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

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES FLOFENICOL AND ENROFLOXACIN RESISTANCE IN HETEROTROPHIC BACTERIA ISOLATED FROM SNAKEHEAD (Channa striatus) AND CLIMBING PERCH (Anabas testudineus) FARMS IN THE MEKONG DELTA By NGUYEN DAI DUONG A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of science in Aquaculture Can Tho, January, 19 2012 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES FLORFENICOL AND ENROFLOXACIN RESISTANCE IN HETEROTROPHIC BACTERIA ISOLATED FROM SNAKEHEAD (Channa striatus) AND CLIMBING PERCH (Anabas testudineus) FARMS IN THE MEKONG DELTA By NGUYEN DAI DUONG A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of science in Aquaculture Supervisor Assoc Prof Dr DANG THI HOANH OANH Can Tho, January, 19 2013 Acknowledgements I wish to express my sincere gratitude to my parents for always encouraging me when I face the problems, supporting me with the best conditions for my studies in four and a half years at Cantho University With my high appreciation and sincere gratitude, I would like to thank my supervisor, Assoc Prof Dr Dang Thi Hoang Oanh for her wholehearted guidance and helping me to access to the facilities as well as materials for my study I also want to thank Mrs Nguyen Thi Thu Hang and Ms Truong Quynh Nhu not only for their constant mentorship but also for their encouragement during my study I am thankful to all staffs and students in the Department of Aquatic Pathology, College of Aquaculture and Fisheries, for helping me and making the good conditions for me to finish my study Special thanks to all my classmates in advanced aquaculture class, course 34 for their constant help in my study duration i Abstract The purpose of this study was to identify the heterotrophic bacteria which were isolated from climbing perch (Anabas testudineus) and snakehead (Channa striatus) farms in the Mekong Delta at genus level by basic morphological, physiological and biochemical tests In each bacterial genus, two isolates were chosen to test with 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) ii 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 Background of the study 1.2 Objectives of the study 1.3 Contents of the study .2 CHAPTER II: LITERATURE REVIEW 2.1 Status of snakehead (Channa striatus) and climbing perch (Anabas testudineus) farming in the Mekong Delta 2.2 General information about the cause of diseases 2.3 Some common bacterial diseases in freshwater fish .5 2.3.1 Columnaris disease 2.3.2 Edwardsiella Septicemia or Edwardsiellosis 2.3.3 Motile Aeromonad Septicemia 2.3.4 Pseudomonad Septicemia or Red Spot Disease .6 2.3.5 Streptococcal infection 2.4 Antibiotic drug resistance 2.5 Classification of some common antibiotics used in aquaculture 2.5.1 Quinolones .8 2.5.2 Fenicol group 2.5.3 Beta-lactams 2.5.4 Tetracyclines 2.5.5 Sulphonamides 2.6 Studies related to antibiotic resistance in aquaculture CHAPTER III: METHODOLOGY 12 3.1 Research place .12 iii 3.2 Materials 12 3.3 The source of bacteria 12 3.4 Methods .12 3.4.1 Bacterial identification 12 3.4.2 Antibiotic susceptibility test and Minimal Inhibitory Concentration .13 3.4.2.1 Antibiotic Susceptibility Test .13 3.4.2.2 Minimal Inhibitory Concentration – MIC 13 CHATER IV: RESULTS AND DISCUSSIONS 14 4.1 The results of bacterial identification 14 4.1.1 Edwardsiella genus 14 4.1.2 Aeromonas genus 16 4.1.3 Pseudomonas genus 17 4.1.4 Staphylococcus genus 18 4.1.5 Streptococcus genus .19 4.2 Antimicrobial susceptibility testing results 20 4.3 Minimal inhibitory concentration (MIC) testing results .22 CHAPTER V: CONCLUSIONS AND RECOMMENDATIONS 24 5.1 Conclusions 24 5.2 Recommendations .24 References 25 Appendices 32 iv List of tables Table 4.1: The results of antimicrobial susceptibility test on 10 isolates 20 Table 4.2: The MIC value of enroflorxacin and florfenicol on tested Isolates 22 v List of figures Figure 4.1: Appearance frequency (%) of genera .14 Figure 4.2: Edwardsiella bacteria isolated from snakehead fish and climbingperch fish farms .15 Figure 4.3: Aeromonas bacteria isolated from snakehead fish and climbing perch fish farms .16 Figure 4.4: : Pseudomonas bacteria isolated from snakehead fish and climbing perch fish farms .17 Figure 4.5: Staphylococcus bacteria isolated from snakehead fish and climbing perch fish farms .19 Figure 4.6: