flofenicol and enrofloxacin resistance in heterotrophic bacteria isolated from snakehead (channa striatus) and climbing perch (anabas testudineus) farms in the mekong delta

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

FLOFENICOL AND ENROFLOXACIN RESISTANCE
IN HETEROTROPHIC BACTERIA ISOLATED FROM
SNAKEHEAD (Channa striatus) AND CLIMBING
PERCH (Anabas testudineus) FARMS IN THE MEKONG
DELTA

By

NGUYEN DAI DUONG

A thesis submitted in partial fulfillment of the requirements for
the degree of Bachelor of science in Aquaculture

Can Tho, January, 19 2012


CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

FLORFENICOL AND ENROFLOXACIN RESISTANCE
IN HETEROTROPHIC BACTERIA ISOLATED FROM
SNAKEHEAD (Channa striatus) AND CLIMBING
PERCH (Anabas testudineus) FARMS IN THE MEKONG
DELTA

By

NGUYEN DAI DUONG

A thesis submitted in partial fulfillment of the requirements for
the degree of Bachelor of science in Aquaculture

Supervisor
Assoc. Prof. Dr. DANG THI HOANH OANH

Can Tho, January, 19 2013


Acknowledgements
I wish to express my sincere gratitude to my parents for always encouraging
me when I face the problems, supporting me with the best conditions for my
studies in four and a half years at Cantho University.
With my high appreciation and sincere gratitude, I would like to thank my
supervisor, Assoc. Prof. Dr. Dang Thi Hoang Oanh for her wholehearted
guidance and helping me to access to the facilities as well as materials for my
study.
I also want to thank Mrs. Nguyen Thi Thu Hang and Ms. Truong Quynh Nhu
not only for their constant mentorship but also for their encouragement during
my study.
I am thankful to all staffs and students in the Department of Aquatic
Pathology, College of Aquaculture and Fisheries, for helping me and making
the good conditions for me to finish my study. Special thanks to all my
classmates in advanced aquaculture class, course 34 for their constant help in
my study duration.

i

Abstract
The purpose of this study was to identify the heterotrophic bacteria which
were isolated from climbing perch (Anabas testudineus) and snakehead
(Channa striatus) farms in the Mekong Delta at genus level by basic
morphological, physiological and biochemical tests. In each bacterial genus,
two isolates were chosen to test with 4 antimicrobial agents by using disk
diffusion method and to determine minimal inhibitory concentration (MIC).
Five genera had been identified including Edwarsiella, Aeromonas,
Pseudomonas, Staphylococcus and Streptococcus from the total of 84 isolates.
The results of antibiotic susceptibility test indicated the majority isolates were
sensitive to florfenicol (9 isolates) and doxycycline (7 isolates) in the total of
10 isolates tested. Highest resistance was detected to amoxicillin (7 isolates),
followed by enroflorxacin (2 isolates). The MIC values of florfenicol and
enroflorxacin were low in most of the cases. The MIC value of florfenicol was
from 0.25-0.5 ppm while the range value of enroflorxacin was slightly higher
(1-32 ppm).

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Table of Contents
Acknowledgements .......................................................................................... i
Abstract ........................................................................................................... ii
Table of contents ............................................................................................ iii
List of tables.....................................................................................................v
List of figures ................................................................................................. vi
List of abbreviations ..................................................................................... vii
CHAPTER I: INTRODUCTION .................................................................1
1.1 Background of the study........................................................................1
1.2 Objectives of the study ..........................................................................2

1.3 Contents of the study .............................................................................2
CHAPTER II: LITERATURE REVIEW ...................................................3
2.1 Status of snakehead (Channa striatus) and climbing perch (Anabas
testudineus) farming in the Mekong Delta ..................................................3
2.2 General information about the cause of diseases ..................................4
2.3 Some common bacterial diseases in freshwater fish .............................5
2.3.1 Columnaris disease ........................................................................5
2.3.2 Edwardsiella Septicemia or Edwardsiellosis .................................5
2.3.3 Motile Aeromonad Septicemia ......................................................6
2.3.4 Pseudomonad Septicemia or Red Spot Disease .............................6
2.3.5 Streptococcal infection ..................................................................7
2.4 Antibiotic drug resistance ......................................................................7
2.5 Classification of some common antibiotics used in aquaculture ..........8
2.5.1 Quinolones .....................................................................................8
2.5.2 Fenicol group ................................................................................8
2.5.3 Beta-lactams ..................................................................................8
2.5.4 Tetracyclines ..................................................................................9
2.5.5 Sulphonamides ..............................................................................9
2.6 Studies related to antibiotic resistance in aquaculture ..........................9
CHAPTER III: METHODOLOGY ..........................................................12
3.1 Research place .....................................................................................12

iii


3.2 Materials ..............................................................................................12
3.3 The source of bacteria ........................................................................12
3.4 Methods ...............................................................................................12
3.4.1 Bacterial identification .................................................................12
3.4.2 Antibiotic susceptibility test and Minimal Inhibitory

