Indian Journal of Applied Microbiology

Indian Journal of Applied Microbiology Biannual Journal of the Indian Association of Applied Microbiologists

Volume 15 Number 2 July-December 2012

Editor -in-Chief Prof. Dr.Usha Anand Rao Director (Research) Raagas Dental College and Research Institute, Uthandi, East Coast Road, Chennai - 600 119, India IJAM Contact Address 2A, Coral Sudha Apartments, No. 16, Eldams Road, Alwarpet, Chennai - 600 018, India Phone: +91 44 2435 0260, Cell: +91 9840238823 E-mail: [email protected] Associate Editor Dr. S. Thiyagarajan

India

Advisory Board Prof. P. Rajendran Dr. B.L. Sarkar Dr. Udhay K. Shankar Prof. S.M. Muthukaruppan Dr. (Mrs.) Madhu Khanna Dr. Dharmalingam Subramaniam

India India USA India India USA

Managing Editor Dr. R. Rajan

India

Prof. S. Kumanan Dr. S. Dhamodaran Dr. K. Gopalakrishnan Dr. A. Michael Dr. M. Sundararaman Dr. I. Seethalakshmi

India Singapore Malaysia India India India

Print ISSN 2249-8400 Volume 15 Number 2 July-December 2012  2012 Indian Association of Applied Microbniologists Indian Association of Applied Microbiologists 2A, Coral Sudha Apartments, No. 16, Eldams Road, Alwarpet, Chennai - 600 018, India Wisdom Academic Publishers No. 10\8, Dr. Nammalvar Street, Triplicane, Chennai - 600 005, India Phone: +91 44 2844 7276 • E-mail: [email protected] The Editor-in-Chief, IAAM and Wisdom Academic Publishers assume no responsibility for the views expressed by the authors of material printed in the Indian Journal of Applied Microbiology.

It is a condition of publication that manuscripts submitted to the Indian Journal of Applied Microbiology have not been published and will not be simultaneously submitted or published elsewhere. By submitting a manuscript, the authors agree that the copyright for their work is transferred to the Indian Association of Applied Microbiologists, if and when the work is accepted for publication. The copyright covers the exclusive reprints, photographic reproductions, microform or any other reproductions of similar nature and transactions. No part of the publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means - electronic, electrostatic, magnetic tape, mechanical photocopying, recording or otherwise, now known or developed in the future - without prior written permission from the copyright holder. Indian Journal of Applied Microbiology is a biannual journal published by Wisdom Academic Publishers, Chennai on behalf of the Indian Association of Applied Microbiologists, Chennai. Printed in India.

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Copyright  2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, pp. iii-iv.

Contents From the Editor’s Desk

v

1.

Vaccine for HIV - a ray of light but a long way to go Vijayakumar Velu, Subhadra Nandakumar, Usha Anand Rao, Sadras Panchatcharam Thyagarajan

1

2.

Production and Molecular characterization of Bacteriocin synthesized by Food borne Lactobacillus sp. YA2 and Phylogenetic studies on the Isolate S. Jamesammal and S. Thiyagarajan

3. Microbial synthesis of Silver Nanoparticles (AgNPs) and evaluation of their biological activity D. Priya Dharshini, P. Balaji, T. Shanmugasundaram, P. Rajendran and R. Balagurunathan

7

21

4. Effect of Phyllanthus amarus extract on SphH gene of Leptospira autumnalis studied by an in-house PCR R. Saravanan, P. Saradhai and E. Rani

40

5.

Characterisation and Preliminary screening of Biosurfactant producing Bacteria isolated from hydrocarbon contaminated soils P. Saminathan and P. Rajendran

46

6.

Nanotechnology in Biology: A Brief Overview Sivaprakash Rathinavelu*

7.

In vitro Antibacterial activity of Cardiospermum halicacabum extracts against Bacterial isolates from Pus samples N. Manikandan, B. Radha, N. Vijaykanth, M.R. Ramesh kumar and N. Arunagirinathan

65

8.

Screening of Asymptomatic carriers for MRSA in the sports personnel and evaluation of Syzygium aromaticum (Clove oil) against MRSA. S.K. Jasmine Shahina, M. Durga Priyadarshini and Padma Krishnan

72

54

iv Contents 9. Antibacterial activity of Pyocyanin pigment produced by Clinical isolate, Pseudomonas aeruginosa WS1 T. Sudhakar, S. Karpagam and M. Lakshmipathy

80

10. Assessment of Organic waste composting and designing of consortia to improve the compost efficiency as a Biofertilizer J. Jelin Ilayaraja and M.S. Dhanarajan

86

11. Bioprospecting of Marine Micromonospora with special reference to Antibacterial metabolite production R. Balagurunathan and M. Radhakrishnan

94

12. Bacteriological examination of Backwater from Fishing Harbour of Ennore Creek in Chennai coast C. Ganga Baheerathi and K. Revathi

100

13. Antibacterial studies of Smilax zeilanica against Rifampicin and Methicillin resistant Staphylococcus aureus 107 K. Kavitha and K. Murugan 14. Isolation of Vibrio spp. and other Predominant bacteria from Green Mussels in Muttukadu, Tamilnadu V. Gayathri and K. Revathi

114

Biography of a Renowned Microbiologist: Martinus Beijerinck

120

Instructions to Authors

122

Author’s Declaration Form

126

Forthcoming Events

127

Application for Membership

129

Subscription Information

130

INDIAN JOURNAL OF APPLIED MICROBIOLOGY

Vol. 15 No. 1 Jan.-Jun. 2012

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Copyright  2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, p. v.

From the Editors Desk The discoveries and inventions are the stepping stones of the development of science and technology. By virtue of the discoveries the human life has not only modernized but also understood the ways and means to overcome the hurdles and attain success. Just as the revolution in therapeutic medicine caused by the discovery of the wonder drug penicillin, many such significant discoveries are considered as boon to human kind. Every discipline of science has its own principle and pride. But the infusions of different branches of science - in the form of interdisciplinary approach - result in wonderful discoveries. Although the discovery of electricity made our life comfortable in every respect, the recent discovery of biological wire or the so called ‘nano-wire’ is a significant contribution in the fields of electrophysics and nanotechnology. When one thinks of an electrical wire we remember a metal, plastic or alloy. In a recent report published in Nature, a marine bacteria which conducts electrons by forming nanowire has been discovered. Is it not astonishing? It not only demonstrates the thinking power of genius but also talks about what a team work can do to conquer nature! This discovery is based on the theory that organisms gain energy by metabolism. Typically the energy is obtained by oxidation, but the anaerobic organisms use alternative methods. A marine surface bacteria of the family Desulphobalbaceae has been observed to possess a spectacular characteristic of energy generation and conductivity. These bacteria, living in groups, form filaments, transport the electrons liberated and carry them to oxygen at the top by forming a live wire. Is it not a fantastic finding? But, it is the inevitable fact that with ecstasy comes along the agony. The malarial vaccines are still evading the effect. Malaria vaccine trials in infants and 12 month follow up study in Africa has shown disappointing results as there is a drastic reduction in the efficacy. Some drastic measures have to be found as malaria is raising its ugly head again and again. Being Microbiologists we have the responsibility to come together and strive to circumvent this precarious agent by suggesting some remedy thus to save the precious human life. The Journal is in good shape and progressing well and I owe this to the efforts of my Editorial team and contributors. We hope to achieve the heights of success and serve better with the noble support of all of you. Warm greetings, Dr. Usha Anand Rao Editor-in-Chief, IJAM

Office Bearers of IAAM President Prof. Dr. P. Rajendran Sri Ramachandra University, Porur, Chennai – 600 116 General Secretary Dr. N. Arunagirinathan Presidency College, Kamarajar Salai, Chepauk, Chennai – 600 005 Joint Secretary Dr. (Mrs.) Lata Sriram Madras Medical College, E.V.R. Periyar Salai, Park Town, Chennai – 600 003 Editor-in-Chief, IJAM Prof. Dr. Usha Anand Rao Raagas Dental College and Research Institute, Uthandi, Chennai – 600 119 Associate Editor, IJAM Dr. S. Thiyagarajan Asan Memorial College of Arts and Science, Jaladampet, Chennai – 600 100 Treasurer Dr. S. Anbalagan Muthayammal College of Arts and Science, Rasipuram – 637 408, Namakkal District Executive Members Dr. A.K. Kathiresan Vels University, Pallavaram, Chennai – 600 117 Mr. Joseph C. Daniel St. Joseph’s College of Arts and Science, Cuddalore – 607 001 Dr. A. Michael P.S.G. College of Arts and Science, Coimbatore – 641 014 Dr. R. Balagurunathan Periyar University, Salem – 636 011 Dr. I. Seethalakshmi Lifeteck Research Centre, Vadapalani, Chennai – 600 026 Ms. Kavitha Madras Christian College, Tambaram, Chennai – 600 059 Mr. T. Murugan S.R.M. Arts and Science College, Kanchipuram District, Kattankulathur – 603 203


Copyright 2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, pp. 1-6.

Vaccine for HIV - a ray of light but a long way to go Vijayakumar Velu1*, Subhadra Nandakumar2,3, Usha Anand Rao4 Sadras Panchatcharam Thyagarajan5 Emory Vaccine Center, Emory University, Atlanta, Georgia, USA Department of Microbiology, James H. Quillen, College of Medicine, East Tennessee State University, Johnson City, Tennessee, USA 3 Current Address: Division of TB Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. 4 Ragas Dental College, Perungudi, Chennai 600 096, India. 5 Sir Ramachandra University, Porur, Chennai 600 113, India. 1 2

Abstract: The development of a safe and effective human immunodeficiency virus (HIV)-1 vaccine is a critically important global health priority. This mini review discusses selected recent data that suggest that indeed it is possible to make a clinically useful preventive vaccine for HIV-1 and outline some of the remaining obstacles that stand in the way of success. Studies with broad neutralizing antibodies for mucosal simian HIV challenges, in non-human primates have suggested that lower doses of neutralizing antibodies than previously thought may be effective in preventing HIV infection in passive protection studies. The successes of the RV144 Thai HIV-1 efficacy trail showing 31% of efficacy has given hope that indeed a protective HIV-1 vaccine can be made. Keywords: HIV, Vaccine, CD4 T-cells, RV 144.

Introduction Human Immunodeficiency Virus (HIV/AIDS) is the leading cause of mortality worldwide, more than 25 years since the discovery of the virus, there is still no completely successful vaccine or effective treatment to control the viral infection. The number of infection increases every year and currently 33.4 million people are living with HIV in the world. The development of safe and effective vaccine would undoubtedly be the best solution for the ultimate control of worldwide AIDS pandemic [1]. *

Author for Correspondence; E-mail: [email protected]

2 Vijayakumar Vel et al

However the HIV vaccine development efforts have not yet proven to be effective, the extraordinary diversity of HIV-1, the capacity of the virus to evade the immune system, the inability to induce broadly neutralizing antibody responses, the early establishment of latent viral reservoirs and the lack of immune correlates of protection represents unprecedented challenges for the vaccine development. The recent RV144 Thai trail represents a new hope for the field that a preventive HIV vaccine can be made and there is a big debate ongoing in the field as to whether HIV vaccine is feasible or not. This review discusses selected data that suggest that indeed it is possible to make a clinically useful preventive vaccine for HIV and outlines some of the remaining obstacles that stand in the way of success and one day the world will get effective HIV/AIDS vaccine to control the virus.

Challenges in the development of a prophylactic HIV-1 vaccine The goal of a HIV-1 vaccine would be either to prevent infection or to reduce viral loads and clinical disease progression after infection. An ideal vaccine would completely block infection and provide sterilizing immunity, however there are many unique challenges for the successful vaccine development that includes; the extensive viral clade and sequence diversity, early establishment of latent viral reservoirs, lack of immune correlate of protection and viral evasion of humoral and cellular immune responses. The key challenge is the lack of clear immune correlates of protection in humans as HIV-1 infected patients are unable to eradicate the virus [2]. Suggestive evidence regarding the immune correlates of protection might be obtained from viral challenge studies in nonhuman primates and from studies of HIV-1 infected individuals who spontaneously control viral replication to very low levels. However, definitive immune correlates of protection will probably only emerge in the context of successful vaccine efficacy studies in humans [3].

Correlates of Immune protection What we know; is the principal correlates of immune protection for most infections and vaccines is the adaptive immune responses in the form of neutralizing antibodies and virus-specific CD4+ and CD8+ T cells. Neutralizing antibodies are the immune correlates of protection from infection. Cellmediated immune responses are the immune correlates of protection from disease. Studies of passive immunization and immune depletion in animal models strongly suggest that both neutralizing antibodies and cell-mediated immune responses may provide effective protection from infection and from disease progression in HIV-1 infection. The effectiveness of antiviral memory T cells is dependent upon both the quality and the magnitude of antigen-specific T cells. Poly-functional (IL-2 and IFN-g) CD4+ and CD8+ T-cell responses, and not mono-functional IFN-g responses, are associated with at least partial virus control. Immunologic monitoring of vaccine-induced T-cell immune responses cannot be limited to cells that secrete IFN-g and should be extended to include cells that secrete IL-2. What we don’t know; is how to induce high titers of neutralizing antibodies, whether any of the vaccines being currently developed will elicit cellular immune responses that will correlate with protection from infection or disease progression.


Vol. 15 No. 2 July-Dec. 2012

Vaccine for HIV - a ray of light but a long way to go 3

What we should know; is the type (poly- or mono-functional) of CD4+ and CD8+ T-cell responses elicited by the vaccines currently being developed. The breadth of the vaccine-induced CD4+ and CD8+ T-cell responses, the best combination of vaccines, that, in the prime-boost immunization strategies will stimulate an immune response similar to that thought to confer protection from disease progression. Therefore for a vaccine to be successful in preventing HIV infection, it should satisfy the following criteria of making effective virus specific cellular and humoral immunity [4].

A ray of light for vaccine development The failure of the first two antibody-based vaccines of clade B gp120 s (VAX004) and clades B and E gp120 s (VAX003), coupled with the failure of the T-cell vaccine based on the recombinant type 5 adenovirus vector (Step) trial induced a degree of skepticism in the HIV-1 vaccine development field that a vaccine could be made. With the outcome of the Step trial in which, there not only was no efficacy, but also a trend toward vaccine-induced acquisition of HIV-1, the field reassessed the state of research once again and doubled the commitment to basic research. Studies of the earliest viral and immunologic events following HIV-1 transmission have demonstrated that for a preventive vaccine to be successful, protective immunity will need to be present before infection. The field is hopeful that the RV144 results signify a proof of concept that indeed a vaccine for HIV-1 is possible. Although the RV144 Thai trial represents a new hope for the field that a preventive HIV-1 vaccine can be made, 31% vaccine efficacy is not sufficient for widespread deployment of the current RV144 immunogen, nor did the protective effect last sufficiently long to be optimally useful. Moreover, many questions remain regarding the path for follow up of the RV144 trial. The gp120 B/E boost proteins are the same immunogen that showed no efficacy in high-risk intravenous drug users in Thailand, raising the question of whether it was the ALVAC prime that made the difference or the heterosexual (presumed lower-risk) population which the RV144 trial utilized. Because CD8 T cells responses were low in phase II trials utilizing the RV144 vaccine [5] and there was no impact on viral load, and the protective efficacy declined over time, the notion is that the correlate of protection in RV144 vaccines will either be traditional neutralizing antibodies or virus inhibitory antibodies capable of blocking HIV-1 transmission [6]. However the durability of this anti-HIV antibody response and variability of antibody response among individuals is still an issue. Thus, T cell help and engaging the full arsenal of innate, cellular and humoral responses to provide a durable humoral response to block transmission as well as a robust cellular response to limit virus replication for those that do become infected will be critical. Cellular immunity is important for direct antiviral functions to decrease virus replication and for providing help for a durable and effective humoral response.

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

4 Vijayakumar Vel et al

Fig.1. Schematic representation of the effective components of the antiviral immune response against different forms of HIV-1 [Neutralizing antibodies are effective in blocking virus particles but poorly effective against cell associated virus, such as virus-infected cells but not against free virus particles. Neither antibodies nor cytotoxic CD8 T cells are effective against latently infected cells. “The ideal vaccine should target eradicating latent viral reservoirs”]

Other considerations for correlates of protective immunity in RV144 are CD8 T-cell responses (though these responses were infrequent in phase I/II trials and in the initial immunogenicity data from RV144) and CD4 T-cell responses that undoubtedly are critical for robust antibody responses. With recent reports of natural killer (NK) cell memory induced by certain infections and the potential for [beta]-chemokine inhibition of HIV-1 [7], innate mechanisms are being studied as well. Genetic studies are being evaluated to follow up on observations of the role of Fc receptor polymorphisms and other genetic factors that might play a role in salutary immune responses [8]. Finally, antibodies capable of inhibiting HIV-1 envelope interactions with integrin alpha 4 beta 7 on mucosal CD4 T cells are being considered [9]. Thus, it is critical for the field to consider all components of adaptive and innate immune responses as synergistic immune responses that should be harnessed for an optimally effective vaccine [10]. Moreover, detailed studies of how each of these immune responses are induced and maintained at mucosal surfaces so that they can be preexisting to prevent or extinguish transmitted/founder virus infection are needed. Identification of the correlates of protection within the vaccine trials will certainly depend upon the quality of the candidate vaccines. For example, weak vaccines are unlikely to provide protection, and as such will not generate any INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Vaccine for HIV - a ray of light but a long way to go 5

immune correlate. In contrast, cellular responses stimulated by potent vaccines are more likely to provide protection and a measurable correlate. Therefore, a detailed qualitative and functional characterization, rather than the simple detection of the vaccine-specific immune response, will be required to determine the immune correlates of protection against HIV.

Conclusion Recent years have been an important and exciting years in HIV-1 vaccine development. The field has made remarkable progress in working together in collaborative teams and sharing of data. The positive results of the RV144 trial coupled with multiple insights into new avenues of T, B and NK cell biology as they pertain to HIV-1, have energized the field. Focus now should be on the earliest events involved in HIV-1 and SIV transmission at mucosal sites, and on the types of mucosal innate, T and B cell responses that can be present before the transmission event or can arise within hours of transmission and prevent or extinguish the HIV-1 transmitted/founder virus. Although formidable roadblocks and tasks remain, the prospects for successful development of a well-tolerated and effective HIV-1 vaccine are promising. What is needed is continued intense and coordinated basic, and translational clinical research with iterative testing of promising concepts in both nonhuman primates and human vaccine trials.

References 1.

Barouch, H. Dan., 2008, “Challenges in the development of an HIV-1 vaccine”, Nature , 455, pp. 613-619.

2.

Anthony L. DeVico and Robert C. Gallo, 2004, “Control of HIV-1 infection by soluble factors of the immune response”, Nat Rev Microbiol, 2 pp.401–413.

3. James Arthos, Claudia Cicala, Elena Martinelli, et al., 2008, “HIV-1. Envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells”, Nature Immunol, 9 pp.301–309. 4.

Giuseppe Pantaleo and Richard A Koup, 2004, “Correlates of immune protection in HIV-1 infection: what we know, what we don’t know, what we should know”, Nature Med, 10, pp.806-810.

5.

Norman G. Jones, Allan DeCamp, Peter Gilbert, et al. , 2009, “AIDS-VAX immunization induces HIV-specific CD8+ T-cell responses in high-risk, HIV-negative volunteers who subsequently acquire HIV infection”, Vaccine 27, pp.1136–1140.

6. Punneep Pitisuttithum, Peter Gilbert, March Gurwith, et al., 2006, “ Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand”, J Infect Dis, 194, pp.1661–1671. 7.

