ORIGINAL ARTICLE. B. Vaseeharan 1, P. Ramasamy 2 and J.C. Chen 3. Abstract

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Antibacterial activity of silver nanoparticles (AgNps) synthesized by tea leaf extracts against pathogenic Vibrio harveyi and its protective efficacy on juvenile Feneropenaeus indicus B. Vaseeharan 1, P. Ramasamy 2 and J.C. Chen 3 1 Department of Animal Health and Management, Alagappa University, Karaikudi, Tamil Nadu, India 2 Alagappa University, Karaikudi, Tamil Nadu, India 3 Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan, Republic of China Keywords antibacterial, Feneropenaeus indicus, silver nanoparticles, tea leaf extract, V. harveyi. Correspondence Baskaralingam Vaseeharan, Department of Animal Health and Management, Alagappa University, Karaikudi 63 3, Tamil Nadu, India. vaseeharanb@gmail.com : received 16 November 29, revised 21 December 29 and accepted 23 December 29 doi:1.1111/j x x Abstract Aims: To determine the antibacterial potential of silver nanoparticles (AgNps) synthesized by tea leaf extract against Vibrio harveyi and its protective effect on juvenile Feneropenaeus indicus. Methods and Results: AgNps were synthesized by a simple procedure using tea leaf extract as the reducing agent. Bacteriological tests were performed in Luria Bertani medium on solid agar plates and in liquid systems supplemented with V. harveyi against different concentrations of AgNps. AgNps synthesized in the present study were shown to be effective against V. harveyi isolated from F. indicus. The combined results of long- and short-term treatment of AgNps synthesized by tea leaf extract showed a 71% reduction in accumulated mortality. Conclusions: The long-term administration of AgNps synthesized by tea leaf extracts at the concentration of 1 lg significantly reduced the mortalities in F. indicus from V. harveyi infections. Significance and Impact of the Study: The AgNps synthesized by tea leaf extract may be an alternative to antibiotics in controlling V. harveyi infections. Introduction Shrimp aquaculture has witnessed tremendous growth worldwide because of an increasing demand. Vibrio species has been recognized as primary and opportunistic pathogen and one of the most virulent bacterial causative agents for the large-scale mortality in all stages of cultured penaeid shrimps (Vaseeharan and Ramasamy 23; Austin and Austin 27). Improper administration of antibiotics against controlling Vibrio outbreaks could result in the development of resistant bacteria strains, and there is much interest in finding ways to formulate new types of safe and cost-effective biocidal materials. In aquaculture, various approaches undertaken have already shown to control the pathogenic Vibrio strains, including chemicals, antibiotics, probiotics, plant-based products and plant oils. Recently, silver or its compounds have been recognized for their broad-spectrum of antimicrobial activities. The antimicrobial properties of silver nanoparticles (AgNps) are well established, and several mechanisms for their bactericidal effects have been proposed (Sondi and Sondi 24). However, the antimicrobial effects of AgNps have not been fully investigated. Alexander et al. (28) demonstrated that highly reactive metal oxide nanoparticles exhibit excellent biocidal action against Gram positive and Gram negative. However, no reports are available for the control of Vibrio spp. isolated from aquaculture environments using plantmediated AgNps. Use of biological organisms such as micro-organisms, plant extract or plant biomass could be an alternative to chemical and physical methods for the production of nanoparticles in an eco-friendly manner 352 Journal compilation ª 21 The Society for Applied Microbiology, Letters in Applied Microbiology 5 (21)

