Characterization of bacteria isolated from silver pomfret (pampus argenteus euphrasen, 1788) cultured in nhatrang bay, vietnam

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CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES CHARACTERIZATION OF BACTERIA ISOLATED FROM SILVER POMFRET (Pampus argenteus Euphrasen, 1788) CULTURED IN NHATRANG BAY, VIETNAM By NGUYEN NHUT THANH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Supervisor Ms. Tran Thi My Duyen Assoc. Prof. Dr. Dang Thi Hoang Oanh Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES CHARACTERIZATION OF BACTERIA ISOLATED FROM SILVER POMFRET (Pampus argenteus Euphrasen, 1788) CULTURED IN NHATRANG BAY, VIETNAM By NGUYEN NHUT THANH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Supervisor Ms. Tran Thi My Duyen Assoc. Prof. Dr. Dang Thi Hoang Oanh Can Tho, December 2013 APPROVED BY SUPERVISOR The thesis “Characterization of bacteria isolated from silver pomfret (Pampus argenteus Euphrasen, 1788) cultured in Nhatrang bay, Vietnam” which edited and passed by the committee, was defended by Nguyen Nhut Thanh in 27/12/2013. Student sign Nguyen Nhut Thanh Supervisor sign Ass. Prof. Dang Thi Hoang Oanh ACKNOWLEDGEMENT First of all, I would like to express my deep and sincere gratitude to my supervisor, Dr. Dang Thi Hoang Oanh for her guidance throughout my degree program.. Many thanks are also given to all other doctors, lecturer of the college of aquaculture and fisheries, and especially to those of the department of aquatic biology and pathology for providing me with great working and learning conditions. I wish to thank all of my friends for helping me get through the difficult times, and for all the emotional support and encouragement, especially Vo Le Thanh Truc, Nguyen Trong Nghia, Au Thi Kim Ngoc, Bui Thi Diem My, Le Thanh Can. Finally, I would like to give thank my academic adviser, Dr Duong Thuy Yen, for her guiding and encouraged me, also my parents and all my family members for their love, understanding and support me more than four years in university. i ABSTRACT The purpose of this study was to isolate and characterize bacterial isolates which were recovered from diseased Silver pomfret cultured in Nha Trang Bay, Khanh Hoa province. A total of eights diseased speciments which were collected in October, 2012. Diseased fish displayed lethargic swimming, hemorrhagic on skin, tumor on the body and whitish granules in internal organs. Bacterial isolates were examined for morphology, selected biochemical characteristics as well as susceptibility to common used antibiotics in aquaculture. These isolates were identified as Photobacterium damselae, Vibrio parahaemolyticus, and Vibrio alginoticus, by using API 20E test kit. Result of antibiotic sensitivity tests showed that these isolates were completely resistance with amoxycilline, bicomarin, ampicillin; and they were all susceptible with norfloxacin, ciprofloxacin, florenicol and tetracycline. ii TABLES OF CONTENTS ACKNOWLEDGEMENT ................................................................................................ i ABSTRACT .................................................................................................................... ii TABLES OF CONTENTS ............................................................................................. iii LIST OF FIGURES ......................................................................................................... v LIST OF TABLES ......................................................................................................... vi CHAPTER 1 INTRODUCTION ..................................................................................... 1 1.1 Introduction ........................................................................................................ 1 1.2 Research objective .............................................................................................. 2 1.3 Research activities .............................................................................................. 2 CHAPTER 2 LITERATURE REVIEW .......................................................................... 3 2.1 Silver pomfret ........................................................................................................ 3 2.2 Cause of diseases ................................................................................................... 3 2.3 Some common bacterial diseases in brackish and marine fishes ............................. 