Joesph C. Sun, Joshua N Beilke and Lweis L.Lanier, 2009, “ Adap-tive immune features of natural killer cells”, Nature, 457, pp.557–561.


6 Vijayakumar Vel et al 8. Donald N. Forthal, Peter B. Gilbert, Gary Landucci and TranPhan, 2007, “Recombinant gp120 vaccine-induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptor-bearing effector cells and correlate inversely with HIV infection rate”, J Immunol , 178, pp.6596–6603. 9. Claudia Cicala, Elena Martinelli, Jonathan P. McNally, et al., 2009, “ The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1”, Proc Natl Acad Sci., 106, pp.20877–20882. 10. Kim J Ha, Rerks-Ngram, Excler Supachaib et al., 2010, “ HIV vac-cines: Lessons learned and the way forward”, Current Opinion in HIV & AIDS, 5, pp.428-434.





Production and Molecular characterization of Bacteriocin synthesized by Food borne Lactobacillus sp. YA2 and Phylogenetic studies on the Isolate S. Jamesammal and S. Thiyagarajan* P.G. & Research Department of Microbiology, Asan Memorial College of Arts & Science, Jaladampet, Chennai – 600 100 Abstract: The probiotic microorganisms belonging to the genus Lactobacillus have been long known to confer various nutritional and therapeutic benefits to the consumers. The bacteriocins produced by probiotic bacteria possess antimicrobial property by which they combat the invading pathogenic organism. For the purpose of isolating potential probiotic bacteria three different types of food samples, namely yoghurt, meat and tomato were processed by standard bacteriological procedures using MRSc agar. Twenty four isolates of bacteria of genus Lactobacillus were obtained. The screening of these isolates for antimicrobial property revealed two isolates exhibiting better antimicrobial activity. These isolates, namely YA1 and YA2 when subjected to acid and bile tolerance tests, Lactobacillus sp. YA2 was observed to own higher probiotic properties. Production of bacteriocin from the potential isolate was carried out by a standard method and the parameters of culture condition such as a temperature of 25°C, pH of 9, incubation time of 48h and NaCl concentration of 2.0% were optimised. The crude bacteriocin was subjected to sequential steps of purification such as ammonium sulphate precipitation, dialysis and Ion- exchange chromatography. The bacteriocin activity of the compound produced by Lactobacillus sp. YA2, as determined in terms of arbitrary units, was observed to be increasing in each step of the purification. Molecular characterization of bacteriocin using SDS-PAGE indicated the bacteriocin thus produced to possess a molecular weight of 43KDa. Sequencing of 16s rRNA of Lactobacillus sp. YA2 and subjecting it to BLAST similarity search and phylogenetic characterization deciphered the identity and maximal similarity of the bacteria with Lactobacillus paracasei. Key words: Probiotic, Lactobacillus sp., bacteriocin, phylogenetic characterization

*

Author for Corresponding; E-mail: [email protected]

8 Jamesammal and Thiyagarajan

Introduction Probiotics are the microorganisms associated with the beneficial effects for humans and animals. These microorganisms contribute to intestinal microbial balance and play an important role in maintaining health. The term ‘probiotic’ was first used by Lilly and Stillwell in 1965 to describe the ‘substances secreted by one microorganism that stimulate the growth of another. The history of probiotics began with the history of man; cheese and fermented milk were well known to the Greeks and Romans, who recommended their consumption, especially for children and convalescents [1]. The origin of cultured dairy products dates back to the dawn of civilization; they are mentioned in the Bible and the sacred books of Hinduism. There has been the development of many of the traditional soured milk or cultured dairy products such as kefir, koumiss, leben and dahi, many of which are still widely consumed, had often been used therapeutically before the existence of bacteria was recognized [2]. Probiotics can be found in both dairy and non dairy products. They are usually consumed after the antibiotic therapy (for some illnesses), which destroys the microbial flora present in the digestive tract (both the useful and the targeted harmful microbes). Regular consumption of food containing probiotic microorganisms is recommended to establish a positive balance of the population of useful or beneficial microbes in the intestinal flora [3]. Probiotics may exert a beneficial effect on allergic reaction by improving mucosal barrier function. In addition, probiotic consumption by young children may beneficially affect immune system development. The probiotic microorganisms consist mostly of the strains of the genera Lactobacillus and Bifidobacterium, but strains of Bacillus, Pediococcus and some yeast have also been found as suitable candidates [4]. More frequently the lactic acid bacteria (LAB) are the organisms of first choice for the production of most of the dairy products owing to their potential probiotic property. Most LAB strains used in the food supply are non-pathogenic, non-virulent, and non-toxigenic microorganisms. Many strains of Lactobacilli and Bifidobacteria are traditional food-grade organisms generally recognized as safe for use in the food supply. Probiotics such as Lactobacillus may be helpful in alleviating some of the symptoms of food allergies such as those associated with milk protein. As the probiotics are gaining more importance from the view points of food safety and healthy dietics, there is always a demand for isolating a potential bacteria which can confer a wide range of nutritional and health benefits. The present study is an attempt to isolate a potential probiotic bacterium from natural and fermented foods and to suggest it as a dietery additive especially for malnourished children and patients with alimentary ailments.

Materials and Methods Isolation of probiotic bacteria Three different types of samples comprising of a fermented food (yoghurt), and two raw natural foods (meat and tomato) were utilised as sources for isolation of probiotic bacteria. A total of fifteen samples of each category were collected from five different shops located in the suburban areas of INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Production and Molecular characterization of Bacteriocin of Lactobacillus sp. YA2 9

Chennai city over a period of three months. The samples were collected in sterile bags, transported and processed in the Department of Microbiology of Asan Memorial College of Arts and Science, Chennai. Probiotic bacteria (Lactobacillus sp.) were isolated by direct qualitative method on MRS Agar. Samples were serially diluted in distilled water and plated on MRSc agar by spread plate technique and incubated at 37°C for 24 h. Among the different colonies grown on the media colonies giving rise to the bacteria resembling the microscopic morphology of Lactobacillus were cultured and purified on onto Lactobacillus agar. These colonies were further subjected to preliminary identification of bacteria by their macroscopic (Colony morphology) and microscopic morphology (Gram staining), and biochemical characteristics (IMViC and Carbohydrate fermentation) by adopting standard procedures [5]. The probiotic bacterial isolates were identified and selected based on the results of phenotypic, cultural and biochemical characterization.

Screening of isolates for antimicrobial property As antimicrobial activity is the principal property expected of a probiotic bacteria, the isolates of Lactobacillus sp. were screened for this purpose. The isolates were inoculated into MRSc broth and incubated aerobically for 24 h at 37°C. The indicator strains used for antimicrobial screening test were Staphylococcus aureus (MTCC 96), Listeria monocytogenes (MTCC 657), Vibrio parahaemolyticus (MTCC 451), Salmonella enterica (MTCC 3231), Escherichia coli (MTCC 40), Trichophyton rubrum (MTCC 296), Epidermophyton floccosum (MTCC 613), Aspergillus flavus (MTCC 8834) and Candida albicans (MTCC 227). Each test culture was inoculated into Brain Heart Infusion broth and incubated at an appropriate temperature for 24 h. Sterile petridishes containing Muller Hinton agar were inoculated with 0.1ml of test culture. Once solidified the dishes were stored for 2 h in a refrigerator. Followed by this four wells were made on the agar and filled using 100µl of cell- free filtrate of Lactobacillus culture previously prepared and incubated at 37°C for 24 h. The antimicrobial activity was determined by measuring the clear zone of growth inhibition around the wells. Isolates of Lactobacillus sp. showing significant inhibitory activity were selected for further studies.

Determination of Probiotic Property of Bacterial Isolates All the selected bacterial isolates were tested for their probiotic property by specific methods-Acid tolerance test and Bile tolerance test [6]. Acid tolerance test: Bacterial cultures were grown in MRSc broth at 37°C overnight, then subculture into fresh MRSc broth and incubated for another 24 h. The cultures were centrifuged at 3000 rpm for 10 min. The pellets were washed sterile in phosphate buffered saline (PBS) and resuspended in PBS. Cell suspension (0.5ml) was diluted up to 50ml using sterile PBS and pH of these series culture aliquots in tubes were adjusted to 2,3,4,5 and 6. Incubation times were 1 to 5 h. Then the cultures were inoculated (0.5ml) into MRSc broth and incubate at 37°C growths and monitored by measuring the absorbance using a spectrophotometer at 600 nm. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


Bile tolerance test: Freshly prepared cultures of Lactobacillus sp. were inoculated (1%) into series of MRSc broth tubes containing 0.1% to 0.3% concentrations of bile salt namely Glycolic acid. These tubes were incubated at 37°C and monitored for growth for hourly intervals up to 5 h by measuring the absorbance using a spectrophotometer at 600 nm, after which the absorbance values were plotted against the incubation time.

Production and assay of Bacteriocins The production of crude bacteriocin was performed by the procedure described by Ogunbanwo et al. [7]. Pure isolates of Lactobacillus sp. were grown in MRSc broth seeded with 5% inoculums of overnight culture and maintained aerobically under shaking at 37°C for 48 h. After incubation, cells were removed from the growth medium by centrifugation (10,000g for 15 min). The cell-free supernatant was adjusted to pH 6.0 using 1N NaOH, filter sterilized by passage through a 0.45 µm pore size membrane filter and it was used as crude bacteriocin. The antibacterial spectrum of the bacteriocin from Lactobacillus sp. was determined using the well diffusion method. Aliquots (50µl) of the crude bacteriocin were placed in 4-mm diameter wells that had been cut in Mueller-Hinton agar plates previously seeded with the indicator organism. After 12-18 h of incubation, the diameters of the zones of growth inhibition were measured.

Optimization of Culture Condition for bacteriocin production The optimization of parameters for the production of bacteriocin by the potential bacterial isolate was carried out by the procedure suggested by Ivanova et al. [8]. The different parameters standardized were concentration of sodium chloride (0.5, 1.0, 2.0 and 3.0%), pH (4,5,6,7,8 and 9), temperature (25°C, 30°C, 35°C and 40°C) and incubation time (6,12,18,24,30,36,42 and 48 h). Each time of standardization of parameters the medium was inoculated with the potential strain and incubated aerobically. The optimal parameter was determined by the significant antimicrobial activity (agar- well diffusion method) exhibited by the supernatant of the culture.

Assay of bacteriocin activity The bacteriocin activity was quantified in terms of its in vitro antimicrobial activity as described by Ivanova et al. [8]. The potential strain was cultured under optimized conditions and subjected to centrifugation at 14,000×g for 30 min at 4°C. The supernatant was collected and filter sterilized (0.22 µm pore size) to eliminate the viable cells and the pH of the supernatant was adjusted to 6.5 with 5M of NaOH. Then the neutralized cell free supernatant was diluted by two fold dilution with sterile deionised water and 100µl of each dilution was added into the wells made on MHA plate. The titre was defined as 2n, where ‘n’ is the highest dilution that result in inhibition of growth of indicator strains. Thus the antimicrobial activity of bacteriocin per millilitre was determined in terms of arbitrary units (AU) by the formula mentioned bellow: One Arbitrary unit = 2n × 1000µl/100µl




Purification and molecular characterization of Bacteriocin The method described by Rajaram et al. [9] was adopted partial purification of bacteriocin. The culture supernatant of the potential strain was subjected to sequential steps of purification such as ammonium sulphate (80%) precipitation, dialysis in 20 mM Potassium Phosphate (pH 7) at 4°C and Ion- exchange chromatography. The elute were collected in every 30 min. Concentration of the protein i.e., bacteriocin was determined by Lowry’s method using bovine serum albumin as Standard. The molecular weight of the bacteriocin was determined by 15% Sodium dodecylsulfate polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with Comassie Brilliant Blue R-250. Medium range moleculer markers (29-200 kDa) with five polypeptides was used as a marker.

Molecular identification and phylogenetic characterization The potential strain Lactobacillus sp. YA2 was subjected to molecular identification by sequencing its 16s rRNA. The partial sequence of 16s rRNA was obtained by the method as described by Jaya Prasad et al. [10]. Then this sequence was submitted for similarly search with GenBank data using Basic Logic Alignment Search Tool (BLAST) – Clustal W of DDBJ available in http:blast.ncbi.nih.gov/blast. This search tool enabled the comparison of the nucleotide sequence of the strain with that of those available in the database. Based on the results the algorithm was obtained and the phylogenetic tree was constructed.

Results Isolation of Lactobacillus and selection of potential probiotic isolate Subsequent to the inoculation of samples and incubation four morphologically different types of colonial growth were observed on MRSc media. They were categorised as colony type A, type B, type C and type D (Table 1). Among the different foods employed higher percentage of isolation of Lactobacillus was achieved with yoghurt (Fig.1). Table 1. Morphological features of colonies isolated on MRSc Agar S.No

Colony

Morphological Features

1.

A

Small, smooth, opaque, white colonies.

2.

B

Large, rough margin, opaque, milky white colonies.

3.

C

Medium sized, rough margin, opaque, white colonies.

4.

D

Large, irregular, smooth, floury white colonies.

Further microscopic and biochemical studies resulted in the preliminary identification of twenty four isolates of colonies belonging to as Lactobacillus sp. The culture supernatants of these isolates were subjected to screening studies on antimicrobial property. Based on the inhibition on maximum number of test organisms, two bacterial isolates namely YA1 and YA2 were selected. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


Fig.1. Frequency of Isolation of Probiotic bacteria

The acid tolerance and bile tolerance tests conducted on the isolates of Lactobacillus sp. YA1 and YA2 are presented in tables 2 and 3. These results indicated that the isolate YA2 posses higher potency by withstanding the acidic pH up to 5 h and could tolerate the bile concentration of 0.3% up to 5 h. Therefore the Lactobacillus sp.YA2 was selected as potential isolate and further studies were concentrated on this isolate. Table 2. Results of Acid tolerance test

Isolates YA1

YA2

Duration Hours

O.D Values at 600 nm for different acidic pH values PH2

PH3

PH4

PH5

PH6

1 hr

2.192

2.098

2.495

3.507

4.312

2 hr

2.625

2.120

2.559

3.741

4.365

3 hr

2.783

2.300

2.653

3.860

4.824

4 hr

2.824

3.853

3.876

4.369

5.566

5 hr

2.194

3.935

3.883

4.736

5.807

1 hr

2.284

3.534

4.395

5.369

5.822

2 hr

2.319

3.672

4.397

5.374

5.992

3 hr

2.480

3.954

4.412

5.377

6.082

4 hr

2.614

3.980

4.414

5.407

6.478

5 hr

2.921

3.983

4.775

5.540

6.527



Production and Molecular characterization of Bacteriocin of Lactobacillus sp. YA2 13 Table 3. Results of Bile tolerance test Isolates

Duration Hours

YA1

YA2

O.D Values at 600 nm for different concentrations of Glycolic Acid 0.1%

0.2%

0.3%

1 hr

0.042

0.023

0.022

2 hr

0.003

0.003

0.023

3 hr

0.105

0.061

0.043

4 hr

0.613

0.198

0.045

5 hr

0.842

0.128

0.218

1 hr

0.026

0.027

0.023

2 hr

0.023

0.003

0.003

3 hr

0.169

0.064

0.059

4 hr

0.593

0.380

0.093

5 hr

0.588

0.613

0.131

Optimization of culture conditions for Bacteriocin production The fermentation conditions required for maximal bacteriocin production of the potential strain were standardized by optimization studies [9]. The optimum bacteriocin production was determined in terms of antimicrobial activity exhibited by the crude bacteriocin of culture filtrate (10µl). These optimized parameters were a temperature of 25°C, pH of 9, incubation time of 48 h and NaCl concentration of 2.0%. Parallel tests with similar concentrations of commercial bacteriocin nisin indicated that the crude bacteriocin of Lactobacillus sp. YA2 exhibited more or less similar antimicrobial activity at par with nisin (Table 4 and 5). Table 4. Assay of Antibacterial activity of crude Bacteriocin and Nisin Drug Tested

Zone of Growth Inhibition of Bacteria S. aureus

E. coli

L. monocytogenes

S.entrica

Crude Bacteriocin

11 mm

10 mm

14 mm

12 mm

Nisin

14 mm

14 mm

16 mm

14 mm

Table 5. Assay of Antifungal activity of crude Bacteriocin and Nisin Drug Tested

Zone of Growth Inhibition of Fungi C. albicans

E. floccosum

T. rubrum

A.flavus

Crude Bacteriocin

10 mm

9 mm

8 mm

8 mm

Nisin

12 mm

13 mm

10 mm

10 mm



Purification, assay and molecular characterization of bacteriocin The fermentation was carried out under optimized conditions. At end of the fermentation, the culture supernatant was filtered and clarified so as to separate the supernatant containing crude bacteriocin. Subsequent to the precipitation and dialysis for 24 h partially purified bacteriocin was separated. The dialysate was further subjected to Ion-exchange chromatography for the purification of bacteriocin. The purified compound thus obtained by sequential precipitation, dialysis and Ion-exchange chromatography was tested for bacteriocin activity [11]. The Arbitrary unit of the compound was determined by considering the highest dilution of the compound (reciprocal) causing clear zone of growth inhibition of indicator organisms. In every step of the purification there was a proportionate increase in bacteriocin activity (Table 6 and 7). The bacteriocin of Lactobacillus sp. YA2 which was purified and confirmed for its activity was then subjected to molecular characterization by SDS-PAGE. The electrophoresis yielded a protein of low molecular weight in the range of 43 kDa corresponding to that of standard molecular weight marker (Fig.2). Table 6. Determination of Bacteriocin activity against Bacteria Arbitrary Units AU/ml Compound

Crude compound

Partially purified compound (after dialysis)

Purified compound (after Ion-exchange chromatography)

Dilution

S.aureus

S.enterica

E.coli

L.monocytogenes

1:1

3200

3200

1600

1600

1:2

3200

1600

1600

1600

1:4

3200

1600

1600

1600

1:8

1600

800

1600

800

1:16

800

800

1600

-

1:1

3200

3200

3200

3200

1:2

1600

3200

3200

1600

1:4

1600

-

3200

1600

1:8

800

1600

3200

800

1:16

800

800

1600

800

1:1

6400

6400

6400

6400

1:2

6400

6400

6400

6400

1:4

3200

3200

6400

6400

1:8

3200

3200

6400

6400

1:16

1600

3200

3200

6400



Production and Molecular characterization of Bacteriocin of Lactobacillus sp. YA2 15 Table 7. Determination of Bacteriocin activity against Fungi Arbitrary Units AU/ml

Compound

Dilution

A.flavus

C.albicans

E.floccosum

T.rubrum

Crude compound

1:1

3200

1600

1600

1600

1:2

3200

1600

1600

1600

1:4

3200

800

800

800

1:8

1600

800

-

-

1:16

-

800

-

-

1:1

-

3200

3200

1600

1:2

-

3200

3200

1600

1:4

3200

3200

1600

800

1:8

3200

1600

800

800

1:16

1600

800

800

800

1:1

6400

6400

6400

6400

1:2

6400

6400

3200

6400

1:4

3200

3200

3200

3200

1:8

3200

3200

1600

3200

1:16

1600

1600

1600

3200

Partially purified compound (after dialysis)

Purified compound (after Ion-exchange chromatography)

Fig 2. Bacteriocin separated by SDS-PAGE



Phylogenetic Characterization of potential strain In order to identify the potential strain Lactobacillus sp. YA2 at the molecular level its 16s rRNA was sequenced as per the original procedure described by Jaya Prasad et al. [10]. From this study a partial sequence of 16s rRNA of the potential isolate with a size of 1360 base pairs was obtained (Fig. 3). Subsequent to the analysis by BLAST and phylogenetic studies, the potential isolate was observed to show 100% similarity with the sequence of the bacteria Lactobacillus paracasei available in GenBank (Fig 4).