2 B. Vaseeharan et al. Silver nanoparticles effect against Vibrio harveyi (Mohanpuri et al. 28). Accordingly, the present study focused to determine the antimicrobial activity of AgNps synthesized by tea leaf extract against the pathogenic Vibrio harveyi and its protective effects on Feneropenaeus indicus obtained in shrimp culture environments. Materials and methods Bacterial strains The pathogenic V. harveyi (LD 5 value of 1 5 CFU ml )1 in experimental infection of F. indicus juveniles) were isolated from the hepatopancreas of black gill disease infected F. indicus by the use of V. harveyi agar (Harris et al. 1996) and identified further using standard biochemical methods (Holt et al. 1994) and confirmed by PCR and 16S rrna typing methods (Oakey et al. 23). Synthesis of AgNps with tea leaf extracts Silver nitrate (AgNO 3 ), purchased from Sigma Aldrich company product no (St Louis, MO, USA), was used for the synthesis of AgNps. The tea leaf extracts were prepared from tea leaves (Camellia sinensis) collected from wild (Ooty, Tamil Nadu, India) and shadow-dried in room temperature. The method of preparation followed the standard protocols described in Begum et al. (29). For the tea leaf broth, 18 g of dried tea leaves was boiled in 4 ml of water in a clean conical flask. The resulting infusion was then filtered repeatedly until no insoluble material appeared. Nanoparticles were synthesized by adding aqueous solutions of AgNO 3 to tea leaf broth extract. In a typical synthesis, 75 ml of Æ1 mol l )1 AgNO 3 was added to 1 ml of tea leaf extract with continuous stirring, at 4 C. Within 3 min, a yellow colouration appeared, indicating the onset of AgNps formation. Synthesized AgNps were confirmed using contact mode using Atomic Force Microscope (Solver P47H; NT-MDT. CO. Moscow, Russia) long silicon nitride probes (1 lm). Bactericidal test To examine the bactericidal effect of AgNps on the selected V. harveyi, approximately 1 5 colony forming units (CFU) of V. harveyi (counted by spread plate method) were cultured on Luria Bertani (LB) agar plates supplemented with AgNps in concentrations of 5 35 lg cm )3. Silver-free LB plates cultured under the same conditions were used as a control. The plates were incubated for 24 h at 37 C, and the numbers of colonies were counted in triplicate. Determination of CFU on the inhibitory effects of AgNps Cultures of V. harveyi at seven different colony forming units (1, 1Æ5, 2, 2Æ5, 3, 3Æ5 and CFU prepared by diluting the stock culture containing CFU V. harveyi) were inoculated in LB agar plates seeded with 3 lg cm )3 AgNps and incubated for 12 h at 37 C. The number of colony forming units was counted and recorded. Growth of Vibrio harveyi against AgNps To observe the growth rate of V. harveyi and to determine the growth curve in the presence of AgNps, Vibrio harveyi bacteria were grown in 1 cm )3 of liquid LB medium supplemented with 5, 1, 15, 2 and 3 lg of AgNps cm )3 of medium. Growth rates and bacterial concentrations were determined by measuring optical density (OD) at 58 nm each 3 min (OD of Æ1 corresponds to a concentration of 1 7 cells cm )3 ). Experimental infections of Feneropenaeus indicus with Vibrio harveyi and AgNps treatment Disease-free healthy F. indicus (with the body weights of 9 11 g) produced from specific pathogen-free brooders were obtained in a commercial farm (Chidambaram, Tamil Nadu, India). The shrimps were maintained in the laboratory conditions for 15 days to ensure the health status and fed with commercial pellet feed twice daily for 2 weeks (Tairoun Feed Company, Taipei, Taiwan). To evaluate the effects of AgNps on the experimental infection of F. indicus with V. harveyi, three diets of standard shrimp meal (shrimp meal, vitamin mix, mineral mix, dextrin, wheat flour, a-starch, cellulose, cod liver oil corn oil and energy kcal 1 g) were prepared containing (control), 5 and 1 lg AgNps, respectively. The ingredients were mixed thoroughly; an adequate quantity of water (3% for 1 g of mixed ingredients) and oil (cod liver oil and corn oil in the ratio 2 : 1) was added. The resulting dough was passed through an extruder and dried at 35 C for 8 h. The dried diet was packaged into a plastic bag and stored