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 Anab

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

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)

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

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

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

List of figures

Figure 4.1: Appearance frequency (%) of 5 genera 14 Figure 4.2: Edwardsiella bacteria isolated from snakehead fish and climbing- perch 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

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 (http://www.agroviet.gov.vn)

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

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

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

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

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

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 Gram-negative, 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

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

Gram-negative, 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)

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

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, penicillinase-producing Staphylococcus species, and most trains of

non-Clostridia Also effective against Gram-negative bacteria, such as Escherichia coli, Salmonella and Pasteurella multocida (Bui Kim Tung,

2001)

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

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,

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

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)

- 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 (20<CFU<200) was chosen for selection of colonies

3.4 Methods

3.4.1 Bacterial identification

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)

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

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

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,

ulcerations, fin rot, body discoloration, mouth sore, eye opacity,

exophthalmia…(Yambot, 1998) In China, Nielsen et al (2001) indicated that

A hydrophila was the dominant motile Aeromonas species that cause disease

outbreaks in aquaculture production in the Zhejiang province It was

remarkable that A hydrophila was responsible for more than 50% of the aeromonands isolated from crucian carp (Carassius carassius) and Wuchang bream (Megalobrama amblycephala) with hemorrhagic septicemia In Vietnam, many bacterial infection cases in fish in association with A

hydrophila were recorded Loan et al (2009) and her partners showed that A hydrophila was the main causative organism which caused the hemorrhagic

disease on cultured tra catfish (Pangasianodon hypophthalmus) in the MD delta With the same result, Lu Tri Tai (2010) indicated that A hydrophila also infected on snakehead fish (Channa striatus) and recently, A hydrophila has been isolated from diseased rice eels (Monopterus albus) that displayed symptoms of hemorrhagic septicemia (Oanh et al., 2012) Comparing with

these details described above, 20% bacterial isolates in this study were

presumptively identified as Aeromonas genus

4.1.3 Pseudomonas genus

In this study, there were 2 genus identified as Pseudomonas and they are

accounted for 2% of the 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 They oxidized glucose but did not ferment it Moreover, 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

Pseudomonas genus which belongs to Pseudomonadaceae family and Pseudomonadales order

Figure 4.4: Pseudomonas bacteria isolated from snakehead and climbing perch (A) Pseudomonas colonies on NA (B) Pseudomonas was Gram-negative rod bacteria (100x) (C) Pseudomonas bacteria oxidized but did not ferment glucose