Fig 3. Sequence of 16s rRNA of the Potential Isolate Lactobacillus paracasei YA2




Fig 4. Results of Phylogenetic studies on Potential strain L. paracasei YA2

Discussion Probiotic bacteria have constituted a vital part of the research in the field of microbiology since the beginning of 19th century. Among the probiotic bacteria the genus Lactobacillus is considered to be superior in conferring the probiotics properties. In recent years, there has been a growing interest in exploring various biological activities of these bacteria, which include antimicrobial activity, anticancerous activity, antimutagenic activity, anticholestrol activity. The present study was under taken to isolate and identify a potential Lactobacillus sp. which will have dominant probiotic characteristics as well as the ability to grow and withstand adverse conditions there by benefiting the consumer. Among the different food samples employed in this study, the Lactobacillus was isolated in higher percentage in yoghurt. The studies by Shaw and Hardling [12] and Barakat et al. [13] had recommended yoghurt as the principal food for the isolation of Lactobacillus. However, few other studies had employed fermented food products such as curd, idli batter and pickle [14], mango pulp [15] and whey [16]. The isolation procedure yielded the growth of 24 isolates of bacteria belonging to 4 predominant morphological groups (types A, B, C and D). The overall occurrences of these bacteria were in the order of 62.5% (Yoghurt), 57.1% (Meat) and 55.5% (Tomato). The rate of occurrence of Lactobacillus among the isolates was 62.5%. The present study further screened these isolates on antimicrobial property and selected two isolates namely, YA1 and YA2. As the acid and bile tolerance characteristics are considered to be the most essential for probiotic bacteria [17-19], based INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


on display of better activity the isolate of Lactobacillus sp. YA2 was selected as potential strain. Most of the earlier studies conducted on isolation and screening of probiotic Lactobacillus had employed similar techniques [20-25]. However Edna T. Lima et al. [26] had applied mainly the preliminary screening technique i.e., test for antimicrobial property for the screening of Lactobacillus isolated from crop and caecum. The present study further concentrated on the production of extracellular bacteriocin by the potential strain Lactobacillus sp. YA2. The optimization of parameters for production of bacteriocin was determined in terms of better efficiency of corresponding culture filtrate to inhibit maximum number of indicator organisms (antimicrobial activity). The culture conditions thus standardized were a sodium chloride concentration of 2.0%, pH of 9, temperature of 25°C, incubation time of 48 h. These findings are in agreement with the reports of Rajaram et al. [9]. Subsequent to the optimization of the parameters of bacteriocin production, a separate fermentation was carried out to produce the same under optimized conditions. The quantitative assay of optimized supernatant for the bacteriocin activity was determined by critical dilution method as described by Manivasagan et al. [11]. The bacteriocin thus produced was subjected to sequential steps of purification such as precipitation by ammonium sulphate, dialysis and ion exchange chromatography. There was a marked increase in its activity at various stages of purification of bacteriocin through that of crude extract, precipitated supernatant, dialysate and ion-exchange elute. While few earlier researchers had employed the partial purification [8,27] of bacteriocin others had performed solid phase extraction and high performance liquid chromatography [28]. The characterization of bacteriocin of Lactobacillus sp. YA2. at the molecular level as determined by SDS-PAGE indicated its molecular weight corresponding to 43kDa of the marker protein. The protein band separated from the compound purified in the present study was confirmed to of bacteriocin category with reference to the reports of earlier studies. Bacteriocins possessing molecular weights ranging from 18kDa of Lactobacillus fermentum KN02 [29] and L. lactis CWBI-B1410 [30] and 94 kDa of Lactococcus lactis [9] had been reported. Besides the molecular characterization of bacteriocin few other studies had determined surfactant property and resistance to enzymes such as amylase; DNase, RNase, lipase, Proteinase K and pepsin [7,8]. The gram positive, rod shaped bacteria Lactobacillus sp. YA2 isolated in the present study, besides biochemical characterization, was further confirmed by elucidating the sequence of its 16srRNA measuring a size of 1360 base pairs. In order to decipher its phylogentic characteristic and identification this partial sequence of the potential isolate was subjected to BLAST and similarity search. Based on the construction of phylogenetic tree the potential strain Lactobacillus sp. YA2 was identified to be Lactobacillus paracasei as it exhibited 100% similarity with the corresponding sequence of the organism available in GenBank . Since the bacteria Lactobacillus sp. YA2, isolated from yoghurt in the present study has been proved to possess significant potential of antimicrobial and probiotic property, further studies have been proposed to manipulate the organism for enhanced production of bacteriocin and to carryout comprehensive determination of physico-chemical and biological properties of bacteriocin through INDIAN JOURNAL OF APPLIED MICROBIOLOGY



in vitro and in vivo studies. Once the bacteria with phenomenal probiotic properties is established, its industrial production and supply to the consumers would be helpful in alleviating the complications of preventing and controlling digestive tract ailments.

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9. Rajaram, D. Halami, P.M. Chandrashekar, A. and Josephm, R., 2000, “Characterization of bacteriocinogenic strains of lactic acid bacteria in fowl and fish intestines and mushroom”, Food Biotech, 13, pp.121-136. 10. Jaya Prasad, R.W., Tagg, J.R. and Bibek Ray., 1999, “Bacteriocins of gram positive bacteria”, Microbio Reviews, 59, pp.171-200. 11. Manivasagam, E, Kumar, C.G, and Anand, S.K. 2010, “Significance of microbial biofilms in food industry: a review”, Inter J Food Microbio, 42, pp. 9-27. 12. Shaw Hardling, N.P., 1984, “Functional cultures and health benefits”. Intern Dairy J, 17, pp.1262– 1277. 13. Barakat, R., Moreno, I. and Oliveira, J., 2000, “Isolation of bacteriocin producing Lactic acid bacteria from meat and meat products and its spectrum inhibitory activity”, Brazilian J Microbiol, 35, pp. 137-144. 14. De Man, L., Avonts, L., Neysens, P., Hoste, B., Vancanneyt, M., Swings, J. And Callewaert, R., 1960, “Applicability and performance of the bacteriocin producer Lactobacillus amylovorus DCE 471 in type II cereal fermentations”, Inter J Food Microbiol, 90, pp. 93–106.


20 Jamesammal and Thiyagarajan 15. Hussain, J.N., Chung, Y. and Liu, W., 2003, “Biosynthesis and mechanism of the action of nisin and subtilin”, ESCOM Science Publishers In: Nisin and novel lantibiotics, Leiden., 10,pp. 287-302. 16. Duraisamy Senbagam, P.M. Chandrashekar, A. and Josephm, R. 2011, “Characterization of bacteriocinogenic strains of lactic acid bacteria in fowl and fish intestines and mushroom”, Food Biotech, 13, pp.121-136. 17. Barat Weimer, P., and Lan-szu Chou, W., 1999, “Molecular and cellular aspects of the secretory immunoglobulin system”, APMIS, 103, pp. 1-19. 18. Tambekar, S. and Bhutada, T. 2010, “Biopreservatives and probiotics for dry sausages”, Int J Food Microbiol, 83, pp. 233–244. 19. Singhai, S., Von Wright, A., Morelli, L., Marteau, P., Brassart, D., de Vos, V.M., Fonden, R., Saxelin, M., Collins, K., Mogensen, G., Birkeland, S.E. and Mattila-Sandholm, T., 2010, “Demonstration of safety of probiotics – A review”, Int J Food Microbiol, 44, pp. 93–106. 20. Larsen, J.J., Pot, B., Christensen, H., Rusul, G., Olsen, J.E., Wee, B.W., Muhamed, K. and Ghazali, H.M., 1993, “Identification of lactic acid bacteria from chilli Boa Malaysian Food Ingredient”, App Env Microbiol, 599-605. 21. Alpay, S., Jood, S. and Khetarpaul, N., 1999, “Effect of germination and probiotic fermentation on nutrient composition of barley based food mixtures”, Food Chem, 119, pp. 779–784. 22. Sablon, E., Contreras, B. and Vandamme, E., 2000, “Antimicrobial peptides of Lactic acid bacteria: mode of action, genetics and biosynthesis”, Adv Biochem Engine Biotech, 68, pp. 21-60. 23. Danielsen, M. and Wind, A., 2000, “Susceptibility of Lactobacillus sp. to antimicrobial agents”, Int J of Food Microbl, 82, pp. 1-11. 24. Ogunshe, A.A.O., Omotoso, M.A and Adeyeye, J.A., 2007, “In vitro antimicrobial characteristics of bacteriocins producing Lactobacillus strains from Nigrian indigenous fermented foods”, Afr J Biotech, 6, pp. 445-453. 25. Singhal, K., Joshi, H. and Chaudhary, B.L., 2009, “Antimicrobial activity, antibiotic resistance and bile salt hydrolase activity of Lactobacillus spp.”, Env Biol Conserv, 14, pp. 1-6. 26. Edna, D.I., Harrigan, G.G. and Goodacre, R., 2007, “Metabolic Profiling: Its Role in Biomarker Discovery and Gene Function Analysis, Kluwer Academic Publishers, Boston”. 27. Wanda, I. and Bonita, R., 1991, Dairy, Food and Environmental Sanitation, 18, pp. 499-503 28. Katrin strom, J., Jorgen Sjogren, M., Anders Broberg, A and Johan Schnuer, J., 2002, “Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides”, J App Microbiol, 68, pp. 4322. 29. Kanagaraj, S, and Nitiya, D., 2011, “Selection and design probiotics”, Int J Food Microbiol, 50, pp. 45-57. 30. Michel Bakar Diop, J., Robin Dubois-Dauphin, E., Emmanuel Tine, A., Abib Ngom, J., Jacqueline Destain, J and Philippe Thonart, J., 2007, “Bacteriocin producers from traditional food products”, Biotech Agron Social Envi, 11, pp. 275–281.





Microbial synthesis of Silver Nanoparticles (AgNPs) and evaluation of their biological activity D. Priya Dharshini1, P. Balaji1, T. Shanmugasundaram2, P. Rajendran3 and R. Balagurunathan2* Department of Microbiology, Vysya college of Arts and Science, Salem - 103, Tamil Nadu, India. Actinobacterial Research Laboratory, Department of Microbiology, Periyar University, Salem - 11, Tamil Nadu, India. 3 Sri Ramachandra Medical College & Research Institute, Porur, Chennai – 600 116, Tamil Nadu, India. 1 2

Abstract: The present study investigated the optimal conditions for maximum synthesis of silver nanoparticles through reduction of silver nitrate (AgNO3) to silver (Ag+) ions by the culture supernatant of Klebsiella pneumoniae, Pseudomonas fluorescence, Proteus vulgaris and Proteus mirabilis isolated from the soil samples of Kumaragiri Hills, Salem, Tamilnadu, India. The microbially synthesized silver nanoparticles were analyzed by visual observation and Fouier Transform Infra Red (FT-IR) spectrophotometer. The biosynthesized silver nanoparticles were further subjected to antibacterial activity and anti-oxidant activity. From the obtained results, the silver nanoparticles showed maximum peaks in FT-IR and the silver nanoparticles of K. pneumoniae and P. mirabilis showed a significant antibacterial effect along with anti-oxidant activity. As these silver nanoparticles are considered as the potential sources for treating the infections caused by drug resistant bacterial pathogens, further work is in progress on these aspects. Key words: Silver nanoparticles, biosynthesis, anti-oxidant.

Introduction Development of reliable and eco-friendly processes for the synthesis of metallic nanoparticles is an important step in the field of application of Nanotechnology. Metal nanoparticles have been widely employed in various fields because of their distinctive features such as catalytic,

*


22 Priya Dharshini et al

optical, magnetic and electrical properties [1, 2]. The non-magnetic metal nanoparticles have fluorescent and scattering properties, which make them more attractive for optical imaging. Metal nanoparticles can be synthesized by both chemical and biological method. Chemical synthesis of metal nanoparticles experiences several drawbacks such as, they are more hazardous. To overcome this, synthesis of metal nanoparticles using microorganisms were introduced, which is compatible with the green chemistry principles. The microorganisms such as bacteria, actinobacteria, fungi and yeast are known to produces a clean, non-toxic, ecofriendly nanostructure mineral crystals and metallic nanoparticles [3]. These nanoparticles are similar to the chemically synthesized nanoparticles in their strict control over size, shape and composition of the particles. Synthesis of silver nanoparticles can be categorized into intracellular and extracellular synthesis based on the place where nanoparticles are formed [1, 3]. In the intracellular method the ions are transported into the microbial cells to form the nanoparticles in the presence of enzymes. Whereas, in extracellular method trapping of metal ions on the surface of the cells and reducing ions in the presence of enzymes. This extracellular production of silver nanoparticles has more commercial importance in various fields. The present work reported the rapid microbial synthesis and characterization of silver nanoparticles by the culture supernatants of Klebsiella pneumoniae, Pseudomonas fluorescence, Proteus vulgaris and Proteus mirabilis isolated from the soil samples of Kumaragiri Hills, Salem area and its anti-bacterial activity, anti-oxidant activity were tested.

Materials and methods Collection of samples Soil samples were collected randomly from different locations [Lat: 11° 38’35’’ N; Long: 78° 11’6’’ E] around Kumaragiri Hills, Salem, Tamilnadu, India (Top layer 0 – 15cm).

Isolation of microorganisms The collected soil samples were processed further for the isolation of microbes by serial dilution method and by using nutrient agar medium. 100µl of the serially diluted soil sample was spread over the medium and incubated at 28°C for 24 h.

Identification of microorganisms From the above nutrient agar plate, colonies were selected based on their colony morphology and the zone of clearance around the colonies especially. The selected colonies were streaked on the nutrient agar plates and frequently sub-cultured. For the identification of the organisms, Gram’s staining and biochemical analysis were performed according to Bergey’s Manual of Determinative Bacteriology [4]. INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Microbial synthesis of Silver Nanoparticles and evaluation of their biological activity 23

Microbial Synthesis of nanoparticles of silver Test organisms were inoculated into flasks containing sterile nutrient broth and the flasks were incubated at 28°C for 24 h in 220 rpm. After the incubation period the culture was centrifuged at 8000 ×g and the supernatant used for the synthesis of silver nanoparticles. Three Erlenmeyer flasks, one containing supernatant with silver nitrate (AgNO3) (Merck, Germany, 99.9% pure) at a concentration of 0.1 g/L and the second containing only the supernatant and the third containing only silver nitrate solution, were incubated for 24 h [5, 6].

Synthesis of silver nanoparticles without microorganisms 100ml of solution of 1mM concentration silver nitrate was prepared for reduction into silver ions. The solution was taken in a 250ml Erlenmeyer flask and heated on water bath at 75ºC for 60 min. Reduction of silver nitrate to silver ions was confirmed by the colour change from colourless to brown. The formation of silver nanoparticles was also confirmed by visual observation [7]. The fully reduced solution was centrifuged at 5000 rpm for 30 min. The supernatant liquid was discarded and the pellet obtained was re-dispersed in de-ionized water. The centrifugation process was repeated two to three times to wash off any absorbed substances on the surface of the silver nanoparticles.

Synthesis of nanoparticles of sodium borohydride Silver nanoparticles were synthesized by chemical reduction [8] of silver nitrate using sodium borohydride as a reducing agent in aqueous solution without organic stabilizers. The process was carried out in 0.5 and 1 L flasks prewashed in concentrated nitric acid. The remains of the acid were removed from the glass walls by abundant amounts of de-ionized water. A 1 mM concentration of AgNO3 solution (room temperature) was mixed with fresh, ice-cold sodium borohydride solution of different concentrations under vigorous stirring. In the first 20 min, the mixture turned bright yellow. After complete injection (less than 2 min) of silver nitrate solution, stirring was stopped immediately and the final color of the mixture changed to dark yellow-bright brownish. The reaction mixture was kept in darkness to avoid the influence of daylight. The synthesis was conducted by varying volumes or molar concentrations of the reagents. 1 mM concentration of AgNO3 and 1 mM NaBH4 were taken in amounts according to the ratios 1:3. For a 1:3 ratio, the concentration of sodium borohydride was 1 mM.

Minimum inhibitory concentration The lowest concentration of agent that inhibited the visible growth of bacteria was considered as the minimum inhibitory concentration (MIC). Different concentrations (10µl - 50µl) of nanoparticles were tested against the bacteria to determine the MIC value [9]. The fresh cultures were obtained by inoculating the nutrient broth with test organisms and incubated at 37°C for 24 h in a laboratory shaker at 200 rpm. After incubation, the organisms are tested against the Ag nanoparticles (1 mg/ml). INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


Anti-bacterial assay The antibacterial assay was performed using 20 different human pathogenic organisms obtained from Madurai Kamaraj University, Madurai, Tamilnadu, India and assay was done by standard disc diffusion method [9]. These test organisms included Acinetobacter sp. 362 (Ab), Citrobacter sp. 377 (Cb), Citrobacter sp. 357 (Cb-1), Citrobacter sp. 378 (Cb-2), Citrobacter sp. 368 (Cb-3), E.coli 361 (Ec), E.coli 389 (Ec-1), E.coli 370 (Ec-2), E.coli 349 (Ec-3), E.coli 367 (Ec-4), K. rhinoscleromatis 350 (Kr), K. rhinoscleromatis 358 (Kr-1), K. rhinoscleromatis 356 (Kr-2), K. oxysporum 353 (Ko), K. oxysporum 351 (Ko-1), Aeromonas hydrolyticus (Ah), Bacillus subtilis (Bs), Pseudomonas aeruginosa (Pa), Proteus vulgaris (Pv), Staphylococcus aureus (Sa), Serratia marcescens (Sm), Vibrio parahaemolyticus (Vp). Mueller Hinton (MH) broth medium was used to cultivate bacteria. Fresh overnight cultures were taken and 100µl of culture was spread on the MH agar plates. Sterile paper discs of 5 mm diameter (containing 30 μg/ml silver nanoparticles) along with four standard antibiotic containing discs (30 μg/ml) were placed in each plate.

Anti-oxidant activity DPPH free radical scavenging assay The effect of Ag nanoparticle on DPPH radical was estimated according to the described procedure [10]. Two milliliters of 0.03mM (11.86 mg/l) methanolic solution of DPPH (Sigma) were added to 50 µl of a methanolic solution (1 mg/ml) of the antioxidant. The decrease in the absorbance at 515 nm was continuously recorded in a spectrophotometer for 16 min at room temperature. The scavenging effect (decrease of absorbance at 515 nm) was plotted against the time and percentage of DPPH radical scavenging ability of the sample was calculated from the absorbance value at the end of 16 min duration.

DPPH free radical scavenging assay at different concentration The antioxidant activity of Ag nanoparticles were measured in terms of hydrogen donating or radical scavenging ability [11], using the stable radical, DPPH method and modified DPPH method developed [12]. A methanolic solution (100µl) of sample extract at various concentrations (10 – 100 µg/ml) was added to 3.9ml (0.025 gL-1) of DPPH solution. The decrease in absorbance at 515 nm was determined continuously recorded in a spectrophotometer for 16 mins. The decrease in the absorbance was related to the concentration of the antioxidant and the radical, the molecular structure of the antioxidant, and its kinetic behavior. The scavenging effect (decrease of absorbance at 515 nm) was plotted against the time and percentage of DPPH radical scavenging ability of the sample was calculated from the absorbance value at the end of 16 min duration as follows: % DPPH activity = control OD - sample OD/ control OD × 100

Determination of reducing power The reducing power of silver nanoparticles was determined according to the standard method followed [13, 14]. The silver nanoparticles (1 mg) in 1ml of methanol was mixed with a phosphate INDIAN JOURNAL OF APPLIED MICROBIOLOGY



buffer (5ml, 0.2 M, pH 6.6) and potassium ferric cyanide (5ml, 1.0%); the mixture was incubated at 50°C for 20 min. A portion (5ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 650 g for 10 min. The upper layer of the solution (5ml) was mixed with distilled water (5ml) and ferric chloride (1ml, 0.1%), and then the absorbance was read using spectrophotometer at 700 nm. A higher absorbance of the reaction mixture indicated maximum reducing power.