Concentration ...............................................................................13
3.4.2.1 Antibiotic Susceptibility Test ...............................................13
3.4.2.2 Minimal Inhibitory Concentration – MIC ............................13
CHATER IV: RESULTS AND DISCUSSIONS ............................... 14
4.1 The results of bacterial identification ..................................................14
4.1.1 Edwardsiella genus ......................................................................14
4.1.2 Aeromonas genus .........................................................................16

4.1.3 Pseudomonas genus ....................................................................17
4.1.4 Staphylococcus genus ..................................................................18
4.1.5 Streptococcus genus .....................................................................19
4.2 Antimicrobial susceptibility testing results .........................................20
4.3 Minimal inhibitory concentration (MIC) testing results .....................22
CHAPTER V: CONCLUSIONS AND RECOMMENDATIONS .........24
5.1 Conclusions .........................................................................................24
5.2 Recommendations ...............................................................................24
References ......................................................................................................25
Appendices.....................................................................................................32

iv


List of tables
Table 4.1: The results of antimicrobial susceptibility test on 10 isolates ......20
Table 4.2: The MIC value of enroflorxacin and florfenicol on 5 tested
Isolates ...........................................................................................................22

v

List of figures
Figure 4.1: Appearance frequency (%) of 5 genera .......................................14
Figure 4.2: Edwardsiella bacteria isolated from snakehead fish and climbingperch fish farms .............................................................................................15
Figure 4.3: Aeromonas bacteria isolated from snakehead fish and climbing
perch fish farms .............................................................................................16
Figure 4.4: : Pseudomonas bacteria isolated from snakehead fish and climbing
perch fish farms .............................................................................................17
Figure 4.5: Staphylococcus bacteria isolated from snakehead fish and climbing
perch fish farms .............................................................................................19
Figure 4.6: Streptococcus bacteria isolated from snakehead fish and climbing
perch fish farms .............................................................................................20
Figure 4.7: The MIC value of ENR on one tested isolate .............................23

vi


List of abbreviations
AMX

Amoxiciline

DO

Doxycyline

ENR

Enroflorxacin

FFC

Florfenicol

MD

Mekong Delta

vii


CHAPTER I
INTRODUCTION
1.1 Background of the study
The Mekong Delta (MD) has more than 1 million ha of water surface (60%
freshwater and 40% brackish water) that gives a great potential to develop
aquaculture. In fact, aquaculture in the MD is expanded every year. In 2000,
the total area for aquaculture in the MD was 445,300 ha and the total
aquaculture production was 365,141 tons. Continuously, in 2008, aquaculture
farming in the MD was sharply increased, the total aquaculture area was
752,000 ha, and the production was 1.83 millions tons (it was accounted for
70% of total area and total production of aquaculture in whole country). In
2012, estimated total aquaculture area will be about 795,000 ha and the total
production can be reached to 2.4 millions tons ().
Given the fact that striped catfish (Pangasianodon hypophthalmus) is the most
common cultured species in the MD. Besides, some other high value species
such as snakehead (Channa striatus) and climbing perch (Anabas testudineus)
become more popularly farmed in freshwater provinces such as Angiang,
Dongthap, Cantho, Kiengiang and Vinhlong. In these culture systems, fish are
being stocked with very high density, over-feeding and bad water quality
management. This can lead to outbreak and spread of infectious diseases.

Bacterial diseases have been reported as one of the significant problems
causing up to 100% mortality of cultured fish (Bui Quang Te, 2006).
Up to now, treatment by using antibiotics is the most popular method that is
being applied to treat infectious diseases such as bacterial diseases. Among
these, enforxacin and flofenicol are the two common antimicrobials using to
treat bacterial diseases in snakehead and climbing perch farms. Nevertheless,
improper using of antibiotics can lead to the increasing of antibiotic resistance
in bacterial pathogens. That will cause difficulty in treatment when diseases
spreading out in fish ponds. Resistance to antibiotics is not limited to bacterial
pathogens, it also extends to environmental bacteria in culture system and can
be transferred to human pathogens; hence, these pathogens will also cause
problems to human health. Thus, this thesis: “Florfenicol and enrofloxacin
resistance in heterotrophic bacteria isolated from snakehead (Channa striatus)
and climbing perch (Anabas testudineus) farms in the Mekong Delta” was

