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 thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Science in Aquaculture Can Tho City, January 16, 2013 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 Can Tho City, January 16, 2013 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 i 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 out of 23 isolates were identified as Vibrio vulnificus, 13 isolates were confirmed as Streptococcus iniae, and the rest 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 ii TABLES OF CONTENTS ACKNOWLEDGEMENT i ABSTRACT ii TABLES OF CONTENTS iii LIST OF FIGURES v LIST OF TABLES vi CHAPTER 1 1.1 Background of study 1.2 Research objectives 1.3 Research contents CHAPTER 2 2.1 Asian sea bass (Lates calcarifer) 2.2 Major diseases in sea bass 2.2.1 Vibriosis 2.2.2 Bacterial hemorrhagic septicemia 2.2.3 Streptococcosis 2.3 Antibiotics 2.3.1 Antibiotics in aquaculture 2.3.2 Antimicrobial resistance CHAPTER 3 3 4 5 3.1 Time and sites of study 3.2 Materials 3.3 Methods 3.3.1 Fish sampling 3.3.2 Bacterial isolation 3.3.3 Bacterial identification 3.3.4 Antimicrobial susceptibility testing 3.3.5 Minimal inhibitory concentration (MIC) test 3.3.6 Data analysis CHAPTER 4.1 Fish sampling and bacterial isolations 4.2 Bacterial identification 4.3 Antimicrobial susceptibility testing 4.3.1 Disk diffusion method 4.3.2 Broth dilution method iii 7 7 8 10 11 11 12 13 13 15 CHAPTER 16 5.1 Conclusions 5.2 Recommendations REFERENCES 16 16 17 APPENDICES 20 Appendix 1.Major antimicrobial drugs used in aquaculture Appendix 2.Fish Disease Diagnosis Form Appendix 3.Some biochemical tests used in bacterial identification Appendix 4.Preparation of culture dilution series per isolate Appendix 5.API 20E and API 20STREP kit results iv 20 21 22 24 25 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 v 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 vi CHAPTER 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 RESULTS AND DISCUSSION 4.1 Fish sampling and bacterial isolations Fish were sampled four times in provinces, including Soc Trang, Dong Nai, and Vung Tau from June to October, 2012 From each farm, both fish with normal external appearance and diseased fish with abnormal swimming, exophthalmia and/or hemorrhage on skin were sampled The internal organs of those healthy fish also had normal display, while diseased fish were having pale liver and swollen kidney and spleen (Figure 4.1) A B Figure 4.1 Diseased signs of sea bass A Exophthalmic eye and hemorrhagic skin B Pale liver, and swollen kidney and spleen A total of 23 bacterial isolates recovered from liver, kidney, spleen, eye, and brain of fish were acquired in this research After 24 hours of incubation at 280C, three types of colony morphology were observed: - Five out of 23 isolates formed smoothly round colonies in green color on TCBS agar media Single colonies of these isolates were in medium sizes, ranging from 12mm (Figure 4.2A) - Five other isolates formed round colonies in yellow color on TCBS agar, having medium sizes of 1-2mm (Figure 4.2B) - The other 13 bacterial isolates formed small, opaque colonies on BHI agar media (Figure 4.2C) 11 A B C Figure 4.2 – Different colony morphologies of bacterial isolates: A Medium-sized, green colonies on TCBS agar B Medium-sized, yellow on TCBS agar C Small, opaque colonies on BHI agar 4.2 Bacterial identification Basing on the colony morphology and biochemical characteristics of bacterial isolates, they were divided into three groups as follows: Group 1: included those five isolates forming green colonies on TCBS agar Bacteria of this group were Gram-negative, short rod (Figure 4.3A) and motile In addition, they gave positive results in oxidase, catalase, and oxidative-fermentative tests The ability to grow on selective TCBS agar and high sensitivity in O/129 test proved that isolates of this group belonged to genus Vibrio