Antimicrobial susceptibility of edwardsiella tarda isolated from clown knifefish (chitala chitala)

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CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES ANTIMICROBIAL SUSCEPTIBILITY OF Edwardsiella tarda ISOLATED FROM CLOWN KNIFEFISH (Chitala chitala) By NGUYEN MINH THUAT A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES ANTIMICROBIAL SUSCEPTIBILITY OF Edwardsiella tarda ISOLATED FROM CLOWN KNIFEFISH (Chitala chitala) By NGUYEN MINH THUAT A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Aquaculture Supervisor Dr. TU THANH DUNG Can Tho, December 2013 APPROVEMENT The thesis “Antimicrobial susceptibility of Edwardsiella tarda isolated from clown knifefish (Chitala chitala)” defended by Nguyen Minh Thuat, which was edited and passed by the committee on 27-12-2013. Sign of Supervisor Sign of Student Dr. Tu Thanh Dung Nguyen Minh Thuat i ACKNOWLEDGEMENT First of all, I would like to express my sincere gratitude to my supervisor, Dr. Tu Thanh Dung for her enthusiastic guidance, advice, and encouragement during the thesis implementation. Many thanks are also given to all other staffs of the College of Aquaculture and Fisheries, and especially to those of the Department of Aquatic Pathology for providing the great working and learning conditions. Special thanks to many of my friends, especially Nguyen Khanh Linh, Pham Dang Hoa Hiep, Nguyen Bao Trung, and Tran Thi My Han for their kindly help throughout the experimental period. Thanks for my academic advisor, Dr. Duong Thuy Yen, who was guiding and giving me a lot of useful advices over the last four years. Above all, I am grateful to my parents and other members in my family for their greatly support and encouragement during studying period. Nguyen Minh Thuat ii ABSTRACT This study was conducted to access the antimicrobial susceptibility of Edwardsiella tarda isolated from clown knifefish (Chitala chitala) cultured in the Mekong Delta, Vietnam. Fish samples were collected in Haugiang and Dongthap provinces. A total number of 17 bacterial isolates were obtained from diseased fishes in different commercial farms. Naturally infected clown knifefish showed clinical signs of petechial hemorrhages on body, fins and the mandible. Internally, ascites, hepatomegaly and splenomegaly were also found. The conventional and rapid identification system were used to identify these isolates as Edwardsiella tarda. All of E. tarda isolates were tested with 16 different antibiotics by using disk diffusion method. The results showed that most of isolates were sensitive to amoxicillin/clavulanic acid, florfenicol, cefotaxime, doxycycline, cefalexin, and cefazolin. However, most of E. tarda isolates were highly resistant against sulfamethoxazole/trimethoprim, norfloxacin, enrofloxacin, oxytetracycline, ampicillin, rifampicin, and novobiocin. Especially, in this study, all of isolates showed multiantibiotic resistance. Besides, three different antibiotics were also used to determine minimal inhibitory concentration (MIC) by using dilution method. iii TABLE OF CONTENTS Acknowledgement ......................................................................................................... ii Abstract ......................................................................................................................... iii Table of contents........................................................................................................... iv List of tables ................................................................................................................. vi List of figures ............................................................................................................... vii List of abbreviations .................................................................................................. viii Chapter 1: Introduction .............................................................................................. 1 1.1 General introduction ....................................................... .............................. 1 1.2 Research objectives ........................................................................................ 2 1.3 Research contents .......................................................................................... 2 Chapter 2: Literature review ...................................................................................... 3 2.1 Clown knifefish (Chitala Chitala) ................................................................. 