antimicrobial susceptibility testing of bacterial agents isolated from asian seabass (lates calcarifer)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES ANTIMICROBIAL SUSCEPTIBILITY TESTING OF BACTERIAL AGENTS ISOLATED FROM ASIAN SEABASS Lates calcarifer By TRAN HUU TINH A thesi

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

ANTIMICROBIAL SUSCEPTIBILITY TESTING OF

BACTERIAL AGENTS ISOLATED FROM

ASIAN SEABASS (Lates calcarifer)

By TRAN HUU TINH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Science in Aquaculture

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

ANTIMICROBIAL SUSCEPTIBILITY TESTING OF

BACTERIAL AGENTS ISOLATED FROM

ASIAN SEABASS (Lates calcarifer)

By TRAN HUU TINH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Science in Aquaculture

Supervisor

Dr TU THANH DUNG

ACKNOWLEDGEMENT

First of all, the author wishes to express special thanks to his supervisor, Dr Tu Thanh Dung, for her valuable guidance, advice, and encouragement He would also like to dedicate his great appreciation to Dr Tran Thi Tuyet Hoa for her kind help in finishing the research

Many thanks are also given to all other doctors of the College of Aquaculture and Fisheries, and especially to those at the Department of Aquatic Pathology for providing him with great working and learning conditions

The author would love to express his sincere appreciation to many of his friends, especially Nguyen Minh Tri, Nguyen Bao Trung, Tran Hoa Cuc, and Tran My Han for their kind help throughout the experiment period

Last but not least, the author really wishes to thank his academic adviser, Dr Pham Minh Duc, who was guiding and encouraging him over the last four years, and his family for their great lifetime support which makes everything possible for him

The author,

Tran HuuTinh

ABSTRACT

The purpose of this study was to isolate some bacterial agents from Asian sea bass, and then perform antimicrobial susceptibility tests Bacterial samples were collected in Soc Trang, Dong Nai, and Vung Tau during June to October – 2012 A total number of

23 bacterial isolates were isolated from both healthy and diseased fish with hemorrhages on skin, and exophthalmia The results showed that 5 out of 23 isolates

were identified as Vibrio vulnificus, 13 isolates were confirmed as Streptococcus iniae, and the rest 5 isolates were of Aeromonas hydrophila Most of the isolates were tested

for susceptibility with 11 antimicrobial agents with both disk diffusion and broth dilution methods The results showed that all bacterial isolates were completely

resistant to colistin Isolates of Vibrio vulnificus were sensitive to most other types of

antibiotics used, but showed intermediate susceptibility to ampicillin and cefotaxime

A hydrophila isolates were resistant to not only colistin, but also flumequine,

norfloxacin, and enrofloxacin, and were susceptible to other antibiotics used Isolates

of Streptococcus iniae were resistant to flumequine, but very sensitive to the rest types

of antibiotics The minimal inhibitory concentration (MIC) of colistin to Streptococcus iniae was highest (>512µm/ml), and that of erythromycin was lowest (0.0325µm/ml)

of the six antibiotics used in this experiment Besides, MICs of the other four antibiotics were also low, ranging from 0.125 to 4µm/ml

Appendix 1.Major antimicrobial drugs used in aquaculture 20

Appendix 3.Some biochemical tests used in bacterial identification 22Appendix 4.Preparation of culture dilution series per isolate 24

LIST OF FIGURES Figure 4.1 Diseased signs of sea bass……… Page 11 Figure 4.2 Different colony morphologies of bacterial isolates……… Page 12

Figure 4.3 Gram stains of V vulnificus, A hydrophila, and S iniae…………Page 13

LIST OF TABLES Table 4.1 Susceptibility pattern of three bacterial species……… Page 14

Table 4.2 MIC values of six antibiotics for two S iniae isolates………Page 15

CHAPTER 1 INTRODUCTION 1.1 Background of study

Sea bass (Lates calcarifer) is a large, euryhaline member of the family Centropomidae

that is widely distributed in the Indo-West Pacific Ocean This species can tolerate crowding and has wide physiological tolerances, and rapid growth, reaching harvestable size (350g - 3kg) in six months to two years Aquaculture of sea bass commenced in the 1970s in Thailand, and rapidly spread throughout much of Southeast Asia because of high market value and demand of this species (FAO, 2012)

In Vietnam, sea bass culture was started in the early 1990s in small scales due to the lack of seeds, but has started to develop quickly in the recent years since fingerlings

can be artificially spawned (Tuan et al., 2000)

Like any other species, sea bass, cultured either ponds or cages, often get diseases such

as viral nervous necrosis, lymphocystis disease caused by viruses, vibriosis by Vibrio spp., bacterial hemorrhagic septicemia by Aeromonas spp., columnaris disease by Flavobacterium spp (FAO, 2012) which can greatly reduce the profitability of the systems Vibriosis caused by Vibrio spp is the most common bacterial disease

affecting culture Sea bass (Wee and Leong, 1986, cited by Chan, 1997) Diseased fish usually have darkening, lethargy, anorexia, reddened ulcerations on body, and reddened abdominal fluid (FAO, 2012)