The results of biochemical tests in API 20E kit with these isolates were compared with those of reference strain ATCC33187 of Buller, 2004 (Table 4.2) Although some tests showed different results, the majority of test results were congruent between the two The profile number 5146105 generated by rapid identification system corresponded to Vibrio vulnificus Group 2: contained bacterial isolates forming yellow colonies on TCBS agar These bacteria were also Gram-negative, short rod (Figure 4.3B) and motile, positive to oxidase, catalase and oxidative-fermentative tests However, all isolates of this group were resistant in O/129 test which suggested bacteria in this group belonged to the genus Aeromonas API 20E kit results (Table 4.2) were not different from those of reference strain ATCC7966 (Buller, 2004) A profile number of 5047127 obtained from API 20E system corresponded to Aeromonas hydrophyla 12 Group 3: consisted of thirteen isolates forming small opaque colonies on BHI agar in addition of sodium chloride Gram staining showed that these bacteria were Gram positive cocci, appeared in pairs or short chains (Figure 4.3C).Results of primary tests revealed that isolates of this group were non-motile, negative to oxidase, catalase, and oxidative-fermentative tests API 20STREP kit was used to determine some biochemical characteristics of this bacterial species The identification result with rapid identification system was confirmed with the result of 16S rRNA partial gene sequencing, and identified isolates of this group as Streptococcus iniae A B C Figure 4.3Gram stains of three bacterial isolates A Gram-negative, curved rod B Gram-negative short rod C Gram-positive cocci 4.3 Antimicrobial susceptibility testing 4.3.1 Disk diffusion method Isolates of each identified species were tested for their susceptibility to ten antimicrobial agents Of the ten antibiotics used, isolates of V vulnificus showed complete resistance to colistin Their susceptibility to ampicillin and cefotaxime was in the intermediate range They were sensitive to rifampicin, doxycycline, enrofloxacin, norfloxacin, sulphamethoxazole/trimethoprim, and created the largest zone of inhibition when tested with florfenicol Susceptibility pattern of A hydrophila was different from that of V vulnificus Isolates of this species were resistant to four types of antimicrobial agents, including colistin, flumequine, enrofloxacin, and norfloxacin However, their sensitivity to cefotaxime, doxycycline, rifampicin, ampicillin, and sulphamethoxazole/trimethoprim was also recorded, and florfenicol appeared to be the most efficient antibiotics in inhibiting the 13 growth of this bacterial species in this study This result is surprising since A hydrophila is said to be resistant to ampicillin, and sensitive to enrofloxacin Although isolates of Streptococcus iniae in our study also showed complete resistant to colistin, they appeared to be the most sensitive species because large zones of inhibition were created when this species was tested with the other types of antibiotics Moreover, isolates of this species were also susceptible to erythromycin used in another previous test Susceptibility testing of this species with cefotaxime gave the largest inhibitory zone Antibiotics V vulnificus A hydrophila S iniae Colistin (30µg) R R R Flumequine (30µg) S R R Enrofloxacin (5µg) S R S Norfloxacin (5µg) S R S Cefotaxime (30µg) I S S Ampicillin (25µg) I S S Doxycycline (30µg) S S S Florfenicol (30µg) S S S Rifampicin (30µg) S S S Sulphamethoxazole/ trimethoprim (19:1, 25µg) S S S Erythromycin (15µg) - - S Note: S – susceptible, R –Resistant, I - Intermediate Table 4.1Susceptibility pattern of three bacterial species 14 4.3.2 Broth dilution method Isolates of Streptococcus iniae obtained from fish farm having high number of dead fish were used in minimal inhibitory concentration test with six antibiotics As shown in