Streptococcus bacteria isolated from snakehead fish and climbing perch fish farms .20 Figure 4.7: The MIC value of ENR on one tested isolate .23 vi List of abbreviations AMX Amoxiciline DO Doxycyline ENR Enroflorxacin FFC Florfenicol MD Mekong Delta vii CHAPTER I INTRODUCTION 1.1 Background of the study The Mekong Delta (MD) has more than million 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 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 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 agalactiae Proceeding of National Fisheries Research Workshop No 4: 289-301 (Read in Vietnamese) Oanh, D.T.H and Nguyen Duc Hien, 2012 Isolation and pathogenicity of Aeromonas hydrophila on rice eel (Monopterus albus) Journal of science Cantho University Volume: 22c – 2012 173-182 (Read in Vietnamese) Oanh, D T H., T Q Nhu, and N D Hien, 2012, Isolation and pathogenicity of Streptococcus agalactiae biotype on climbing perch (Anabas testudineus) Journey of Science Cantho University Volume: 22c – 2012 194-202 (Read in Vietnamese) Oanh, D T H and N T Phuong, 2012 Isolation and characterization of Streptococcus agalaciae from red tilapia cultured in the MK Delta Journey of Science Cantho University Volume: 22c 203-2012 (Read in Vietnamese) Rajkumarbharathi, M., N Sukumaran, A.G Murugesan, 2011 Antibiotic sensitivity of bacterial Flora isolation from gill and gut of snakehead fish “Channa striatus” Volume 3, No Journal for Bloomers of Research 137-141 Reger, p.J., D.F Mockler, and M.A Miller, 1993 Comparison of antimicrobial susceptibility beta-lactamase production, plasmid analysis and serum bacterial activity in E tarda, and E hoshinae J Med Microbital 39: 273-281 Robinson, J A and F p Meyer, 1996 Streptococcal fish pathogen Journal of Bacteriology 92-512 Sae-Oui, D., K Muroga, and T Nakai 1984 A case of Edwardsiella tarda infection in cultured colored carp Cyprinus carpio Fish Pathol 19(3):197-199 Sinh, L.X., and D.M Chung, 2010 Current situation and Challenges for snakehead farming (Channa micropeltes and Channa striatus) in the MD Agriculture and Rural Development College of Aquaculture and Fisheries Cantho University Instalment 2: 56 – 63 (Read in Vietnamese) Shotts, E B and R Rimler 1973 Medium for the isolation of Aeromonas hydrophila Journal of Applied Microbiology 26: 550 - 553 Shu-Peng H., H Tain-Yao, C Ming-Hui and W Way-Shyan, 2000 Antibacterial effect of chloramphenicol, thiamphenicol and flofenicol against aquatic animal bacteria Journal Veterinary Medicine Science 62: 479-485 Stock, I., and B Wiedemann, 2001 Natural antimicrobial susceptibility of Edwardsiella tarda, E ictaluri and E hoshinae Antimicrobial Agents Chemotherapy 45: 2245-2255 29 Sugiyama, A., & Kusuda, R (1981) Studies on the characters of Staphylococcus epidermidis isolated from diseased fishes Fish Pathology, 16, 35 - 41 Tai, L.T., 2010 Identification of common diseases on intensively famred snakehead (Channa striatus) Master thesis Cantho University (Read in Vietnamese) Thịnh H N., and T T Loan, 2007 Isolation and investigation for antibiotic resistance of Edwardsiella ictaluri from intensively farmed Tra catfish, Pangasius hypophthalmus Science and Technology Research 1st and 2nd 175-179 (Read in Vietnamese) Thanh, H.T.N., 2012 Histopathological characteristics of “black body” diseased climbing perch (Anabas testudineus) Master thesis Cantho University (Read in Vietnamese) Thinh, N.H., B.T Kim, and D.V Phuong, 2011 One case of heavy Trypanosoma infection in intensive culture climbing perch (Anabas testudineus) Proceeding of National Fisheries Research Workshop No 207 – 216 (Read in Vietnamese) Truong Thu Khoa and Tran Thi Thu Huong, 1993 Identification freshwater fish species in the MD College of Aquaculture and fisheries Cantho Univeristy (Read in Vietnamese) Thy, D.T.M., et al., 2012 Histopathology of Climbing perch (Anabas testudineus) infected with Aeromonas hydrophila and Streptococcus sp Journal of Science Volume: 22c 183-193 (Read in Vietnamese) Truong Thy Ho, Nontawith Areechon, Prapansak Srisapoome and Songsri Mahasawasde, 2008 Identification and Antibiotic Sensitivity Test of the bacteria isolated from Tra Catfish (Pangasianodon hypophthalmus) [Sauvage, 1878]) cultured in Pond in Vietnam Kasetsart J (Nay Sei.) 42 54-60 Wakabayashi, H & Egusa, S 1972 Characteristics of a Pseudomonas and pond cultured eels (Anguilla japonica) Bulletin of the Japanese Society of Scientific Fisheries, 38, 577 – 87 Wood, J.W., 1974 Disease of pacific Salmon: Their prevention and Treatment, 2nd ed., state of Washington Department of Fisheries Hatchery Division, 82 p Wrzesinsk, C., L Crouch, P Gaunt, D Holifield, N Bertrand, and R Endris 2005 Florfenicol residue depletion in channel catfish, Ictalurus punctatus (Rafinesque) Aquaculture 253:309–316 Wyatt, L.E., R Nickelson II & C Vanderzant, 1979 Edwardsiella tarda in freshwater catfish and their environment Applied and Environmental Microbiology 38: 710 – 714 30 Yambot V A., 1998 Isolation of Aeromonas hydrophila from Oreochromis niloticus during Fish Disease Outbreaks in the Philippines Asian Fisheries Science 10: 347-354 Ministry of Agriculture and Rural Development, 2012 Elimination of Cypermethrin, Deltamethrin, Enroflorxacin in aquaculture (Read in Vietnamese) Retrieved from: http://www.agroviet.gov.vn/Pages/news_detail.aspx?NewsId=20904&Pa ge=5 31 Appendices Appendix 1: Plate inoculation procedure for culture and purification of bacteria  Inoculate sample on a small segment of the surface of the culture medium  Flame inoculation loop until red hot, allow to cool and to touch loop on edge of un-inoculated area of medium to ensure coolness  Spread part of sample over about ¼ of the plate by making 3-4 parallel streaks with the loop  Repeat streaking procedure as shown, flaming and cooling the loop between each sequence Label underside (not lid) of plate and incubate Appendix 2: Gram-staining method:  Apply ammonium oxalate/crystal violet solution to heat-fixed smear for  Wash with water  Apply iodine solution for  Tip off iodine solution  Decolorise with alcohol/acetone (95% :5%) until no more violet color emanates from the smear  Wash thoroughly with water  Apply safranin solution for  Wash with water, drain and/or blot dry and examine Gram-positive organisms- blue/purple Gram-negative organisms – pink/red Appendix 3: Motility  Ring the outside edge of a coverslip with Vaseline  Place a loopful of liquid culture on the centre of the coverslip within the vaseline ring  Lower a microscope slide onto the Vaseline and press lightly to ensure a good seal  Carefully invert the slide and attached coverslip and examine under the microscope Focus first onto the edge of the hanging drop with a lowpower objective before progressing through higher power objectives  True motility is non-random and must not be confused with vibratory Brownian movement or convection currents A motile organism is one 32 which actively moves to change its position relative to other organisms present Appendix 4: Catalase Test  A plate of nutrient agar is streaked and incubated at the optimum temperature for 24 hr (or longer if required to see good growth)  Scrape a sample of bacterial culture off the plate with a glass rod or platinum wire and transfer to a drop of 3% H2O2 on a clean glass slide A positive test is the almost immediate production of gas bubbles Appendix 5: Oxidase Test  Wet a small piece of filter paper with oxidase reagent  The test organism (grown on a media free from glucose and nitrate) is removed with a platinum wire of glass rod and smeared across the surface of wet paper A positive reaction is shown by the development of a dark purplecolor within 10-30 sec Appendix 6: Glucose Oxidation-Fermentation (O-F) Test  Inoculate two tubes by stabbing with needle carrying bacteria  Overlay the medium in one tub with sterile liquid paraffin to a depth of cm  Examine daily for up to 7days Result Open tube Covered tube Oxidative Yellow (+) Green (-) Fermentative Yellow (+) Yellow (+) No reaction Blue/Green (-) Green (-) Note: Negative (-) has to wait until days Appendix 7: Saline tolerance test  Prepare four tubes of Nutrient Broth with different concentration of NaCl at: 0%, 1.5%, 3% and 6% and one NB tube for negative control  Inoculate four tubes by stabbing needle with carrying bacteria  After 24 or 48 hours incubated at 28oC, the positive reaction is recoded in the more turbid comparing to control tube Appendix 8: 0/129 sensitivity  This test is mainly used to distinguish 0/129 resistant Aeromonas sp from 0/129 sentitive Vibrio sp., most of which have a requirement to salt in the 33 growth medium A blood agar base is often recommended although TSA is ususlly equally suitable for the test Both