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
- Xem thêm -