table 4.2, the minimal inhibitory concentration value of colistin was the highest, even greater than 512µg/ml Other antibiotics had a lot lower MIC values, ranging from 4µg/ml to even 0.0325µg/ml which was the lowest value, acquired with erythromycin The result of this test was congruent with the previous test, using disk diffusion method, in the way that Streptococcus iniae was resistant to colistin, and sensitive to other antibiotics, thus resulting in high MIC value of colistin and low MIC values of others Antibiotics Erythromycin Enrofloxacin Oxytetracycline Trimethoprim Florfenicol Colistin MIC (µg/ml) S iniae isolates 0.25 T15S 0.0325 T17L 0.25 T15S 0.25 T17L 0.5 T15S T17L 0.125 T15S T17L T15S 128 T17L >512 T15S Table 4.2 MIC values of six antibiotics for two S iniae isolates 15 CHAPTER CONCLUSIONS AND RECOMMENDATION 5.1 Conclusions The results showed that the three bacterial species, including Streptococcus iniae, Vibrio vulnificus, and Aeromonas hydrophila can be isolated from Asian sea bass (Lates calcarifer) Sea bass infected with S iniae often displayed erratic swimming External diseased signs may include bilateral or unilateral exophthalmia, and hemorrhagic on skin Internally, diseased fish may get pale liver, and the spleen and kidney were swollen All three bacterial species were resistant to colistin, while only A hydrophila and S iniae were resistant to flumequine Except for A hydrophila being resistant to enrofloxacin and norfloxacin, isolates of three species were sensitive to all other antibiotics used In this study, S iniae isolates were susceptible to enrofloxacin, oxytetracycline, trimethoprim, florfenicol, and were the most sensitive to erythromycin with an MIC = 0.25µg/ml On the other hand, they were resistant to colistin, with MIC value > 125µg/ml 5.2 Recommendations Antibiotics such as erythromycin and florfenicol can be used to control S iniae, V vulnificus, and A hydrophila when they are causing disease outbreak in fish farm However, the type and dosage of antibiotics to be used, and timeline of usage need to be put in careful consideration Further researches need to be carried out in order to understand more about the pathogenicity of the three bacterial species, with a special attention to S iniae on Asian sea bass (Lates calcarifer) 16 REFERENCES Austin, B., and D Austin, 2007 Bacterial Fish Pathogens – Diseases of farmed and wild fish, Fourth edition Praxis Publishing United Kingdom 552pp Bromage, E.S., A Thomas, and L Owens, 1999.Streptococcus iniae, a bacterial infection of barramundi Lates calcarifer Diseases of Aquatic Organisms, Volume 36: 177 – 181 Chan, S M., 1997 Identification of Vibrio spp from culture sea bass Lates calcarifer and their antibiogram relationship with plasmid profile Universiti Putra Malaysia Cheong, L., 1989 Status of knowledge on farming of seabass (Latescalcarifer) in South East Asia.AQUACOP.IFREMER.Actes de Colloque, 8pp.: 421-428 Clinical and Laboratory Standards Institute Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Proposed Guideline.CLSI document M49-P [ISBN 1-56238-577-1] Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005 Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Ninth Edition Clinical and Laboratory Standards Institute document M2-A9 [ISBN 1-56238-586-0] Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006 Creeper, J H., and N B Buller, 2006.An outbreak of Streptococcus iniae in barramundi (Latescalcarifer) in freshwater cage culture Australian Veterinary Journal, Volume 84, No 11, 2006: 408-411 Dung, T T., P T Huong, and N A Tuan, 2010 Multiple resistance pattern of Edwardsiella ictaluri isolated from Tra catfish Pangasianodon hypophthalmus in the Mekong Delta FAO.© 2006-2012.Cultured Aquatic Species Information Program.Lates calcarifer Cultured Aquatic Species Information Program Text by Rimmer, M.A In: FAO 17 Fisheries and Aquaculture Department [online] Rome Updated June 2006.