media can be supplemented with NaCl if required  Sensitivity is determined using discs containing 150g and 10g vibriostat The discs are placed on the surface of an agar plate which has been spread with a suspension of the organism will show a clear zone of growth inhibition round the disc Appendix 9: Antibiotic susceptibility test (Geert Huys, 2002) Bacterial cultivation and material preparations (DAY 1)  The organism to be tested will be cultivated on TSA medium at 280C under aerobic atmosphere Streak out the pure culture on a TSA plate in a way that distinct colonies will be obtained  Preparing the desired volume of TSA medium according to the manufacturer‟s instructions Before pouring the agar medium, bottles should be cooled to 40-500C in a water bath All plates are poured on a flat, horizontal surface each to an identical depth of mm + mm (corresponding to 20 mL + mL of medium in 10 cm radius petri dishes) Preparing tubes containing mL sterile physiological solution (PHY-SOL) Inoculation of the antibiogram (DAY 2)  Harvesting a number of distinct colonies from the fresh grown plate culture to suspend in a tube containing PHY-SOL until turbidity (visually) corresponding to 1.0 McFarland standard is reached It remains important to take more than one colony in order to obtain a representative sample  Using a micropipet, spot 100 uL of the standardized suspension on the surface of a TSA plate Spread plate the suspension using a sterile glass triangle rod Allow to dry the plates for max 15 minutes Longer drying times allow pre-incubation of the cells which should be avoided Where the room temperature is more than 50C above or below the incubation temperature, plates must be incubated within 10 minutes of the application of the discs Where room temperature lies within 0C of the incubation temperature, plates should be incubated as soon as possible after the application of the discs  Manually using sterile forceps applied the discs onto the agar surface Discs must not be relocated once they have made contact with the agar surface Incubate the plates at 280C for 24 hours Reading of the antibiogram (DAY 3)  The diameter of the inhibition zones are measured to the nearest mm from the point of abrupt inhibition of growth (using a callipers or mm ruler) 34 Where there is any doubt the point of 80% inhibition should be taken as the zero edge  If the plates are not sufficiently grown, read again after 48 h incubation Plates on which the growth of the test isolate produces isolated colonies (less than semi-confluent growth) should not be read If zones of inhibition produced by adjacent discs overlap to the extent that two measurements at right angles cannot be made, the zones around these discs should not be recorded Equally, zones demonstrating significant distortion from circular should not be reported  If the zones of inhibition produced on plates inoculated with control isolate E coli LMG 8223 are not within the tolerance limits set (within 3mm), then all data collected in that particular set must be rejected After the validation study performed by all partners, it was decided that a maximum between batch variation of mm is allowed as a quality control for zones determined with the control isolate Table 1: The inhibitory zone diameter (mm) limit of antimicrobial agents Antibiotics Resistant Intermediate Susceptible Amoxicillin (AMX/25g) 13 14-17 18 Doxycyline (DO/30g) 12 13-15 16 Enroflorxacin (ENR/5g) 16 17-22 23 Florfenicol (FFC/30g) 16 17-19 20 Source: Clinical and Laboratory Standards Institute (CLSI), 2011 Appendix 10: Minimal Inhibitory Concentration (Geert Huys, 2002) Bacterial cultivation and material preparations (DAY 1-2)  The organism to be tested will be cultivated on TSA medium at 280C under aerobic atmosphere Streak out the pure culture on a TSA plate in a way that distinct colonies will be obtained  After overnight incubation, the streak cultures are checked for purity A number of pure colonies (app or more if the isolate is a suspected fastidious organism) are introduced into a glass culture tube containing 10 mL TSB and incubated at 290C overnight Ideally, the culture tubes should fit into a portable spectrophotometer In this way, the overnight grown culture can be easily adjusted to a standardized cell density by dilution with sterile TSB  Prepare a bottle with the necessary volume of