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Characterization and Antimicrobial Susceptibility of Vibrio Spp. Isolated From Different EnvironmentsINTRODUCTION The genus Vibrio comprises of Gram-negative straight or curved rods, motile by means of a single polar flagellum. Vibrios are capable of both respiratory and fermentative metabolism. O2 is a universal electron acceptor; they do not denitrify. Most species are oxidase-positive. In most ways vibrios are affiliated to enteric bacteria, but they portion a good deal of properties with pseudomonads a well. The Family Vibrionaceae is found in the "Facultatively Anaerobic Gram-negative Rods" in Bergey's Manual (1986), on the level with the Family Enterobacteriaceae. In the revisionist taxonomy of 2001 (Bergey's Manual), based on phylogenetic analysis, Vibrionaceae, Pseudomonadaceae and Enterobacteriaceae are all landed in the Gammaproteobacteria. Vibrios are discerned from enterics by being oxidase-positive and motile by means of polar flagella. Vibrios are discerned from pseudomonads by being fermentative as well as oxidative in their metabolism. Of the vibrios that are clinically substantial to humans, Vibrio cholerae,the agent of cholera, is the most important. Most vibrios have comparatively simple growth element necessaries and will grow in synthetic media with glucose as a sole source of carbon and energy. However, since vibrios are specifically marine organisms, most species require 2-3% NaCl or a sea water base for optimal growth. Vibrios vary in their nutritional versatility, but a good deal of species will grow on more than 150 dissimilar organic compounds as carbon and energy sources, occupying the same level of metabolic skillfulness as Pseudomonas. In liquid media vibrios are motile by polar flagella that are enclosed in a sheath neverending with the outer membrane of the cell wall. On solid media they may synthesize a great deal of lateral flagella which are not sheathed. Vibrios are one of the most mutual organisms in surface waters of the world. They take place in both marine and freshwater habitats and in associations with aquatic animals. Some species are bioluminescent and live in mutualistic associations with fish and other marine life. Other species are pathogenic for fish, eels, and frogs, as well as other vertebrates and invertebrates. V. cholerae and V. parahaemolyticus are pathogens of humans. Both construct diarrhea, but in ways that are altogether different. V. parahaemolyticus is an invasive organism affecting mainly the colon; Cholerae is noninvasive, affecting the little intestine through secretion of an enterotoxin. Vibrio vulnificus is an emergent pathogen of humans. This organism causes wound infections, gastroenteritis, or a syndrome known as "primary septicemia." METHODOLOGY EXPERIMENT: 1 Collection of Samples (Collins et al, 1973): Marine Samples were collected in dissimilar emplacements of Rameshwaram marine region (Palk bay) at the depth of 1 – 2 m at respective locations. Sewage and soil samples were accumulated in and around G.R.D. College campus. EXPERIMENT:2 2.1. Bacterial Enumeration (Mary et al, 1985): Number of culturable, aerobic, heterotrophic bacteria present in water and sediment samples was determined by plating on marine agar and nutrient agar. For marine isolates, the media were prepared by the 3.5% sodium chloride (NaCl). Then the plated were incubated at suitable time and temperature. EXPERIMENT: 3 3.1. Isolation of vibrio spp from water and sediment samples (Mary et al, 1985): Different selective media were applied for the isolation of vibrio sp from marine source. For marine isolates, the media were prepared by the 3.5% sodium chloride (NaCl). Media applied for the isolation of vibrio sp are 1. Thiosulfate Citrate Bile Sucrose Agar (with 3.5% NaCl) 2. Marine agar medium; EXPERIMENT: 4 4.1 Identification Microscopy: Gram-staining characteristics and cell morphologies were determined by general methods (Gerhardt et al., 1981). Motility was observed in wet mount using microscope. Cultural characteristics : Colony morphology of respective isolates of vibrio sp were observed on Nutrient agar, Thiosulfate Citrate Bile Sucrose Agar (TCBS), Blood agar, Mac conkey agar, Mannitol salt agar and results were tabulated. Physiological characterization: Preliminary physiological characterization such as catalase test, starch hydrolysis test, indole test, MRVP test, citrate test, oxidase test, string test, carbohydrate fermentation test (sugars used-sucrose, lactose, glucose, maltose, Mannitol) , cholera red reaction were carried out and results were tabulated. EXPERIMENT: 5 Anti microbial susceptibility test Kirby-Bauer method Procedure: 1. Make a suspension at an suitable turbidity of the bacterial culture to be tested. 