[Cited 27 May 2012] http://www.fao.org/fishery/culturedspecies/Lates_calcarifer/en#tcNA0019 Hai, T N., and N T Phuong, 2006 Juvenile Production and Marine Fish Culture College of Aquaculture and Fisheries, Can Tho University, Can Tho, Vietnam 62pp Kim, Y.R., S E Lee, C M Kim, S Y Kim, E Y Shin, D H Shin, S S Chung, H E Choy, A P Fox, J D Hillman, M Handfield, and J H Rhee 2003 Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients INFECTION ANDIMMUNITY, Vol 71, No 10, Oct 2003: 5461–5471 Kumarasamy, K K., M A Toleman, T R Walsh, J Bagaria, F Butt, R Balakrishnan, U Chaudhary, M Doumith, C G Giske, S Irfan, P Krishnan, A V Kumar, S Maharjan, S Mushtaq, T Noorie, D L Paterson, A Pearson, C Perry, R Pike, B Rao, U Ray, J B Sarma, M Sharma, E Sheridan, M A Thirunarayan, J Turton, S Upadhyay, M Warner, W Welfare, D M Livermore, N Woodford, 2010 Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study Noga, E G., 2010 Fish Diseases - Diagnosis and Treatment, Second Edtion Blackwell Publishing.USA 519pp Plumb, J A.,and L A Hanson, 2011 Health Maintenance and Principal Microbial Diseases of Cultured Fishes, Third Edition Blackwell Publishing, Iowa, USA 492pp Prescott, J F., J D Baggot, and R D Walker 2000 Antimicrobial Therapy in Veterinary Medicine, Third Edition Iowa State University Press, Iowa, USA, 796pp Roberts, R., 2012 Fish Pathology, Fourth Edition Blackwell Publishing, Iowa, USA 581pp 18 Tenover, F C., 2006 Mechanisms of antimicrobial resistance in bacteria American Journal of Medicine, Vol.119 (6A), June 2006 Toranzo, A E., B Magariños, and J L Romalde, 2005.A review of the main bacterial fish diseases in mariculture systems Aquaculture, volume 246, Aquaculture, Volume 246, Issues 1–4, 18 May 2005: 37-61 Tuan, L A., N.T Nho, and J Hambrey 2000 Current practices of marine cage culture in Vietnam In: Reports in marine cage culture conference 26 to 27 June, 2000, NhaTrang NhaTrang University:1-15 Woo, P T K, and D W Bruno 1998, Fish Diseases and Disorders, Volume 3.Viral, Bacterial, and Fungal Infections.CABI Publishing, United Kingdom 19 APPENDICES Appendix Major antimicrobial drugs used in aquaculture (Prescott et al., 2000) Route Dose (mg/kg fish/day) Oral 50-80mg/kg, 10 days Neomycin Oral 50-80mg/kg, 10 days Kanamycin Bath 20mg/l Tetracycline Oral 50-80mg/kg, 10 days Bath 20mg/l Oral 50-80mg/kg, 10 days Bath 20mg/l Product Indication Antibiotics Ampicillin Beta-lactams Amoxicillin Benzyl penicillin (with streptomycin) Aminoglycosides Tetracyclines Gram (-) bacteria Oxytetracycline Doxycycline Macrolides Erythromycin Chloramphenicol group Florfenicol Oral 50-80mg/kg, 10 days Thiamphenicol Bath 20mg/l Oral 200mg/kg, 10days Bacterial kidney disease Synthetic Antimicrobial Agents Sulfamethazine Sulfonamides Sulfadimethoxine Sulfaguanidine Potentiated sulfonamides Trimethoprim/ sulfadiazine(1:5) Ormetoprim/ sulfadimethoxine 50mg/kg, 10days Oral 50mg/kg, 5days Oxilinic acid Quinolones Flumequine Gram (-) bacteria Oral Sarafloxcin 10-30mg/kg, 10days 10mg/kg, 5days Note: Only oxytetracycline and ormetoprim-sulfadiazine are approved for use with food fish in the United Staes 20 Appendix Fish Disease Diagnosis Form Date: Case No.: _ Name: _ Phone: _ Address: _ HISTORY Freshwater: _ Marine: System size: l/ha Species affected: _ Species in system: No fish in system: % fish affected: _ Average fish size: _ g/cm Age(s) of affected fish: When morbidity started: _ When mortality started: _ When morbidity ended: When mortality ended: How long has the system been set up? Temperature: _ Types of life support present: _ Water source: Water appearance (cloudy, colored): _ History of maintenance, including water exchange and water quality checks: _ Behavioral changes? Y- N- Describe: Respiratory rate: (normal _, faster _, slower ): _ Appearance of fish: _ _ Appetite (normal: _, less: _, more: ): _ Other clinical signs: _ 21 Appendix Some biochemical