1


carried out to provide information for proper use of antibiotics and
management in the above mentioned culture systems.
1.2 Objectives of the study
This thesis is carried out to classify and determine the susceptibility of
heterotrophic bacteria isolated from snakehead fish (Channa striatus) and
climbing perch (Anabas testudineus) farms to florfenicol and enrofloxacin.
1.3 Contens of the study
This thesis is focused on the following contents:
1. Classification to genus level of heterotrophic bacteria isolates that were
isolated from snakehead and climbing perch farms
2. Florfenicol and enrofloxacin susceptibility testing and determination of
minimal inhibitory concentration of those antibiotics to bacterial isolates.

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CHAPTER II
LITERATURE REVIEW
2.1 Status of snakehead (Channa striatus) and climbing perch (Anabas
testudineus) farming in the Mekong Delta
In the MD, cage culture of giant snakehead fish (Channa micropeltes) was
started in 1960s while the cultivation of common snakehead (Channa striatus)
was started in 1990s and mainly farmed in some provinces such as: Dongthap,
Angiang, Cantho and Haugiang. Family Channaidea had 4 species: Channa
gachua, Channa lucius, Channa striatus and Channa micropeltes in the MD
(Truong Thu Khoa and Tran Thi Thu Huong, 1994); however, Channa
striatus and Channa micropeltes were the two main species commonly farmed
in the MD.
The total production of snakehead fish farming was increased rapidly in the
recent years. In 2002, the total production of snakehead was about 5,294 tons
(Long et al., 2004), mainly cultured in Angiang, Dongthap, Cantho and
Kiengiang. Sinh et al. (2010) estimated that the total production in the MD
was increased up to 30,000 tons, 6 times higher than in 2003. According to
the survey of Le Xuan Sinh and Do Minh Chung (2010), culture period of
snakehead was about 4-5 months; therefore, farmers could have 2 crops per
year. With this benefit, snakehead farming can contribute to the food source
of local areas and help farmers to improve their income during the blooding
season.
Besides benefits, Sinh et al. (2010) also warmed that snakehead fish farmers
could face some major problems such as lack of capital, pollution of cultured
area, unstable price of fish, increasing price of trash fish, and fish diseases.
Of these, fish diseases were the most important factor causing high mortality

and made farmers to loss their profits (Bui Quang Te, 2006). The study of Le
Xuan Sinh and Do Minh Chung (2010) on diseases of snake head fish also
showed that the pathogenic agents on snakehead fish including parasites,
bacteria and fungi. Duc et al. (2012) reported 4 genera of bacteria which
were identified on collected fish samples in which Aeromonas comprised of
54.3%, Edwardsiella comprised of 17.3%, Streptococcus comprised of
14.8% and Pseudomonas comprised of 13.6%.
Besides snakehead (Channa striatus), climbing perch (Anabas testudineus) is
also a fish species which is commonly cultured in the MD. Climbing perch

3


farming was started in 2000s and rapidly developed in La Nga River of
Dongnai province. The production at that time could be reached to 80-100
tons/ha. When the artificial propagation of climbing perch (Anabas
testudineus) became more popular in the MD, farming of climbing perch was
more developed in Haugiang, Longxuyen, Cantho and in some other
provinces in the recent years. Nowadays, the total production of intensive
culture system of climbing perch (Anabas testudineus) can reach to 150-200
tons/ha with 70-150 individual/m2 (Thinh et al., 2011).
However, with the rapid development of climbing perch farming in
unpromoted areas as well as stoking with very high density, the outbreak of
diseases have spread and caused the great loss to the farmers (Bui Quang Te,
2006). Pal et al. (1990) and Dash et al. (2009) (quoted by Thinh et al., 2011)
showed that when climbing perch had Epizootic ulcerative syndrome caused
by Aphanomyces invadans, there was also a presence of Aeromonas sp.,
Pseudomonas sp., and Flaxibacter columnaris. Besides, the result of the study
of Thinh et al. (2011) also indicated that the main causative agent causing
“black body” disease on climbing perch could be Trypanosoma sp. Moreover,

A. hydrophila, Edwardsiella ictaluri and Streptococcus were also found out as
the facultative causative agents on this species.
Both snakehead (Channa striatus) and climbing perch (Anabas testudineus)
are popularly cultured in the Mekong Delta. However, researches on diseases,
especially bacterial diseases, on both species are very few. Therefore, study on
bacterial diseases which are isolated from both species is of obsolute necessity
to farmers.
2.2 General information about the cause of disease
According to Plumb (1999), disease can be caused by two agents: an
etiological agent (specific cause) and a nonetiological agent (contributing
cause). 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.
4