4 2.3.1 Vibriosis .......................................................................................................... 4 2.3.2 Bacterial hemorrhagic septicaemia................................................................... 5 2.3.3 Streptococcosis ................................................................................................ 5 2.4 Some commonly used antibiotics ........................................................................... 6 2.4.1 Oxytetracycline................................................................................................ 7 2.4.2 Ciprofloxacin ................................................................................................... 8 2.4.3 Florfenicol ....................................................................................................... 8 CHAPTER 3 MATERIALS AND METHODS ............................................................... 9 3.1 Time and sites of study........................................................................................... 9 3.2 Materials ................................................................................................................ 9 3.3 Methods ................................................................................................................. 9 3.3.1 Fish sampling .................................................................................................. 9 3.3.2 Bacterial isolation .......................................................................................... 10 3.3.3 Bacterial identification ................................................................................... 10 3.3.4 Antimicrobial susceptibility testing ................................................................ 10 3.3.5 Data collection, calculation and analysis ........................................................ 11 CHAPTER 4.................................................................................................................. 12 RESULT AND DISCUSSION ...................................................................................... 12 iii 4.1 Fish sampling and clinical signs ........................................................................... 12 4.2 Bacterial identification ......................................................................................... 13 4.3 Antimicrobial susceptibility testing ...................................................................... 15 CHAPTER 5 CONCLUSIONS AND RECOMMENDATION ...................................... 17 5.1 Conclusions.......................................................................................................... 17 5.2 Recommendation ................................................................................................. 17 REFERENCE ................................................................................................................ 18 Appendix 1.Some biochemical tests used in bacterial identification ........................... 20 Appendix 2. API 20E kit results ................................................................................. 22 Appendix 3. The diameters of inhibition zone of the antibiotic susceptible test .......... 23 iv LIST OF FIGURES Figure 4.1 Fish sampled Figure 4.2 Internal of diseased fish Figure 4.3 Gram staining, O/F test and bacterial colonies on NA Figure 4.4 API 20E identification test result of Photobacterium damselae Figure 4.5 API 20E identification test result of Vibrio parahaemolyticus Figure 4.6 API 20E identification test result of Vibrio alginoticus Figure 4.7 API 20E identification test result of Aeromonas hydrophyla v LIST OF TABLES Table 4.1 The percentages of distinct strains Table 4.2 Susceptibility pattern of bacterial species vi CHAPTER 1 INTRODUCTION 1.1 Introduction With more than 3200 kilometers of coastline and large area of water surface, Vietnam has great conditions to develop aquaculture (both fresh and marine aquaculture). In fact, aquaculture in Vietnam is developing year by year, especially farmed stripped catfish (Pangasianodon hypophthalmus) and penaeid shrimp (black tiger and white leg shrimp), Vietnam was ranked the world’s third of aquaculture (FAO 2010). Besides, marine aquaculture is contributed an important part for aquaculture with high value species such as sea bass, cobia (Rachycentron Canadum), grouper (Serranidae), silver pomfret (Pampus argenteus)… Silver pomfret is one of a new cultured species is Vietnam as it has high nutrition contain and quality of flesh meat. Silver pomfret is easy to find in Northern gulf and