3 2.2 Common disease on clown knifefish ............................................................ 3 2.3 Edwardsiella tarda infection ......................................................................... 4 2.4 Antimicrobial used in aquaculture ................................................................ 6 2.5 Common antibiotic groups and modes of action ........................................... 7 2.5.1 Beta-lactams ........................................................................................... 8 2.5.2 Tetracyclines .......................................................................................... 9 2.5.3 Phenicols ................................................................................................ 9 2.5.4 Quinolones and fluoroquinolones ........................................................ 10 2.5.5 Sulfonamides ........................................................................................ 10 2.5.6 Aminoglycosides .................................................................................. 10 2.6 Antimicrobial resistance .............................................................................. 11 2.7 Antimicrobial susceptibility testing of E. tarda ........................................... 12 Chapter 3: Materials and methods ............................................................................ 14 3.1 Time and places ......................................................................................... 14 3.2 Materials ...................................................................................................... 14 3.3 Methods ....................................................................................................... 14 iv 3.3.1 Fish sampling ....................................................................................... 14 3.3.2 Bacterial isolation ............................................................................... 16 3.3.3 Bacterial identification ........................................................................ 16 3.3.4 Disk diffusion method ......................................................................... 17 3.3.5 Minimal inhibitory concentration (MIC) test ...................................... 18 Chapter 4: Results and discussion ............................................................................. 19 4.1 Fish sampling and bacterial isolation ........................................................... 19 4.2 Bacterial identification ................................................................................. 21 4.3 Antimicrobial susceptibility testing ............................................................. 22 4.4 Minimal inhibitory concentration (MIC) .................................................... 29 Chapter 5: Conclusions and recommendations........................................................ 31 5.1 Conclusions .................................................................................................. 31 5.2 Recommendations ........................................................................................ 31 References ................................................................................................................... 32 Appendix 1: Major antimicrobial drugs used in aquaculture ...................................... 37 Appendix 2 List of antimicrobials and drugs are banned ............................................ 38 Appendix 3 List of antimicrobials and drugs are limited ............................................ 49 Appendix 4: Fish disease diagnosis form ................................................................... 40 Appendix 5: Some biochemical tests used in bacterial identification ........................ 41 Appendix 6: Bacterial identification scheme .............................................................. 45 Appendix 7: List of solvents and diluents ................................................................... 45 Appendix 8: Preparation of culture dilution series ..................................................... 46 Appendix 9: Species of fish infected with Edwardsiella tarda .................................. 47 Appendix 10: Biochemical characteristics of E. tarda ............................................... 49 Appendix 11: Zone diameter and MIC interpretive criteria ....................................... 50 Appendix 12: Some types of multi-antibiotics resistance ........................................... 50 Appendix 13: Sensitivity test of 17 isolates of E. tarda ............................................. 