tests used in bacterial identification Gram’s Stain Method: - Place a loopful of sterile saline onto a clean glass slide, using a sterile loop - Use a sterile loop to evenly spread a small amount of bacteria on the slide - Naturally dry the slide - Use forceps to hold the slide with the film upwards and slowly pass through a flame three times to fix the film - Let the slide cool, and place it in a staining dish - Leave the slide immersing in the crystal violet solution for about minute - Wash off the residual stain with water, and then immerse the slide in iodine for minute - Gently mix the slide in alcohol/acetone mixture for 10 seconds - Carefully wash the slide with water and then let it dry - Place the slide in Saffranin solution for minutes - Use water to wash the slide, let it dry, and clean its bottom - Observe the stained slide under the microscope at x40 objective, and then x100 objective with oil immersion - Gram positive bacteria have the color of blue/purple, while Gram negative bacteria are red/pink when being observed under the microscope Motility Test: - Place Vaseline on the four corners of a coverslip - Put the coverslip, Vaseline up, on the bench - Place a loopful of sterile saline on the coverslip, using a sterile loop - Sterilize the loop again, and take a tiny amount of bacteria to gently spread it out in the saline - Gently lower a microscope slide onto the Vaseline mounds without allowing the slide to touch the drop of culture - Quickly, but gently, invert the slide so that the drop is hanging from the coverslip - Adjust the lens to see bacterial motility 22 Oxidase Test: Place the tip of the oxidase stick onto a bacterial colony, and observe the stick for about 30 second to see if there are any changes on color The appearance of blue/purple indicates a positive result Oxidative-Fermentative (O-F) Test: - Use the tip of a sterile straight wire to pick up an inoculum of a pure growth from a culture plate, and make a single stab into the agar tube - Inoculate the second O-F tube, and aseptically cover the agar with 1mm layer of liquid paraffin - Incubate both tubes for up to days, observe and read the results: Open tube Green Blue at top Yellow Yellow Closed tube Green Green Green Yellow Result No reaction on glucose Alkaline reaction Oxidative Fermentative O/129 Sensitivity: - Remove several colonies of growth using a loop and inoculate a small quantity of sterile saline with the culture - With the bottle cap replaced tightly, suspend the culture in the saline by gently shaking - Using a sterile pipette, remove small volume of the suspension and carefully pipette drops of suspension onto the surface of the agar - Sterilize a glass spreader by immersing it in 70% ethanol, and then pass the spreader through an alcohol flame to burn off the excess ethanol - Gently spread the suspension over the whole surface of the agar plate - Replace the lid and allow the plate to dry for minute - Place one 10µg disc and one 150µg disc of O/129 onto the plate as illustrated - Invert the plate and incubate at a suitable temperature 23 Appendix Preparation of culture dilution series per isolate Tube Final concentration (µg/ml) Antimicrobial solution Bacterial solution 1024 2ml (stock solution) 2ml 512 2ml (1024µg/ml) 2ml 256 2ml (512µg/ml) 2ml 128 2ml (256µg/ml) 2ml 64 2ml (128µg/ml) 2ml 32 2ml (64µg/ml) 2ml 16 2ml (32µg/ml) 2ml 8 2ml (16µg/ml) 2ml 2ml (8µg/ml) 2ml 10 2ml (4µg/ml) 2ml 11 2ml (2µg/ml) 2ml 12 0.5 2ml (1µg/ml) 2ml 13 0.25 2ml (0.5µg/ml) 2ml 14 0.125 2ml (0.25µg/ml) 2ml 15 0.0625 2ml (0.125µg/ml) 2ml 16 0.03125 2ml (0.0625µg/ml) 2ml 24 Appendix API 20E and API 20STREP kit results API 20E V Tests vulnificusAT CC33187 API 20STREP V vulnificusi solate A hydrophila ATCC7966 A hydrophila isolate Tests S iniae (Buller, 2004) S iniae isolate ONPG + + + + VP - - ADH - - + - HIP - - LDC + + + + ESC + + ODC + + - - PYRA + + CIT + - - - GAL - - H2 S - - - - GUR + + URE - - - - GAL - - TDA - - - - PAL + + IND - + + + LAP + + VP - - + + ADH - -/+ GEL + + + + RIB + + GLU + + + + ARE - - MAN - + + + MAN + + INO - - - - SOR - - SOR - - - - LAC - - RHA - - - - TRE + + SAC - - + + INU - - MEL - - - - RAF - - AMY + + - + AMD + + ARA - - + + GLYG + + 25 [...]