Visual observation During biosynthesis, all the flasks were observed daily for visual colour change from white to brown, which indicates the biosynthesis of silver nanoparticles [15]. After colour change, the whole mixture was centrifuged at 5000 rpm for 15 mins and the cells and supernatant was observed to determine the intra (or) extra cellular synthesis of silver nanoparticles. If the cells are brown in colour, the silver nanoparticle synthesis is intracellular and if the supernatant is brown in colour, the silver nanoparticle synthesized extracellularly.

FT-IR analysis of dried mass after bio-reduction To remove any free biomass residue or compound that is not the capping ligand of the nanoparticles, the residual solution of 100ml after reaction was centrifuged at 5000 rpm for 10 min and the supernatant liquid was decanted. The resulting suspension was re-dispersed in 10ml sterile distilled water and centrifugation process was repeated for three times. Then the purified suspension was freeze dried to obtain dry powder. Finally, the dried nanoparticles were analyzed by FT-IR-JASCO 4100 Spectrophotometer [16].

Results Isolation and identification of microorganisms The microorganisms from the soil samples were isolated and identified based on Gram’s staining and biochemical methods which was determined with the Bergey’s Manual of Determinative Bacteriology. Table 1 shows the detailed characteristics of isolated organisms. The isolated organisms were K. pneumoniae, P. mirabilis, P. fluorescence and P. vulgaris.

Synthesis of silver nanoparticles Formation of silver nanoparticles from different microorganisms was confirmed by the reduction of silver nitrate to silver ions by the color change from colorless to brown in 1 mM aqueous silver nitrate solution. This was confirmed by Visual observation and FT-IR spectrophotometer.



Characterization of silver nanoparticles Visual Observation The colour change from white to brown colour was observed after the incubation period. The suspension was centrifuged at 5000 rpm for 15 min. As the colour of the supernatant changed to brown colour, it indicated extracellular synthesis of silver nanoparticles. Table.1. Biochemical characteristics of microorganisms isolated from soil samples Characteristic

Klebsiella pneumoniae

Pseudomonas fluorescence

Proteus vulgaris

Morphology

Straight rods arranged Straight rods singly or pairs

Straight or slightly curved rods but not helical

Straight rods

Size

0.3-1.0µm in diameter and 0.66.0µm in length

0.4-0.8µm in diameter and 1-30µm in length

0.5-1.08µm in diameter and 1.55.0 µm in length

0.4-0.8µm in diameter and 1-30µm in length

Gram Staining

Gram negative

Gram negative

Gram negative

Gram negative Motile with Peritrichous flagella

Proteus mirabilis

Motility

Non-motile

Motile with Peritrichous flagella

Motile with one or several polar flagella

Capsule

Capsulated

Non-capsulated

Non-capsulated

Non-capsulated

Indole Test

-

-

-

+

Methyl red Test

-

+

-

+

Voges Proskauer Test

-

-

-

Citrate + utilization Test

-

-

+

Urease Test

+

+

-

+

TSI test

A/A G+

A/K

K/K G-

A/K H2S +

Oxidase Test

-

-

+

-

Catalase Test

+

+

+

+

FT-IR spectrum Fig.1 shows the FT-IR spectrum of the silver nanoparticles of different microorganisms. Table 2 shows the absorption peaks and the related absorption bands for 1mM concentration of silver nanoparticles.




(a) Klebsiella pneumonia (b) Proteus mirabilis Fig. 1(a). FT-IR Spectrum of Nanoparticles of soil bacteria



(c) Pseudomonas fluorescence (d) Proteus vulgaris Fig. 1(b). FT-IR Spectrum of Nanoparticles of soil bacteria



Microbial synthesis of Silver Nanoparticles and evaluation of their biological activity 29 Table. 2 FT-IR absorption peak and the related absorption bands for 1mM concentration of silver nanoparticles Silver nanoparticles (1mM Concentration)

K. pneumoniae

P.fluorescence

Absorption peak

Absorption band

3852.5-3903.6 cm–1

C-H stretching vibration of alkenes

3750 cm–1

Asymmetrical O-H stretching

2962 cm–1

C-H stretching peaks of methyl groups

2345.1-2363.9 cm–1

Carbonyl group along with ammonium band

1650 cm–1

N-O stretch transisomer (Nitrate)

1399.5 cm–1

Polysaccharides (CO, CHO vibrations)

1113.1 cm–1

C-O stretchings

668.6-669.3 cm–1

C-H alkynes group

3852.5 – 3903.6 cm–1


3751.3 cm–1

C-H stretching vibration

2345.4 – 2364.8 cm-1 and 2931.7 cm–1

Cyclic alkanes

1648.6 cm–1

unconjugated linear alkanes

1392.4 cm

sulfates (organic)

1112.5 cm–1

C-O stretching vibrations in alcohol

–1

668.6 – 669.3 cm–1

C-H bending vibration to the alkynes group

3852.5 – 3903.6 cm


–1

3751.3cm-1& 2364.8 cm–1 P.mirabilis

2345.4

–


3434.0 cm–1

Free hydroxyl groups of alcohol and phenol

1648.9 cm–1

Unconjugated linear alkanes

1398.7 cm–1

Sulfates (organic)

1113.1 cm


668.6 – 669.3 cm–1


–1



P.vulgaris

3852.5 – 3903.6cm–1


3751.3 cm–1 & 2345.4 cm–1


3449.8 cm–1

N-H stretching compounds

2931.7 cm–1

Methyl group

1654.5 cm

Asymmetrical stretching in NO2 groups

1545.1 cm–1

Nitroso compounds (aliphatic)

1400.2 cm–1

Alkene C-H bending vibration

1114.1 cm–1


668.6 – 670.2 cm–1


–1

N=O

absorption

Minimum inhibitory concentration of silver nanoparticles Preliminary screening for minimum inhibitory concentration against microorganisms including Gram positive and Gram negative bacteria was carried out to determine whether the silver nanoparticles synthesized from microorganisms possessed any biological activity. Table 3 shows the MIC of silver nanoparticles of microorganisms against pathogenic microorganisms. Among the concentration tested, 1 mM concentration of silver nanoparticles of K. pneumoniae exhibited higher zone of inhibition against E. coli, K. oxysporum and P. aeruginosa with 14 mm at 50 µg/ml concentration. Similarly less zone of inhibition against Citrobacter sp., E. coli, K. rhinoscleromatis, P. vulgaris, S. aureus, S. marcescens with 6 mm at 10 µg/ml concentration. 1 mM concentration of silver nanoparticles of P. fluorescens exhibited higher zone of inhibition against E. coli with 16 mm at 50 µg/ml concentration, similarly with less zone of inhibition against Citrobacter, E. coli, V. parahaemolyticus with 5 mm at 10 µg/ml concentration. 1 mM concentration of silver nanoparticles of P. mirabilis exhibited higher zone of inhibition against Citrobacter sp., A. hydrolyticus, V. parahaemolyticus with 17 mm at 50 µg/ml concentration, similarly less zone of inhibition against E. coli, K. oxysporum with 5 mm at 10 µg/ml concentration. 1 mM concentration of silver nanoparticles of P. vulgaris exhibited higher zone of inhibition against E. coli, K. oxysporum with 16 mm at 50 µg/ml concentration, similarly less zone of inhibition was observed against Citrobacter with 6 mm at 10 µg/ml concentration.



Microbial synthesis of Silver Nanoparticles and evaluation of their biological activity 31 Table 3. Minimal inhibitory concentration of silver nanoparticles against pathogenic microorganisms Zone of Inhibition (mm) 1 mM nanoparticles used

Pathogens

Concentration of Klebsiella

used

pneumoniae (µg/ml)

Concentration of Pseudomonas

Concentration of Proteus

Concentration of

mirabilis (µg/ml)

Proteus vulgaris (µg/ml)

fluorescence (µg/ml)

10

20

30

40

50

10

20

30

40

50

10

20

30

40

50

10

20

30

40

50

Ab

7

7

9

10

12

7

9

10

12

12

6

8

9

11

12

7

8

12

11

12

Cb

8

9

11

11

13

9

11

11

13

15

9

10

12

16

17

7

10

11

14

15

Cb-1

6

5

7

11

13

5

7

11

13

12

6

7

11

12

14

8

10

10

11

14

Cb-2

7

7

8

11

11

7

8

11

11

12

8

7

9

13

12

6

9

9

10

11

Cb-3

8

5

7

11

12

5

7

11

12

15

8

8

11

14

15

8

10

12

13

14

Ec

8

6

7

10

11

6

7

10

11

14

5

9

12

14

15

8

10

11

12

14

Ec-1

6

5

12

8

8

5

12

8

8

10

6

7

10

11

13

8

10

13

14

15

Ec-2

7

6

10

12

13

6

10

12

13

14

7

10

10

12

14

8

11

12

13

16

Ec-3

8

9

11

13

14

9

11

13

14

16

9

11

12

14

15

10

12

13

13

13

Ec-4

8

6

11

12

12

6

11

12

12

13

8

11

10

12

13

8

9

11

14

16

Kr

6

8

10

11

12

8

10

11

12

12

8

10

10

12

13

8

10

11

13

14

Kr-1

8

7

11

12

13

7

11

12

13

15

9

11

11

15

16

9

11

12

12

13

Kr-2

8

9

11

11

12

9

11

11

12

13

8

11

15

16

16

9

11

12

12

14

Ko

8

6

9

12

14

6

9

12

14

14

5

9

11

12

13

7

10

13

15

16

Ko-1

9

7

11

10

12

7

11

10

12

15

9

11

11

14

15

7

9

10

12

14

Ah

8

6

11

9

10

6

11

9

10

12

9

11

13

15

17

9

11

11

12

15

Bs

7

6

10

11

12

6

10

11

12

13

9

10

13

14

14

7

8

10

11

12

Pa

9

6

10

12

13

6

10

12

13

14

8

10

12

12

14

8

12

12

13

14

Pv

6

6

9

8

11

6

9

8

11

13

7

9

10

11

11

6

8

10

11

12

Sa

6

7

7

8

10

7

7

8

10

11

6

7

7

9

11

7

9

10

11

13

Sm

6

6

8

7

9

6

8

7

9

10

7

8

8

10

11

8

10

10

11

13

Vp

9

5

10

10

10

5

10

10

10

12

8

10

10

15

17

10

11

12

13

15

Acinetobacter sp. 362 (Ab), Citrobacter sp. 377 (Cb), Citrobacter sp. 357 (Cb-1), Citrobacter sp. 378 (Cb-2), Citrobacter sp. 368 (Cb-3), E.coli 361 (Ec), E.coli 389 (Ec-1), E.coli 370 (Ec-2), E.coli 349 (Ec-3), E.coli 367 (Ec4), K. rhinoscleromatis 350 (Kr), K. rhinoscleromatis 358 (Kr-1), K. rhinoscleromatis 356 (Kr-2), K. oxysporum 353 (Ko), K. oxysporum 351 (Ko-1), Aeromonas hydrolyticus (Ah), Bacillus subtilis (Bs), Pseudomonas aeruginosa (Pa), Proteus vulgaris (Pv), Staphylococcus aureus (Sa), Serratia marcescens (Sm), Vibrio parahaemolyticus (Vp)


32 Priya Dharshini et al Table 4. Comparison of antimicrobial activity of silver nanoparticles and standard antibiotics against pathogenic microorganisms Concentration

Zone of Inhibition (mm)

of nanoparticle/

1mM concentration of silver nanoparticles

standard antibiotic

Klebsiella

Pseudomonas

Proteus

Proteus

used (µg/ml)

pneumoniae

fluorescence

mirabilis

vulgaris

Ab

10

10

12

Cb

11

13

12

Cb-1

9

10

Cb-2

9

Cb-3

13

Ec

Pathogens used

Standard antibiotic discs (µg/ml) K

Do

Cf

Ak

N

11

18

14

10

23

22

9

13

11

-

30

23

10

11

13

8

9

21

21

10

11

11

-

7

15

-

13

11

10

12

21

16

30

25

23

11

12

11

12

13

13

9

22

25

Ec-1

13

7

10

12

28

16

30

19

22

Ec-2

7

11

8

-

16

11

9

20

21

Ec-3

11

9

13

12

11

15

11

24

18

Ec-4

7

11

8

7

18

12

17

22

21

Kr

9

10

7

10

14

7

12

23

23

Kr-1

11

10

11

12

10

9

12

14

20

Kr-2

11

13

10

11

12

10

14

23

20

Ko

14

9

10

11

14

7

10

22

20

Ko-1

11

11

10

11

15

7

11

21

23

Ah

10

7

12

9

20

21

31

28

24

Bs

30

11

8

11

10

22

11

-

24

23

Pa

10

11

10

9

21

22

17

22

18

Pv

7

10

6

11

-

7

16

20

12

Sa

8

6

6

6

22

12

35

23

23

Sm

7

8

7

-

22

10

-

24

21

8

-

8

7

22

25

30

32

25

Vp

K- Kanamycin; Do- Doxycycline; Cf- Ciprofloxacin; Ak- Amikacin; N- Neomycin Acinetobacter sp. 362 (Ab), Citrobacter sp. 377 (Cb), Citrobacter sp. 357 (Cb-1), Citrobacter sp. 378 (Cb-2), Citrobacter sp. 368 (Cb-3), E.coli 361 (Ec), E.coli 389 (Ec-1), E.coli 370 (Ec-2), E.coli 349 (Ec-3), E.coli 367 (Ec4), K. rhinoscleromatis 350 (Kr), K. rhinoscleromatis 358 (Kr-1), K. rhinoscleromatis 356 (Kr-2), K. oxysporum 353 (Ko), K. oxysporum 351 (Ko-1), Aeromonas hydrolyticus (Ah), Bacillus subtilis (Bs), Pseudomonas aeruginosa (Pa), Proteus vulgaris (Pv), Staphylococcus aureus (Sa), Serratia marcescens (Sm), Vibrio parahaemolyticus (Vp).

Anti-microbial activity Antimicrobial activity of different microbial silver nanoparticles tested against pathogenic microorganisms is shown in Table 4. 1mM concentration of K. pneumoniae silver INDIAN JOURNAL OF APPLIED MICROBIOLOGY



nanoparticles showed significantly higher zone of inhibition of 14 mm against K. oxysporum at 30 µg/ml concentration followed by Citrobacter sp. and E. coli with 13 mm zone of inhibition. 1mM concentration of P. fluorescence and P. mirabilis showed maximum 13 mm zone of inhibition against Citrobacter sp. and K. rhinoscleromatis each and E. coli. P. vulgaris silver nanoparticles were the least effective with only 12 mm zone of inhibition for many of the pathogens tested at 30 µg/ml concentration. The antimicrobial activity of silver nanoparticles was compared with the known broad spectrum antibiotics with 30 mg/ ml of concentration

Antioxidant activity DPPH free radical scavenging assay The radical scavenging activity using a DPPH generated radical was tested with a fixed concentration of 50 µg/ml in 1mM of silver nanoparticles of different microorganisms along with ascorbic acid (Table 5). All the silver nanoparticles of microorganisms exhibited a very high radical scavenging activity. In terms of percentage, the inhibiting activity at 16 min was calculated to be in the following order ascorbic acid (17.54), 1mM concentration of K. pneumoniae silver nanoparticles (28.07), 1mM concentration of P. fluorescence (5.20), 1mM concentration of P. mirabilis (21.05) and 1mM concentration of P. vulgaris silver nanoparticles (10.52). Table 5. 1,1 – Diphenyl-2-picryl hydrazyl radical scavenging capacity of ascorbic acid and silver nanoparticles of different microorganisms Silver nanoparticle

OD value at 515nm

DPPH radical scavenging capacity (%)

Negative control ( Methanolic DPPH)

0.057

-

Positive control (Ascorbic acid)

0.047

17.54

Klebsiella pneumoniae

0.041

28.07


0.054

5.20

Proteus mirabilis

0.045

21.05

Proteus vulgaris

0.051

10.52

DPPH free radical scavenging assay at different concentration The scavenging activity of 1mM concentration of silver nanoparticles of different microorganisms and nanoparticles of silver nitrate and borohydride at different concentration was determined by DPPH assay and the results are shown in Table. 6. The 1mM concentration silver nanoparticles of P. fluorescence and P. mirabilis had a very high radical scavenging activity with a maximum of 61.40% and 54.38% at 100 µg/ml concentration. The 1mM concentration silver nanoparticles of P. vulgaris and K. pneumoniae had a low radical scavenging activity with a maximum of 50.87% and 40.35% at 100 µg/ml concentration. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


Similar experiment was carried out with nanoparticles of silver nitrate and sodium borohydride which resulted in a maximum of 36.84% at 100 µg/ml concentration in 1mM concentration of nanoparticles of silver nitrate Table 7. Nanoparticles of sodium borohydride maximum of 56.14% at 100 µg/ml concentration in 1mM concentration of nanoparticles. Similarly DPPH free radicals scavenging activity ascorbic acid experiment resulted in a maximum of 52.63% at 100 µg/ml concentration. Table 6. 1,1 – Diphenyl-2-picryl hydrazyl radical scavenging capacity of silver nanoparticles of different

microorganisms at different concentrations Conc. of silver nanoparticle (µg/ml)

Control OD value at 515nm

OD value at 515nm


Kp

Pf

Pm

Pv

Kp

Pf

Pm

Pv

10

0.063

0.067

0.068

0.072

-

-

-

-

20

0.060

0.064

0.062

0.066

-

-

-

-

30

0.064

0.056

0.056

0.063

-

17.54

1.75

-

40

0.061

0.052

0.050

0.062

-

8.77

12.28

-

50

0.055

0.043

0.049

0.057

3.50

24.56

14.03

0.00

60

0.048

0.039

0.044

0.054

15.78

31.57

22.80

5.26

70

0.047

0.036

0.040

0.047

17.54

36.84

29.82

17.54

80

0.044

0.032

0.036

0.039

22.80

43.85

36.84

31.57

90

0.037

0.028

0.032

0.033

35.08

50.87

43.85

42.10

100

0.034

0.022

0.026

0.028

40.35

61.40

54.38

50.87

0.057

Kp – Klebsiella pneumonia; Pf – Pseudomonas fluorescence; Pm – Proteus mirabilis; Pv – Proteus vulgaris

Determination of reducing power The reducing power of 1mM concentration of silver nanoparticles of different microorganisms is shown in Table 8. The reducing power of the nanoparticles increased with an increasing concentration. The 1mM concentration of silver nanoparticles of P. mirabilis and P. vulgaris appears to be less effective on reducing power with optical density value of 0.085 and 0.108. 1mM concentration of silver nanoparticles of K. pneumoniae and P. fluorescence appeared to be more effective on reducing power with optical density value of 0.137 and 0.115 at 700nm. The reducing power of the nanoparticles of silver nitrate and sodium borohydride is shown in Table 9. The 1mM concentration of nanoparticles of silver nitrate showed lesser reducing power with optical density of about 0.924 while the 1mM concentration of nanoparticles of sodium borohydride possessed the optical density of about 1.069 at 700nm. INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Microbial synthesis of Silver Nanoparticles and evaluation of their biological activity 35 Table 7. 1,1 – Diphenyl- 2-picryl hydrazyl radical scavenging capacity of nanoparticles of silver nitrateand sodium borohydride of different microorganisms Sample concentrations