Antibiotics, widely used for treatment of bacterial infections, including vibriosis, can result in antimicrobial resistance of bacteria if misused Bacterial resistance often causes treatment failure since antibiotics normally used for treatment of specific disease are no longer effective (Tenover, 2006) Higher dosages of antibiotics may be then applied, and hopefully can cure the illness However, residues in food product are not favorable to consumers Besides, resistant characteristics can readily spread

through bacterial populations (Kumarasamy et al., 2010) The continuous use of

antibiotics increases the risks of antibiotic residues in fish meat and fish products Presently, antibiotic resistance, especially to prohibited agents, is a big concern not only to farmers, but also to public health managers because it can greatly reduce the

effectiveness of treatment, and contains potential risks to human health (Dung et al.,

2010) Therefore, this research is done in order to provide the most updated information about the antimicrobial susceptibility of some bacterial agents, which will hopefully aid in disease treatment

1.2 Research objectives

The research was aimed to investigate the antimicrobial susceptibility of bacterial

agents isolated from infected Asian Sea bass (Lates calcarifer), and to find out the

most effective antibiotics for treating the disease

1.3 Research contents

- Isolating and identifying bacterial isolates

- Performing antimicrobial susceptibility tests, using disk diffusion and broth dilution methods

CHAPTER 2 LITERATURE REVIEW

2.1 Asian sea bass (Lates calcarifer)

Lates calcarifer, commonly called the giant sea perch, sea bass or barramundi, is an

important coastal, estuarine, and freshwater fish in the Indo-West pacific region (Cheong, 1989) Sea bass juveniles naturally grow in rivers or lakes, and then migrate

to estuary, and coastal areas, when reaching maturity, for spawning According to Hai and Phuong, 2006, the optimal salinity for sea bass to spawn is 30-32ppt, but sea bass can tolerate a wide range of salinity fluctuation Sea bass may be farmed in ponds, or net cages, with the later being more predominant Places with little industrial pollutions and water velocity of 0.2-0.6m/s are suitable for cage construction Water quality parameters are properly maintained at: 4-6mg/l of dissolved oxygen, temperature of 25-300C, and salinity of 27-30ppt for grow-out culture

2.2 Major diseases in sea bass

According to FAO (2006), sea bass are susceptible to many types of viral, bacterial, and fungal diseases such as lymphocytis, vibriosis, bacterial hemorrhagic septicemia, streptococcosis, and columnaris disease

2.2.1 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 serious economic losses in marine culture are Listonella (Vibrio) anguillarum, Vibrio ordalii, Vibrio salmonicida, Vibrio vulnificus biotype 2 (Toranzo et al., 2004), and Vibrio harveyi

(FAO, 2006) V anguillarum is the most common fish-pathogenic vibrio (Noga,

2010)

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

2.2.2 Bacterial hemorrhagic septicemia

Bacterial hemorrhagic septicemia, also referred to as motile aeromonad infection, infectious dropsy, red pest, red disease, 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 resistance to many types of antibiotics, including ampicillin, chloramphenicol, erythromycin, nitrofurentoin, novobiocin, streptomycin, sulphonamides, tetracycline, oxytetracycline (Aoki, 1988;

De Paola et al., 1988, cited by Austin and Austin, 2007), but were highly sensitive to

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

2.2.3 Streptococcosis

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

is common This disease can cause darkening, pale gill, reddened fluid and organs in infected fish (Noga, 2010) Many host species have been reported with streptococcus

infection, including rainbow trout, tilapia, hybrid striped 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 pyogenes, S

parauberis, and S equi, have been reported from fish, S iniae and S agalactiae are

the two that most frequently 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 (Stoffregen et al., 1996, cited by Austin and Austin, 2007)

Besides, laboratory studies also showed the efficacy of oxytetracycline and amoxicillin

in controlling S iniae (Darwish et al., 2002; Darwish and Ismaiel, 2003, cited by

Austin and Austin, 2007)

2.3 Antibiotics

2.3.1 Antibiotics in aquaculture

Antibiotic is a chemotherapeutics agent that can inhibit the growth of micro-organisms such as bacteria, fungi, and protozoa (Kummerer, 2009) Antibiotics have been used extensively in human, veterinary medicine, agriculture, and aquaculture, and have

steadily increased especially in developing countries (Kumar et al., 2009) Nowadays,

there are many types of antibiotics available A list of major antimicrobial agents used

in aquaculture is shown in Appendix 1

2.3.2 Antimicrobial resistance

Antimicrobial resistance is the ability of a microorganism to resist an antimicrobial medicine, to which it may be previously sensitive The increase in resistance of bacteria, especially Gram-negative type, is due to mobile genes on plasmid that can

readily spread through bacterial populations (Kumarasamy et al., 2010) Antimicrobial

resistance can be divided into natural resistance and acquired resistance Natural resistance means that the bacteria are innately resistant For example, Streptomyces has some genes responsible for resistance to its own antibiotic Other examples include

organisms that lack a transport system or a target for the antibiotics In other cases, the resistance can be due to increased efflux activity Acquired resistance refers to bacteria that are usually sensitive to antibiotics, but are liable to develop resistance under the selective pressure of use of an antibiotic Acquired resistance is often caused by mutations in chromosomal genes, or by the acquisition of mobile genetic elements, such as plasmids or transposons, which carry the antibiotic resistance genes