... Owens, 1999.Streptococcus iniae, a bacterial infection of barramundi Lates calcarifer Diseases of Aquatic Organisms, Volume 36: 177 – 181 Chan, S M., 1997 Identification of Vibrio spp from culture sea bass Lates calcarifer and their antibiogram relationship with plasmid profile Universiti Putra Malaysia Cheong, L., 1989 Status of knowledge on farming of seabass (Latescalcarifer) in South East Asia.AQUACOP.IFREMER.Actes... enrofloxacin, norfloxacin, sulphamethoxazole/trimethoprim, and created the largest zone of inhibition when tested with florfenicol Susceptibility pattern of A hydrophila was different from that of V vulnificus Isolates of this species were resistant to four types of antimicrobial agents, including colistin, flumequine, enrofloxacin, and norfloxacin However, their sensitivity to cefotaxime, doxycycline, rifampicin,... purity before being used Bacterial colonies are then put into 10ml tube of BHB, and incubated at 300C, for another 24 hours The concentration of bacteria is determined, using spectrophotometer, and diluted to the acquired concentration of 5 x 105 CFU/ml Volumes of 2ml of bacterial solution are put into tubes containing 3ml antimicrobial solution of different concentrations Tubes of two control treatments... 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. .. type and dosage of antibiotics to be used, and timeline of usage need to be put in careful consideration Further researches need to be carried out in order to understand more about the pathogenicity of the three bacterial species, with a special attention to S iniae on Asian sea bass (Lates calcarifer) 16 REFERENCES Austin, B., and D Austin, 2007 Bacterial Fish Pathogens – Diseases of farmed and wild... characteristics of this bacterial species The identification result with rapid identification system was confirmed with the result of 16S rRNA partial gene sequencing, and identified isolates of this group as Streptococcus iniae A B C Figure 4.3Gram stains of three bacterial isolates A Gram-negative, curved rod B Gram-negative short rod C Gram-positive cocci 4.3 Antimicrobial susceptibility testing 4.3.1... 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... testing 4.3.1 Disk diffusion method Isolates of each identified species were tested for their susceptibility to ten antimicrobial agents Of the ten antibiotics used, isolates of V vulnificus showed complete resistance to colistin Their susceptibility to ampicillin and cefotaxime was in the intermediate range They were sensitive to rifampicin, doxycycline, enrofloxacin, norfloxacin, sulphamethoxazole/trimethoprim,... elimination of the upper part, 25ml of saline solution is added, and the solution is well mixed The density of bacteria is determined, using spectrophotometer (OD= 600nm) Bacterial solution is then diluted to the approximate concentration of 1-2 x 108 colony-forming units (CFU)/ml The standardized bacterial suspension is evenly spread on the surface of the agar plates with a cotton swab The surface of the... T15S 128 T17L >512 T15S Table 4.2 MIC values of six antibiotics for two S iniae isolates 15 CHAPTER 5 CONCLUSIONS AND RECOMMENDATION 5.1 Conclusions The results showed that the three bacterial species, including Streptococcus iniae, Vibrio vulnificus, and Aeromonas hydrophila can be isolated from Asian sea bass (Lates calcarifer) Sea bass infected with S iniae often displayed erratic swimming External ...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... Tran HuuTinh i 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... 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