distilled water Preparation and inoculation of the dilution series (DAY 3) 35  The optical density of the overnight culture of the isolate is determined spectrophotometrically at 590 nm and is standardized at 0.1 + 0.02 (i.e app 10E8 CFU/mL) by diluting with sterile TSB  For each batch of 10 isolates (i.e test isolates and the reference isolate), two 50 mL sterile stock solutions of enrofloxacin or flofenicol (i.e stock solution of 1024 ppm and stock solution of 256 ppm) will be prepared in the suitable solvent However, subsequent dilutions of these stock solutions can be made up in sterile water The use of two stock solutions is recommended to minimize quantitative errors in the low-concentration range of the serial dilution series  For each set of 10 isolates, a dilution series of two-fold antibiotic concentrations (4-1024 ppm) is prepared in sterile tubes of 50 mL according to the scheme outlined in Table In the first step, the antibiotic concentrations 512 and 256 ppm are prepared from stock solution (1024 ppm) by adding equal volumes of sterile mQ Antibiotic concentrations 128, 64, 32, 16, 8, and ppm are obtained by making serial dilutions from stock solution (256 ppm) in a second step It is absolutely crucial to thoroughly mix every freshly prepared antibiotic dilution prior to using it to prepare the next dilution It should be noted that each antibiotic dilution undergoes a final 1:2 dilution when the broth culture is added (Table 3) Table 2: Scheme for preparation of antibiotic dilution series (per 10 isolates) Tube Concentration obtained (ppm) Volume of AB solution 1024 ppm 25 mL (stock solution 1) - 512 ppm 25 mL (1024 ppm) 25 mL 256 ppm 25 mL (512 ppm) 25 mL 128 ppm 25 mL (stock solution 2) 25 mL 64 ppm 25 mL (128 ppm) 25 mL 32 ppm 25 mL (64 ppm) 25 mL 16 ppm 25 mL (32 ppm) 25 mL 8 ppm 25 mL (16 ppm) 25 mL ppm 25 mL (8 ppm) 25 mL No (mL) Volume of sterile mQ Stock solution = 1024 ppm; stock solution = 256 ppm  Following the preparation of the serial antibiotic dilutions, mL of freshly standardized broth culture of the isolate is inoculated in each tube of the dilution series according to the scheme outlined in Table In this regard, it is important to note that the standardized cultures should be processed within the hour after preparation Dilutions and broth cultures should be well homogenized prior to mixing 36 Table 3: Scheme for preparation of culture dilution series (per isolate) MIC Final concentration Volume of AB solution No (ppm) (mL) Volume of broth culture (mL) 512 ppm mL (stock solution 1; tube 1) mL 256 ppm mL (512 ppm; tube 2) mL 128 ppm mL (256 ppm, tube 3) mL 64 ppm mL (128 ppm; tube 4) mL 32 ppm mL (64 ppm; tube 5) mL 16 ppm mL (32 ppm; tube 6) mL ppm mL (16 ppm; tube 7) mL ppm mL (8 ppm; tube 8) mL ppm mL (4 ppm; tube 9) mL  For each batch of MIC determinations, a blank tube (i.e mL noninoculated TSB mixed with mL water) will be included In addition, a positive control will be included for each isolate The positive control is made up by mixing mL adjusted broth culture with mL sterile distilled water  All MIC tubes (concentration range 2-512 ppm) and control tubes of the test isolates and the control isolate as well as the blank are incubated aerobically at 280C for 24h  Each isolate will also be checked for purity by plating a drop of the adjusted culture onto TSA medium, and this plate will be incubated under the same conditions as the MIC test itself Reading of the MIC (DAY 4)  The purity of the broth culture is checked on TSA on the basis of uniform colonial morphology If contamination is noted than all data generated from the involved isolate will be rejected  Following a 24h, growth is determined visually among the different tubes of the serial dilution by comparing with the positive control and with the blank Any series where discontinuity in growth is observed (e.g growth in tubes and but not in tube 6) should be discarded The end-point is defined as the lowest antibiotic concentration for which there is no visual growth This concentration should be reproted as the MIC of that antibiotic for that particular isolate If trailing end-points are observed, this should be reported as a remark and a 80% reduction in growth should be reported as end-point Note: Sterile water mQ (deionized) water or double-distilled water containing 0.85% NaCl 37 Appendix 11: The results