frozen at )2 C until use. V. harveyi was grown for 24 h in tryptone soy broth supplemented with 2% NaCl. Two hundred F. indicus, each approximately 5 6 g, were divided equally into eight groups, each housed in a 25-l tank. Four of the 25-l tanks were fed with the diet containing 1 lg of AgNps for 5 days after which the shrimp were fed with normal feed without AgNps (long-term treatment). On the 5th day, the F. indicus in all of the four tanks were infected with V. harveyi ( cfu ml) for 1 h. Following this, Journal compilation ª 21 The Society for Applied Microbiology, Letters in Applied Microbiology 5 (21)

3 Silver nanoparticles effect against Vibrio harveyi B. Vaseeharan et al. the shrimp in two of the tanks were again fed with the diet containing 1 lg of AgNps (combined treatment). Feneropenaeus indicus in two of the remaining four tanks were fed with the diet containing 1 lg of AgNps for 2 days, and the animals were then transferred to a separate tank, in which V. harveyi ( cfu ml) was maintained for 1 h (short-term treatment); the remaining two tanks were infected with V. harveyi ( cfu ml) alone as a positive control. The same experiment was repeated using F. indicus fed with the diet containing 5 lg of AgNps. The cumulative mortality of the F. indicus fed with the diet containing 5 lg and 1 lg of AgNps was recorded and analysed using analysis of variance (anova). Number of V harveyi colonies CFU applied in experiment 1 6 Results Antibacterial activity of AgNps against Vibrio harveyi The number of V. harveyi colonies grown on LB plates as a function of the concentration of AgNps when approximately 1 6 CFU were applied to the plates is shown in Fig. 1. The presence of these particles at a concentration of 1 lg cm )3 inhibited bacterial growth by 7%. The size of bacterial colonies grown on plates with more than 2 lg cm )3 of nanoparticles was significantly reduced (Fig. 2). Number of V. harveyi colonies as a function of the concentration of AgNps in LB agar plates expressed as a percentage of the number of colonies grown on silver-free control plates. Figure 2 Number of Vibrio harveyi colonies grown up on Luria Bertani agar plates containing 3 lg cm )3 of silver nanoparticles as a role of the number of colony forming units (CFU) applied in the experiments. Growth of Vibrio harveyi at different concentrations of AgNps Figure 3 illustrates the comparative growth performance of V. harveyi at different experimental concentrations of AgNps supplemented in liquid LB broth. Higher concentration of AgNps (35 lg 1 ml) arrested the growth of V. harveyi, and no growth was observed in terms of increment in OD of broth. It was observed that the arrest in growth of V. harveyi present in media was directly proportional to the concentration of AgNps. Number of Vibrio harveyi CFU (%) µg 1 µg 2 µg 25 µg 3 µg 35 µg Concentration of silver nanoparticles (µg/ml) Figure 1 Number of Vibrio harveyi colonies as a role of the concentration of silver nanoparticles in Luria Bertani agar plates expressed as a percentage of the number of CFU grown on silver-free control plates. Experimental infections of Feneropenaeus indicus with Vibrio harveyi and the treatment of AgNps The comparative mortality rates (in percentage) of F. indicus juveniles in the six experimental groups (treated O D value Hours Figure 3 Growth of Vibrio harveyi by silver nanoparticles synthesized by tea leaf extract at different concentrations. ( ) Control; ( ) 5lg; ( )1lg; ( ) 15lg; (h) 2lg; ( ) 25lg and (+) 3 lg. 354 Journal compilation ª 21 The Society for Applied Microbiology, Letters in Applied Microbiology 5 (21)