51 Appendix 14: Sample collection information ............................................................. 52 v LIST OF TABLES Table 4.1: Sample collection sites and number of bacterial isolates............................ 19 Table 4.2: Primary tests of E. tarda ............................................................................. 21 Table 4.3: The number and percentages of susceptible, intermediate and resistant E. tarda isolates ................................................................................................................. 23 Table 4.4: The MIC values (µg/ml) of 3 antibiotics with 4 E. tarda isolates .............. 29 vi LIST OF FIGURES Figure 3.1: The political map of Haugiang province ................................................... 15 Figure 3.2: The political map of Hongngu district ....................................................... 15 Figure 4.1: External and internal signs of diseased fish .............................................. 20 Figure 4.2: Colonies morphology on TSA media and gram staining of E. tarda ........ 20 Figure 4.3: Percentages of susceptible, intermediate and resistant isolates ................ 23 Figure 4.4: Sensitivity of E. tarda with NOR (1), KZ (2), DO (3), ENR (4) ............. 24 Figure 4.5: The percentage of isolate with multi-antibiotics resistance ...................... 27 Figure 4.6: The MIC value of enrofloxacin (isolate EHG4) (16µg/ml)....................... 29 vii LIST OF ABBREVIATIONS AMC Amoxicillin/clavulanic acid, AMP Ampicillin, BHIA Brain Heart Infusion Agar, CFU Colony forming unit, CLSI Clinical and Laboratory Standards Institute, CTX Cefotaxime, DO Doxycycline, ENR Enrofloxacin, FDA Food and Drug Administration, FFC Florfenicol, KZ Cefazolin, OT Oxytetracycline, MHA Mueller-Hinton Agar, MIC Minimal inhibitory concentration, NAFQAD National Agro-Forestry-Fisheries Quality Assurance Department, NOR Norfloxacin , NV Novobiocin, PBPs Penicillin binding proteins, RD Rifampicin, S Streptomycin, SXT Trimethoprim+sulfamethoxazole, TE Tetracycline, TSA Tryptic soya agar, UB Flumequine, VASEP Vietnam Association of Seafood Exporters and Producers. viii CHAPTER 1 INTRODUCTION 1.1 General introduction The Mekong Delta is the area which has highest aquaculture production in Vietnam, contributing over 90% of Vietnam aquaculture production. In there, Pangasius catfish industry has played a leading role in domestic consumption and foreign export. However, in recent years, Pangasius catfish culture had to face to some problems such as product price instability, technical barriers from imported countries and particularly serious diseases. In this situation, many provinces have changed to culture the other species and clown knifefish (Chitala chitala) is one of the favorable species for farmer’s selection. Because of high economic value and easy consumption, clown knifefish has become an important cultured species in the Mekong Delta. Besides, they are also favourable ornamental fish due to black spot on the flanks body. In Vietnam, clown knifefish have been cultured in some provinces in Mekong Delta, particularly in Dongthap and Haugiang provinces. In there, Haugiang had cultured at the area of over 168 ha in 2010 and will expand to 494 ha in 2020, supplying 3000 tons of fish production annually (Haugiang Portal). Because of high meat yield with delicious flavor, fast growth rate, good adaption to new living conditions, clown knifefish culture was not only limited in term of ornamental fish but also expanded into intensive model for commercial food demand. Therefore, the development of fish farming were concerned and promoted in many provinces in recent years. It requires more research on the seed production, nutrition, environmental and fish health management, especially disease on clown knifefish. Currently, there are some researches about artificial seed production, culture technique in earthen pond, polyculture and nutrition. However, research on clown knifefish disease was limited and not significant. On the other hand, to improve production, farmers have been tried to increase stocking density so it led to disease outbreak easily. Intensive fish farming has promoted the growth of several bacterial diseases, which has led to an increase in the use of antimicrobials. Meanwhile, the farmers have no information about the causes of disease as well as proper treatments, they just use and combine some antibiotics and drugs as use for some other fish species. It is not only ineffective but also results in antimicrobial resistance if misused. The continuous use of antibiotics increases the risks for the presence of antibiotic residues in fish meat and fish products. Nowadays, antibiotic resistance, especially to prohibited agents, is a big concern to farmers and consumer healthcare because it can greatly reduce the effectiveness of treatment, and contains potential risks to human health. Thus, this 1 research was done to provide the newest information about bacterial agent infected on clown knifefish and to investigate their antimicrobial susceptibility. 