OD value at 515nm

1Mm nanoparticles used

(µg/ml)

Silver nitrate

Sodium borohydride

10

0.086

0.061

0.057

-

-

20

0.081

0.059

-

-

30

0.089

0.058

-

-

40

0.080

0.055

-

3.50

50

0.076

0.041

-

28.07

60

0.061

0.031

-

45.61

70

0.056

0.029

1.75

49.12

80

0.051

0.027

10.52

52.63

90

0.049

0.025

14.03

56.14

100

0.036

0.025

36.84

56.14

Silver nitrate Sodium borohydride

Control OD at 515 nm

Table 8. Reducing power of the 1mM silver nanoparticle of microorganisms at different concentrations OD value at 700 nm Concentration of silver nanoparticle (µg/ml)

1mM Nanoparticles Klebsiella pneumoniae


Proteus mirabilis

Proteus vulgaris

10

0.078

0.073

0.066

0.069

20

0.079

0.085

0.067

0.069

30

0.083

0.062

0.071

0.072

40

0.085

0.077

0.066

0.075

50

0.089

0.086

0.070

0.079

60

0.093

0.094

0.071

0.085

70

0.097

0.098

0.075

0.087

80

0.097

0.114

0.079

0.089

90

0.108

0.128

0.083

0.095

100

0.115

0.137

0.085

0.108


36 Priya Dharshini et al Table 9. Reducing power of 1mM nanoparticles silver nitrate and sodium borohydride at different

concentration OD value at 700nm Sample concentration (µg/ml)

1mM nanoparticles used Silver nitrate

Sodium borohydride

10

0.701

0.981

20

0.753

1.049

30

0.794

1.050

40

0.813

1.065

50

0.827

1.052

60

0.845

1.053

70

0.899

1.055

80

0.907

1.058

90

0.917

1.062

100

0.924

1.069

Discussion A study on extra-cellular biosynthesis of silver nanoparticles (AgNPs) by the culture supernatants of K. pneumoniae, P. fluorescence, P. mirabilis and P. vulgaris were carried out [3, 5, 6]. Visual observation of the culture supernatant incubated with AgNO3 showed a colour change from yellow to brown whereas no colour change was observed in culture supernatant without AgNO3 or media with AgNO3 solution alone. The appearance of a yellowish brown colour in AgNO3-treated culture supernatant suggested the formation of silver nanoparticles (AgNPs). This is also compared with the nanoparticles of silver nitrate and sodium borohydride which appears to brown in colour [8, 15]. The silver nanoparticles with their unique chemical and physical properties are proving as an alternative for the development of new antibacterial agents. The silver nanoparticles have also found diverse applications in the form of wound dressings; coatings for medical devices, silver nanoparticles impregnated textile fabrics, etc. The advantage of using silver nanoparticles for impregnation is that there is continuous release of silver ions and the devices can be coated by both the outer and inner side hence, enhancing its antimicrobial efficacy [17]. The burn wounds treated with silver nanoparticles shows better cosmetic appearance and scarless healing. Thus, it can be concluded that metallic silver has been in use since ancient times. However, with the advent of silver nanoparticles and its major use as an antimicrobial agent, much experimental trials are needed to understand the toxicity [18, 19]. The concentration of AgNO3 plays an important role in the synthesis and size reduction of nanoparticles; different concentrations of AgNO3 were used. The maximum synthesis of AgNPs was observed at 1mM concentration. The control experiments involving different concentrations of AgNO3 showed no synthesis of nanoparticles. Similar results INDIAN JOURNAL OF APPLIED MICROBIOLOGY



were obtained for the synthesis AgNPs. The results clearly indicated that a 1mM concentration of Ag+ ions was most appropriate for the maximum synthesis of AgNPs indicating that the sizes of AgNPs decrease with increasing concentrations of AgNO3 [20]. However, when the concentration of AgNO3 is more than 5mM, the sizes of AgNPs were altered. The confirmation of the particle synthesis by visual observation [15] and IR spectroscopy analysis [21] (after completion of the reaction) reveals the presence of silver nanoparticles. The nanoparticle synthesis by microbial route has been proved to be highly effective against multi-drug resistant pathogenic bacteria [22]. Antibacterial activity of silver nanoparticles against pathogenic bacteria were investigated and compared with the standard antibiotic discs. Antibacterial effects of Ag nanoparticles obeyed a dual action mechanism of antibacterial activity, i.e., the bactericidal effect of Ag+ and membrane-disrupting effect of the polymer subunits [23]. There is an increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body, and to prevent the deterioration of fats and other constituents of foodstuffs. In both cases, there is a preference for antioxidants from natural sources rather than from synthetic sources [11]. Therefore, there is a parallel increase in the use of methods for estimating the efficiency of anti-oxidants [12]. One such method that is currently popular is based upon the use of the stable free radical diphenylpicrylhydrazyl (DPPH). The DPPH radical has been widely used to test the ability of compounds as free-radical scavengers or hydrogen donors and to evaluate the antioxidative activity of silver nanoparticles [13, 14]. DPPH radical scavenging activities of all the extracts were dose dependent. The decrease in absorbance of the DPPH radical caused by antioxidant was due to the scavenging of the radical by hydrogen donation. The silver nanoparticles of different microorganisms were examined for their antioxidant activities. Among the four different samples P. mirabilis showed the highest antioxidant activity than the other three silver nanoparticles of different microorganisms at 100 µg /ml concentration. Reducing power of the silver nanoparticles of different microorganisms is also a supporting feature for its antioxidant activity [14]. The reducing properties are generally associated with the presence of reductones, which have been shown to exhibit antioxidant action by breaking the chain reactions by donating a hydrogen atom. The 1mM concentration of silver nanoparticles of P. mirabilis and P. vulgaris appear to be less effective on reducing power with optical density value of 0.085 and 0.108. 1mM concentration of silver nanoparticles of K. pneumoniae and P. fluorescence appear to be more effective on reducing power with optical density value of 0.137 and 0.115 at 700nm. Similarly, reducing power of the nanoparticles of silver nitrate and sodium borohydride was reported by this study [8, 24]. The 1mM concentration of nanoparticles of silver nitrate has less reducing power with optical density of about 0.924 while the 1mM concentration of nanoparticles of sodium borohydride has the optical density of about 1.069 at 700nm.



Conclusion The present study is a report on the synthesis of silver nanoparticles from certain soil bacteria, K. pneumoniae, P. mirabilis, P. fluorescence and P. vulgaris, possessing significant antibacterial and anti-oxidant activities. UV–visible absorbance spectral analysis confirmed the surface plasma resonance of microbially synthesized silver nanoparticles, which can be considered as an efficient, eco-friendly and simple process. Further characterization by XRD, EDAX, SEM, TEM etc. of the silver nanoparticles are in progress. Acknowledgement: The authors thank the Principal and Head of Department of Microbiology, Vysya College, Salem, Tamilnadu, India for providing the research facilities.

References 1.

Kalishwaralal, K., Deepak, V., Ramkumar Pandian S. and Gurunathan, S., 2009, “Biosynthesis of silver and gold nanoparticles using Brevibacterium casei”, Bioresour Technol, 100, pp.5356–5358.

2. Tanja, K., Ralph, J., Eva, O. and Claes-Goran, G., 2010, “Silver-based crystalline nanoparticles, microbially fabricated”, Proc Natl Acad Sci, 96, pp.13611–13614. 3.

Minacian, S., Shahverdi, A.R., Nohi, A.S. and Shahverdi, H.R., 2008, “Extracellular biosynthesis of silver nanoparticles by some bacteria”, J Sci IAU, 17(66), pp.1-4.

4.

Holt, J.C., Kreig, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T., 1994, “Bergey’s Manual of Determinative Bacteriology”, 9, pp.259-274.

5.

Jeevan, P., Ramya, K. and Edith Rena, A., 2011, “Extracellular biosynthesis of silver nanoparticles by culture supernatant of Pseudomonas aeruginosa”, Ind J Biotechnol, 11, pp.72–76.

6. Kalishwaralal, K., Deepak, V., Ramkumarpandian, S., Nellaiah, H. and Sangiliyandi, G., 2008, “Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis”. J Matlet, 10, pp.52-54. 7.

Mann, S. and Ozin, G. A., 1996, “Synthesis of inorganic materials with complex form”, Nature, 382, pp.313 – 318.

8.

Mandal, S., Arumugam, S.K., Pasricha, R. and Sastry, M., 2005, “Silver nanoparticles of variable morphology synthesized in aqueous foams as novel templates”, Bull Mater Sci, 28 (5), pp.503–510.

9. Singh, M., Singh, S., Prasad, S. and Gambhir, I.S., 2008, “Nanotechnology in medicine and antibacterial effect of silver nanoparticles”, Dig J Nanomater Bios, 3, pp.115-122. 10. Brand-Williams,W., Cuvelier, M. E.and Berset, C., 1995, “Use of a free radical method to evaluate antioxidant activity”, Lebensm Wiss Technol, 28, pp.25-30. 11. Moure, A., Franco, D., Sineiro, J., Dominguez, H., Nunez, M. J. and Lema, J. M., 2000 “Evaluation of extracts from Gevuina avellana hulls as antioxidants”, J Agri Food Chem, 48, pp.3890–3897. 12. Oyaizu, M., 1986, “Studies on products of browning reaction: anti-oxidative activity of products



Microbial synthesis of Silver Nanoparticles and evaluation of their biological activity 39 browning reaction prepared from glucosamine”, Japan J Nutr, 44, pp.307-315. 13. Yen, G. C., Duh P. D. an P. D. Chuang, D. Y., 2000, “Antioxidant activity of anthroquinones and anthrone”, Food Chem, 70, pp.437-441. 14. Sanchez-Moreno, C., Larrauri J. A. and Saura-Calixto, F. A., 1998, “A procedure to measure the antiradical efficiency of polyphenols”, J Sci Food Agri, 76, pp.270-276 15. Senapati, S., Ahmad, A., Khan, M.I., Sastry, M. and Kumar, R., 2005, “Extracellular biosynthesis of bimetallic Au- Ag alloy nanoparticles”, Small, 1, pp.517-520. 16. Zhang, Y., Peng, H., Huang, W., Zhou, Y. and Yan, D., 2008, “Facile preparation and characterization of highly antimicrobial colloid Ag or Au nanoparticles”, J Colloid Interface Sci, 325, pp.371-382. 17. Parikh, R.Y., Singh,S., Prasad, B.L.V., Patole, M.S., Sastry, M. and Shouche, Y.S., 2008, “Extracellular synthesis of crystalline silver nanoparticles and molecular evidence of silver resistance from Morganella sp.: Towards understanding biochemical synthesis mechanism”, 110, pp.162-68. 18. Verma, V.C., Kharwar, R.N. and Gange, A.C., 2010, “Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus”, Nanomedicine, 5(1), pp.33-40. 19. Rai, M., Ingle, A., Gade, A. and Bawaskar, M., 2009, “Fusarium solani: a novel biological agent for the extracellular synthesis of silver nanoparticles”, J Nanopart Res, 11, pp.2079-2085. 20. Sastry, M., Ahmad, A., Khan, M.I. and Kumar, R., 2003, “Biosynthesis of metal nanoparticles using fungi and actinomycetes”, Curr Sci, 85, pp.162-170. 21. Tiwari, D.K., Behari, J. and Sen, P., 2008, “Time and dose-dependent antimicrobial potential of Ag nanoparticles synthesized by top-down approach”. Curr Sci, 95, pp.647- 655. 22. Ragunathan, R., Rani, C. and Prasanna Kumar, K., 2010, “Biosynthesis of Silver Nanoparticles Using Lactobacillus acidophilus (Probiotic Bacteria) and its Application”, Intern J Nanotechnol Appli, 4, pp.217-222. 23. Venkataraman, A., Raghunnandan, D., Mahesh, B. D., Balaji, S. D., Basavaraja, S. and Manjunath,S.Y., 2010, “Microwave- assisted rapid extracellular synthesis of stable bio- functionalized silver nanoparticles from guava (Psidium guajava) leaf extract”, J Nanopart Res, 12, pp.2163- 2177. 24. Lewis, L.N., 1993, “Chemical catalysis by colloids and clusters”, Chem Rev., 93, pp.2693.



Copyright 2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, pp. 40-45

Effect of Phyllanthus amarus extract on SphH gene of Leptospira autumnalis studied by an in-house PCR R. Saravanan*, P. Saradhai and E. Rani Department of Biotechnology, Biomedical Engineering Research Foundation, Affiliated to Periyar University, Salem – 636 122 , Tamil Nadu, India. Abstract: Leptospirosis is an important re-emerging infectious disease in human resulting from direct or indirect contact with the infected animals’ urine. Pathogenic Leptospira requires ions for their growth and these spirochetes use their hemolysins such as the SphHingomyelinases to obtain ions from host red blood cells during their infection which results in anemia. Due to the side effects encountered in most cases of chemotherapy today, people around the globe show more interest in alternative medicines especially from herbal sources. The present study was designed to evaluate the effect of Phyllanthus amarus extract on SphH gene of Leptospira autumnalis using an in-house PCR. During the observation of the amplified products from the reaction mixture, the L. autumnalis, which were treated with the plant extract, were not showing the specific DNA band as that of the negative control (triple distilled water). Interestingly the positive control samples (extract untreated L. autumnalis) evinced specific DNA band with respect to the SphH gene amplification. Keywords: Leptospira autumnalis, SphH gene, Phyllanthus amarus, PCR

Introduction Leptospirosis is considered as an important re-emerging infectious disease worldwide [1]. The disease in human results from direct or indirect contacts with the infected animal’s urine. The pathogenic Leptospira cause diverse damage in humans due to the virulence factors produced by the bacteria and among them the hemolysins are the most important virulence factor. Hemolysin

*


Effect of Phyllanthus amarus extract on SphH gene of Leptospira autumnalis 41

SphH (sphingomyelinases) gene has been reported in most of the Leptospiral serovars. Pathogenic Leptospira requires ions for their growth and these spirochetes probably use their hemolysins such as the sphingomyelinases to obtain ions from host red blood cells during infection which results in erythrocyte lysis in host [2]. Doxycycline may be used to prevent infection but it shows some side effects such as diarrhea or loose stools, nausea, abdominal pain, vomiting and may cause tooth discoloration if used in person below 8 years of age. Penicillin may cause a temporary exacerbation of the symptoms [3]. Due to the side effects produced by the chemotherapy today, people around the globe are showing growing interest and preference to alternative medicines especially of herbal extracts. An effective course of treating leptospirosis still remains unsolved problem. To overcome the adverse reaction by the above drugs, herbal-based therapeutics had been used in treating leptospirosis [4]. Phyllanthus amarus (L.) belongs to the family Euphorbiaceae commonly called as Bahupatra in India. Traditionally, these plants are ayurvedic herbs used in southern India for the treatment of liver diseases [5]. Studies on the herbal extracts effect on molecular level damage of Leptospira are scanty. In view of these drawbacks and to emphasize the importance of alternative medicine for this dangerous disease this research work was designed to evaluate the effect of P. amarus extract on SphH gene of Leptospira autumnalis using an in-house PCR.

Materials and Methods Leptospira Culture Leptospira autumnalis cultures in EMJH semisolid medium (Difco, Sparks, UK) were procured from the WHO Center for Reference and Research on leptospirosis, Brisbane, Australia.

Collection of Plant material The plant Phyllanthus amarus were collected from ABS medicinal plant research center, Karippatti, Salem, Tamilnadu, India during the month of December 2011. Fresh plant material were washed under running tap water, air dried and then homogenized to fine powder and stored in airtight bottles.

Preparation of aqueous extract Ten grams of air-dried powder was added to distilled water and boiled on slow heat for 2 h [6]. It was then filtered through 8 layers of muslin cloth and centrifuged at 5000g for 10 min. The supernatant was collected. This procedure was repeated twice. After 6 h, the supernatant collected at an interval of every 2 h, was pooled together and concentrated to make the final volume one-fourth of the original volume [7].

Leptospira autumnalis DNA extraction 1ml of L. autumnalis culture was exposed with 50µl of P. amarus extract and incubated at room temperature for 24 h [8]. In the positive control vials instead of P. amarus extract 50µl sterile triple INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

42 Saravanan et al

distilled water was added. After incubation period the respective DNA samples were isolated from both the set of vials by using the QIAamp DNA extraction kit (QIAGEN, Hilden, Germany).

Amplification of Leptospira autumnalis SphH gene The in-house PCR was attempted using the following primers for the amplification of SphH gene [9]: 5’- GGCTCGAGATGCGAAACATTTTCCGAAA-3’ (Forward) 5’- CCAGATCTTCGACTTTAGGATCGTTAT-3’ (Reverse) In a sterile 0.5ml microfuge tube, the following contents were added: 4µl of 10X PCR buffer, 7µl of Template DNA, 1 µl of forward and reverse primer, 2µl of 25 mM magnesium chloride, 4µl of dNTPs, 2µl of Taq DNA polymerase and 29µl of triple distilled water. The tubes were vortexed gently and spun the tubes for ten seconds to settle the contents and the tubes were placed in a controlled temperature heat block of the thermocycler. The thermal profiler involved 30 cycles of each of the following: Denaturation at 94ºC for 1 min 30 sec, Primer annealing at 56ºC for 1 min and Polymerization at 72ºC for 1 min. In the final step, the set up was kept at 72ºC for 3 min extension to ensure the complete polymerization [10]. After the amplification the PCR products were taken in gel loading buffer and run by electrophoresis in 1.5% agarose gel along with the untreated leptospiral DNA as positive control and plain triple distilled water as the negative control. The DNA bands were visualized and documented (BIO-RAD, USA) [11].

Fig . 1 Amplification of SphH gene of Leptospira autumnalis



Effect of Phyllanthus amarus extract on SphH gene of Leptospira autumnalis 43

Results Polymerase chain reaction of the P. amarus extract treated and untreated (positive control) leptospiral DNA along with the sterile plain triple distilled water (negative control) yielded interesting results. The positive and negative controls were consistently giving concordant results by the in-house PCR for more than five repetitions. The extract treated leptospiral DNA template did not amplify for the SphH gene in spite of eight repeated attempts. Successful demonstration of the specific DNA band of approximately 1.6 kbp in the positive control indicated the reliability of the standardized in-house PCR. The damage caused by the extract of P. amarus on the leptospiral DNA prevented its amplification and the result was similar to that of the negative control (Fig. 1). Thus the absence of DNA band of leptospiral DNA on the agarose gel indicated that the active principle of P. amarus was effective in damaging the SphH (sphingomyelinase) gene.