central southern of Vietnam, in the past, people mainly catch silver pomfret in the wild. In recent years, people started to culture silver pomfret in cages with intensive model. In this system, fish are stocked at high density, usually over feeding. This can make the fish get stress and diseases, especially this can lead to disease spread out easily on the sea to wild fish, or the others cages. Bacterial diseases have been reported as one of the worst problems causing up to 100% mortality of cultured fishes (Bui Quang Te, 2008). This is the new specie, there is a few research about silver pomfret are conducted on over the world. Antibiotics are known as the useful treatment for the bacterial disease, but can make bad result if not use at correct ways or dosage. Bacterial resistance often happens and cause treatment failure. People prefer using higher dosages of antibiotics when the previous dosages do not have effect; they hope to heal illness completely, but residues in flesh product are not favorable to consumers. In addition, resistance characteristic could readily and quickly spread out in bacterial populations (Kumarasamy et al., 2010). The uncontrolled of using antibiotics can make the presence of antibiotic residues in fish meat and fish products and this also lead to a disease which cause by resistance bacteria and hard to treat. Thus, this thesis “Characterization of bacteria isolated from silver pomfret (Stromateoides argenteus Euphrasen, 1788) cultured in Nhatrang bay, Vietnam” is carried out to provide more information about disease on this species. 1 1.2 Research objective The aim of this research is to investigate the bacterial pathogen which are isolated from silver pomfret (Pampus argenteus) in Nhatrang bay, then find out the antibiotic which is susceptible to these bacteria. 1.3 Research activities This thesis focuses on the following contents: 1. Classification to species level of bacteria that was isolated from silver pomfret. 2. Antibiotics susceptibility testing. 2 CHAPTER 2 LITERATURE REVIEW 2.1 Silver pomfret Silver pomfret (Pampuss argenteus) is a valuable food fish with a wide geographical distribution from East China Sea to Southest Asia, the India Ocean, Arabian Gulf and the North Sea (Davis and Wheeler 1985). Silver pomfret is found in coastal water from 5 to 100 meters depth, it has maximum size 80 centimeters but usually see at 30 centimeters (FAO). In the past, it was only caught on the sea, but in recent years, it is stared being culture in cages. Cages are placed in clean area, with viscosity about 0.20.6m/s. This is new species cultured in Vietnam, it has high economic value base on its nutrition value, high flesh meat quality. There is not many research about this species have been reported in Vietnam. 2.2 Cause of diseases Aquatic organisms are sensitive with the diseases, because they exposure directly with the water where they live. Diseases can be caused by an etiological (specific cause) or a nonetiological (contributing cause) agent. Etiological agents can be classified as either inanimate or animate. Inanimate etiological agents are factors without life of their own and can originate within a host (endogenous) or outside of a host (exogenous). Endogenous, inanimate factors are those associated with genetic and/or metabolic disorders of the host. Exogenous, inanimate agents include trauma, temperature shock, electrical shock, chemical toxicity, and dietary deficiencies. These etiological agents may serve as sublethal stressors that predispose fish to infectious disease. Animate etiologies are living communicable infectious agents, which include viruses, bacteria, fungi, protozoa, helminthes and copepods (Plumb, 1999). Nonetiological causes of disease are characterized as extrinsic (from outside the body) or intrinsic (within the body). Extrinsic factors are usually associated with environmental conditions or dietary problems, and intrinsic factors include age, gender, heredity, and fish species. Both fish species and strain of fish are important because all are not equally susceptible to a specific disease organism. Feed quality, 3 water quality factors, and water temperature extremes can be classified as either etiological or nonetiological extrinsic factors and can contribute to infectious disease (Plumb, 1999). 