4 B. Vaseeharan et al. Silver nanoparticles effect against Vibrio harveyi with AgNps) along with control group after being challenged with V. harveyi are shown in Fig. 4. Combined treatment with 1 lg AgNps containing diet (i.e. 71% reduction in mortality) group showed a significant reduction in mortality, when compared with all the other experimental groups (P <Æ5). Similar trends were observed in the groups of combined 5 lg AgNps, long-term 1 lg AgNps and long-term 5 lg AgNps, as these groups showed significant reduction in mortality after V. harveyi challenges (P <Æ5). Both short-term 1 lg AgNps and short-term 5 lg AgNps showed no significant reduction in mortality between them (P > Æ5). Control groups had the highest value of mortality (91%) after the 14th day of experimental challenge. Discussion AgNps have received increasing attention, because they are able to control pathogenic microbes of various origins (Morones et al. 25). Synthesis based on the biotransformation of silver salts into AgNps has also been reported recently, where micro-organisms and plant extracts were employed to obtain AgNps. It has been well documented that plant extracts of geranium leaves (Shankar et al. 23), neem leaves (Shankar et al. 24), black tea leaves (Begum et al. 29) and other fruits have the potential to synthesize AgNps. In this study, we have explored the synthesis of AgNps with tea leaf extracts and its application to control V. harveyi infections in aquaculture. In vivo results obtained from the growth of V. harveyi at different AgNps concentration showed that higher concentration (35 lg cm )3 ) present in the LB agar plates effectively reduces the colony forming units into 5% when compared to the control. It is also observed that the concentration of AgNps is directly proportional to the reduction in CFU of V. harveyi in LB agar plates. Pal et al. (27) stated that inhibition of bacterial growth is directly proportional to the concentration of AgNps. Morones et al. (25) demonstrated that the bactericidal activity of AgNps is species specific. In their study, Vibrio cholera and Pseudomonas aeruginosa were observed to be more resistant than Escherichia coli and Salmonella typhus. The total reduction in Vibrios in the media required more than 75 lg ml )1 AgNps in the growth media. In the present study, lower concentrations of AgNps showed negligible bactericidal activity against V. harveyi. It was also observed that the effective bactericidal activity also depends upon the CFU load of the V. harveyi in the media. Our results were analogous with the results obtained by Sondi and Sondi (24), where the authors illustrated that the inhibitory effect of AgNps depends on the CFU of bacteria used in the experiments. The bacterial growth rate as observed at 58 nm was significantly regulated by the presence of AgNps (Fig. 4). No growth (increase in OD value) was observed at 35 lg Ag nanoparticles concentration present in LB broth. Several mechanisms of growth inhibition by AgNps were postulated in bacteria viz., binding on the sulfur-rich proteins present in the membrane surfaces, DNA binding properties (Feng et al. 2) and inhibition of cell cycle (Pommerville and Edward Alcamo 26). Although no work has been reported yet on the application of AgNps on controlling Vibrios in aquaculture, it appears that aquatic animals can be employed to evaluate Figure 4 Mortality (%) in different experimental groups of Feneropenaeus indicus treated with 5 lg and 1 lg of silver nanoparticles synthesized by tea leaf extract during experimental infection with Vibrio harveyi. ( ) Control; ( ) Short-term 5 lg; ( ) Short-term 1 lg; ( ) Combined 5 lg; (h) Combined 1 lg; ( ) Long-term 5 lg and (+) Long-term 1 lg. % mortality Time after nanoparticle treatment (h) Journal compilation ª 21 The Society for Applied Microbiology, Letters in Applied Microbiology 5 (21)