2. Place a sterile cotton swab in the bacterial suspension and remove the excess liquid by pressing and rotating the cotton versus the inside of the tube above the liquid level. The swab is streaked in at least three directions over the surface of the Mueller-Hinton agar and TCBS agars obtain uniform growth. A final sweep is made around the rim of the agar. Be sure to streak for confluency. 3. Allow the plates to arid for five minutes. 4. Using sterile forceps, place disks containing the following antibiotics on the plate: penicillin G, ampicillin, cephalothin, erythromycin, tetracycline, methicillin, streptomycin or other suitable antibiotic disks. 5. Incubate the plates within 15 minutes after applying the disks. The plates ought to be incubated soon after placing the disks since the test is standardized underneath conditions where diffusion of the antibiotic and bacterial growth begin at approximately the same time. 6. Following during one night incubation, measure the diameter of the zone of growth inhibition around each disk to the nearest whole mm. Examine the plates cautiously for well-developed colonies within the zone of inhibition. 7. Using a usual table of antibiotic susceptibilities, determine if the strain is resistant, intermediate, or susceptible to the antibiotics tested. RESULT AND DISCUSSION Totally 35 samples were assembled in dissimilar emplacements of marine region, garden soil, sewage water and domestic water.Vibrio bacteria are gram-negative and for the most part halophilic. Vibrio is rod-shaped, and may be straight or curved. They are motile organisms, using a single polar flagellum to travel. Vibrios are one of the most mutual organisms in surface waters of the world. They take place in both marine and freshwater habitats and in associations with aquatic animals. Some species are bioluminescent and live in mutualistic associations with fish and other marine life. These samples were processed through the commonly used procedures such as selective media (listed below), Gram’s staining, wet mount observation for motility and bio chemical tests includes catalase test, starch hydrolysis test, indole test, MRVP test, citrate test, oxidase test, string test, carbohydrate fermentation test (sugars used-sucrose, lactose, glucose, maltose, Mannitol) , cholera red reaction were carried out and results were tabulated for identification of vibrio sp from the above samples, and that may be processed, the details of the description as shown COLONY MORPHOLOGY OF vibrio sp. MEDIA DETAILS Nutrient agar Moist, translucent, regular, disc shaped, 1-2mm in size, bluish tinge may be seen in transmitted light as distinguishable colour Mac Conkey agar Colorless colonies after, prolonged incubation pink colour colonies were seen. (plate:2) Blood agar Colonies were surrounded by a zone of hemolysis Thiosulphate citrate bile salt agar Colonies are yellow in colour due to fermentation. Mannitol salt agar No prominent growth was observed. The colony morphology of vibrio strains was varying for the duration of the isolation in the selective media. The colonies were very clear, moist, disc shaped, yellow and pink colonies were observed from respective sources. The mother culture was sub cultured in the same media for culture maintenance. All the detached vibrio strains were numbered for the easy identification and convenience. Gram staining reaction was recorded from heat fixed smears of vibrio culture. Motility and cell shape were determined by direct observation of wet mounts of fresh broth culture using microscopy. The chacteristics of Vibrio on TCBS medium shows that most of isolates were motile and capable of formulating yellow and green pigmentation. Thiosulphate-citrate-bile salts-sucrose (TCBS) agar (Difco) is a selective medium commonly applied to isolate members of the genus Vibrio from estuarine environments. The high concentration of thiosulphate and citrate and the strong alkalinity of the medium for the most part inhibit the growth of Enterbacteriaceas. Oxbile and cholate suppress necessary enterococci .Any coliform bacteria, which may grow, can not metabolize sucrose. Only a few sucrose Protease strains may grow to from yellow, vibriod like colonies. The mixed indicator thymol blue, bromothymol blue changes it is color to yellow, when acid is formed even in this strongly alkaline medium Out of 35 dissimilar samples collected, only 17 samples were showing the presence of vibrio sp, most of the marine samples collected from respective marine