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. Intrinsic
factors include age, gender, heredity, and fish species. Both fish species and
isolate of fish are important because all are not equally susceptible to a
specific disease organism. Feed quality, extreme water quality parameters
such as temperature 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 freshwater fish
Bacteria are one of the most important-causative agents causing adverse
diseases to fish. Most bacterial agents causing diseases to fish are Gramnegative, some of them are Gram-positive. They are ubiquitous in the
environment (sea, lakes, rivers, canals, ponds…) and considered as the
primary pathogen or the opportunistic pathogens. They are usually chronic,
acute or sub-acute diseases. In some cases, bacterial diseases can cause 100%
of mortality (Bui Quang Te, 2006).
2.3.1 Columnaris disease
Causative agents: According to Bernardet et al. (1996), Flavobacterium
columnare, F. branchiophilum and F. psychrophila are Gram-negative,
motile by gliding, produce yellow colonies on agar and they are facultative
anaerobe. They produce acid from glucose, produce H2S, and give positive
catalase. (Quoted by Plumb, 1999).
Gross signs: The disease may be the appearance of discolored gray, patchy
areas in the area of the dorsal fin. These characteristic “saddleback” lesions
may progress until skin erosion exposes underlying muscle tissue. These
lesions may become yellow and cratered and are often prominent in the
mouth and head regions. Virulent isolates of F. columnaris may attack gill
tissue and cause a “gill rot” condition (Wood, 1974). Systemic infections due
to less virulent isolates may occur with no apparent external signs. However,
cutaneous infection seems to be more prevalent in most species of fish.
(Quoted by Noga, 2010).
2.3.2 Edwardsiella septicemia or Edwardsiellosis
Causative agents: Edwardsiella tarda and Edwardsiella ictaluri are short,
motile, Gram-negative rods (0.8 x 1 to 3m). They are cytochrome oxidase

5

negative, ferment and oxidize glucose while producing gas (Wyatt et al.,
1979). Colonies are grey and smooth, with good growth on BHIA (Amandi
et al., 1982) or TSA (Meyer and Bullock, 1973).
Gross signs: Fish infected with E. tarda sometimes become lethargic,
“hang” at the surface, and swim in a spiraling or erratic pattern. Gross
external lesions vary with species. Channel catfish often develop small,
cutaneous ulcerations. In advanced cases, however, larger depigmented areas
mark the sites of deep muscle abscesses (Meyer and Bullock, 1973). The
flounder Paralichthys olivaceus and the cichlid Tilapia nilotica develop
swollen abdomens due to ascites (Nakatsugawa, 1983), and the bream Evynis
Japonicus develops ulcers on the head (Kusuda et al., 1977). Diseased
common carp (Cyprinus carpio), Japanese eel, and striped bass (Monrone
saxatilis) show hemorrhages on the body and fins (Miyazaki and Egusa,
1976). In eels, lesions on internal organs may perforate the body wall, and in
striped bass, epithelial hyperplasia sometimes gives the fish a tattered
appearance. (Quoted by Bullock et al., 1985).
2.3.3 Motile Aeromonad septicemia (MAS)
Causative agents: Aeromonas hydrophila, A. caviae, A. sobria are Gramnegative, short, motile rods that are cytochrome oxidase positive and
ferments glucose. These bacteria measure 0.8-0.9 x 1.5 m, are polar
flagellated and produce no soluble pigments (Shotts and Rimler, 1973;
Popoff, 1984) (Quoted by Rocco C. Cipriano, 2001).
Gross Signs: External signs of MAS disease vary from darkening in color,
enlargement of the abdominal area to an extensive superficial reddening of a
large area of the body, often with necrosis of fins or tail and extensive
ulceration over a considerable portion of the flanks or dorsum. The ulcers are
usually shallow and the surface may turn into brown as it necrotizes or
decays. Other disease signs are scales loss or mouth sores (Bui Quang Te,
2006).
2.3.4 Pseudomonad septicemia or red spot disease
Causative agents: Pseudomonas flourescens is a Gram-negative, slightly

motile bacillus that is cytochrome oxidase positive. Colonies of P.
fluorescens on EIM are small and black. It does not produce gas and a
diffusible fluorescent pigment on agar media (Bui Quang Te, 2006).