2.3 Some common bacterial diseases in brackish and marine fishes Bacteria are one of the most important-causative agents causing adverse diseases to fish. Most bacterial agents causing diseases to fish are Gram-negative; some of them are Gram-positive. They are ubiquitous in the environment (sea, lakes, rivers, canals, ponds…) and are considered as the primary pathogen or the opportunistic pathogen. They are usually chronic, acute or subacute diseases. In some cases, bacterial diseases can cause 100% of mortality (Bui Quang Te, 2006). 2.3.1 Vibriosis Vibriosis, also known as salt-water furunculosis, boil-disease, or ulcer-disease (Austin and Austin, 2007), is among the most prevalent fish diseases caused by bacteria of genus Vibrio (Woo and Bruno, 1998).This type of disease is commonly considered stress mediated with the predisposing factors of handling, moving from fresh to salt water (Plumb and Hanson, 2011), high temperature, crowding, and organic pollution (Noga, 2010). Vibriosis has been reported on many fish species, including salmon, rainbow trout, turbot, sea bass, sea bream, striped bass, cod, and eel (Actis et al., 1999 cited by Toranzo, 2005).Within Vibrionaceae, the species causing the most economically serious diseases in marine culture are Vibrio anguillarum, Vibrio ordalii, Vibrio salmonicida, Vibrio vulnificus biotype 2 (Toranzo et al., 2004), and Vibrio harveyi (FAO, 2006).V.anguillarumis the most common fish-pathogenic vibrio (Noga, 2010). Infected fish may havered areas on body, skin ulcers, depression, exophthalmos, corneal ulcers, and swollen abdomen (Noga, 2010).Vibriosis, as with other bacterial septicaemias, can be controlled by maintaining good water quality, but where outcreaks occur, treatment with an oral antibiotic is the only option (Woo and Brono, 1998) 4 2.3.2 Bacterial hemorrhagic septicaemia Bacterial hemorrhagic septicaemia, also referred to as motile aeromonad infection, infectious dropsy, red pest, reddisease, red sore, rubella, and others (Plumb and Hanson, 2011), has been associated with several members of genus Aeromonas such as A. hydrophila,A. sobria, A. caviae, and Pseudomonas sp.(FAO, 2006). Disease syndromes may include lethargy, anorexia, irregular reddened skin ulcerations, reddened abdominal fluid, and pale gills (Austin and Austin, 2007). By far the most significant fish pathogen is A. hydrophila (Noga, 2010). A. hydrophila is widely distributed in the aquatic environment (Roberts, 2012). This species is a pathogenic species mainly to fresh water fish, and occasionally to marine fish (Austin and Austin, 2007). They occur as Gram-negative, motile, straight rods (0.3-1.0 x 1.03.5µm) (Roberts, 2012). This bacteria species showed resistant to many types of antibiotics, including ampicillin, chloramphenicol, erythromycin, nitrofurentoin, novobiocin, streptomycin, sulphonamides, tetracycline, oxytetracycline (Aoki, 1988; De Paoplaet al., 1988, cited by Austin and Austin, 2007), but were very sensitive to enrofloxacin (Brag and Todd, 1988, cited by Austin and Austin, 2007). 2.3.3 Streptococcosis Streptococcosis is sometimes called “popeye” because exophthalmos (exophthalmia) is very common. This disease can cause darkening, pale gill, reddened fluid and organs in infected fish (Noga, 2010).Diverse host species have been reported with streptococcus infection, including rainbow trout, tilapia, hybrid triped bass (Eldar et al., 1994) and sea bass (Bromage et at., 1999; Creeper and Buller, 2006). Even though, many species of Streptococcosis, including S. agalactiae, S. iniae, S. dysqalactiae, S. dysgalactiae, S. pyogenes, S. parauberis, and S. equi, have been reported from fish,S. iniaeand S. agalactiaeare the two that mostfrequently cause serious disease in tilapia. Streptococcus iniae are small, Gram-positive, facultative anaerobic cocci, appearing in chains (Roberts, 2012). Although S.iniae can affect various freshwater and coastal fish species (Austin and Austin, 2007), this bacterial species is more commonly isolated from fresh-water fish such as rainbow trout and tilapias than from marine fish such as flounders and sardines (Kusada and Salati, 1999, cited by Roberts, 2012).Fish infected by this species often get damaged brain, exophthalmia, surface and internal. 5 More importantly, fish pathogen S.iniae can cause disease in human hemorrhaging (Austin and Austin, 2007). This bacterial agent can be treated with fluoroquinolone compound, enrofloxacin (Stoffregenet al., 1996, cited by Austin and Austin, 2007). Besides, laboratory studies also showed the efficacy of oxytetracycline and amoxicillin in controlling S.iniae (Darwishet al., 2002; Darwish and Ismaiel, 2003, cited by Austin and Austin, 2007). 