region shows positive results for vibrio sp, when equated to other samples such as sewage water and soil samples. And in case of domestic water sample (tap water and solid homogeneous inorgani substance water) shows absence of vibrio strain. Vibrios are inhabitants of aquatic environments. They once in a while contaminate humans, causing intestinal or extraintestinal diseases. The most prevalent diseases caused by vibrios are those that are well reported, including cholera and other forms of acute gastroenteritis. In addition, there may be a heap of other vibrio-caused diseases that go unreported a great deal of of the virulence determinants of Vibrio spp. are well characterized. ENUMERATION OF BACTERIAL POPULATION The total bacterial population were observed and enumerated on the marine agar plates and nutrient agar plates. The vibrio colonies were isolated and enumerated in a TCBS medium. Both populations were counted and shown Showing the total bacterial population and vibrio count. S.NO LOCATION Total bacterial Population Vibrio Population 1 Marine region 108 × 105 65 × 102 2 Garden soil 142 × 106 21 × 102 3 Sewage water 161 × 106 42 × 102 4 Tap water TFTC 0 5 Mineral water TFTC 0 Physiological characteristics of vibrio sp BIO CHEMICAL TEST NO OF POSITIVES NO OF NEGATIVES OXIDASE TEST 16 1 NITRATE TEST 16 1 CATALASE TEST 8 9 INDOLE TEST 17 0 METHYL RED TEST 17 0 VOGES PROSKEUAR 5 12 STRING TEST 17 0 CHOLERA RED RXN 4 13 CITRATE TEST 7 10 STARCH HYDROLYSIS 0 17 HEAMOLYSIS 5 12 SUCROSE 15 2 MANNITOL, ACID 16 1 LACTOSE 0 17 GLUCOSE, ACID 17 0 GLUCOSE, GAS 1 16 MALTOSE 16 1 By observing the results of physiological tests, most of the tests were found to be positive for vibrio cholerae.The isolates which showed positive results for vibrio cholerae were principally isolated from marine surroundings when equated to other samples such as sewage water and soil samples. Through this result we observed that Vibrios are inhabitants of aquatic environments. Marine animals may injure humans in various ways. Some animals cause injury by inducing infection. These contagions result when oral bacteria are introduced in to the tissues of victims who are bitten. Bacteria present within the tissues of marine animals may cause infection when they are ingested. In addition, seawater itself holds bacteria, so that skin and soft tissue injuries exposed to seawater may become secondarily injected. Kirby-Bauer Disk-Diffusion Method: Antibiotic Disk Susceptibilities Most of the isolates from marine source were highly incidence of antibiotic resistance was apparent versus Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rifampin and Streptomycin over soil and sewage samples. However, antibiotic resistance was lower versus Chloramphenicol, Tetracycline, penicillin G, Nalidixicacid, Gentamycin Sulphafurazole, Trimethoprim, Neomycin and Amikacin. This may be due to the fact that terrestrial bacteria entering into seawater with antibiotic immune plasmids may be responsible for the prevalence of the resistance in genes in the marine environment. However, there are few reports available on acquired antibiotic resistance versus ampicillin (68%), cefuroxime (66.7%), amikacin (55%), kanamycin (58.8%) and trimethoprim (76.7%) in Sparus sarba in China. It may be presumed that anthropogenic constituents (hospital effluents) might have influenced in acquiring resistance in Vibrio spp due to these antibiotics, as there are no reports available on the use of these drugs for aquaculture in India. However, the results of our present study serve as a baseline info for future exploration on the extent of antibiotic resistance, which may be revealed through isolation of plasmids, their transformation efficacy and conjugation experiments. Results of incidence of multiple antibiotic resistance in Vibrios may improve our cognition on drug immune strains and it is effect on future therapy of shrimp as well as humane diseases. Therefore, unscrupulous use of antibiotics versus diseases will have to be obviated and limitations for the use of antibiotics may be enforced by a nationwide antibiotic policy for India. REFERENCES 1. Richard J., and Bennett N T.1993.Infections Caused by Halophilic Marine Vibrio Bacteria. From the Department of Surgery, University of Florida, Gainesville, Florida Vol. 217, No. 5, 525-531 2. Colwell, R. R., and W. M. Spira. 1992. The ecology of V. cholerae, p. 107–127. In D. Barua and W. B. Greenough III (ed.), Cholera. Plenum Medical Book Co., New York, N.Y. 3. West, P. A., and J. V. Lee. 1982. Ecology of Vibrio spp. including V. cholerae in natural waters of Kent, England. J. Appl. Bacteriol. 52:435–448. 