6


Gross Signs: The external disease signs of pseudomonad septicemia are
similar to those caused by other Gram-negative bacterial pathogen of fish.
The disease causes small hemorrhages in the skin around the mouth and
opercula and along the ventral or abdominal surfaces. The body surface may
ooze blood and slime in severe cases but there is no reddening of the fins and
anus (Tu Thanh Dung, 2005).
2.3.5 Streptococcal infection
Causative agents: Streptococcus spp. are Gram-positive, non-motile,
spherical or ovoid cells occurring singly or in chains, with a cell diameter of
0.6-0.9 m. They are catalase negative, oxidase-negative, facultatively
anaerobic chemo-organotrophs with a fermentative metabolism (Bui Quang
Te, 2006).
Gross Signs: Pathological changes vary with species affected; however,
unilateral or bilateral exophthalmia and hemorrhages in the eye chamber,
inside opercula, and at the base of fins are common. Golden shiners
developed numerous raised lesions on the dorsolateral body areas, but no
internal signs (Robinson and Meyer, 1966), yellowtails exhibited congestion
in intestine, liver, spleen, and kidney (kusuda et al., 1976); and a variety of
saltwater species showed hemorrhagic enteritis, bloody peritoneal fluid, and
pale livers, but macroscopically normal kidneys (Plumb et al., 1974).
Infected cultured eels showed numerous diffuse hemorrhages on the ventral
body surface (kusuda et al., 1978).
2.4 Antibiotic drug resistance

Antimicrobial resistance is resistance of a microorganism to an antimicrobial
medicine to which it may be previously sensitive. Resistant organisms (they
include bacteria, viruses and some parasites) are able to withstand attack by
antimicrobial medicines, such as antibiotics, antivirals and antimalarials, so
that standard treatments become ineffective and infections persist and may
spread to others. Antimicrobial resistance is a consequence of the use,
particularly the misuse of antimicrobial medicines and develops when a
microorganism mutates or acquires a resistance gene (Bui Thi Tho, 2003).
There are 2 types of bacterial resistance to antimicrobials:
Inherent (natural) resistance: Bacteria may be inherently resistant to an
antibiotic. For example, an organism lacks a transport system for an
antibiotic or an organism lacks the target of the antibiotic molecule; or, as in
7


the case of Gram-negative bacteria, the cell wall is covered with an outer
membrane that establishes a permeability barrier against the antibiotic.
Acquired resistance: Several mechanisms are developed by bacteria in order
to acquire resistance to antibiotics. All require either the modification of
existing genetic material or the acquisition of new genetic material from
another source.
2.5 Classification of some common antibiotics used in aquaculture
2.5.1 Quinolones
This group includes enrofloxacin, sarafloxaxin and oxolinic acid.
Enrofloxacin has broad spectrum, bactericidal at relatively low
concentrations, highly bio-available following either oral or parenteral
administration in most species and achieves good penetration of body tissues
and fluids. Sarafloxaxin is used to treat Escherichia coli infections whereas
oxolinic acid is used against Gram-negative bacteria (Tu Thanh Dung, 2005).
In 2012, enroflorxacin is banned in Vietnam„s aquaculture.

2.5.2 Fenicol group
Florfenicol has broad spectrum, primarily bacteriostatic. Activity similar to
chloramphenicol, including many Gram-positives and Gram-negatives and
without the risk of including human aplastic anaemia associated with
chloramphenicol (Bui Thi Tho, 2003).
2.5.3 Beta-lactams
This group includes amoxicillin and ampicillin. Amoxicillin is active against
penicillin-sensitive Gram-positive bacteria and some Gram-negative bacteria.
Gram-positive spectrum includes alpha- and beta- haemolytic Streptococci,
some Staphylococci species and Clostridia species. Gram-negatives:
Escherichia coli, many isolates of Salmonell, and Pasteurella multocida are
susceptible to destruction by beta-lactamases. Ampicillin is active against
alpha- and beta- haemolytic streptococci, including Streptococcus equi, nonpenicillinase-producing Staphylococcus species, and most trains of
Clostridia. Also effective against Gram-negative bacteria, such as
Escherichia coli, Salmonella and Pasteurella multocida (Bui Kim Tung,
2001).