Several mechanisms of antimicrobial resistance are readily spread to a variety of bacterial genera First, the organism may acquire genes encoding enzymes, such as β-lactamases, that destroy the antibacterial agent before it can have an effect Second, bacteria may acquire efflux pumps that extrude the antibacterial agent from the cell before it can reach its target site and exert its effect Third, bacteria may acquire several genes for a metabolic pathway which ultimately produces altered bacterial cell walls that no longer contain the binding sites of the antimicrobial agent, or bacteria may acquire mutations that limit access of antimicrobial agent to the intracellular site via down-regulation of porin genes (Tenover, 2006)

CHAPTER 3 MATERIALS AND METHODS 3.1 Time and sites of study

Time: The research was conducted June to October, 2012

Locations: Fish samples were obtained from Soc Trang, Dong Nai, and Vung Tau provinces Experiments were performed in the laboratory of Department of Aquatic Pathology, College of Aquaculture and Fisheries, Can Tho University

3.2 Materials

Equipment: incubator, refrigerator, laminar flow hood, centrifuge, spectrophotometer,

autoclaves, drying oven, microscopes, magnetic stirrer, electronic scale, aluminum foils, pipettes, centrifuge tubes, experimental tubes, paraffin film

Chemicals: absolute alcohol, distilled water, sodium chloride, glycerol, liquid

paraffin, crystal violet solution, iodine solution, decolorizing agents (acetone, ethanol), safranin (red counterstain)

Culture media: TCBS (Thiosulfate-Citrate-Bile Salt-Sucrose Agar), BHIA (Brain

Heart Infusion Agar), BHIB (Brain Heart Infusion Broth), TSA (Tryptic Soy Agar),

NA (Nutrient Agar)

Antibiotics:

- Antibiotic disc: cefotaxime (30µg), rifampicin (30µg), norfloxacin (5µg), flumequine (30µg), doxycycline (30µg), colistin (10µg), enrofloxacin (5µg), erythromycin (15µg), florfenicol (30µg), sulphamethoxazole/trimethoprim (25µg, 19:1) (produced by Oxoid Inc., England)

- Antibiotic powder: colistin, erythromycin, enrofloxacin, florfenicol, oxytetracycline, trimethoprim

3.3 Methods

3.3.1 Fish sampling

Sea bass samples were collected from culture ponds in Soc Trang, Dong Nai, and Vung Tau provinces A number of 4-8 diseased and 1-2 healthy (with no clinical

signs) fish, with the weight ranging from 250-600gr/fish, were collected from each pond and transported live to the laboratory of the College of Aquaculture and Fisheries, Can Tho University for analysis If fish farms were far from the laboratory, diseased fish were also sampled on-farm to avoid death and decomposition of samples during transportation

3.3.2 Bacterial isolation

Fish samples were first put on clean trays for observing and recording of external signs into a sampling sheet as shown Appendix 2 They were disinfected with 700 alcohols, and then carefully dissected to avoid damaging internal organs, and reduce the risk of contamination Internal signs of fish were also observed and recorded

Bacterial samples from liver, kidney, spleen, brain, and eyes were inoculated on agar plates supplemented with sodium chloride to acquire the salinity of 15‰, and incubated at 280C After 24 hours of incubation, bacterial growth was checked, and representative bacterial colonies were sub-cultured for purity

3.3.3 Bacterial identification

Pure culture of bacterial isolates obtained were used in primary tests, including Gram staining, motility, oxidase, catalase, oxidative-fermentative, O/129 tests, following the method of Frerichs and Millar (1993), and Buller (2004) Detailed procedures for each specific test are shown in Appendix 3 Bacterial strains were fully identified by using API 20E, and API 20STREP, following the instruction of the suppliers

3.3.4 Antimicrobial susceptibility testing

The susceptibility patterns of the identified isolates were performed by using disk diffusion method described in Clinical and Laboratory Standards Institute document M2-A09 (CLSI, 2006)

Incubating loops are used to take 1-2 colonies from pure culture, and put into bottle with about 30ml sterile BHI to vortex at 200 rounds/minute for 24 hours Bacterial solution is now transferred into 50ml falcon tube for centrifugation (4000 rounds/minute for 15 minutes) The upper solution part is eliminated, and bacteria are washed 2-3 times under sterile saline solution