Available online at www.sciencedirect.com
ScienceDirect Procedia Chemistry 18 (2016) 12 – 17
Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and Structural Biology 2015 Conference, MCLS 2015
Extracellular Enzymes Produced by Vibrio alginolyticus Isolated from Environments and Diseased Aquatic Animals Supansa Bunpaa, Natthawan Sermwittayawonga*, Varaporn Vuddhakula a
Food safety and health research unit, Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand
Abstract A total of 17 Vibrio alginolyticus isolates were obtained from environments, diseased fish and shrimp using CHROMagar Vibrio and confirmed by biochemical tests and PCR targeted to the gyrB gene. They were investigated for production of exoenzymes. All of the isolates from diseased fish and shrimp (n = 8) showed gelatinase, lecithinase, and caseinase activities, while 75% (6/8) of them possessed amylase and lipase activities. In environmental isolates (n = 9), the gelatinase and lecithinase activities were detected in all isolates. Four out of nine isolates (44%) possessed lipase, while 67% (6/9) of environmental isolates were positive for both caseinase and amylase activities. Interestingly, Į-hemolysin activity was detected in all V. alginolyticus isolates from diseased fish and shrimp but it was detected in only 44% (4/9) of the environmental isolates, suggesting that they might be involved in bacterial pathogenesis. The arbitrarily primed polymerase chain reaction (AP-PCR) technique showed distinct DNA profiles of all isolates consisting of 7 to 11 bands ranging from 0.3 – 6.0 Kb (with a common band of 1.2 Kb). Fourteen DNA profiles were obtained from a dendrogram analysis with 20% maximum similarity. These results indicate genetically heterogenicity among V. alginolyticus. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license © 2015 The Authors. Published by Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-reviewunder under responsibility of organizing the organizing committee of the Molecular and Life Cellular Life Infectious Sciences: Diseases, Infectious Diseases, Peer-review responsibility of the committee of the Molecular and Cellular Sciences: Biochemistry and Structural Biology 2015 (MCLS 2015). Biochemistry and Structural Biology 2015 (MCLS 2015) Keywords: Vibrio alginolyticus; exoenzymes; AP-PCR
* Corresponding author. Tel.: +66 841 725 271; fax: +66 744 46 661. E-mail address:
[email protected]
1876-6196 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and Structural Biology 2015 (MCLS 2015) doi:10.1016/j.proche.2016.01.002
Supansa Bunpa et al. / Procedia Chemistry 18 (2016) 12 – 17
Nomenclature AP-PCR ECPs PCR ng ȝl rpm
Arbitrarily primed polymerase chain reaction Extracellular products Polymerase chain reaction nanogram microliter revolutions per minute
1. Introduction Vibrio alginolyticus is a member of the family Vibrionaceae, which are Gram negative bacteria with a curved rod shape, that are motile, non-spore-forming and that grow in 10% NaCl. It can be isolated from diseased marinecultured animals with clinical symptoms of bacterial septicaemia and skin ulcer1. This bacterium is also an important opportunistic bacterial pathogen associated with seafood-borne infections in humans and is a normal inhabitant of estuarine and marine environments2. The pathogenesis for marine animals and humans following infection has been associated with only the virulent strains of V. alginolyticus. However, the avirulent strains do not infect and do not cause disease. Some genes may determine strain-specific characteristics such as virulence factors3. Extracellular products (EPSs) such as chitinases, hemolysins, alkaline proteases, cysteine proteases, alkaline metalchelator-sensitive proteases, serine proteases and metalloproteases have been isolated from cell-free culture supernatants (CFS) of V. harveyi, V. anguillarum, V. alginolyticus and other species. These ECP have been proposed as virulence factors for fish and other marine organisms4. However, the structural and functional characteristics of the other genes of V. alginolyticus that encode exotoxins associated with fish and shrimp diseases are poorly known and represented in the DNA and Amino Acid International databases5. Characterization of pathogenic V. alginolyticus strains and their toxic ECP is a prerequisite for better understanding of pathogenesis, mechanisms of infection and control. Arbitrarily primed polymerase chain reaction (AP-PCR) also referred to as the random amplified polymorphic DNA is a PCR-based technique for typing bacterial genomic DNA. This technique was first developed by John Welsh in 19906. The technique is used for molecular epidemiological analysis because it is easy and fast, and it is also used for identification of strain-specific variations in DNA or specific fingerprints of bacteria7. AP-PCR is a PCR-based method that uses a short single primer (usually 10 bp) to amplify anonymous stretches of DNA. With this technique, there