2.4 Some commonly used antibiotics Antibiotics are known as the best treatment when fish get bacterial diseases. People usually take over dosages for sure that they treat the disease completely; this is not only wasting of money but also make opportunities for bacterial resistance. The residual of antibiotic in fish (aquatic species) could affect to human health. The antibiotics most frequently used in aquaculture to combat bacterial diseases include oxytetracycline, florfenicol, sarafloxacin, and enrofloxacin (Roque et al., 2001; SotoRodríguez et al., 2006). Globally, other antibiotics such as chlortetracycline, quinolones, ciprofloxacin, norfloxacin, oxolinic acid, perfloxacin, sulfamethazine, gentamicin, and tiamulin are used (Holmstrom et al., 2003). Major antimicrobial drugs used in aquaculture (Prescott et al., 2000) Route Dose (mg/kg fish/day) Oral 50-80mg/kg, 10 days Neomycin Oral 50-80mg/kg, 10 days Kanamycin Bath 20mg/l Tetracycline Oral 50-80mg/kg, 10 days Bath 20mg/l Oral 50-80mg/kg, 10 days Bath 20mg/l Product Indication Antibiotics Ampicillin Beta-lactams Amoxicillin Benzyl penicillin (with streptomycin) Aminoglycosides Tetracyclines Oxytetracycline Doxycycline Macrolides Gram (-) bacteria Erythromycin 6 Bacterial kidney disease Chloramphenicol group Florfenicol Oral 50-80mg/kg, 10 days Thiamphenicol Bath 20mg/l Oral 200mg/kg, 10days Synthetic Antimicrobial Agents Sulfamethazine Sulfonamides Sulfadimethoxine Sulfaguanidine Potentiated sulfonamides Trimethoprim/ sulfadiazine(1:5) 50mg/kg, 10days Oral Ormetoprim/ sulfadimethoxine 50mg/kg, 5days Oxilinic acid Quinolones Gram (-) bacteria Flumequine Oral Sarafloxcin 10-30mg/kg, 10days 10mg/kg, 5days Note: Only oxytetracycline and ormetoprim-sulfadiazine are approved for use with food fish in the United Staes. 2.4.1 Oxytetracycline Oxytetracycline is widely employed to treat bacterial infections in aquaculture farms, such as vibriosis and furunculosis (Capone et al., 1996; Prescott et al., 2000; Reed et al., 2006; Wang et al., 2004). It belongs to the tetracycline group, which exerts antimicrobial action against both Gram (-) and (+) bacteria, ricksettsias, mycoplasmas, and others (Gómez-Gil et al., 2001). Tetracyclines are produced by Streptomyces spp., which possess determinants for resistance to this class of antibiotics. Oxytetracycline is a bacteriostatic antibiotic that exerts its antimicrobial effect against protein synthesis, by bonding directly to the S7 protein of the 30S subunit of the bacterial ribosome, thereby impeding the bonding of aminoacyl-tRNA (aminoacyl transfer RNA) to the A-site of the ribosome. This prevents the addition of amino acids to the growing peptide chain (Chambers, 2004; Isidori et al., 2005; Jara, 2007). In order for oxytetracycline to interact with its target site, it needs to pass through the external membrane via passive diffusion through the OmpF and OmpC pores, and through the cytoplasm membrane via an energy dependent process (Jara, 2007). 7 2.4.2 Ciprofloxacin Ciprofloxacin is the main metabolite of Enrofloxacin and is active against a broad spectrum of aerobic Gram (-) bacteria, including enteric pathogens such as Pseudomonas and Serratia marcescens. It is also active against Gram (+) pathogens, even when these bacteria have developed resistance to other antibiotics, such as penicillin (Wen et al., 2007). It is not active against anaerobic bacteria and may be used occasionally, in combination with other antibacterial agents, for the treatment of mycobacterial infections. The antibacterial effects of ciprofloxacin arise from its inhibition of Topoisomerase IV and bacterial DNA gyrase, which act by cleaving the DNA of the bacterial chromosome and rejoining the ends once a superhelix is formed (Banerjee et al., 2007). When these enzymes are inhibited, bacterial cell multiplication is interrupted. 2.4.3 Florfenicol This fluorinated antibiotic, derived from thiamphenicol, is a potent and broadly acting bacteriostatic agent. It is effective in the treatment of infections caused by Pasteurella piscicida, Aeromonas salmonicida, Vibrio anguillarum, and Edwardsiella tarda. Its chemicalstructure is very similar to that of chloramphenicol, and florfenicol is effective against bacteria that have developed the ability to deactivate other drugs, such as thiamphenicol and chloramphenicol. Pharmacokinetically, florfenicol use has been reported among some species of fish such as Atlantic salmon (Salmo salar), in which a bioavailability of more than 95% is present, exhibiting a good distribution among all of the organs and tissues. Its halflife in fish is less than 15 h (Yanong & Curtis, 2005). However, published information for shrimp is scarce, meaning that the kinetic behavior of this compound among these crustaceans has not yet been completely elucidated. 