1.2 Research objectives The research was conducted to investigate the antimicrobial susceptibility of E. tarda isolated from clown knifefish (Chitala Chitala) and find out the most effective antibiotics for treatment. 1.3 Research contents  Isolation and identification of Edwardsiella tarda isolates.  Investigation of antimicrobial susceptibility of isolated E. tarda and determination of minimal inhibitory concentration (MIC). 2 CHAPTER 2 LITERATURE REVIEW 2.1 Clown knifefish (Chitala Chitala) Clown knifefish is a fish of genus Chitala and order Osteoglossiformes with different common names: Chital (Bangladesh); Humped featherback (English); clown knifefish (Fishbase). The body is elongated and strongly compressed laterally. Dorsal profile is highly convex. Scales are very minute and short dorsal fin. Anal fin is long and confluent with caudal fin. Pectoral fins are reduced. Dorsal portion is coppery green colored and silvery at sides and below. There are 15 silvery bars present on each side of dorsal ridge and 5-9 small black spots near the end of the caudal fin. Lateral line is complete and the maximum length reported about 120 cm. This species prefer to live in environments with large aquatic plants, the pH in the water 6.5 -7, temperature is about 26 – 28oC. In the proper environment, fish can live for over 10 years and can be reach over 90 cm. Comparing among species in the family, clown knifefish has faster growth rate. With normal knifefish, they can reach 100 g after 12 months meanwhile clown nifefish can reach 400g-500g in 6 months and reach 1-1.2 kg in 12 months (Bangladesh Fisheries Information Share Home). This species is naturally distributed in the African region and Asian countries including India, Pakistan, Bangladesh, Sri Lanka, Nepal, Indonesia, Malaysia, Philippines and some countries in the MeKong River basin like Myanmar, Thailand, Cambodia, Laos and Vietnam. They are commonly found in estuaries, ponds, fields, canals and rivers. Naturally, clown knifefish can live in the middle and the bottom zone where low oxygen or brackish water with low salinity basing on air-breathing organ. This is a carnivorous, predator fish and they are nocturnal feeder, fish often hide among aquatic plants at daytime and more active at night. The main food resources are fish, small crustaceans, algae, organic decay, mollusc…(Poulsen et al., 2004). 2.2 Common disease on clown knikefish At the present, the research on clown knifefish is limited and not significant. However, in 2012, Tu Thanh Dung and Tran Thi My Han performed the ―Study of agent causing haemorrhagic disease on clown knifefish‖. Fish samples were collected from 17 commercial clown knifefish farms in the Mekong Delta provinces: Haugiang, Dongthap and Cantho. Diseased fish showed gross signs of reddening of abdomen, ascites and hemorrhagic internal organs. After isolation and identification, the authors 3 concluded that Aeromonas hydrophila was the causative agent of haemorrhagic disease in clown knifefish. 2.3 Edwardsiella tarda infection Edwardsiella tarda is one of the members of genus Edwardsiella and family Enterobacteriaceae which has 20 genera and more than 100 species of falcultatively anaerobic Gram-negative rods (0.3-1.2 x 1-6.3µm). They are motile by piritrichous flagella, or non motile, non-sporing and chemoorganotrophic with the respiratory and fermentative metabolism, cytochrome oxidase-negative and catalase-positive. Beside E. tarda, another member of genus Edwardsiella is Edwardsiella ictaluri that causes enteric septicaemia in catfish. That is the serious disease affecting commercial catfish culture in the Southern United State and causing epizootics with the mortality rates of 10-50% (Inglis et al.., 1993). In recent years, this disease has become one of the big concerns in Pangasius catfish in Mekong Delta, Vietnam. The remaining species of this genus is Edwardsiella hoshinae. Fish are usually infected with E. tarda or E. ictaluri, whereas E. hoshinae infection is usually