is no specific target DNA, so each particular primer will randomly anneal to the template DNA8. The DNA fragments generated are then separated and detected by gel electrophoresis. In this study, 17 V. alginolyticus strains associated with diseased fish and shrimp in aquaculture farms and environments were isolated using CHROMagar Vibrio and confirmed by biochemical tests and PCR targeted to the gyrB gene. These strains were investigated for exoenzyme production and their genetic diversity was evaluated by AP-PCR. 2. Methods 2.1 Isolation of V. alginolyticus For environmental isolates, six samples of water and three samples of sediments were collected from a fish aquaculture area at Khong-Jilhad, Krabi province, Thailand. The samples were enriched in alkaline peptone water (APW) and incubated at 37°C for 6-8 h. A loopful of culture broth was spread on CHROMagar Vibrio and incubated at 37°C for 18-24 h. After incubation, the presumptive milky white colonies on CV agar were randomly selected and then subcultured on thiosulphate-citrate-bile salt-sucrose agar (TCBS) and incubated overnight at 30qC. To isolate the bacteria from diseased fish, a group of seven cultured juvenile tiger groupers Epinephelus fuscoguttatus (with clinical signs of vibriosis including darkened body colour, white nodular skin lesions, haemorrhagic visceral organs, and usually pale gills and sudden death) were collected from a fish aquaculture area at
13
14
Supansa Bunpa et al. / Procedia Chemistry 18 (2016) 12 – 17
Khong-Jilhad, Krabi province, Thailand. Bacteria were isolated from white nodular skin lesions and from the internal organs (brain, spleen, liver and kidney). All samples were streaked onto TCBS and incubated at 30°C for 18-24 h. After incubation, yellow colonies were randomly selected, then subcultured on CHROMagar Vibrio, and incubated under the same condition. V. alginolyticus isolate 8 from diseased shrimp was obtained from the culture collection of the coastal aquatic animal health and research institute, Songkhla province, Thailand. All V. alginolyticus isolates were stored at -80°C in Luria-Bertani (LB) broth supplemented with 1% NaCl and 20 % glycerol. 2.2 Biochemical test All isolates were preliminarily identified by the following biochemical tests: motility, oxidase, growth and colony color on TCBS and CHROMagar Vibrio, production of arginine dihydrolase, lysine and ornithine decarboxylase, glucose fermentation, indole, MR-VP, and growth at different salinities (0, 3, 6, 8 and 10%). 2.3 Molecular identification of V. alginolyticus The tested isolates were grown in LB broth containing 1% NaCl and incubated overnight at 30°C with shaking at 150 rpm. One ml of broth culture was boiled for 10 minutes and the tube was immediately placed on ice for 10 minutes. The supernatant was obtained by centrifugation at 14,000 rpm for 5 minutes. Then it was 10-fold diluted in distilled water and used as DNA template for PCR. To investigate the gyrB gene, PCR was performed using primers 1F and 4R to detect a 747-bp amplicon. Briefly, 20 μL of reaction mixture consisted of 1X Pfu polymerase buffer, 0.2 mM deoxyribonucleotide triphosphate, 0.25 μM of each primer, 1 unit of Pfu DNA polymerase (a gift from Dr. Decha Sermwittayawong), and 2 μl of DNA template. The PCR amplification condition consisted of 94°C for 4 minutes followed by 25 cycles of denaturation at 94°C for 45 sec, annealing at 60°C for 45 sec, extension at 72°C for 1 minute, and a final extension at 72°C for 4 minutes. Amplification was conducted in a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The PCR products were then electrophoresed on 1% agarose gel to detect the amplicon of the expected size. The gel was stained with ethidium bromide for 5 minutes and destained with distilled water for 20 minutes. DNA fragments were visualized using a UV transilluminator (Synegene, Model Gene Genius). 2.4 Production of extracellular products (ECPs) The glycerol stock was re-streaked on TSA supplemented with 1% NaCl and incubated at 37°C for 18-24 h. A single colony of each isolate was picked and tested for lipase, haemolysin and gelatinase activities on TSA media with 1% NaCl containing 1% (v/v) Tween 80, 5% Sheep red blood cell and 12% gelatin, respectively. Amylase, caseinase and lecithinase activities were detected on TSA supplemented with 1% NaCl containing 0.5 % (v/v) starch, 1% (v/v) skim milk, and 1% (v/v) egg yolk emulsion9 , respectively. Agar plates were then incubated at 30 °C for 2 days. The presence of opalescence around the bacterial colonies were recorded as positive10. 