Discussion The available therapies for leptospirosis in modern medicine are very limited, potential alternatives from traditional medicine and their respective mechanisms of the action are worth investigating [12]. Recently, significant attention has been focused on plant extracts and biologically active compounds isolated from popular plant species [13]. However, the effect active principle of of plant extracts on the pathogen’s at molecular level is poorly studied. In the present study Phyllanthus amarus was observed to be effectively damaging the leptospiral DNA. Therefore the template DNA could not give any amplification when targeted for this most virulent gene (SphH). An earlier study revealed antileptospiral effect of P. amarus and Eclipta alba against L. icterohaemorrhagiae. In their study both aqueous and methanol extracts at the concentration of 1μg/ml was reported to cleave Leptospira DNA completely [14]. The complete DNA damage in the present study this phenomenon of complete DNA damage could be attributed for the failure in the amplification of the SphH gene in spite of many repeated trials. Indian medicinal plants such as Plectranthus amboinicus extracts are reported to be not only hapato-renal protective but also they did possess anti-leptospiral effects [8]. Another herbal extract study also reported antileptospiral activity of Eclipta alba when tested by both tube dilution and micro dilution technique [15]. An in- vitro experiment conducted using Zingiber officinale (Ginger), Ocimum sanctum (Tulasi) and Piper nigrum (Black pepper) against various Leptospira serogroups like L. australis, L. pomona, L. autmnalis, L. icterohaemorragiae, L. canicola, L. copenhageni and L. semaranga had revealed that except L. autmnalis all the other serogroups were susceptible to Ocimum sanctum (Tulasi) and Piper nigrum (Black pepper). Further, the L. autmnalis a most dominant serovar especially in west Chennai was susceptible only to the extracts of Z. officinale [16]. The present PCR study has an advantage of utilizing the tool for more accurate diagnosis of leptospirosis this is in full agreement with a previous study stating that the cross reactions caused by exposure to leptospires of the same group can occur for example infection by L. balcanica and L. medenensis can produce false positive L. hardjo reactions. This technique was earlier used by Truccolo et al. [17] with a single set of arbitrary primers in quantitative PCR to evaluate the INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

44 Saravanan et al

ampicillin, afloxacin and doxycycline for the treatment of experimental leptospirosis. The study is in full agreement with the present PCR study. Therefore the present study suggests that the use of Phyllanthus amarus could play a vital role in covering the basic health needs in developing countries and could offer a new source of herbal medicine for leptospirosis.

References 1.

Kamath, S.A. and Joshi S.R., 2003, “Re-emerging infections in urban India-focus Leptospirosis”, J. Assoc Physicians India, 51, pp. 247-248.

2.

Carvalho, E., Barbosa, A.S., Gómez, R.M., Oliveira, M.L.S., Romero, E.C., Gonçales, A.P., Morais, Z.M., Vasconcellos, S.A. and Ho, P.L., 2010, “ Evaluation of the expression and protective potential of leptospiral sphingomyelinases”, Current Microbiol, 60, pp. 134-142.

3.

Shenoy, V.S., Chowdhury, A.A., Bhalgat., P.S. and Juvale, N.I., 2006, “Pulmonary leptospirosis: an excellent response to bolus methylprednisolone”, Postgrad Med J, 82, pp. 602-606.

4.

Emmanouilides, C.E., Kohn, O.F. and Garibaldi, R., 1994, “Leptospirosis complicated by a JarischHerxheimer reaction of adult respiratory distress syndrome: Case report”, Clin Infect Dis, 18, pp. 1004-1006.

5. Portillo, V.R., Freixa, B., Adzet, T. and Canigueral, S., 2001, “Antifungal activity of Paraguyan plants used in traditional medicine”, J Ethnopharmocol., 76, pp. 93-98. 6.

Sadiaque, J., Rqobab, N.A., Bughaith, M.F. and E.L-Gindey, A.R., 1989, “The bioactivity of certain medicinal plants on the stabilization of RBC membrane system”, Fitoterapia LX., 01, pp. 525-532.

7.

Parekh, J., Nair, R. and Chanda, S., 2005, “Preliminary screening of some folklore medicinal plants from western India for potential antimicrobial activity”, Ind J Pharmacol., 37, pp. 408-409.

8.

Nirmaladevi, K. and Periyanayagam, K., 2011, “Nephroprotective effect of Plectranthus ambonicus spreng on glycerol induced acute renal failure”, Herbal Tech Industry, pp. 15-16.

9.

Seoung, H.L., Kyung, A.K., Yong, K.P., Inwha, S., Min, J.A.K. and Yong, J.L., 2000, “Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovars”, Gene, 254, pp. 19-28.

10. Gravekamp, C., Van de Kemp, K.H., Franzen, D. M., Carrington, G.J., Schoone, G.J., Van Eys, C.O., Everard, R.A., Hartskeerl. A. and Terpstra, W.J., 1993, “Detection of seven species of pathogenic leptospires by PCR using two sets of primers”, J Gen Microbiol, 139, pp. 1691-1700. 11. Sambrook, J., Fritsch, E.F. and Maniatis, T., 1987, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NewYork. 12. Coelho de S.G., Haas, A.P.S., Von P.G.L., Schapoval, E.E.S. and Elisabetsky, E., 2004, “Ethnopharmacological studies of antimicrobial remedies in the south of Brazil”, J Ethnopharmacol, 90, pp. 135-143. 13. Nikaido, H., 1999, “Microdermatology: Cell surface in the interaction of microbes with the external world”, J Bacteriol, 181, pp. 4-8.



Effect of Phyllanthus amarus extract on SphH gene of Leptospira autumnalis 45 14. Chandan, S., Umesha, S. and Balamurugan, V., 2012, “Antileptospiral, Antioxidant and DNA damaging properties of Eclipta alba and Phyllanthus amarus”, Sci Report, 231, pp. 1-8. 15. Prabu, N, Joseph Pushpa Innocent, P, Chinnaswamy, K., Nataraja S., Lakshmi S., 2008, “In-Vitro evaluation of Eclipta alba against serogroups of Leptospira interrogans”, Ind J Pharma Sci, 70, pp. 788 – 791. 16. Yuwvaranni, S. and Thiruvengadam, S., 2010, “Distribution, isolation, identification, characterization and effect of chlorine, antibiotics and herbs on bacterial genus Leptospira in Chennai”, Int J Chem Sci, 8, pp. 520-526. 17. Truccolo, J., Charavay, F., Merlen, F. and Perolat, P., 2002, “Quantitative PCR assay to evaluate ampicillin, afloxacin and doxycycline for treatment of experimental leptospirosis”, Antimicrob Agents Chemother, 46, pp. 848 – 853.



Copyright 2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, pp.46-53.

Characterisation and Preliminary screening of Biosurfactant producing Bacteria isolated from Hydrocarbon contaminated soils P. Saminathan1* and P. Rajendran2 1 2

Department of Microbiology, Karpagam University, Coimbatore - 641 021, Tamilnadu, India. Department of Microbiology, Sri Ramachandra University, Porur, Chennai, Tamilnadu, India

Abstract: Biosurfactants are surface active compounds produced by microorganisms. These molecules reduce surface tension between aqueous solutions and hydrocarbon mixtures. In this study one hundred samples of hydrocarbon saturated soil were collected from thirty different areas in Chennai, Tamilnadu, India. Seventy two bacterial strains were isolated and cultured by enriching carbon (Glycerol) source. Each culture medium was subjected to screening so as to confirm biosurfactant production. Studies were conducted to determine surface tension and emulsification activity. The result revealed that fifty four strains of bacteria showed surface tension in the range between 0.072Nm-1 to 0.026Nm-1. The emulsifying capacity evaluated by the E24 emulsification index ranged from 85- 36 % EA. Proposed to carry out further investigation on its efficacy in the bioremediation of hydrocarbon contaminated soils. Keywords: Biosurfactant, Surface tension, Emulsification Index, Bioremediation.

Introduction Pollution is the phenomenon of introduction of contaminants into a natural environment that causes instability, disorder, harm or discomfort to the ecosystem. Among the pollutants, oil pollution is the major significant. Accidental and deliberate crude oil spills have been still continue to be a significant source of environmental pollution and pose a serious environmental problem due to the possibility of soil contamination [1]. Oil contamination in soil has been a major threat to the environment because of poor solubility. Soil contamination with petrol, diesel and engine oils are becoming one of the major environmental issues. To remove soil *

Author for Correspondence; E.mail:[email protected]

Characterisation and Preliminary screening of Biosurfactant producing Bacteria �� 47

contaminated with oils, bioremediation provide an effective and efficient strategy to speed up the clean-up processes. One of the approaches to enhance biodegradation of oil is to use biosurfactants. Biosurfactants are a structurally diverse group of surface-active molecules synthesized by Microorganisms. These molecules reduce surface and interfacial tensions in both aqueous solutions and hydrocarbon mixtures, which makes them potential candidates for enhancing oil recovery [2, 3]. The biosurfactants have unique properties of structural diversity, possibility of cost effective production and biodegradability make them a promising choice for application in enhancing hydrocarbon bioremediation. The first step in the microbial degradation of oil is to use biosurfactants which could increase solubility of oil in water to enhance the bioavailability of the hydrophobic substrates, leading to higher oil degradation rates. The present study aimed to isolate an effective biosurfactant producing bacteria which can be used for bioremediation of oil spills and to idntify a potential strain of bacteria for bioremediation of oil spills in earth space. In order to characterize and preliminarily screen biosurfactant producing bacteria, they were isolated from hydrocarbon saturated soils i.e., the soils in the petroleum filling station and automobile units.

Materials and Methods Sampling area Composite soil samples saturated with hydrocarbons were collected from various gas stations (Petrol pumps) and automobile stations located in the city of Chennai, Tamilnadu, India.

Sampling Thirty different areas of soil, having contamination with different types of petroleum fractions i.e., Petrol, Diesel and Kerosene, were selected for sampling purpose. Soil samples were collected in sterilized plastic bags for the isolation studies.

Isolation and enumeration of bacterial isolates from the samples One gram of soil sample was diluted with 99ml of sterile distilled water. The samples were kept in shaker at 200 rpm for 24-48 h. After incubation, samples were serially diluted from 10-1 to 10-6 in sterile distilled water. From the dilutions 0.1ml was spreaded over that 20ml of sterile Nutrient agar, Cetrimide agar, Trypticase soy agar [4] (37oC for 24 h) and Starch casein agar [5] (30oC for 7-10 days).The number of total aerobic bacteria were recorded and colonies showing promising growth on selective media were then subsequently pure cultured and maintained in the same media as slants. The bacterial isolates were then characterized by morphologically and using different biochemical tests.

Extraction of biosurfactants The growth, biosurfactant production and screening of carbon sources were studied using mineral salt media. Erlenmeyer flasks of 1000ml capacity containing 250ml of the mineral salt medium INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

48 Saminathan

and

Rajendran

with glycerol as the carbon source were individually inoculated with 5ml of each inoculum [6]. The flasks were incubated in rotary shaker incubator at 30oC for 24-48 h. The culture obtained was used for the extraction of biosurfactants. The culture medium was centrifuged at 350g for 20 min and then the isolated supernatant was adjusted to pH of 2.0 by adding 5 mol/H2SO4 for the biosurfactant precipitation. The precipitates were extracted with two volumes of diethely ether/methanol (1:1,v/v) mixture. Evaporation of the solvent yielded crude biosurfactants [7].

Preliminary Screening of biosurfactant producing organisms The identified colonies were tested for their biosurfactant production by the following methods.

Surface Tension test Based on the surface tension reducing capacity, the isolated colonies were screened for biosurfactant production and it was calculated by using standard drop weight method [8]. Surface tension (T) = mg/3.8rNm-1 Where, m: Mass of one drop of the liquid; g: acceleration due to gravity; r: radius of the capillary tube To determine the surface tension, mass of the medium has to be calculated and simply weighing the drop of the medium. Mass of one drop of the medium, m= W2-W1/total droplet Where, W2- Weight of the sample with beaker; W1- Weight of the empty beaker.

Emulsification Index (E24) Biosurfactants has the ability to emulsify various hydrocarbons. The emulsifying property of the biosurfactant was carried out with petrol, diesel and kerosene. The emulsification index on hydrocarbons was calculated by standard method [9]. E24= (Height of the emulsified layer/Total height of the hydrocarbon) × 100.

Results A total of 72 isolates belonging to Pseudomonas spp. and Actinomycetes spp. were isolated from 100 oil contaminated soil samples (Fig-1). The density of Pseudomonas spp. and Actinomycetes spp. were in the range of 5.2 × 104 to 4.2 × 106. The occurrence of different bacterial isolates from soils were identified as follows: 32 isolates (44.4%) of Pseudomonas aeruginosa, 25 isolates (34.7%) INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Characterisation and Preliminary screening of Biosurfactant producing Bacteria �� 49

of Pseudomonas fluorescence, 7 isolates (9.7%) of Pseudomonas putida and 8 isolates (11.1%) of Actinomycetes spp. (Table 1; Fig. 2) Area wise incidence indicated that 90% (27/30) of sampling area were positive (Table 2). Extracted biosurfactants were found to be turbid and yellowish brown in colour. The surface tensions of the medium were reduced from the range of 0.072 Nm-1 to 0.026 Nm-1. Among the 72 isolates of bacteria, 54 were found to effectively reduce the surface tension (Table 3a & 3b). Emulsification index were also calculated and tabulated (Table 4). Table 1. Percentage of isolation of bacteria from hydrocarbon contaminated soils Bacteria

No. isolated

% of Isolation

P. aeruginosa

32

44.4

P. fluorescence

25

34.7

P. putida

7

9.7

Actinomycetes spp.

8

11.1

Fig. 1. Areas chosen for isolation of bacteria from hydrocarbon contaminated soils


50 Saminathan

and

Rajendran

Table 2. Area wise incidence of bacterial isolates from hydrocarbon contaminated soils. Bacterial isolates

No.of isolates out of 30 areas

Percentage (%)

P.aeruginosa

23

76.6

P. fluorescence

21

70.0

P. putida

7

23.3

Actinomycetes spp.

8

26.6

Fig. 2. Number of organisms isolated from hydrocarbon contaminated soils. Table 3a. Results of reduction of surface tensions values of the samples Name of the sample

Surface tension Nm-1

Control

0.072

P. aeruginosa-2642

0.026

P. fluorescence-1749

0.028

P. putida-7525

0.030

Rhodococcus rhodochorous-3552.

0.028

P. aeruginosa Samples (25)

0.026 to 0.049

P. fluorescence Samples (19)

0.028 to 0.049

P. putida Samples (5)

0.030 to 0.044

Actinomycetes spp. Samples (5)

0.028 to 0.048



Characterisation and Preliminary screening of Biosurfactant producing Bacteria �� 51 Table 3b. Results of non-reduced surface tension values of the samples Name of the sample

Surface tension Nm-1

Control

0.072


0.071 to 0.072

P.fluorescence Samples (6)

0.071


0.071 to 0.072


0.071

Discussion The present study was conducted with an objective to isolate a potential beneficial bacteria from contaminated soils located in the city of Chennai, India. A higher prevalence of Pseudomonas species compared with other genera of bacteria isolated from the same sample was recorded. This may be attributed to moisture and warm condition and other characteristics of the soil samples. Some species of Pseudomonas have been recently use as an agent for bioremediation which could clear the environmental pollution and cause partial or complete degradation of pollutants [10]. The fermentation of biosurfactant was carried out using 30 g/L glycerol as the sole carbon source [11]. The fermentation of P. aeruginosa was first investigated by using vartious carbon sources. Rhamnolipid production induced by glycerol was much higher than that of other substrates including glucose, vegetable oil and liquid paraffin. Therefore the glycerol was concluded as the most effective substrate for the production of biosurfactants. Table 4. Determination of Emulsification index (E24) of the samples. Name of the samples

Emulsification Index(%) Petrol

Diesel

Kerosene

Control –Tween -80

85

82

83

P. aeruginosa-2642

82

80

78

P. fluorescence-1749

80

79

75

P. putida-7525

80

82

81

Rhodococcus rhodochorous-3552 .

79

82

81


36-82

42-79

38-75

P. fluorescence Samples (19)

37-79

44-73

43-74


49-70

51-69

49-71


42-73

49-71

52-65


52 Saminathan

and

Rajendran

Biodegradation of crude oil by microbial agents is a natural process, in which the higher amount of the polluting oil is used as an organic carbon source. This causes the breakdown of petroleum components to lower molecular compounds or transformed into the other organic compounds such as biosurfactants [12]. Biosurfactant production is associated with uptake of water soluble carbon substrates such as glycerol by bacteria. The bacteria isolated in the present study did not require any hydrophobic substrates, however, they needed soluble carbohydrates in the form of glycerol for their growth. The biosurfactant producing ability of the strain Streptomyces spp. VITDDK3 was tested by different screening methods. Both Gram positive and Gram negative bacteria are capable of producing surface active agents and they are amphipathic extracellular lipopeptides[13]. Production of lipopeptide biosurfactant producing bacterium Rhodococcus sp. TW53 was reported from pacific ocean deep sea sediments[14]. Biosurfactant activity of free fatty acids and glycolipids extracted from R. erythropolis (3C-9-Strain) isolated from sea shore soil had been demonstrated by earlier studies [15]. The surface tension values of the culture supernatants ranged from 0.026 Nm-1 to 0.072 Nm-1. The decrease in surface tension indicated the production of extracellular surface active compounds. Further investigation showed that among the 72 isolates, 54 of them were effectively reduce the surface tension. They were also observed to effectively emulsify the petrol than diesel and kerosene. Among these 54 bacterial isolates, nine of them displayed maximal activity as determined by emulsification index method. Future studies involving identification of bacteria, optimization of production conditions and the efficacy of bacteria on bioremediation of hydrocarbon contaminated soil have been proposed.

References 1.

Trindade, P.V.O., Sobral, L.G., Rizzo, A.C.L., Leite, S.G.F., Soriano, AV., 2005, “Bioremediation of a weathered and recently oil-contaminated soils from Brazil: a comparison study”, Chemosphere, 58, pp. 515-522.

2. Sarkar, A.K., Goursand, J.C., Sharma, N.M. and Georgiou, G.,1989, “A Critical evaluation of MEOR Process”, In situ , 13, pp.207-238 3.

Singer, M., 1985, “Microbial biosurfactants”, Microbes oil Recovery, 1, pp.19-38.

4.

Lowbury, E.J.L. and Collins, A.G., 1955, “The use of a new Cetrimide product in a selective medium for Pseudomonas aeruginosa”, J Clin Pathol, 8, pp.47

5. Deepika, T.L., Kannabiran and Dhanasekaran, D., 2009, “Diversity of antidermatophytic Streptomyces in the coastal region of Chennai, Tamilnadu, India”, J Pharm res, 2(1), pp. 22-26. 6. Sekar, K.V., Sarita Kumar, Nagasathya, A., Palanivel, S. and Subramanyam Nambaru, 2010, “Effective biosurfactant production by Pseudomonas aeruginosa and its efficacy on different oils”, JALRB, 1:1, pp.40-44.



Characterisation and Preliminary screening of Biosurfactant producing Bacteria �� 53 7.

Zhang Guo-liang, W.U., Yue-ting, Qian Xin-ping, Meng Qin, 2005, “Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of Rhamnolipids”, J Zhejiang Univ Sci, 6 B (8), pp.725730.

8.

Tadros T., 2005, “Adsorption of surfactants at the air/liquid and liquid/liquid interfaces. In. Applied surfactants: Principles & Applications”, Weinheim: Wiley VCH, pp.81-82.

9.

Cooper, D., Goldenberg, B., 1987, “Surface active agents from 2 Bacillus species”, Appl Environ Microbiol, 53(2), pp.224-229.

10. Haas, D. and Defago, G., 2005, “Biological control of Soil-borne pathogens by Pseudomonas fluorescens”, Nature Rev Microbiol, 3, pp.307-319. 11. Matsufuji, M., Nakata, K. and Yoshimoto,A., 1997, “High production of Rhamnolipids by Pseudomonas aeruginosa growing on ethanol”, Biotechnology letters, 19, pp.1213-1215. 12. Chhatre, S.A., Purohit, H.J., Shanker, R., Chakrabarti, T. and Khanna, P., 1996, “Bacterial consortia for crude oil spill remediation”, Wat Sci Tech, 34, pp.187-193. 13. Molitt,M.C. and Neilan, B.A., 2000, “The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides”, FEMS Microbiol Lett, 191, pp.159-167. 14. Peng, F., Wang, Y., Sun, F., Z.Liu,Q.Lai and Shao, Z., 2008, “A novel lipopeptides produced by a Pacific Ocean deep-sea bacterium, Rhodococcus sp. TW 53”, J Applied Microbiol, 105: 698-705. 15. Peng, F., Liu, Z., Wang, L. and Shao, Z., 2007, “An oil degrading bacterium: Rhodococcus erythropolis strain 3C-9 and its biosurfactants”, J Applied Microbiol, 102, pp.1603-1611. 16. Ron, E.Z. and Rosenberg, E., 2001, “A Review of natural roles of Biosurfactants”, Environ Microbiol, 3(4), pp.229-236. 17. Desai, J. and Banat,I.M., 1997, “Microbial production of surfactant and their commercial potential”, Am Soc Microbiol, 61(1), pp.47-64. 18. Anyanwu and Chukwudi, 2010, “Surface activity of extracellular products of Pseudomonas aeruginosa isolated from petroleum-contaminated soil”, Int J Envir. Sci, 1(2), pp. 225-235. 19. Prince, R.C., 1997, Bioremediation of oil spills,Trends Biotechnol, 15, pp.158-160.




