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

LIST OF FIGURES Figure 4.1 Fish sampled Figure 4.2 Internal of diseased fish Figure 4.3 Gram staining, O/F test and bacterial colonies on NA Figure 4.4 API 20E identification test resu

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

CHARACTERIZATION OF BACTERIA ISOLATED FROM

CULTURED IN NHATRANG BAY, VIETNAM

By NGUYEN NHUT THANH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

CHARACTERIZATION OF BACTERIA ISOLATED FROM

CULTURED IN NHATRANG BAY, VIETNAM

By NGUYEN NHUT THANH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

APPROVED BY SUPERVISOR

The thesis “Characterization of bacteria isolated from silver pomfret (Pampus argenteus

Euphrasen, 1788) cultured in Nhatrang bay, Vietnam” which edited and passed by the committee, was defended by Nguyen Nhut Thanh in 27/12/2013

I wish to thank all of my friends for helping me get through the difficult times, and for all the emotional support and encouragement, especially Vo Le Thanh Truc, Nguyen Trong Nghia, Au Thi Kim Ngoc, Bui Thi Diem My, Le Thanh Can

Finally, I would like to give thank my academic adviser, Dr Duong Thuy Yen, for her guiding and encouraged me, also my parents and all my family members for their love, understanding and support me more than four years in university

aquaculture These isolates were identified as Photobacterium damselae, Vibrio parahaemolyticus, and Vibrio alginoticus, by using API 20E test kit Result of antibiotic

sensitivity tests showed that these isolates were completely resistance with amoxycilline, bicomarin, ampicillin; and they were all susceptible with norfloxacin, ciprofloxacin,

florenicol and tetracycline

TABLES OF CONTENTS

ACKNOWLEDGEMENT i

ABSTRACT ii

TABLES OF CONTENTS iii

LIST OF FIGURES v

LIST OF TABLES vi

CHAPTER 1 INTRODUCTION 1

1.1 Introduction 1

1.2 Research objective 2

1.3 Research activities 2

CHAPTER 2 LITERATURE REVIEW 3

2.1 Silver pomfret 3

2.2 Cause of diseases 3

2.3 Some common bacterial diseases in brackish and marine fishes 4

2.3.1 Vibriosis 4

2.3.2 Bacterial hemorrhagic septicaemia 5

2.3.3 Streptococcosis 5

2.4 Some commonly used antibiotics 6

2.4.1 Oxytetracycline 7

2.4.2 Ciprofloxacin 8

2.4.3 Florfenicol 8

CHAPTER 3 MATERIALS AND METHODS 9

3.1 Time and sites of study 9

3.2 Materials 9

3.3 Methods 9

3.3.1 Fish sampling 9

3.3.2 Bacterial isolation 10

3.3.3 Bacterial identification 10

3.3.4 Antimicrobial susceptibility testing 10

3.3.5 Data collection, calculation and analysis 11

CHAPTER 4 12

RESULT AND DISCUSSION 12

4.1 Fish sampling and clinical signs 12

4.2 Bacterial identification 13

4.3 Antimicrobial susceptibility testing 15

CHAPTER 5 CONCLUSIONS AND RECOMMENDATION 17

5.1 Conclusions 17

5.2 Recommendation 17

REFERENCE 18

Appendix 1.Some biochemical tests used in bacterial identification 20

Appendix 2 API 20E kit results 22

Appendix 3 The diameters of inhibition zone of the antibiotic susceptible test 23

LIST OF FIGURES Figure 4.1 Fish sampled

Figure 4.2 Internal of diseased fish

Figure 4.3 Gram staining, O/F test and bacterial colonies on NA

Figure 4.4 API 20E identification test result of Photobacterium damselae Figure 4.5 API 20E identification test result of Vibrio parahaemolyticus Figure 4.6 API 20E identification test result of Vibrio alginoticus

Figure 4.7 API 20E identification test result of Aeromonas hydrophyla

LIST OF TABLES Table 4.1 The percentages of distinct strains

Table 4.2 Susceptibility pattern of bacterial species

CHAPTER 1 INTRODUCTION 1.1 Introduction

With more than 3200 kilometers of coastline and large area of water surface, Vietnam has great conditions to develop aquaculture (both fresh and marine aquaculture) In fact, aquaculture in Vietnam is developing year by year, especially farmed stripped catfish

(Pangasianodon hypophthalmus) and penaeid shrimp (black tiger and white leg shrimp),

Vietnam was ranked the world’s third of aquaculture (FAO 2010) Besides, marine aquaculture is contributed an important part for aquaculture with high value species such

as sea bass, cobia (Rachycentron Canadum), grouper (Serranidae), silver pomfret (Pampus argenteus)… Silver pomfret is one of a new cultured species is Vietnam as it

has high nutrition contain and quality of flesh meat

Silver pomfret is easy to find in Northern gulf and central southern of Vietnam, in the past, people mainly catch silver pomfret in the wild In recent years, people started to culture silver pomfret in cages with intensive model In this system, fish are stocked at high density, usually over feeding This can make the fish get stress and diseases, especially this can lead to disease spread out easily on the sea to wild fish, or the others cages Bacterial diseases have been reported as one of the worst problems causing up to 100% mortality of cultured fishes (Bui Quang Te, 2008) This is the new specie, there is a few research about silver pomfret are conducted on over the world