2.5 Arbitrarily primed polymerase chain reaction (AP-PCR) All isolated V. alginolyticus strains were grown on TSA supplemented with 1% NaCl at 37°C for 24 h, and were then transferred to 3 ml of TSB supplemented with 1% NaCl and incubated overnight at 37°C with shaking at 150 rpm. Bacterial cells were harvested by centrifugation and genomic DNA was extracted with the Presto Mini genomic DNA Bacteria Kit (Geneaid). DNA was diluted to 10 ȝg/ȝl and used as the DNA template. AP-PCR was performed using primer 4 (5ƍ-AAGAGCCCGT-3ƍ)11. The 30 ȝl of PCR mixture consisted of 2.5 ȝl of DNA template (10 ȝg/ȝl), 1X Ex taq buffer (20 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Nonidet P-40 and 50% glycerol), 0.33 mM dNTPs, 0.83 ȝM primer and 2.5 U Ex Taq DNA polymerase (TaKaRa, Japan). The PCR was performed in a T100 Thermal Cycler (Bio-Rad). The thermo cycle was started with a cycle at 95°C for 4 minutes, followed by 45 cycles of denaturation at 95°C for 1 minute, annealing at 36°C for 1 minute, extension at 72°C for 2 minutes, and finally, an additional 72°C for 7 minutes. The amplicons were detected by 1.5% agarose gel electrophoresis. After completion of the electrophoresis, the gel was stained with ethidium bromide for 5 minutes, destained in distilled water for 1 h, and photographed under an UV transilluminator (Synegene, Model Gene Genius). A dendrogram was constructed using a Bioprofile image analysis system (Viber Lourmat).
15
Supansa Bunpa et al. / Procedia Chemistry 18 (2016) 12 – 17
3. Results and discussion Seventeen isolates of V. alginolyticus were obtained from the environment, diseased fish and shrimp. All isolates showed biochemical characteristics of V. alginolyticus with positive results on the oxidase test, Voges-Proskauer test, sucrose fermentation on TCBS agar and growth in the presence of 10% NaCl. These isolates then were positive for PCR targeted to the gyrB gene (data not shown). The enzymatic activities of V. alginolyticus are shown in Table 1. All of the isolates from diseased fish and shrimp (n = 8) showed gelatinase, lecithinase, and caseinase activities, while amylase and lipase activities were found in 75% (6/8). As for the environmental isolates (n = 9), the gelatinase and lecithinase activities were detected in all isolates. Four out of nine isolates (44%) possessed lipase activity, while both caseinase and amylase activities were found in 67% (6/9) of the environmental isolates. These results were the same as found by Balebona and his team12. These activities might allow Vibrio species to adhere to the epithelial cells of the host, to evade the first barrier of natural defence and to colonise all internal organs. To investigate any haemolytic activity, the tested isolates were inoculated onto 5% sheep red blood cell agar. In this study, the Į-hemolysin activity was detected in all (8/8) V. alginolyticus strains isolated from diseased fish and shrimp and in only 44% (4/9) of those from environmental isolates indicating that Į-hemolysin may be involved in bacterial pathogenesis. The DNA patterns obtained from AP-PCR using primer 4 showed distinct DNA profiles of all isolates, regardless to the source of the samples (Fig. 1). In this study, a high level of reproducible DNA patterns was observed. All isolates showed reproducible patterns consisting of 7 to 11 bands ranging from 0.3 – 6.0 Kb (with a common band of 1.2 Kb). Furthermore, a dendrogram analysis revealed 14 DNA fingerprint patterns: seven from diseased fish and shrimp, two from sediments and five from seawater, placing these isolates in different clusters with 20% maximum similarity (Fig. 2). Each cluster contains one or two strains. Moreover, most V. alginolyticus strains originating from seawater were grouped into the same cluster. In this study we found that V. alginolyticus strains isolated from the same origin (diseased fish) were different as they were grouped in different clusters. These results were the same as obtained by other researchers who found that genetic variation can occur even within V. alginolyticus strains isolated from diseased fish Sparus aurata and there was a high level of genetic diversity among strains irrespective of their source of recovery13. Table 1. Exoenzyme production of the 17 Vibrio alginolyticus strains identified from diseased fish and shrimp, sediments and seawater Name of isolates 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Total Notes:
Origin
Hemolysis On BA+ 5% Sheep RBC*
Gelatinase (12% gelatin)
Lecithinase (1% egg yolk )
Caseinase (1% skim milk)
Amylase (0.5% starch )
Lipase (1% tributilin)
Diseased fish
Į Į Į Į Į Į Į
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + +
+ + + + +
Į
+
+
+
+
+
Sediments
Ȗ Ȗ Į
+ + +
+ + +
+ +
+ +
+ + -
Seawater
Ȗ Ȗ Į Ȗ Į Į
+ + + + + +
+ + + + + +
+ + + +
+ + + + -
+ +
17/17
17/17
14/17
12/17
10/17
Diseased shrimp
(*) Į = Į – haemolytic ; Ȗ = Ȗ-haemolytic
16
Supansa Bunpa et al. / Procedia Chemistry 18 (2016) 12 – 17
Fig. 1. DNA fingerprints of V. alginolyticus generated by AP-PCR using primer 4. Lane. M: 1 Kb ladder (New England BioLabs). Number of isolates: Lanes 1-7: V. alginolyticus isolates 1-7 from diseased fish, Lane 8: V. alginolyticus isolate 8 from diseased shrimp, Lanes 9-11: V. alginolyticus isolates 9-11 from sediments and Lanes 12-17: V. alginolyticus isolates 12-17 from seawater.