8


2.5.4 Tetracyclines
This group includes tetracyclines, chlortetracyline and oxytetracycline.
Tetracycline is broad spectrum with activity against Gram-positives and
Gram- negatives, including some anaerobics. Oxytetracycline is active also
against Chlamydia, Mycoplasma, protozoa and several Ricketsiae, including
Ehrlichia and Haemonartonella, actively against to Escherichia coli,
Klebsiella species, Salmonella species, Staphylococus species and
Streptococcus species. However, resistance has been acquired by coliforms,
mycoplasma, Streptococci and Staphylococci (Bui Kim Tung, 2001).
2.5.5 Sulphonamides

This group includes sulphonamides, sulfanilamide, sulfaquinoxaline and
sulfathiazole. They are antibacterial; antiprotozoal; broad spectrum;
inhibiting Gram- postives and Gram-negatives and some protozoa, such as
Coccidia. However, they are ineffective against most obligate anaerobes and
should not be used to treat serious anaerobic infections. Resistance of animal
pathogens to sulphonamides is widespread as a result of more than 50 years
of therapeutic use. Nevertheless, it still used in combination with other
medications (Bui Thi Tho, 2003).
2.6 Studies related to antibiotic resistance in aquaculture
In the world
In 1979, Hawke is the first scientist who did antimicrobial susceptibility test
on 10 isolates of E. ictaluri. Afterward, Shotts and Watman (1986) also did
antibiotic susceptibility tests on 118 E. ictaluri with 37 types of antibiotics in
US, the result showed that most of Gram-negative bacteria were susceptible
to those antibiotics. However, there were more than 90% of bacterial isolates
which are resistant to Colistin and Sulfamids. Reger et al. (1993) also
showed that E. ictaluri isolates in US were sensitive to enrofloxacin,
gentamicine and doxycyline.
In the research of Stock et al. (2001), three species of Edwardsiella bacteria
were naturally sensitive to: Tetracyclines, aminiglycosides, -lactam,
quinolones, chloramphenicol, nitrofurantoin, and fosfomycin. Besides, they
were also naturally resistant to macrolides, lincosamides, glycopeptides,
rafampin and fusidic Acid. However, E. tarda was naturally resistant to
oxacilin, and benzylpenicilin.

9


Miranda et al. (2003) (quoted by Tran Duy Phuong, 2009) conducted a
survey in for fish farms in Chile and isolated 25 different isolates of bacteria;

they were all resistant to oxytetraxyline.
In Banglades, Rahman et al. (2010) did the tests with tail fin rot disease fish
(Indian major carp, catla (Catla catla) and climbing perch (Anabas
testudineus) which were collected from different fish farms in Bangladesh.
After carrying out biochemical characterization tests, Flavobacterium
columnare was identified as the main causative agent of tail and fin rot
disease occurring in those fish. Besides, all isolates were screened again
some kinds of antibiotics. The result showed that these isolates exhibited
sensitivity to antibiotics: Chloramphenicol, oxytetracycline, erythromycin,
streptomycin, but some of them were resistant to sulphamethoxazole and all
were resistant to gentamicin and cefradine.
In India, Rajkumarbharathi et al. (2011) carried out a research on several
bacterial populations which were isolated from the gills and gut of Channa
striatus, these bacterial species were Vibrio sp, Lactobacilli sp, Proteus sp,
Pseudomonas sp, Salmonella sp, and Aeromonas sp. The prevailing colonies
of gills and gut microbes were studied for their resistance to 22 antibiotics.
The result showed that the bacterial floras of the gill and gut have maximum
sensitivity to chloramphenicol, gentamycin, kanomycin, levofloxacin, and
neomycin.
In Vietnam
Oanh et al. (2005) isolated 169 bacterial isolates in different fish ponds in the
MD. In these isolates, there were 34 % bacterial isolates which were multiple
resistant to 6 kinds of antibiotics including chloramphenicol, ampicilline,
tetracycline, trimethoprim+sulfamethoxozol and nitrofurantoin.
Thinh et al. (2007) conducted a survey in Can Tho, Dong Thap, Vinh Long,
An Giang and Ben Tre province for bacterial isolation and identification.
Among 97 isolates, 47 were identified as E. ictaluri. The result showed that
all 47 isolates resisted to sulfamethoxazole/trimethoprim and 46 (97.8%) to
colistin. Besides, a number of isolates showed different levels of resistance to
florfenicol, amoxicilin, oxytetracycline and doxycycline which were 20

(42.5%), 19 (40.4%), 15 (31.9%), and 13 (27.7 %) respectively.
The research of Ho et al. (2008) showed that ciprofloxacin, amoxicillin and
ampicillin have good sensitivity against E. ictaluri. Besides, the isolates of
Aeromonas hydrophila were also sensitive to sulphamethoxazole,