Antibiotics are known as the useful treatment for the bacterial disease, but can make bad result if not use at correct ways or dosage Bacterial resistance often happens and cause treatment failure People prefer using higher dosages of antibiotics when the previous dosages do not have effect; they hope to heal illness completely, but residues in flesh product are not favorable to consumers In addition, resistance characteristic could readily and quickly spread out in bacterial populations (Kumarasamy et al., 2010) The uncontrolled of using antibiotics can make the presence of antibiotic residues in fish meat and fish products and this also lead to a disease which cause by resistance bacteria and hard to treat

Thus, this thesis “Characterization of bacteria isolated from silver pomfret

(Stromateoides argenteus Euphrasen, 1788) cultured in Nhatrang bay, Vietnam” is carried out to provide more information about disease on this species

1.2 Research objective

The aim of this research is to investigate the bacterial pathogen which are isolated from

silver pomfret (Pampus argenteus) in Nhatrang bay, then find out the antibiotic which is

susceptible to these bacteria

1.3 Research activities

This thesis focuses on the following contents:

1 Classification to species level of bacteria that was isolated from silver pomfret

2 Antibiotics susceptibility testing

CHAPTER 2 LITERATURE REVIEW 2.1 Silver pomfret

Silver pomfret (Pampuss argenteus) is a valuable food fish with a wide geographical

distribution from East China Sea to Southest Asia, the India Ocean, Arabian Gulf and the North Sea (Davis and Wheeler 1985) Silver pomfret is found in coastal water from 5 to 100 meters depth, it has maximum size 80 centimeters but usually see at 30 centimeters (FAO) In the past, it was only caught on the sea, but in recent years, it is stared being culture in cages Cages are placed in clean area, with viscosity about 0.2-0.6m/s This is new species cultured in Vietnam, it has high economic value base on its nutrition value, high flesh meat quality There is not many research about this species have been reported in Vietnam

Nonetiological causes of disease are characterized as extrinsic (from outside the body)

or intrinsic (within the body) Extrinsic factors are usually associated with environmental conditions or dietary problems, and intrinsic factors include age, gender, heredity, and fish species Both fish species and strain of fish are important because all are not equally susceptible to a specific disease organism Feed quality,

water quality factors, and water temperature extremes can be classified as either etiological or nonetiological extrinsic factors and can contribute to infectious disease (Plumb, 1999)

2.3 Some common bacterial diseases in brackish and marine fishes

Bacteria are one of the most important-causative agents causing adverse diseases to fish Most bacterial agents causing diseases to fish are Gram-negative; some of them are Gram-positive They are ubiquitous in the environment (sea, lakes, rivers, canals, ponds…) and are considered as the primary pathogen or the opportunistic pathogen They are usually chronic, acute or subacute diseases In some cases, bacterial diseases can cause 100% of mortality (Bui Quang Te, 2006)

2.3.1 Vibriosis

Vibriosis, also known as salt-water furunculosis, boil-disease, or ulcer-disease (Austin and Austin, 2007), is among the most prevalent fish diseases caused by bacteria of

genus Vibrio (Woo and Bruno, 1998).This type of disease is commonly considered

stress mediated with the predisposing factors of handling, moving from fresh to salt water (Plumb and Hanson, 2011), high temperature, crowding, and organic pollution (Noga, 2010)

Vibriosis has been reported on many fish species, including salmon, rainbow trout, turbot, sea bass, sea bream, striped bass, cod, and eel (Actis et al., 1999 cited by

Toranzo, 2005).Within Vibrionaceae, the species causing the most economically serious diseases in marine culture are Vibrio anguillarum, Vibrio ordalii, Vibrio salmonicida, Vibrio vulnificus biotype 2 (Toranzo et al., 2004), and Vibrio harveyi (FAO, 2006).V.anguillarumis the most common fish-pathogenic vibrio (Noga, 2010)

Infected fish may havered areas on body, skin ulcers, depression, exophthalmos, corneal ulcers, and swollen abdomen (Noga, 2010).Vibriosis, as with other bacterial septicaemias, can be controlled by maintaining good water quality, but where outcreaks occur, treatment with an oral antibiotic is the only option (Woo and Brono, 1998)

2.3.2 Bacterial hemorrhagic septicaemia

Bacterial hemorrhagic septicaemia, also referred to as motile aeromonad infection, infectious dropsy, red pest, reddisease, red sore, rubella, and others (Plumb and Hanson, 2011), has

been associated with several members of genus Aeromonas such as A hydrophila,A sobria,

A caviae, and Pseudomonas sp.(FAO, 2006) Disease syndromes may include lethargy,

anorexia, irregular reddened skin ulcerations, reddened abdominal fluid, and pale gills (Austin and Austin, 2007)