reported in reptiles and birds (Park et al.., 2012). Edwardsiella tarda is one of the serious fish pathogens, infecting both cultured and wild fish species. Research on Edwardsiellosis (caused by E. tarda) has revealed that E. tarda had a broad host range and geographic distribution, and contains important virulence factors that enhance bacterial survival and pathogenesis in hosts. Edwardsiellosis had been reported worldwide in many economically important fish species. List of fish species reported to be infected with E. tarda (Evan et al, cited by Woo and Bruno, 2011) were showed in Appendix 9. Additionally, this infection also led to serious economic losses in the aquaculture of olive flounder (Japanese flounder; Paralichthys olivaceus), the most important fish species in South Korean aquaculture with production valued at 489.7 billion Korean Won (40,922 MT), which corresponded to 56.5% of total fisheries production in 2010 (Park et al.., 2012). Edwardsiella septicaemia is the currently accepted name for the disease caused by this pathogen, but there are some other names such as fish gangrene, emphysematous putrefactive disease of catfish (Meyer & Bullock, 1973) and red disease of eels (Egusa, 1976). Edwardsiella tarda was first isolated in Japan by Hoshinae (1962) with the name of Paracolabacterium anguillimortiferum. In 1965, Ewing et al described as E. tarda which is now accepted world-wide (cited by Inglis et al., 1993). E. tarda is a Gram-negative, short, rod-shape, facultative anaerobic bacterium that measures about 2–3 μm in length and 1μm in diameter. It is usually motile, but isolates from red sea 4 bream and yellowtail are non-motile. This bacterium can survive at 0–4% sodium chloride, pH 4.0-10, and 14-45°C. The biochemical characteristics of E. tarda are catalase positive, cytochrome oxidase negative, production of indole and hydrogen sulfide, fermentation of glucose, and reduction of nitrate to nitrite. However, several variations of biochemical tests have been found for ornithine decarboxylase, citrate utilization, hydrogen sulfide production, and fermentation of mannitol and arabinose (Park et al., 2012). Based on phenotypic characteristics, E. tarda isolates were grouped into 3 different biogroups (Ewing et al.,1965, Grimont et al,. 1980, Walton et al., 1993, cited by Inglis et al., 1993). Firstly, wild type strains (associated with human and fish infection) which are negative for sucrose (suc), mannitol (manol), and arabinose (ara), and hydrogen sulphide (H2S) positive. Secondly, Biogroup 1 strains which isolated from diseased zoo animals (reptiles and birds) with opposite characteristics to wild type strains. Finally, biogroup 2 strains (only isolates from human) which are negative for sucrose and H2S and positive for manol and ara (Alcaide et al., 2006). The specific biochemial characteristics of E. tarda are shown in Appendix 9. E. tarda can be seperated from E. ictaluri by the salt tolerance, indole production, H2S production and higher temperature tolerance (Inglis et al.., 1993). The clinical signs of infected fish differ from region to region and from fish species to species (Inglis et al.., 1993). Edwardsiellosis in fish usually occurs under imbalanced environmental conditions, such as high water temperature, poor water quality, and high organic content. Fish infected with E. tarda show abnormal swimming behaviors, including spiral movement and floating near the water surface. Beside, infected fishes show loss of pigmentation, exophthalmia, opacity of the eyes, swelling of the abdominal surface, petechial hemorrhage in fin and skin, and rectal hernia. Internally, watery and bloody ascites in the abdominal space and congested liver, spleen, and kidney are found. Histopathological characteristics of Edwardsiellosis in fish are suppurative interstitial nephritis, suppurative hepatitis, and purulent inflammation in the spleen. Abscesses of various sizes, bacterial colonization, and infiltration of neutrophils and macrophages are found in the liver, spleen, and kidney. Some remarkable pathological features have also been demonstrated in fish, such as dorsolateral petechial hemorrhage and abscesses in cutaneous lesions of catfish hyperplasia, necrosis and inflammation in lateral line canals of striped bass; and necrosis and aggregation of bacteria-laden macrophages in red sea bream . However, the symptoms and pathological changes in fish are similar to those of other bacterial infections, including Aeromonas hydrophila, Vibrio anguillarum and Pseudomonas anguilliseptica. Thus, other molecular or biochemical methods are recommended for diagnosis of E. tarda infection (Park et al.., 2012). 