10


ciprofloxacin,
oxytetracycline,
enrofloxacin,
erythromycin,
sulphamethoxazole/trimethoprim, doxycycline and florfenicol.
Huong et al. (2011) with the study on drug resistance of E. ictaluri and A.
hydrophila isolated from tra catfish in the MD. The results showed that E.
ictaluri isolates were still highly sensitive to amoxicillin, highly resistance to
streptomycin, enrofloxacin, chloramphenicol, florfenicol, tetracycline and
doxycyline. Whereas, A. hydrophila isolates in this study displayed high
sensitiveness to fenicol, quinolones, tetracyclines group.
In the research of Dung et al. (2012) with the study about the aetiological
agent causing white patch disease in catfish farm (Pangasianodon
hypophthalmus) and therapy solution, F. columnare was found as the main
causal agent causing white patch disease in catfish. Besides, the result also
showed that there were 60% isolates resisting to enrofloxacin and
chloramphenicol.

11


CHAPTER III

METHODOLOGY
3.1 Research place
The study was carried out in the Department of Aquatic Pathology, College of
Aquaculture and Fisheries, Cantho University.
3.2 Materials
The media, chemicals, antibiotics are used for this study are listed below:
- Nutrient agar (NA)
- Tryptone soya agar (TSA).
- Crystal violet
- Ammonium oxalate
- Iodine
- Potassium iodide
- Safranin
- Chemicals for biochemical tests: O/F, motility, Indole, Oxidase,
Catalase…
- Antibiotics: Amoxiciline (AMX/25g), enflorxacin (ENR/5g),
doxycyline (DO/30g), and florfenicol (FFC/30g). (Biorad)
- Powder antibiotics for MIC: Enroflorxacin and florfenicol (Biorad).
3.3 The source of bacteria:
Samples were collected from water (W), sediment (S) and organism (O) in 3
snakehead fish farms and 3 climbing perch fish farms in Vinhthanh district.
Then, they were kept at 4oC and processed in the lab within 5 hours after
collection as well as pooling of samples per sample type (W, S, O). After that,
the series of pooled samples were diluted in sterilize 0.9% NaCl solution.
They also were inoculated in triplicate on TSA. Afterwards, they were
incubated at 28oC, overnight under aerobic conditions. Countable plates
(203.4 Methods
3.4.1 Bacterial identification

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After recovery, bacteria are inoculated in NA media from 24h-48h. Shape and
color of colonies are observed and recorded. Besides, bacteria are also
identified by some basic biochemical tests based on the scheme of Cowan and
Steel‟s (Barrow and Feltham, 1993) (appendix 11): Gram-staining, motility
test, catalase test, oxidase test, O-F test and saline tolerance test.
3.4.2 Antibiotic susceptibility test and minimal inhibitory concentration
3.4.2.1 Antibiotic susceptibility test
Antimicrobial susceptibility test was carried out by the method of Geert Huys
(2002). Ten isolates in this study were chosen from 5 genera (2 isolates/
genus) to screen 4 antimicrobial agents: AMX, ENR, DO, and FFC (appendix
9).
3.4.2.2 Minimal Inhibitory Concentration (MIC)
In this study, MIC was carried out by the method of Geert Huys (2002). Based
on the results of antimicrobial susceptibility test, 5 isolates (1 isolate/genus)
showed the sensitiveness to ENR and FFC were selected for MIC testing
(appendix 10).

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CHAPTER IV
RESULTS AND DISCUSSIONS
4.1 The results of bacterial identification
A total of 84 isolates in 6 farms were identified to genus level by basic
morphological, physiological and biochemical tests based on the scheme of
Barrow and Feltham (1993). The result showed that 57 isolates were identified
as Edwardsiella, 13 isolates as Aeromonas, 2 isolates as Pseudomonas, 10

isolates as Staphylococcus and 2 isolates as Streptococcus (Figure 4.1,
appendix 11 and 13).

Figure 4.1: Appearance frequency (%) of 5 genera

Testesd isolates formed Gram-negative and Gram-positive bacteria groups. In
the Gram-negative group, the occurrence rate of Edwardsiella genus was the
highest and accounted for 69% of total tested isolates. The next dominant
genus was Aeromonas (15%) and Pseudomonas was the last one (2%). In the
Gram-positive group, the most dominant genus was Staphylococcus (12%)
and Streptococcus was accounted for 2%.
4.1.1 Edwardsiella genus
The genus Edwardsiella was suggested by Ewing et al. (1965) to encompass a
group of enteric bacteria generally described under vernacular names such as
paracolon. In this research, there were 57 isolates (69%) identified as
Edwardsiella bacteria. After 48 hours incubated at 29oC, these isolates
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showed the milky punctuate colonies on the NA or TSA agar plates.
Observing under the microscope, these organisms were Gram-negative motile
rods, they were cytochrome oxidase negative, catalase positive. At 29C, they
fermented and oxidized glucose while producing gas. Besides, they could
develop on the media with 1.5% NaCl. In some cases, they also showed their
tolerance at 3% NaCl. Based on their characteristics and scheme of Cowan
and Steel‟s (Barrow and Feltham, 1993), these isolates were presumtively
identified
as
Edwardsiella
genus,