4. Xu, H. S., N. C. Roberts, F. L. Singleton, R. W. Attwell, D. J. Grimes, and R. R. Colwell. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8:313–323. 5. Baumann, P., and R. H. W. Schubert. 1984. Family II. Vibrionaceae, p. 516–550. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic Bacteriology, vol. 1. Williams & Wilkins, Baltimore, Md. 6. Farmer, J. J., III, F. W. Hickman-Brenner, and M. T. Kelly. 1985. Vibrio, p. 282–301. In E. H. Lennette, A. Balows, W. J. Hausler, and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 7. Kay, B. A., C. A. Bopp, and J. G. Wells. 1994. Isolation and identification of Vibrio cholerae O1 from fecal specimens, p. 3–20. In I. K. Wachsmuth, P. A. Blake, and O. Olsvik (ed.), Vibrio cholerae and cholera: molecular to global perspectives. ASM Press, Washington, D.C. 8. West, P. A., and R. R. Colwell. 1983. Identification and classification of Vibrionaceae–-an overview, p. 285–341. In R. R. Colwell and M. B. Hatem (ed.), Vibrios in the environment. John Wiley & Sons, New York, N.Y. 9. Chun, J., A. Huq, and R. R. Colwell. 1999. Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl. Environ. Microbiol. 65:2202–2208 10. Nandi, B., R. K. Nandy, S. Mukhopadhyay, G. B. Nair, T. Shimada, and A. C. Ghose. 2000. Rapid method for species-specific identification of Vibrio cholerae using primers aimed to the gene of outer membrane protein W. J. Clin. Microbiol. 38:4145–4151. 11.Gabriella caldini, Angela neri, Stefania cresti, Vieri boddi, Gian maria rossolini, and Eudes lanciotti high prevalence of vibrio cholerae non-o1 carrying heat-stable enterotoxin-encoding genes amid vibrio isolates from a temperate-climate river basin of central Italy. Applied and environmental microbiology July 1997, Vol. 63, No. 7 p. 2934–2939 12. Abbott, S.L., Cheung, W.W.K.W. and Janda, J.M. (1993) Evaluation of a new selective agar, thiosulfate-chloride-iodide (TCI), for the growth of pathogenic Vibrio species. Medical Microbiology Letters 2, 362–370. 13 .Morris, G.K. (1982) Media for Vibrio species. In Quality Assurance and Quality Control of Microbiological Culture Media ed. Corry, J.E.L. and Verlay, G.I.T. pp. 169–174. Germany: Darmstadt. 14. Oliver, J.D. (2003) Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples. In Culture Media for Food Microbiology, 2nd Edn eds Corry, J.E., Curtis, G.D.W. and Baird, R.M. Amsterdam: Elsevier Science, in press. 15. Ferguson, G. E., C. W. Lingham, S. K. Love, and R. 0. Vernon. 1947. Springs of Florida, p. 196. Fla. Geol. Bull. no. 31. E. 0. Printing Co., DeLand, Fla. 16. Lee, J. S. 1973. What seafood processors will have to recognise when it comes to Vibrio parahemolyticus. J. Milk Food Technol. 36:405-408. 17.Gopal, S, SK Otta, I Karunasagar, M Nishibuchi, and I Karunasagar. "The occurrence of Vibrio species in tropical shrimp culture environments; significations for feed safety." International Journal of Food Microbiology. 2005 Jul 15;102(2):151-9. 18.Graf, J. "The Light-Organ Symbiosis of Vibrio fischeri and the Hawaiian Squid, Euprymna scolopes." February 2005. Accessed 8 July 2005. 19.Ho, Hoi. "Vibrio infections." eMedicine. 22 May 2005. Accessed 8 July 2005. 20.Oliver, JD. "Wound contagions caused by Vibrio vulnificus and other marine bacteria." Epidemiology and infection. 2005 Jun;133(3):383-91. 21.Sarkar, M, S Das, A Bandyopadhaya, K Ray, and K Chaudhuri. "Upregulation of humane mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis." FEBS letters. 2005 Jun 20;579(16):3449-60. 22.Senoh, Mistutoshi, Shin-Ichi Miyoshi, Keinosuke Okamoto, Belen Fouz, Carmen Amaro, and Sumio Shinoda. "The Cytotoxin-Hemolysin Genes of Human and Eel Pathogenic Vibrio vulificus Strains: Comparison of Nucelotide Sequences and Application to the Genetic Grouping. Microbiology and immunology. 2005;49(6):513-9. 23.Gerhard,P.,R.G.E.Murray,R.N.Costilow,E.W.Nester,W.A.Wood,N.R.Krieg,andG.B.phillips.1981.Manual of method for generalbacteriology.American SOCIETY FOR microbiology, Washington, D.C. 24. Mary. L. G and rita r. colwell, Enumeration, isolation and characterization of N2 fixing bacteria from sea water. Department of microbiology, University of Maryland. Vol 50 no .2. 1985 Other References 1.Dennis Kunkel Microscopy, Inc. 2.Fix, Douglas F. "Vibrio." Accessed 8 July 2005. 3.Todar, Kenneth. Todar's Online Textbook of Bacteriology. 2005. Accessed 8 July 2005. 4.Vibrio fischeri Genome Project. 19 September 2002. Accessed 8 July 2005. 5.http://microbewiki.kenyon.edu/index.php/Vibrio 6.Center for Disease Control. 1973. Vibrio parahaemolyticus gastroenteritis. Morbid. Mortal. Weekly Rep. 22:231-232.
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