5 Silver nanoparticles effect against Vibrio harveyi B. Vaseeharan et al. nanoparticles toxicity (Snell and Hicks 21) and DNA delivery in aquatic animals (Rajesh Kumar et al. 28). But the application of metallic nanoparticles on controlling bacterial pathogens in aquatic animals still remains as an unexplored area of research, and the present study made a successful attempt to demonstrate the ability of AgNps on controlling aquaculture diseases. Silver has a long-standing history as germicide and has little or no toxic impact at low concentrations (Hill 29). Reduction in shrimp mortality was evident during the V. harveyi challenges after the administration of AgNps at the concentration of 1 lg for combined treatment group (shortterm + long-term treatment). The mortality was 29% in this group, whereas control group showed 91% mortality rate after 14th day of experimental challenges. This demonstrates that AgNps can be used as an alternative to antibiotic administration for the control of V. harveyi infections in shrimp culture. In conclusion, AgNps-based diet administration in shrimps showed promising results on controlling V. harveyi infections. Further research is required to evaluate the biological mechanism, toxicological studies and bioaccumulation of AgNps in shrimps. Acknowledgements This work was supported by Indo Taiwan programme of cooperation from Department of Science and Technology, New Delhi, India, (Project code: ITRD3) and National Science Council, (NSC B-19-2) Taiwan. The authors thank Dr Junda Lin (Florida Institute of Technology, Melbourne, FL, USA) for his comments on an earlier version of the manuscript. References Alexander, S., Klabunde Kenneth, J., Marchin George, R. and Sorensen Christopher, M. (28) Biocidal activity of nanocrystalline silver powders and particles. Langmuir 24, Austin, B. and Austin, D.A. (27). Bacterial Fish Pathogens: Disease of Farmed and Wild Fish, 4th edn, Vol. XXVIII, Chichester, UK: Springer Praxis Books. Begum, N.A., Mondal, S., Basu, S., Laskar, R.A. and Mandal, D. (29) Biogenic synthesis of Au and Ag nanoparticles using aqueous solutions of Black Tea Leaf extracts. Colloid Surf B 71, Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N. and Kim, J.O. (2) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52, Harris, L., Owens, L. and Simth, S. (1996) A selective and differential medium for Vibrio harveyi. Appl Environ Microbiol 62, Hill, J. (29) Colloidal Silver: A Literature Review: Medical Uses, Toxicology & Manufacture, 3rd edn. Washington, DC: Clear Springs Press. Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T. (1994) Bergey s Manual of Determinative Bacteriology, 9th edn. Baltimore, MD, USA: The Williams and Wilkins Company. Mohanpuri, P., Rana, N.K. and Yadav, S.K. (28) Biosynthesis of nanoparticles: technological concepts and future applications. J Nanopart Res 1, Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T. and Yacaman, M.J. (25) The bactericidal effect of silver nanoparticles. Nanotechnology 16, Oakey, H.J., Levy, N., Bourne, D.G., Cullen, B. and Thomas, A. (23) The use of PCR to aid in the rapid identification of Vibrio harveyi isolates. J Appl Microbiol 95, Pal, S., Yu Kyung, T. and Song, J.M. (27) Does the Antibacterial activity of silver nanoparticles depend on the Shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Appl Environ Microbiol 73, Pommerville, C. and Edward Alcamo, I. (26) Fundamentals of Microbiology. 8th edn. Sudbury, MA: Jones & Bartlett Publishers. Rajesh Kumar, S., Ishaq Ahmed, V.P., Parameswaran, V., Sudhakaran, R., Sarath Babu, V. and Sahul Hameed, A.S. (28) Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum. Fish Shellfish Immunol 25, Shankar, S.S., Ahmad, A. and Sastry, M. (23) Geranium leaf assisted biosynthesis of silver nanoparticles. Biotechnol Prog 19, Shankar, S., Rai, A., Ahmad, A.C. and Sastry, M. (24) Rapid synthesis of Au, Ag, and bimetallic Au core Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J Colloid Interf Sci 275, Snell, T.W. and Hicks, D.G. (21) Assessing toxicity of nanoparticles using Brachionus manjavacas (Rotifera). Environ Toxicol, in press, doi: 1.12/tox Sondi, I. and Sondi, B.S. (24) Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J Colloid Interface Sci 275, Vaseeharan, B. and Ramasamy, P. (23) Abundance of potentially pathogenic micro-organisms in Penaeus monodon larvae rearing systems in India. Microbiol Res 158, Journal compilation ª 21 The Society for Applied Microbiology, Letters in Applied Microbiology 5 (21)