of bacterial identification Genera Aeromonas Edwardsiella Pseudomonas Staphylococcus Streptococcus Features Gram - - - + + Shape Rod Rod Rod Cocci Cocci Motility + + + - - Oxidase + - + - - Catalase + + + + - O/F +/+ +/+ +/- -/- -/- O/129 - 38 Appendix 12: The results of antimicrobial susceptibility tests Bacterial code The inhibitory zone diameter of antibiotics after testing (mm) Genus FFC ENR DO AMX VT69 Edwarsiella 20 24 18 VT28 VT77 Edwarsiella Aeromonas 33 25 26 27 17 26 10 11 VT34 Aeromonas 27 18 23 VT73 Staphilococcus 18 15 10 VT77 Staphilococcus 24 20 18 12 VT14 Pseudomonas 23 27 30 11 VT16 Pseudomonas 20 14 10 VT47 Streptococcus 25 27 16 15 VT7 Streptococcus 23 21 17 14 39 Appendix 13: Bacterial isolates index Size of colonies (mm) Salt tolerance No Bacterial isolates Genus Colonies shape VT3 - A - - S - 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - VT3 - B - - O - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - VT3 - C - - W- 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - VT3 - B - - W- 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - VT3 - C - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - VT3 - B - - W- 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - VT3 - A - - W- 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - VT3 - B - - W- 04 Edwardsiella round-milky punctate 1-1.5 - Rod + - - - VT3 - B - - S - 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 10 VT3 - C - - W- 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 11 VT3 - A - - O - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 12 VT3 - B - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 13 VT3 - A - - O - 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 14 VT3 - A - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 15 VT3 - A - - S - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 16 VT3 - B - - S - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 17 VT3 - A - - W- 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 18 VT3 - A - - O - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 40 Gram Bacterial shape 0% 1,5 % 3% 6% 19 VT3 - A - - O - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 20 VT3 - A - - S - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 21 VT3 - A - - W- 03 Aeromonas circular, smooth, raised 2-2.5 - Rod + + + - 22 VT3 - B - - O - 02 Pseudomonas circular, smooth 2-4 - Rod + + + - 23 VT3 - B - - W- 02 Pseudomonas circular, smooth 2-4 - Rod + + + - 24 VT3 - C - - O - 03 Staphylococcus small ,convex, round 0.5-1 + sphere + + + - 25 VT3 - C - - S - 03 Staphylococcus small ,convex, round 0.5-1 + sphere + + + - 26 VT3 - C - - O - 04 Staphylococcus small ,convex, round 0.5-1 + sphere + + + - 27 VT3 - C - - O - 02 Staphylococcus small ,convex, round 0.5-1 + sphere + + + - 28 VT3 - C - - S - 04 Staphylococcus small ,convex, , round 0.5-1 + sphere + + + - 29 VT3 - C - - S - 01 Staphylococcus small ,convex, , round 0.5-1 + sphere + + + - 30 VT3 - C - - O - 01 Streptococcus small ,convex, round 0.3-0.5 + sphere + + + - 31 VT2 - C - - W- 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 32 VT2 - B - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 33 VT2 - B - - S - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 34 VT2 - B - - S - 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 35 VT2 - A - - O - 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 36 VT2 - C - - W- 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 37 VT2 - B - - S - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 38 VT2 - C - - S - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 39 VT2 - C - - S - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 41 40 VT2 - C - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 41 VT2 - C - - O - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 42 VT2 - A - - W- 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 43 VT2 - A - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 44 VT2 - B - - W- 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 45 VT2 - A - - W- 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 46 VT2 - B - - W- 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 47 VT2 - B - -W - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 48 VT2 - A - - S - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 49 VT2 - S - - W- 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 