Enterobacteriaceae
family,
Enterobacterales order respectively.
Edwardsiella genus has 2 members which commonly infect on fish: E. tarda
(Ewing et al., 1965) and E. ictaluri (Hawke et al., 1979). Although the most
prominent fish species infected by E. tarda are eels (Egusa, 1976) and channel
catfish (Ictalurus punctatus) (Meyer and Bullock, 1973), this organism has
been isolated from many other freshwater and marine fish species including
common carp (Cyprinus carpio) (Sae-Oui et al., 1984), Japanese flounder
(Paralichthys olivaceus) (Nakatsugawa, 1983), Mullet (Mugil cephelus)
(Kusuda et al., 1976), tilapia (Tilapia nilotica) (Miyashito, 1984) (Quoted by
Bui Quang Te, 2006). In Vietnam, E. tarda has also been isolated from many
freshwater species including tra catfish (Pangasianodon hypophthalmus) (Bui
Quang Te et al., 2006) and snakehead (Channa striatus) (Luu Tri Tai, 2010).

Figure 4.2: Edwarsiella bacteria isolated from snakehead and climbing perch. (A)
Edwardsiella colonies on NA. (B) Edwardsiella was Gram-negative rod bacteria
(100x). (C) Edwardsiella bacteria oxidized and fermented glucose.

In contrast, E. ictaluri can cause the diseases of a few species of warmwater
fishes. E.ictaluri was firstly isolated from channel catfish (Ictalurus
punctatus) by Hawke (1979). Besides, E. ictaluri was found as the etiological
agent causing the diseases on blue catfish (Ictalurus fuscatus) (Hawke, 1981)
and white catfish (Ictalurus catus) (Plumb and Sanchez, 1983). In Vietnam, E.
ictaluri was firstly reported as the causative agent causing Bacillary Necrosis
of Pangasius (BNP) on tra catfish (Pangasianodon hypophthalmus) (Ferguson
et al., 2001). The bio-characteristics of Edwardsiella genus isolates which
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were isolated from snakehead and climbing perch in this study were similar to
the details about Edwardsiella genus described above; therefore 69% bacterial
isolates in this study were presumptively identified as Edwardsiella genus.
4.1.2 Aeromonas genus
Thirteen isolates identified as Aeromonas and they are accounted for 15% of
total isolates. After 24 hours incubated at 29oC, these isolates produced
circular, smooth, raised colonies on TSA or NA. Under microscope
examination, they were observed as short, motile, Gram-negative bacilli.
Phenotypically, they were cytochrome oxidase positive, catalase positive,
fermented and oxidized glucose. In addition, they were resistant to the
vibriostatic agent O/129 (2,4- diamino,6,7-di-isopropyl pteridine). Besides,
they grew on the media with 3% NaCl. Based on the scheme of Cowan and
Steel (Barrow and Feltham, 1993), these isolates were presumptively
identified as Aeromonas genus that belongs to Aeromonadaceae family,
Aeromonadales order.

Figure 4.3: Aeromonas bacteria isolated from snakehead and climbing perch. (A)
Aeromonas colonies on NA. (B) Aeromonas was Gram-negative rod bacteria (100x).
(C) Aeromonas bacteria oxidized and fermented glucose.

The motile aeromonads associated with haemorrhagic septicemia in fresh
water fishes are A. hydrophila, A. caviae and A. sobria (Austin and Austin‟s,
1987). Among those, A. hydrophila has been reported as the main etiological
agent among three species (Plumb, 1999). In the research of Lallier et al.
(1981) about the relative virulence of A. hydrophila and A. sobria on rainbow
trout (Oncorhyncus mykiss), their result performed that strains of A.
hydrophila isolated from either healthy or diseased fish were more virulent
than strains of A. sobria. Moreover, A. hydrophila was also reported as the
main causal agent causing the hemorrhagic septicemia in fish in many
countries. During the fish disease outbreaks in Philippines, A. hydrophila was

isolated from diseased Nile tilapia (Orechromis niloticus) and identified as the
main etiological organism causing disease with signs such as: Skin lesions,

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