5 Furthermore, one of the big concerns about this pathogen is human health effects. E. tarda is an opportunistic pathogen in human. It causes both intestinal and extraintestinal infections, mainly in individuals with impaired immune systems. Gastroenteritis is the most common disease associated with E. tarda, with symptoms ranging from mild secretory enteritis to chronic enterocolitis. Gastroenteritis is more common in children and extraintestinal infections are more common in adults (Public Health Agency of Canada). According to the report of Jordan et al., (1969) about human infection with E.tarda, A case of liver abscess and septicemia due to E. tarda and the clinical information on eight cases of mild diarrhea or wound infection in which the organism was isolated are reported. Moreover, in the report of Clarridge et al.,(1980), three cases of intraintestinal infection caused by E. tarda which were described as typhoid-like illness, peritonitis with sepsis, cellulitis from a wound acquired while fishing. In a retrospective study of Jaruratanasirikul and Kalnauwakul (1991), 45 specimens of E. tarda infection from 44 adult cases at Songklanagarind Hospital during February 1982 to March 1989 were reviewed. There were 24 males and 20 females, with a mean age of 48.20 years. Nearly all of E. tarda were isolated from extraintestinal sources, especially pus and urine and most of them were subsequently found to be nosocomial-acquired infections. Forty one patients were cured of the infection. Three cases died from bacteremia and serious underlying diseases. In 2001, a series of 11 cases of extraintestinal E. tarda infection is presented; including the first reported case of myonecrosis in an immunocompetent patient was reported by Slaven et al in study of Myonecrosis caused by Edwardsiella tarda. 2.4 Antimicrobials used in aquaculture Antimicrobial agents are substances that have ability to kill or inhibit the growth of microorganisms. Antibiotics can be derived from natural sources or have synthetic origins. Antibiotics should be safe (non-toxic) to the host, allowing their use as chemotherapeutic agents for the treatment of bacterial infectious diseases (Romero et al., 2012). List of major antimicrobial drugs used in aquaculture was shown in Appendix 1. The legal antimicrobials used in aquaculture must be approved by the government agency. For example, the Food and Drug Administration (FDA) organization in United State, the following antimicrobials are allowed to use in aquaculture: oxytetracycline, florfenicol, and sulfadimethoxine/ormetoprim. These agencies set rules for antibiotic use, including permissible routes of delivery, dose forms, withdrawal times, tolerances, and use by species, including dose rates and 6 limitations. The most common route for the delivery of antibiotics to fish is mixing the antibiotic with specially formulated feed (Romero et al., 2012). Determination of antimicrobials use in aquaculture in the world is not easy because different countries have different policies, regulations and distribution systems. The amount of antibiotics and other drugs used in aquaculture are significant differences among countries. In the survey of Defoirdt et al., (2011) showed that approximately 500-600 metric tons of antibiotics were used in shrimp farm in Thailand in 1994; he also emphasized the large variation between different countries, with antibiotic use about 1g per metric ton in Norway compare to 700g per metric ton in Vietnam (cited by Romero, 2012). Another survey about antibiotic use in Asia of Mudd (2003, cited by Nhan, 2010) showed very large use of antibiotics in many countries such as China (1,500 tones), Japanese (1,100 tones), Korea (550 tones), Thailand (420 tones), India (400 tones), Philipines (350 tones), Pakistan (200 tones), Taiwan (180 tones), Malaysia (150 tones), Bangladesh (100 tones), Vietnam (50 tones) and Indonesia (20 tones). In Vietnam, according to the National Agro-Forestry-Fisheries Quality Assurance Department (NAFQAD) yearly reports on residues found in the fish farm, the limited antibiotic residues had been detected in aquaculture including: quinolones and sulfonamides which are wildely used in aquaculture. Most of antibiotic residues belong to acceptable limits but sometimes quinolones was found at 18 times allowed limits. Residues of banned antibiotics are rarely detected and the survey found that chloramfenicol contamination in large proportion of water sample. That proved banned drugs were still being use (Kinh, 2010). The lists of chemicals, antibiotics banned & limited for manufacturing, trading in aquaculture in Vietnam were shown in Appendix 2 & 3. 