Fig. 2. Dendrogram based on the DNA fingerprint patterns of V. alginolyticus isolates obtained from AP-PCR using primer 4. Dendrogram was constructed using a Bioprofile image analysis system (Vilber Lourmat, France). Numbers on the horizontal axis indicate the percentage of similarity.
Supansa Bunpa et al. / Procedia Chemistry 18 (2016) 12 – 17
4. Conclusion In this study we found that all V. alginolyticus strains isolated from the environment and diseased fish produced several extracellular enzymes and the results of AP-PCR patterns also demonstrated that these isolates are genetically heterogeneous. However, further research on protein expression of this species may clarify their role in bacterial pathogenesis. Acknowledgements This research work was supported by Department of Microbiology, Faculty of Science, Prince of Songkla Universityand the Fiscal fund year 2014 (grant no. SCI560062S). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13.
Zuo F, Jian J, Wu Z. Characterization of extracellular proteases from Vibrio alginolyticus isolated from maricultured fish. Acta Hydrobiologica Sinica. 2006;30:553-8. George M, John K, Iyappan T, Jeyaseelan M. Genetic heterogeneity among Vibrio alginolyticus isolated from shrimp farms by PCR fingerprinting. Letters in applied microbiology. 2005;40:369-72. Cai SH, Lu YS, Wu ZH, Jian JC, Huang YC. A novel multiplex PCR method for detecting virulent strains of Vibrio alginolyticus. Aquaculture Research. 2009;41:27-34. Lee K, Yu S, Yang T, Liu P, Chen F. Isolation and characterization of Vibrio alginolyticus isolated from diseased kuruma prawn, Penaeus japonicus. Letters in applied microbiology. 1996;22:111-4. Aguirre-Guzmán G, Mejia Ruíz H, Ascencio F. A review of extracellular virulence product of Vibrio species important in diseases of cultivated shrimp. Aquaculture Research. 2004;35:1395-404. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic acids research.1990;18:7213-8. Martinez I, Espelid S, Johansen A, Welsh J, McClelland M. Fast identification of species and strains of Vibrio by amplification of polymorphic DNA. Journal of fish diseases. 1994;17:297-302. Fritsch P, Rieseberg LH. The use of random amplified polymorphic DNA (RAPD) in conservation genetics. Molecular genetic approaches in conservation. 1996;1996:54-73. Hörmansdorfer S, Wentges H, Neugebaur-Büchler K, Bauer J. Isolation of Vibrio alginolyticus from seawater aquaria. International journal of hygiene and environmental health. 2000;203:169-75. Zanetti S, Spanu T, Deriu A, Romano L, Sechi LA, Fadda G. In vitro susceptibility of Vibrio spp. isolated from the environment. International journal of antimicrobial agents. 2001;17:407-9. Okuda J, Ishibashi M, Abbott SL, Janda JM, Nishibuchi M. Analysis of the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes in urease-positive strains of Vibrio parahaemolyticus isolated on the West Coast of the United States. Journal of clinical microbiology. 1997;35:1965-71. Balebona MC, Andreu MJ, Bordas MA, Zorrilla I, Moriñigo MA, Borrego JJ. Pathogenicity of Vibrio alginolyticus for Cultured Gilt-Head Sea Bream (Sparus aurataL.). Applied and environmental microbiology. 1998;64:4269-75. Snoussi M, Hajlaoui H, Noumi E, Zanetti S, Bakhrouf A. Phenotypic and genetic diversity of Vibrio alginolyticus strains recovered from juveniles and olderSparus aurata reared in a Tunisian marine farm. Annals of Microbiology. 2008;58:141-6.
17