By far the most significant fish pathogen is A hydrophila (Noga, 2010) A hydrophila is

widely distributed in the aquatic environment (Roberts, 2012) This species is a pathogenic species mainly to fresh water fish, and occasionally to marine fish (Austin and Austin, 2007) They occur as Gram-negative, motile, straight rods (0.3-1.0 x 1.0-3.5µm) (Roberts, 2012) This bacteria species showed resistant to many types of antibiotics, including ampicillin, chloramphenicol, erythromycin, nitrofurentoin, novobiocin, streptomycin, sulphonamides, tetracycline, oxytetracycline (Aoki, 1988;

De Paoplaet al., 1988, cited by Austin and Austin, 2007), but were very sensitive to

enrofloxacin (Brag and Todd, 1988, cited by Austin and Austin, 2007)

2.3.3 Streptococcosis

Streptococcosis is sometimes called “popeye” because exophthalmos (exophthalmia)

is very common This disease can cause darkening, pale gill, reddened fluid and organs in infected fish (Noga, 2010).Diverse host species have been reported with streptococcus infection, including rainbow trout, tilapia, hybrid triped bass (Eldar et al., 1994) and sea bass (Bromage et at., 1999; Creeper and Buller, 2006) Even

though, many species of Streptococcosis, including S agalactiae, S iniae, S dysqalactiae, S dysgalactiae, S pyogenes, S parauberis, and S equi, have been reported from fish,S iniaeand S agalactiaeare the two that mostfrequently cause

serious disease in tilapia

Streptococcus iniae are small, Gram-positive, facultative anaerobic cocci, appearing

in chains (Roberts, 2012) Although S.iniae can affect various freshwater and coastal

fish species (Austin and Austin, 2007), this bacterial species is more commonly isolated from fresh-water fish such as rainbow trout and tilapias than from marine fish such as flounders and sardines (Kusada and Salati, 1999, cited by Roberts, 2012).Fish infected by this species often get damaged brain, exophthalmia, surface and internal

More importantly, fish pathogen S.iniae can cause disease in human hemorrhaging

(Austin and Austin, 2007) This bacterial agent can be treated with fluoroquinolone

compound, enrofloxacin (Stoffregenet al., 1996, cited by Austin and Austin, 2007)

Besides, laboratory studies also showed the efficacy of oxytetracycline and

amoxicillin in controlling S.iniae (Darwishet al., 2002; Darwish and Ismaiel, 2003,

cited by Austin and Austin, 2007)

2.4 Some commonly used antibiotics

Antibiotics are known as the best treatment when fish get bacterial diseases People usually take over dosages for sure that they treat the disease completely; this is not only wasting of money but also make opportunities for bacterial resistance The residual of antibiotic in fish (aquatic species) could affect to human health The antibiotics most frequently used in aquaculture to combat bacterial diseases include

oxytetracycline, florfenicol, sarafloxacin, and enrofloxacin (Roque et al., 2001; Rodríguez et al., 2006) Globally, other antibiotics such as chlortetracycline,

Soto-quinolones, ciprofloxacin, norfloxacin, oxolinic acid, perfloxacin, sulfamethazine,

gentamicin, and tiamulin are used (Holmstrom et al., 2003)

Major antimicrobial drugs used in aquaculture

(Prescott et al., 2000)

(mg/kg fish/day) Indication Antibiotics

Beta-lactams

Ampicillin

Oral 50-80mg/kg, 10 days

Gram (-) bacteria

Amoxicillin Benzyl penicillin (with streptomycin)

Aminoglycosides Neomycin Oral 50-80mg/kg, 10 days

Chloramphenicol

group

Florfenicol Oral 50-80mg/kg, 10 days

Gram (-) bacteria

sulfadimethoxine 50mg/kg, 5days

Quinolones

Oxilinic acid

Oral 10-30mg/kg, 10days Flumequine

Note: Only oxytetracycline and ormetoprim-sulfadiazine are approved for use with food

fish in the United Staes

2.4.1 Oxytetracycline

Oxytetracycline is widely employed to treat bacterial infections in aquaculture farms,

such as vibriosis and furunculosis (Capone et al., 1996; Prescott et al., 2000; Reed et al., 2006; Wang et al., 2004) It belongs to the tetracycline group, which exerts

antimicrobial action against both Gram (-) and (+) bacteria, ricksettsias,

mycoplasmas, and others (Gómez-Gil et al., 2001) Tetracyclines are produced by Streptomyces spp., which possess determinants for resistance to this class of

antibiotics Oxytetracycline is a bacteriostatic antibiotic that exerts its antimicrobial effect against protein synthesis, by bonding directly to the S7 protein of the 30S subunit of the bacterial ribosome, thereby impeding the bonding of aminoacyl-tRNA (aminoacyl transfer RNA) to the A-site of the ribosome This prevents the addition of

amino acids to the growing peptide chain (Chambers, 2004; Isidori et al., 2005; Jara,

2007) In order for oxytetracycline to interact with its target site, it needs to pass through the external membrane via passive diffusion through the OmpF and OmpC pores, and through the cytoplasm membrane via an energy dependent process (Jara, 2007)