2.5 Common antibiotic groups and modes of action There are some ways to classify antibiotics. However, antibiotics were usually grouped based on their structure or function (modes of action). By structure, they were classified by basing on the molecular structure of antibiotics. For example, betalactams have beta-lactam ring in their structure or aminoglycosides are vary only by side chains attached to basic structure. Meanwhile, regarding on mode of action, antibiotics could be devided into five functional groups: inhibitors of cell wall synthesis; inhibitors of protein synthesis; inhibitors of membrane function; antimetabolites; and inhibitors of nucleic acid synthesis. Another way of antibiotic classification was basing on the antibacterial activities (bactericidal and bacteriostatic). Firstly, bactericidal effect, the antibiotic generally kills the bacteria by interfering with either the formation of the bacterium's cell wall or its cell contents. Examples include 7 penicillin, fluoroquinolones, and metronidazole. Secondly, bacteriostatic effect, the antibiotics stops bacteria from multiplying by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism. Examples include tetracyclines, sulfonamides, chloramphenicol, and macrolides (Romero et al.., 2012). 2.5.1 Beta-lactams This group includes smaller groups: penicillins, cephalosporins, monobactams, carbapenems, but penecillins and cephlosporins were more common used in aquaculture. There were about 50 beta-lactams on the market and they are all bactericidal. Moreover, they are non-toxic which can be administered at high doses and relatively inexpensive. Beta-lactams are organic acid and mostly were soluble in water. They have similar structure (common beta-lactam ring) as well as function (inhibit the cell wall synthesis), particularly all of them are inactivated by bacterialproduced enzymes called beta-lactamase (VITEK-technology, 2008). About their mode of action, beta-lactams interfere with cell wall synthesis block peptidoglycan synthesis and thus are active against growing bacteria. In gram-negative bacteria, they enter the cell through porin channels in the outer membrane and beta-lactam molecules bind to penicillin binding proteins (PBPs) that are enzymes required for cell wall synthesis. The attachment of the beta-lactam molecules to the PBPs, located on the surface of the cytoplasmic membrane, blocks their function. This causes weakened or defective cell walls and leads to cell lysis and death. Meanwhile, in Gram-positive bacteria which have no ounter membrane, beta-lactams diffused through the cell wall and the next steps are similar to Gram-negative bacteria (Coyle, 2005). Penicillins are chemically rather unstable, being decomposed by heat, light, oxidizing and reducing agents. Beside, they can be inactivated by heavy metals. Penicillins are sensitive to hydrolysis by bacterial beta-lactamase enzymes. Therefore, to prevent this situation, people usually combine beta-lactams + beta-lactamase inhibitor (clavulanic acid, sulbactam, tazobactam,...). For example, amoxicillin + clavulanic acid and ampicillin + sulbactam are common used in aquaculture. Benzylpenicillin (Penicillin G) is a natural antibiotic produced by fungus, penicillium notatum. It has a narrow spectrum of action, mainly against Gram-positive bacteria hence is little use in aquaculture. Meanwhile, ampicillin and amoxicillin which are produced by chemical treatment of benzylpenicillin are known as semi-synthesis. They have similar spectra of action but broader than benzylpenicillin and widely used in aquaculture (Treves-Brown, 2000). Cephalosporins have different generations and the spectra of activity is also improved after each generation. For example, cefazolin belongs to the 1st generation of 8 cephalosporins which have narrow spectrum; good Gram-positive activity and relatively modest Gram-negative activity whereas cefotaxime is a member of 3rd generation cephalosporins which have wider spectrum of action and more active against Gram-negative bacteria (VITEK-technology, 2008). 