fish diseases14.
Table 7 showed that, the most effective antibiotic on isolated bacteria was enrofloxacin. Pseudomonas spp. were resistant to many antibiotics. Improper use of many antibiotics in aquaculture may lead to the presence of antibiotic-resistant bacteria10. For example, amoxicillin is a broad-spectrum beta-lactam antibiotic with a higher absorption rate when given orally43 but as shown in Fig. 3 blaTEM gene (B-Lactam resistance gene e.g., ampicillin, amoxicillin) was present in all examined Pseudomonas spp., leading to the development of resistance to B-Lactam group44. However, data in Table 7 and 8 revealed that the isolated bacterial strains were highly sensitive to enrofloxacin in vitro and in vivo. Similar results were also obtained by Riviere et al.43 who reported that fluoroquinolones having a piperazine group at position 7 as enrofloxacin is highly active against many pathogenic aerobic Gram-negative bacteria as Pseudomonas spp.
The increase in serum ALT, AST, creatinine and urea in infected fish may be attributed to the liver, kidney and gill damage induced by infected bacteria. Serum biochemical analysis of the infected fish after treatment with enrofloxacin revealed a significant improvement of liver and kidney functions. That pointed to the effect of enrofloxacin against infected bacteria. Results are in accordance with the results obtained by Koehler and Ashdown45, Laganà1 et al.46, Trevesbrown47 and El-Atta and Tantawy 48 who found that A. hydrophila, most Vibrio strains, P. aeruginosa and P. fluorescens were susceptible to ciprofloxacin. Furthermore, the resistance of Pseudomonas spp. to amoxicillin was noticed (Table 7) as a result of having blaTEM gene (as shown in Fig. 3). This result was supported by El-Hady and Samy49 who mentioned that P. aeruginosa acquired the resistance to amoxicillin due to heavy contamination of marine water by sewages polluted with antibiotics residues. Finally, improper use of antibiotics may lead to the appearance of resistant strains of bacteria. These bacteria become able to adapt to the antibiotic by mutating and developing resistance gen, so many antibiotics lost their power (amoxicillin in this work). Therefore, using of antibiotics in the veterinary field should be under veterinary supervision with therapeutic doses. Moreover, good environmental conditions should be provided in aquaculture to avoid stresses and immune suppression of fish. Immune compromised fish are more susceptible to the infection.
CONCLUSION
It could be concluded that the high mortalities in some marine fish farms in the Damietta governorate could be due to some pathogenic bacteria mainly Aeromonas, Pseudomonas and Vibrio. Administration of enrofloxacin reduced mortalities in these farms; however, improper mass use of antibacterial agents reduced the chance of using many suitable antibiotics as a result of the development of antibiotic-resistant bacterial strains.
SIGNIFICANCE STATEMENT
This study discovers the possible causes of treatment failure with many antibiotics that can be beneficial for antibiotic selection. This study will help the researchers to uncover the critical area of resistant bacterial-gens were not able to explore. Thus, a new theory on the selection of effective antibiotics based on the absence of antibiotics resistant bacterial-gens may be arrived at.