8 CHAPTER 3 MATERIALS AND METHODS 3.1 Time and sites of study - Time: January to June, 2013. - Locations: Fish were sampled at Nhatrang bay, Vietnam. - Place for conducting experiments: Department of Aquatic Pathology, College of Aquaculture and Fisheries, Cantho University. 3.2 Materials The media, chemicals, antibiotics and consumables used for this study are listed below: - Nutrient agar (NA) Tryotic soy agar (TSA). Crystal violet (color Index No.42555) Ammonium oxalate Iodine Potassium iodide Safranin (Color Index No.50240) Chemicals for biochemical tests: O/F, motility, Indole, Oxidase, Catalase… Antibiotics: enflorxacin (ENR/5g), florfenicol (FFC/30g,), amoxycilline (25g), norfloxacin (5g), ciprofloxacin (30g), trim/sulfa (1:19) (25g), tetracyline (30g), neomycin (30g), ampicilline (35g), doxycyline (30g). 3.3 Methods 3.3.1 Fish sampling Silver pomfret samples are collected from culture cages in Nhatrang bay, Nhatrang. Samples of 2-3 diseased fish are collected from each cage, then sampled on-cages. The fish have some clinical sign such as infected with water fleas, tumor on the body, the body cavity and internal organs are bleeding, internal organs has white spots. After that, keep the samples to transport to the laboratory of the College of Aquaculture and Fisheries, Cantho University for analysis. 9 3.3.2 Bacterial isolation Fish samples were first put on clean trays for observing. They were disinfected with 70 0 alcohols, and then carefully dissected to avoid damaging internal organs, and reduce the risk of contamination. Internal signs of fish were also observed and noted. Bacterial samples from liver, kidney, spleen, and brain were inoculated on nutrient agar plates (Merck) supplemented with 1.5% sodium chloride to acquire the salinity of 15‰ and incubated at 280C. After 24 hours of incubation, bacterial growth was checked, and representative bacterial colonies were sub-cultured for purity. 3.3.3 Bacterial identification Pure culture of bacterial isolates after being obtained were used in primary tests, including Gram staining, motility, oxidase, catalase, oxidative-fermentative, O/129 tests, following the method of Frerichs and Millar (1993), and Buller (2004). Detailed procedures for each specific test are shown in Appendix 3. Bacterial strains were fully identified by using API 20E test kit, following the instruction of the suppliers. 3.3.4 Antimicrobial susceptibility testing The susceptibility patterns of the identified isolates were made by using disk diffusion method described in Clinical and Laboratory Standards Institute document M2-A09 (CLSI, 2006). Five strains of bacteria which are identified used for this experiment. Incubating loops are used to take 1-2 colonies from pure culture, and put into bottle with about 30ml sterile BHI to vortex at 200 rounds/minutes for 24 hours. Bacterial solution is now transferred into 50ml falcon tube for centrifugation (4000 rounds/minute for 15 minutes). The upper solution part is eliminated, and bacteria are washed 2-3 times under sterile saline solution. After the final centrifugation, and elimination of the upper part, 25ml of saline solution is added, and the solution is well mixed. The density of bacteria is determined, using spectrophotometer (wavelength = 600nm). Bacterial solution is then diluted to the approximate concentration of 1-2 x 108 colony-forming units (CFU)/ml. The standardized bacterial suspension is evenly spread on the surface of the agar plates with a cotton swab. The surface of the medium is let to dry for 3-5 minutes to allow for the absorption of excess moisture. Antibiotic disks are placed on the surface of the inoculated and dried plate with sterile forceps, and lightly pressed down to ensure complete contact between the disk and the 10 agar surface. Position disks such that the minimum center-center distance is 24mm and no closer than 10-15mm from the edge of the petri dish. A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150mm plate. The zones of inhibition are observed after 24 hours of incubation at 280C. The zone of inhibition is the point at which no growth is visible to the unaided eye. Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standard for veterinary pathogen. 3.3.5 Data collection, calculation and analysis Data was analysis by using Microsoft Excel 2007. 11
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