2.5.2 Tetracyclines They are broad spectrum bacteriostatic drugs which can be natural fermentative or semi-synthesis derivatives. Tetracyclines are yellow crystalline compound, varying solubilities in water but all are soluble in both acids and alkalis (Treves-Brown, 2000). Tetracyclines (e.g. tetracycline, minocycline and doxycycline) bind to the 30S subunit of the ribosome and block the attachment of transfer RNA (tRNA). Since new amino acids cannot be added to the growing protein chain, synthesis of protein is inhibited. The action of tetracyclines is bacteriostatic (Coyle, 2005). Oxytetracycline and chlortetracycline are natural products, have been used in aquaculture. In there, oxytetracycline has been used widely because it is not only available in most market but also cheaper than other broad spectrum antibacterial drugs. Doxycycline have been used in aquaculture but only limited extent because they are more expensive than oxytetracycline (Treves-Brown, 2000). 2.5.3 Phenicols This is broad spectrum and bacteriostatic group which is active against both gram-positive and negative bacteria. Phenicols include chloramphenicol, thiamphenicol and florfenicol. In there, chloramphenicol and florfenicol are commonly used in aquaculture. However, because of high toxicity, causes bone marrow applasia and other hematological abnormalities so it was banned to use for diseases treatment and prevention in human food animals by Ministry of Agriculture and Rural Development (2002). Meanwhile, thiamphenicol is a derivative of chloramphenicol with similar spectra of activity but less toxic. Similarly, florfenicol is a newest generation of phenicol group. It is not only a broad spectrum activity but also overcome the demerits of chloramphenicol. Thiamphenicol and florfenicol differ from chloramphenicol in having a metyl-sulfonate group (CH3SO2) in place of a nitro group in the molecular structure and this is claimed to avoid the cause of aplastic anaemia. That makes these antibiotics acceptable for use in food producing species (TrevesBrown, 2000). Regarding on mode of action, they bind to 50S subunit the ribosome and interferes with binding of amino acids to the elongation step of prottein synthesis (Coyle, 2005). 9 2.5.4 Quinolones and fluoroquinolones Quinolones are the group of chemically related synthetic antibacterial agents which are all carboxylic acids. All of them are bactericidal. Nalidixic acid was the first quinolone to be developed. Its spectrum of activity is mainly against Gram-negative bacteria. However, the sub-group of fluoroquinolones (fluorine atom at position 6 in the molecular) have been developed and enhanced antibacterial activity. Some common fluoroquinolones are enrofloxacin, ciprofloxacin, norfloxacin,...Most of quinolones are amphoteric and sparingly soluble at neutral pH. Sodium salts are soluble and they are absorbed through the fish gills so they can be administered by immersion (Treves-Brown, 2000). About quinolones and fluoroquinolones mode of action, they easily diffuse through the peptidoglycan and cytoplasmic mambrane. They interfere with DNA synthesis by blocking the enzyme DNA gyrase. DNA gyrase helps to wind and unwind DNA during DNA replication. The enzyme binds to DNA and introduces double stranded breaks that allow the DNA to unwind. Fluoroquinolones bind to the DNA gyrase-DNA complex and allow the broken DNA strands to be released into the cell, which lead to the cell death (Coyle, 2005). 2.5.5 Sulfonamides This is a large range of synthetic compounds which are all derivatives of sulfanilamide. Sulfonamides have a broad spectrum of activity and have been used for virtually all bacterial fish diseases. They are bacteriostatic and varying solubilities in water (depend on the pH and buffering capacity of the water). One advantage of sulfonamides is that they are absorbed through the fish gills so it can be administered by immersion. This group includes sulfanilamide, sulfadiazine, sulfamerazine, sulfamethoxazole,.... However, the combination of trimethoprim + sulfamethoxazole (bactericidal) is commonly used in aquaculture (Treves-Brown, 2000). Inhibition of the metabolic pathway for folic acid synthesis (known as antimetabolite) is the mode of action of the sulfonamides and trimethoprim. Trimethoprim and sulfonamides can be used separately or together. When used together they produce a sequential blocking of the folic acid synthesis pathway and have a synergistic effect. Both trimethoprim and the sulfonamides are bacteriostatic (Coyle, 2005). 2.5.6 Aminoglycosides This group includes streptomycin, gentamicin, kanamycin, neomycin,...which have the same basic chemical structure. They are broad spectrum and bactericidal antibiotics. It can be active against both gram negative and positive bacteria. Their 10
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