Nanotechnology in Biology: A Brief Overview Sivaprakash Rathinavelu* Center for Molecular Medicine and Therapeutics, PSG Institute of Medical Sciences & Research, Coimbatore – 641 004, Tamilnadu, India. Abstract: Nanotechnology is a multidisciplinary field that applies principles from engineering, physics, chemistry, and biology for creating novel nano material with unique properties. Nanoparticles exhibit properties that can be applied usefully to a number of fields including biology and medicine. Newer materials at the subcellular level have been identified or are in the process of development for advancing various fields including sciences, imaging, medicine, diagnostics, and disease treatment. However, due to the nanoparticles size, they can pass through cell membranes and induce damage. Their high surface area to volume ratio also makes nanoparticles highly catalytic and reactive raising concerns about their safety. In this review a brief overview of nanotechnology and some of its potential applications in biology and related toxicity are discussed. Key Words: Nanotechnology, Nanoparticles, Nanobiotechnology, Nanomedicine, Quantum Dots.

Introduction Nanotechnology is a significant new area of science that is multidisciplinary, continually evolving and developing different scientific disciplines. It integrates aspects of physics, chemistry, biology, material sciences, biotechnology and chemical engineering and involves working with nanoparticles and devices at the nanoscale level, which is about 1 to 100 nanometers [1]. Nanoscience uses techniques and tools to make high performance nanoscale particles and products that have significant impact on all areas of society and in almost all industries [2]. Interdisciplinary approach at the nanoscale level resulting in the creation of nanomaterials and devices with a wide range of commercial applications is termed Nanotechnology. Theoretical capability of nanotechnology, envisioned as early as 1959 by physicist Richard Feynman, came to the experimental stage only in 1981, when IBM scientists in Zurich, Switzerland,

*Author for Correspondence; E-mail: [email protected]

Nanotechnology in Biology: A Brief Overview 55

built the first scanning tunneling microscope that allowed resolution enough to visualize single atoms [3,4]. Eric Drexler in his first book ‘Engines of Creation’ (1986) introduced the term ‘nanotechnology’ and ways to manufacture extremely high performance miniaturized machines [5]. Since then nanotechnology has evolved widely with designing, modeling, and fabricating molecular structures and devices for advances in biology, chemistry, physics, engineering, computer science, electronics and mathematics. This area of research is currently of intense, due to a wide variety of potential applications in biomedical, optical, and electronic fields. However as evidenced with other fields, emergence of nanotechnology is also associated with numerous social, legal, cultural, ethical, religious, philosophical and political implications [6].

Nanoparticle Properties Nanoparticles are purposefully engineered or naturally occurring particles in which one or more dimensions measure less than 100 nanometers in size. Nanoparticles and nanomaterials significantly differ from the bulk materials in their behavior. At nano scale, nanoparticles possess unusual physical, chemical and optical properties derived from complex atomic and molecular characteristics [7, 8]. Their electronic and optical properties and their chemical reactivity as small clusters are completely different from that of each component in bulk. Nanoparticles are influenced by surface tension, polar and electrostatic interactions or covalent interactions, quantum mechanics, Van der Waals attraction, surface plasmon resonance and superparamagnetism making gravity insignificant [9]. This makes well defined conditions and experiments important in synthesizing nanoparticles and to evaluate complex theoretical model predictions on nanoparticles.

Classification Nanoparticles are presented generally as an aerosol, a suspension or an emulsion. They are generally categorized based on their geometry, morphology, uniformity, composition, and agglomeration [10]. Geometry: Based on nanoparticles geometry, nanoparticles are divided as 1D, 2D and 3D nanomaterials. 1D nanomaterials are particles with one dimension in the nanometer scale. Thin films or surface coatings, circuitry of computer chips, anti-reflection and hard coatings on eyeglasses are examples in this category. They are referred as nanolayers, nanoclays, nanosheets and nanoplates. 2D nanomaterials have two-dimension in the micro or nanometer scale. They form elongated structure and include nanotubes, nanofibers, and nanorods. Carbon nanotubes and carbon fibers are good examples in this category. 3D nanomaterials have all three dimensions in the nanometer scale and are referred as equiaxed nanoparticles, nanogranules and nanocrystals. Fullerenes, dendrimers and QD are examples in this category. Morphology: Flatness, sphericity, and aspect ratio is considered as morphological characteristics. High aspect ratio nanoparticles include nanotubes and nanowires. Small-aspect ratio morphologies INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

56 Sivaprakash Rathinavelu

include spherical, oval, cubic, prism, helical, or pillar shaped nanoparticles. Collections of many particles exist as powders, suspension, or colloids. Composition: Nanoparticles can be composed of a single constituent material or be a composite of several materials. The nanoparticles found in nature are often agglomerations of materials with various compositions, while pure single-composition materials can be easily synthesized by a variety of methods. Agglomeration: Based on their chemistry and electro-magnetic properties, nanoparticles can exist as dispersed aerosols, as suspensions/colloids, emulsions, or in an agglomerate state. Nanoparticles due to their high surface energy can come together to form clusters or agglomerates. For example, magnetic nanoparticles tend to cluster, unless their surfaces are coated with a non-magnetic material. In an agglomerate state, nanoparticles may behave as larger particles, depending on the size of the agglomerate. Uniformity: Uncontrolled agglomeration due to van der Waals forces can result in microstructural in-homogeneities. Interactive forces, either attractive or repulsive, between the nanoparticles determine the fate of individual or collective nanoparticles. This necessitates nanoparticles synthesis at high level of purity and uniformity of structure to be of use in private, industrial and military sectors.

Nanoparticle Synthesis Based on the strategies used to synthesize nanoparticles, they are classified as natural, incidental or engineered. Physical, chemical and biosynthesis methods are commonly employed for synthesizing nanoparticles with the “bottom-up” or “top-down” approaches [11]. In the “bottom-up” approach, materials and devices are built from molecular components which assemble themselves chemically by principles of molecular recognition. This method of nanoparticle fabrication involves condensation of atoms or molecular entities in a gas phase or in solution. In the “top-down” approach, nano-objects are constructed from larger entities without atomic-level control. This approach aims at miniaturizing current technologies in which materials are processed to fabricate microscopic objects. It involves milling or attrition, chemical methods, and volatilization of a solid followed by condensation of the volatilized components. Biosynthesis is a kind of bottom-up eco friendly approach using microorganisms that does not lead to accumulation of toxic chemicals as by products. Using this approach, gold, silver, platinum, mercury, selenium, Cadmium telluride, and a variety of other metal nanoparticles have been produced extracellularly or intracellularly in microorganism [12].

Nanotechnology Applications in Biology Application of nanotechnology principles to biology/biotechnology, by which classical microtechnology approach is employed to manipulate molecular, genetic and cellular processes in INDIAN JOURNAL OF APPLIED MICROBIOLOGY



developing products for use in diverse fields, is termed Nanobiotechnology. With a wide range of potential applications it has become one of the areas in research and development seeing huge growth in the recent years. Nanoparticles are being used or are in different stages of evaluation in sciences, medicine, agriculture, and industry [13-16]. The unique physical, chemical, and biological properties of materials at the nanoscale enable novel applications and functions. Compared to fineparticles, nanoparticles have a dramatic increase in chemical and physical activities, such as ion release, adsorption ability etc. which makes them attractive for use in various fields [9]. Potential applications of nanotechnology in the near future will be focused on, but not restricted to, areas of energy production, storage and conversion; air pollution remediation; agricultural productivity enhancement; vector and pest detection and control; water treatment; imaging, disease diagnosis and monitoring; molecular imaging, drug screening and delivery; and food processing and storage. Biosensors: A sensor that integrates a biological element with a physiochemical transducer to produce an electronic signal proportional to a single analyte which is read by a detector is termed biosensor. Based on the biological element used, biosensors are broadly classified as enzyme biosensors, immunosensors, DNA sensors and microbial sensors [17]. In addition to the biosensors are electrical and electrochemical sensors, optical, thermal and mass sensitive sensors with variable uses. Nanotechnology is enabling the development of sensitive biosensor materials that can detect the presence of very small amounts of toxins, pathogens, and spoilage in foods and food processing facilities, monitoring of diseases, and detection of bioterrorism agents, detection of environmental toxins and assessing effectiveness of remediation processes [18-21]. Diagnostics and imaging: Nanotechnology is expanding the options on currently available diagnostic methods to improve on the cost effectiveness, sensitivity, specificity, miniaturization, multiplex detection and speed and time of diagnosis [22-25]. Microfluidic lab-on-a-chip assays are now available in genomics and proteomics which have added advantages such as smaller dimensions, lower sample consumption, high-throughput ability and ease of automation [26, 27]. This helps in reducing volume and the use of expensive reagents, increase automation thereby reducing inter and intra assay variability, and provide additional information on cellular and molecular interactions. Bead ARray Counter (BARC), is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents [28]. DNA probes are patterned onto the microfabricated chip directly above a GMR sensor, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. Recently researchers have created a chip with a series of nanowires that are coated with DNA sequences of disease-causing bacteria or viruses. If DNA from one of those pathogens is present in INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


an analyte, it binds to the nanowire with the matching sequence, a process that changes the wire’s conductivity [29]. This change in conductivity of a particular nanowire is read out by a transistor on a conventional microchip. This device is a step forward towards a universal handheld diagnosis system that can detect hundreds of pathogens in a single sample giving an instant read out. A new class of optical reporters called quantum dots (QD), which are semiconductor nanocrystals, are used as an alternative to organic dyes and fluorescent proteins. They are generally in the 2-10 nanometer size range and possess controllable, optical and electrical properties, which stem from their composition. They are brighter and more stable against photobleaching and have controllable emission wavelengths, sharper emission profiles, robust signal strength, and a single source for excitation. QD have proved useful in a wide range of applications from multiplexed analysis such as DNA detection, in vivo imaging and diagnostics, and tracking and cell sorting [30-33]. Specially designed QD conjugated to targeting antibodies fluoresce brightly after binding to diseased cells. This fluorescence can then be picked up by new, specially developed, advanced imaging systems, enabling the accurate pinpointing of a disease even at a very early stage. Nanosystems capable of effectively sorting sparse cells from blood and other tissues are significant in the detection and diagnosis of various genetic defects. They take advantage of the unique properties of sparse cells manifested in differences in deformation, surface charges and affinity for specific receptors and/or ligands [34]. Nanotools such as atomic force microscopy (AFM), scanning electron microscopy (SEM), scanning near field optical microscopy (SNOM), transmission electron microscopy (TEM), surface enhanced raman scattering (SERS), surface plasmon resonance (SRP) and fluorescence resonance energy transfer (FRET) are used in imaging diagnostics for nanoscale detection and analysis of nanosized molecules and structures [35-37]. Therapeutics: Nanotechnology based platforms for single cell studies, microcytometry and cell sorting, screening of compound libraries, can aid in lead identification in drug discovery. This increases the potential for efficient and multiple drug screening thereby reducing the cost of drug development. Nanoparticles as therapeutics can be delivered to targeted sites, including locations that cannot be easily reached by standard drugs. Nanodrugs such as organic dendrimers, hollow polymer capsules, and nanoshells are designed to carry an effective dosage of drug needed for the patient that will be released only at times when specific molecules are present or when triggered externally [35]. Similar approaches can also be employed in imaging applications [30]. These nanodelivery systems are capable of targeting different cells and extracellular elements in the body and due to their nano size can also readily traverse membrane boundaries to deliver drugs, genetic materials, and diagnostic agents specifically to the targets. Due to targeted tissue specific delivery, harmful side effects of potent medications can be avoided by reducing the effective dosage needed to treat the patients. Even drugs with poor bioavailability can be used in therapy with the help of nanotechnology. Nanotechnology-based methods to deliver drugs only to cancerous cells have shown promise, but they do not work for all cancers. This method of drug delivery can overcome INDIAN JOURNAL OF APPLIED MICROBIOLOGY



the painful side effects of conventional chemotherapy that can wreak havoc on healthy tissues. Thus nanotechnology can revolutionize therapeutics and newer strategies are being developed for diagnosis and treatment of cancer, neurodegenerative diseases, osteoporosis and a variety of other diseases [38-44].

Nanoparticle Toxicity Nanoparticles beneficial to the industry may also induce biological toxicity. The toxicity depends on size, aggregation, composition, crystallinity, etc. [45-47]. Contact with nanomaterials can likely occur, during production or usage, via the skin, lungs or gastrointestinal tract; and translocation to other vital organs through the bloodstream can potentially disrupt cellular processes causing disease [1, 48]. Particles below 20 nm can be taken up by the skin and those below 10-50 nm can enter cells via receptors. Injections and implants are other possible ways of exposure to nanoparticles. Nanometer sized particles are created in countless physical processes and exposure to them may have potential health risks, ranging from benign to lethal [49]. Many diseases such as cancer and neurodegenerative diseases are associated with dysfunction of basic cell processes such as cell proliferation, metabolism and cell death [46]. Nanoparticles have been found to interfere with these basic cellular functions. Cationic nanoparticles in microarray studies have been found to influence genes controlling cell proliferation, differentiation and apoptosis. Polycationic nanoparticles are used in gene delivery and can induce immediate or delayed cytotoxicity through necrosis or through apoptosis, and compromise transcription and translation. This potentially affects protein expression thereby limiting restoration of gene function [50-52]. Micelles as drug carriers have the ability to solubilize poorly soluble drugs and increase their bioavailability. They can stay in the blood long enough to provide gradual accumulation of drugs in the required pathological areas. However, monomer constituents of polymeric micelles, depending on their nature, can influence cell apoptosis or necrosis, or both [53]. Hence characterization of individual nanoparticles and their dispersions are essential for in vitro evaluation of their biological effects, since each nanoparticle shows unique chemical and physical properties. Certain monomeric constituents act as inhibitors of protein efflux pumps in polarized endothelial cells of the bloodbrain barrier and could potentially interfere with transport of modulators and homeostatic mediators in the central nervous system [54]. Polymeric micelles employed in cisplatin delivery can induce differential gene expression in certain cells compared to free cisplatin alone depicting the influence of nanoparticles on cellular gene expression [55]. Nanoparticles, depending on their composition and size, are able to enter cells and interact with subcellular structures to induce oxidative stress and production of inflammatory cytokines. Inside the cells, nanoparticles are found in the outer-cell membrane, cytoplasm, mitochondria, lipid vesicles, nuclear membrane, or within the nucleus. Depending on their localization inside the cell, the nanoparticles can damage organelles or DNA, or ultimately cause cell death. This interaction may lead to irreversible organ damage; and cause varied pathologies in respiratory, gastrointestinal, nervous and cardiovascular systems [48]. Toxicity of QD used in molecular imaging is directly INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


related to oxidation of the nanoparticle core/shell material, leading to the release of free cadmium ions which are toxic to cells. QD can also damage DNA and disrupt normal cell activity. Nanomolar quantum dot concentrations altered expression of genes involved in the cell cycle regulation. Cadmium selenide QD used in imaging are lethal to cells under UV irradiation, as this releases highly toxic cadmium ions. The intracellular distribution of QDs in cells and the associated nanoscale effects are deleterious to cells [56]. The adverse health effects of nanoparticles on the respiratory tract depend on the residence time, particle size, composition and structure. Smaller particles have higher toxicity and are correlated with impaired macrophage clearance, inflammation, accumulation of particles, and epithelial cell proliferation, followed by fibrosis, emphysema, and tumors. In the GI tract, nanoparticles are associated with inflammatory bowel disease, Crohn’s disease and colon cancer. Nanoparticles can reach central nervous system directly through axons of the olfactory pathway or from systemic circulation by directly crossing the blood-brain barrier or through the olfactory bulb and induce inflammation in the brain. Accumulation of high concentrations of metals like copper, aluminum, zinc and iron in the brain with associated oxidative stress can lead to functional loss and cell damage causing neurodegenerative diseases such as Parkinson’s, Pick’s and Alzheimer’s diseases. Nanoparticles in the circulatory system are associated with the occurrence of arteriosclerosis, blood clots, arrhythmia, heart diseases, and cardiac arrest. Occurrence of autoimmune diseases, such as systemic lupus erythematosus, scleroderma, and rheumatoid arthritis are associated with exposure to some nanoparticles [48].

Conclusion Nanotechnology offers new ways to address challenges in a wide range of settings. With recent development of nanotechnology, engineered nanomaterials are being embraced by the industry for a variety of purposes. It is already having an impact in life sciences, electronics and nanobiotechnology, in particular on diagnostics, imaging and drug delivery. Black soot and mineral powders have been in use as cosmetics for thousands of years in ancient Egypt, and some of them still continue to be used today in products like sun screens. Nanoparticles are also in use in dental fillings, orthopedic products, photovoltaic cells, and water filtration and catalytic systems. The levels of direct and indirect exposure to these nanoparticles and their toxicological and environmental effects have not been completely elucidated. However, with recent research data, there is heightened concern that nanomaterials may negatively impact public health, and that engineered nanomaterials may become a source of nanoparticle pollution when not safely manufactured, handled, disposed or recycled. Uniquely engineered nanomaterials with new sizes, shapes and physicochemical properties can adversely affect human health. Majority of the toxicity studies on nanoparticles have been carried out either in vitro or in animal models. Intra and inter species variations make translating cellular and immunological toxicity results from animal models to humans difficult requiring further validation. Influence of differences in nanoparticle size, surface area, concentration, particle chemistry, and structure, and exposure medium, routes of exposure on toxicity in humans need to be further studied. INDIAN JOURNAL OF APPLIED MICROBIOLOGY



Even though nanoparticles provide us with the ability to reduce cost, miniaturize tests, and are proven efficient than macroparticles in several ways, ethical considerations and strict guidelines with biocompatibility assessments are essential before being approved for extensive use in humans. Acknowledgement: The author is thankful for the support provided by the authorities in PSG Institute of Medical Sciences, Coimbatore during the preparation of this article.