50 VT2 - A - - S - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 51 VT2 - A - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 52 VT2 - C - - W- 01 Edwardsiella round-milky punctate 1-1.5 - Rod + - - - 53 VT2 - A - - W- 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 54 VT2 - B - - S - 03 Edwardsiella round-milky punctate 1-1.5 - Rod + + - - 55 VT2 - B - - S - 04 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 56 VT2 - C - - S - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 57 VT2 - C - - O - 01 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 58 VT2 - C - - O - 02 Edwardsiella round-milky punctate 1-1.5 - Rod + + + - 59 VT2 - C - - O - 03 Edwardsiella round-milky punctate 3.5-4 - Rod + + + - 60 VT2 - C - - O - 04 Edwardsiella round-milky punctate 3.5-4 - Rod + + + - 42 61 VT2 - A - - O - 02 Edwardsiella round-milky punctate 4-5 - Rod + + + - 62 VT2 - C - - S - 04 Edwardsiella round-milky punctate 1.5-2 - Rod + + + - 63 VT2 - C - - - 03 Edwardsiella round-milky punctate 2-3 - Rod + + - - 64 VT2 - C - - S - 01 Edwardsiella round-milky punctate 2-3 - Rod + + - - 65 VT2 - C - - S - 01 Edwardsiella round-milky punctate 1.5-2.5 - Rod + + + - 66 VT2 - A - - W- 01 Edwardsiella round-milky punctate 1-1.2 - Rod + + + - 67 VT2 - A - - W- 03 Edwardsiella round-milky punctate 0.2-0.5 - Rod + + - - 68 VT2 - C - - O - 01 Aeromonas circular, smooth raised 1.5-3.5 - Rod + + + - 69 VT2 - C - - O - 02 Aeromonas circular, smooth, raised 1-1.5 - Rod + + + - 70 VT2 - C - - O - 03 Aeromonas circular, smooth, raised 3.5-4.5 - Rod + + + - 71 VT2 - A - - W- 04 Aeromonas circular, smooth, raised 2.2-5 - Rod + + + - 72 73 74 75 76 77 78 79 80 81 VT2 - A - - S - 03 VT2 - C - - W- 04 VT2 - C - - W- 01 VT2 - C - - W- 02 VT2 - A - - O - 04 VT2 - B - - W- 04 VT2 - B - - W- 01 VT2 - B - - S - 02 VT2 - B - - W- 02 VT2 - B - - O - 01 Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas Aeromonas Staphylococcus Staphylococcus circular, smooth, raised circular, smooth, raised circular, smooth, raised circular, smooth, raised circular, smooth, raised circular, smooth, raised circular, smooth, raised circular, smooth, raised small ,convex, round small ,convex, round 1.5-2 1.2-1.5 3.5-4 3.5-4 1.5-2.5 1.5-2.5 2.5-3 3.5-4 1-1.2 1-1.2 + + Rod Rod Rod Rod Rod Rod Rod Rod sphere sphere + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - 82 VT2 - C - - W- 03 Staphylococcus small ,convex, round 2-2.5 + sphere + + + - 83 84 VT2 - A - - S - 04 VT2 - A - - W- 01 Staphylococcus Streptococcus small ,convex, round small ,convex, round 1.5-2 0.3-0.5 + + sphere sphere + + + + + + - 43 [...]... cultured in the MD Climbing perch 3 farming was started in 2000s and rapidly developed in La Nga River of Dongnai province The production at that time could be reached to 80-100 tons/ha When the artificial propagation of climbing perch (Anabas testudineus) became more popular in the MD, farming of climbing perch was more developed in Haugiang, Longxuyen, Cantho and in some other provinces in the recent... 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 2 CHAPTER II LITERATURE REVIEW 2.1 Status of snakehead (Channa striatus) and climbing perch (Anabas testudineus). .. 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. .. 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. .. in Australia In recent times, in the MD, Streptococcus sp have been isolated from snakehead (Channa striatus) (Lu Tri Tai, 2010), tilapia (Oreochromis sp.) (Thy et al., 2011) and climbing perch (Anabas testudineus) (Oanh et al., 2012) with the external symptoms such as: Erratic swimming, darkening of body color, exophthalmia, hemorrhages on the opercula and bases of fin, and ulceration of body Basing... 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... time (Kha and Hung, 2007) In this study, there was also 2 tested isolates showing resistance to DO and ENR In the previous study, Reger et al (1993) indicated that E ictaluri strains isolated from channel catfish in United State were totally susceptible to DO On the other hand, the studies of Crumlish et al (2002) and Dung et al (2004) indicated that E ictaluri isolated from tra catfish in the MD was... 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... 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... 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