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Nanotechnology in Biology: A Brief Overview 63 33. Xing, Y., and Rao, J., 2008, “Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging”, Cancer Biomarkers, 4, pp. 307–319. 34. A. M. Morawski, P. M. Winter, K. C. Crowder, et al., 2004, “Targeted nanoparticles for quantitative imaging of sparse molecular epitopes with MRI”, Magn Reson Med, 51, pp. 480–486, 2004. 35. McNeil, S.E., 2005, “Nanotechnology for the Biologist’’, J Leukocyte Biology, 78, pp. 585-594. 36. Choi, H.S., and Frangioni, J.V., 2010, “Nanoparticles for Biomedical Imaging: Fundamentals of Clinical Translation”, Mol Imaging, 9, pp. 291–310. 37. Bhushan, B., 2010, “Handbook of Nanotechnology”, Springer, Heidelberg, Dordrecht, London, New York. 38. Helene, A., and Berg Van Den, A., 2004, “Microtechnologies and nanotechnologies for single-cell analysis” Curr Opin Biotechnol, 15, pp. 44-49. 39. Bawa, R., 2008, “Nanoparticle-based Therapeutics in Humans: A Survey”, Nanotechnol Law & Business, pp. 135-155. 40. Rathod, K.B., Patel, M.B., Parmar, P.K., et al., 2011, “Glimpses of current advances of Nanotechnology in Therapeutics”, Int J Pharm Pharm Sci, 3, pp. 8-12. 41. Ochekpe, N.A., Olorunfemi, P.O., and Ngwuluka, N.C., 2009, “Nanotechnology and Drug Delivery Part 1: Background and Applications”, Trop J Pharm Res, 8, pp. 265-274. 42. Ochekpe, N.A., Olorunfemi, P.O., and Ngwuluka, N.C., 2009, “Nanotechnology and Drug Delivery Part 2: Nanostructures for Drug Delivery”, Trop J Pharm Res, 8, pp. 275-287. 43. Srikanth, M., and Kessler, J.A., 2012, “Nanotechnology—novel therapeutics for CNS disorders” Nature Reviews Neurology, 8, 307-318. 44. Sinha, R., Kim, G.J., Nie, S, and Shin, D.M., 2006, “Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery”, Mol Cancer Ther, 5, pp. 1909-1917. 45. Nel, A., Xia, T., Mädler, L., and Li, N., 2006, “Toxic Potential of Materials at the Nanolevel,” Science, 311, pp. 622-627. 46. Albanese, A., Tang, P.S., and Chan, W.C.W., 2012, “The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems”, Ann Rev Biomed Engine, 14, pp. 1-16. 47. Brayner, R., 2008, “The toxicological impact of nanoparticles”, Nano Today, 3, pp. 48-55. 48. Suh Yah, C., Simate, G.S., and Iyuke, S.E., 2012, “Nanoparticles toxicity and their routes of exposures” Pak J Pharm Sci, 25, pp.477-491. 49. Song,Y., Li, X., and Du, X., 2009, “Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma”, Eur Respir J, 2009; 34: 559–567. 50. Pan, Y., Leifert, A., Ruau, D., et al., 2009, “Gold Nanoparticles of Diameter 1.4 nm Trigger Necrosis by Oxidative Stress and Mitochondrial Damage”, Small, 5, 18, 2067–2076. 51. Seob Cho, W., Kim, S., Han, B.S., et al., 2009, “Comparison of gene expression profiles in mice following intravenous injection of 4 and 100 nm-sized PEG-coated gold nanoparticles”, Toxicol Lett, 191, pp. 96–102.


64 Sivaprakash Rathinavelu 52. Jeong Eom, H., and Choi, J., “p38 MAPK Activation, DNA Damage, Cell Cycle Arrest and Apoptosis As Mechanisms of Toxicity of Silver Nanoparticles in Jurkat T Cells”, Environ Sci Technol., 44, pp 8337–8342. 53. Moghimi, S. M., Hunter, A. C., Murray, J. C., and Szewczyk, A., 2004, “Cellular distribution of nonionic micelles”, Science, 303, pp. 626–627. 54. Yang, Z. ., Liu, Z.W., Allaker, R.P., et al., 2010, “A review of nanoparticle functionality and toxicity on the central nervous system” J R Soc Interface, 7, pp. S411-S422. 55. Nishiyama, N., Koizumi, F., Okazaki, S., et al., 2003, “Differential gene expression profile between PC-14 cells treated with free cisplatin and cisplatin-incorporated polymeric micelles”, Bioconjugate Chem, 14, pp. 449-457. 56. Clift, M.J., Brandenberger, C., Rothen-Rutishauser, B., et al., 2011, “The uptake and intracellular fate of a series of different surface coated quantum dots in vitro”, Toxicol, 286, pp. 58-68.





In vitro Antibacterial activity of Cardiospermum halicacabum extracts against Bacterial isolates from Pus samples N. Manikandan, B. Radha, N. Vijaykanth, M. R. Ramesh kumar and N. Arunagirinathan* Post Graduate and Research Department of Microbiology & Biotechnology, Presidency College (Autonomous), Chennai–600 005, Tamil Nadu, India Abstract: The antibacterial effect of aqueous, ethanol, acetone and hexane extracts of leaves of Cardiospermum halicacabum on four bacterial isolates of pus samples viz. Staphylococcus aureus, Klebsiella spp., Proteus spp. and Pseudomonas aeruginosa were determined by Agar well diffusion, Disc diffusion and Microdilution methods. The results revealed that acetone and hexane were the best extractive solvents for antimicrobial properties of leaves of C.halicacabum followed by ethanol and aqueous. For Disc diffusion method the zones of inhibition of 10, 13, 15 and 16 mm against S. aureus, 8, 9, 11 and 13 mm against Klebsiella spp. 8, 10, 14 and 15 mm against Proteus spp. and 8, 12, 14 and 15 mm against P.aeruginosa, were obtained at 0.5, 1.0, 2.0 and 4.0 mg respectively for acetone extract. For hexane 12, 13, 15 and 16 mm against S. aureus, 11, 12, 15, 18 mm against Klebsiella spp. 8, 9, 13, 14 mm against Proteus spp. and 13, 14, 14 and 15 mm against P. aeruginosa were obtained at 0.5, 1.0, 2.0 and 4.0 mg respectively. For well diffusion method the Zones of inhibition of 13, 16.5, 19.5 and 21.5 mm against S. aureus, 13, 14, 15.5 and 17.5 mm against Klebsiella spp. 13, 15, 16.5 and 19.5 mm against Proteus spp. and 11, 13.5, 16 and 18 mm against P. aeruginosa, were obtained at 0.5, 1.0, 2.0 and 4.0 mg respectively for acetone extract. For hexane extract 13, 17, 19 and 21 mm against S. aureus, 13, 14, 16 and 17 mm against Klebsiella spp., 13, 15, 17 and 20 mm against Proteus spp. and 11, 14, 16 and 18 mm against P. aeruginosa, were obtained at 0.5, 1.0, 2.0 and 4.0 mg respectively. For microdilution assay Acetone extract showed bactericidal activity against S. aureus at 400 µg/ml, Klebsiella spp. at 800 µg/ml, against Proteus spp. at 100 µg/ml and P. aeruginosa at 200 µg/ml and for hexane extract against S. aureus at 200µg/ml, Klebsiella spp. and P. aeruginosa at 400 µg/ml and Proteus spp. at 800 µg/ml. C. halicacabum plant product showed significant bactericidal activity against the 4 clinical strains used


66 Manikandan et al because the inhibition obtained by the Acetone and hexane extracts ranged from 100-800 µg/ml. The results therefore established a good support for the use of C. halicacabum in traditional medicine. Key words: Cardiospermum halicacabum, Antibacterial activity, MIC

Introduction The use of plants by man to treat common ailments is time immemorial and many of the traditional medicines are still included as part of the habitual treatment of various maladies [1]. About 60 % of the total global population remains dependent on traditional medicines for their healthcare system [2]. In India thousands of species of plants are known to have medicinal values and the use of different parts of several medicinal plants to cure specific ailments has been in vogue since ancient times [3]. Medicinal plants are valuable natural resources and regarded as potentially safe drugs and have been tested for biological, antimicrobial and hypoglycemic activity [4]. The screening of plant extracts and their products for antimicrobial activity has shown that higher plants represent a potential source of novel antibiotic prototypes. Even though hundreds of plant species have been tested for antimicrobial properties, the vast majority of them have not yet been evaluated [5]. Cardiospermum halicacabum is a climber belongs to the family Sapindaceae. The plant is a twinner, pubescent or nearly glabrous annual or perennial with slender branches, climbing by means of tendrillar hooks. Leaves ternately compound, leaflets membranous, depressed, pyriform capsule wrangled at the angles. Seeds black with a large white shaped aril. It has been widely used in traditional medicines for curing various human ailments. This plant exhibits a wide range of biological and pharmacological properties. It is well known that active constituents contributing extracts and powders from the leaves, roots and seeds of this plant are used in the preparation of syrups and infusions in traditional medicine against diabetics and arthritis.

Materials and Methods Collection of Plant sample Cardiospermum halicacabum leaves were collected from Ponparappipatty village, Namakkal district, Tamil Nadu, India.

Clinical isolates used

Four pathogenic bacteria used in this study as test organisms including Staphylococcus aureus, Klebsiella spp., Proteus spp. and Pseudomonas aeruginosa isolated from pus samples. Pus samples were collected from the Sundar Hospital, Poonamalle, Chennai, India. The isolates were sub cultured on Nutrient agar (Himedia, Mumbai, India) and stored at 4°C. INDIAN JOURNAL OF APPLIED MICROBIOLOGY


In vitro Antibacterial activity of Cardiospermum halicacabum extracts 67

Preparation of extracts Twenty grams of C. halicacabum leaf powder was well dissolved in 100ml of double distilled water (ratio 1:5). The suspension was filtered by using a Seitz filter of pore size 0.2 μm. The sterile extract was then transferred to lyophilization flask and kept in deep freezer at – 80ºC for 4 hours. The frozen extract was then loaded onto the Lyophilizer. The lyophilized powder was then transferred to sterile 5ml vials and stored for further use. The acetone, ethanol and hexane extract were prepared by using 100ml of 70% of Acetone/ Ethanol/Hexane instead of 100ml of double distilled water. The other steps were same as given in the aqueous extract preparation.

Assay of Antibacterial activity Antibacterial activity of C. halicacabum leaf extract was studied by using agar diffusion and disc diffusion methods according to Bauer et al. [6]. A stock solution (1mg/ml) of the extracts and the dilutions of the stock solution containing 0.5, 1.0, 2.0 and 4.0mg/ml were prepared in dimethyl sulfoxide (DMSO). The inoculum was prepared for each strain and adjusted to the Mc Farland standard 0.5 Scale. Lawn culture was made on Muller Hinton agar plates. Prepared extract loaded discs were placed on the swabbed plates. Chloramphenicol, Oxacillin and Gentamycin were used as standard reference drugs (100 μg). The plates were incubated at 37°C for 48 h. The zone of inhibition was measured in mm and compared with standard antibiotic discs.

Determination of MIC Minimum inhibitory concentration (MIC) was determined by microdilution method [7] using Mueller Hinton broth. A stock solution (1mg/ml) of the extracts and the dilutions of the stock solution containing 800, 400, 200, 100, 50, 25 and 12.5 µg/ml were prepared in Nutrient broth. Hundred μl of each dilution was added in the respective wells and 100μl Nutrient broth as control in the microtitre plate. Chloramphenicol, Oxacillin and Gentamycin were used as standard reference drugs (100 μg). The microtitre plates were incubated at 37°C for 18-24 h. The dilutions that showed no growth were termed as bactericidal activity.

Results Antibacterial activity by Disc diffusion method Antibacterial activities of Aqueous, Acetone, Ethanolic and Hexane extracts of C. halicacabum against 4 clinical bacterial isolates by Disc diffusion method are shown in Table 1. Zones of inhibition of 7, 8, 10 and 11 mm against S. aureus and 7, 8, 9 and 10 mm against Klebsiella spp. were noticed for aqueous extract, zones of inhibition of 10, 13, 15 and 16 mm against S. aureus and 8, 9, 11 and 13 mm against Klebsiella spp. for acetone extract, zones of inhibition of 10, 12, 15 and 15 mm against S. aureus and 7, 8, 13 and 17 mm against Klebsiella spp. for ethanolic extract, zones of inhibition of 12, 13, 15 and 16 mm against S. aureus and 11, 12, 15 and 18 mm against Klebsiella spp. for hexane extract were observed at 0.5, 1.0, 2.0 and 4.0 mg of extract respectively. Antibacterial activities of INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

68 Manikandan et al

three standard antibiotics viz, chloramphenicol, Gentamycin and Oxacillin against the 4 clinical isolates were checked by disc diffusion method. Zones of inhibition of 26 mm against S. aureus and 25 mm against Proteus spp. by Oxacillin, 24mm against Klebsiella spp. by chloramphenicol and 21 mm against P. aeruginosa by Gentamycin were noticed. Zones of inhibition of 12.5, 13.5, 15.5 and 16.5 mm against S. aureus, 11.5, 14, 15 and 16 mm against Klebsiella spp. were obtained from aqueous extract. Zones of inhibition of 13, 16.5, 19.5 and 21.5 mm against S. aureus, 13, 14, 15.5 and 17.5 mm against Klebsiella spp. were obtained from acetone extract. Zones of inhibition of 13, 14, 16, 17 mm against S.aureus and 13.5, 14.5, 16.5 and 18 mm against Klebsiella spp. were obtained from ethanolic extract. Zones of inhibition of 13, 17, 19, 21 mm against S.aureus and 13, 14, 16 and 17 mm against Klebsiella spp. were obtained from hexane extract at 0.5, 1.0, 2.0 and 4.0 mg respectively (Table 2).

Agar Well Diffusion Method Microdilution Method Antibacterial activities of four extracts (Aqueous, Acetone, Ethanol and Hexane) against the 4 clinical bacterial isolates by microdilution method are depicted in Fig. 1. Aqueous extract exhibited bactericidal activity against Klebsiella spp. at 200 µg/ml and against Proteus spp. at 200 µg/ml. Acetone extract showed bactericidal activity against S. aureus at 400 µg/ml, against Klebsiella spp. at 800 µg/ml, against Proteus spp. at 100 µg/ml and against P.aeruginosa at 200 µg/ml. Ethanolic extract exhibited bactericidal activity against Proteus spp. at 400 µg/ml. Hexane extract showed bactericidal activity against S. aureus at 200 µg/ml, against Klebsiella spp. at 400 µg/ml, against Proteus spp. at 800 µg/ml and against P.aeruginosa at 400 µg/ml. Antibacterial activities of standard antibiotics chloramphenicol and Oxacillin against 4 clinical isolates were also checked by microdilution Method. Both these antibiotics exhibited bactericidal activity at 1.56 µg/ml against test organisms.

Fig. 1.

Antibacterial Effect of aqueous, acetone, ethanolic and hexane extracts of C. halicacabum by Microdilution method



7 -

Klebsiella spp.

Proteus spp.

P. aeruginosa

2

3

4 6

-

8

8

1.0

9

6

9

10

2.0

Aqueous

10

7

10

11

4.0

8

7

8

10

0.5

12

10

9

13

1.0

14

14

11

15

2.0

Acetone

15

15

13

16

4.0

8

10

7

10

0.5

10

12

8

12

1.0

11

13

13

15

2.0

Ethanol

13

15

17

15

4.0

13

8

11

12

0.5

Zone of inhibition in mm and concentration of drug in mg/ml

14

9

12

13

1.0

14

13

15

15

2.0

Hexane

15

14

18

16

4.0

12.5 11.5 12.0 12.0

S. aureus,

Klebsiella spp.

Proteus spp.

P. aeruginosa

1

2

3

4

0.5

Test Organisms

S.No

14.0

14.0

14.0

13.5

1.0

15.5

15.5

15.0

15.5

2.0

Aqueous

16.5

17.5

16.0

16.5

4.0

11.0

13.0

13.0

13.0

0.5

13.5

15.0

14.0

16.5

1.0

16.0

16.5

15.5

19.5

2.0

Acetone

18.0

19.5

17.5

21.5

4.0

13.0

13.0

13.5

13.0

0.5

13.5

13.5

14.5

14.0

1.0

15.5

15.0

16.5

16.0

2.0

Ethanol

16.5

17.5

18.0

17.0

4.0

Zone of inhibition in mm and concentration of drug in mg/ml

1.0

2.0

4.0

11.0 14.0 16.0 18.0

13.0 15.0 17.0 20.0

13.0 14.0 16.0 17.0

13.0 17.0 19.0 21.0

0.5

Hexane

Table 2. Antibacterial properties of Aqueous, Acetone, Ethanol and Hexane extracts of Cardiospermum halicacabum by Agar well diffusion method.

7

S. aureus

1

0.5

Test Organisms

S.No

Table 1. Antibacterial properties of Aqueous, Acetone, Ethanol and Hexane extracts of Cardiospermum halicacabum by Disc diffusion method

In vitro Antibacterial activity of Cardiospermum halicacabum extracts 69


70 Manikandan et al

Discussion Out of four C. halicacabum extracts used (Aqueous, Acetone, Ethanol and Hexane), Acetone and hexane extract showed maximum bactericidal activity and aqueous extract showed minimum activity against the 4 clinical isolates tested by Well diffusion, Disc diffusion and Microdilution methods. A zone of inhibition of bacterial growth of more than 10 mm by the plant extract indicated significant inhibitory activity [8]. The zones of inhibition of more than 10 mm were observed for solvent extracts than aqueous extracts for the four clinical isolates. Our results agree with Deepan et al. [9] who had reported the phytochemical screening and in-vitro anti-microbial activity leaves of C. halicacabum. The preliminary phytochemical analysis by them revealed the presence of alkaloids, carbohydrates, proteins and saponins. The extracts exhibited marked anti-microbial activity against both Gram positive and Gram negative bacteria. Mariyappan et al. [10] had studied the in vitro antibacterial activity of C. halicacabum by agar well diffusion method. The most susceptible Gram positive bacteria were Bacillus spp. and Brevibacterium spp. and the most susceptible Gram negative bacteria were Borchothrix spp., Clavibacter spp. and Ancylobacter spp. This study also corroborates with the present observation. Berghe et al. [11] reported that if an inhibition is obtained by 1-10 mg plant extract/ml test solution the extract can be considered worthy for further investigation. In the present study acetone and hexane extracts showed Minimum inhibitory concentration ranging from 100-800 µg/ml and thus the C. halicacabum plant product was found to have significant antibacterial activity. The active principle in this plant product can be further studied before using it for effective control of important clinical bacteria.

References 1. Maluventhan, V. and Murugesan, S.,2010, “Phytochemical analysis and Antibacterial activity of medicinal plant Cardiospermum halicacabum Linn”, J Phytology, 2, 68–77. 2.

Balandrin, M.F., Klocke, J.A., Wurtele, E.S. and Bollinger, W.H., 1985, “Natural plant chemicals. Sources of Industrial and Medicinal materials”, Science, 228, 1154-1160.

3.

Parekh, J., Jadeja, D. and Chanda, S., 2005, “Efficacy of Aqueous and Methanol Extracts of Some Medicinal Plants for Potential Antibacterial Activity”, Turk J Biol, 29, 203-210.

4. Hassawi, D. and Kharma, A., 2006, “Antimicrobial activity of medicinal plants against Candida albicans”, J Biological Sci, 6, 104-109. 5. Parek, J., Karathia, N. and Chandra, S. , 2006, “Screening of some traditionally used medicinal plants for potential antibacterial activity”, Ind J Pharma Sci, 68, 832-834. 6. Bauer, R., Kirby, M., Sherris, J. and Turch, M., 1966, “Antibiotic susceptibility testing by a standardized single disc method”, Am J Clin Pathol, 36,439-496. 7. Rajarajan,, S.,2002, “In vitro antibacterial and antifungal properties in the leaf extract of Henna (Lawsonia inermis .L)”, Ind J Appl Microbiol, 2, 59-61.



In vitro Antibacterial activity of Cardiospermum halicacabum extracts 71 8.

Dobrowoiski, M., 1991, “Antibacterial, antifungal, antiamoebic, anti-inflammatory and antipyretic studies on propolis bee products”, J Ethnopharmacol, 35, 77-82.

9.

Deepan, T., Alekhya, V., Saravanakumar, P. and Dhanaraju, M.D.,2012, “Phytochemical and AntiMicrobial Studies on the Leaves Extracts of Cardiospermum halicacabum Linn.”, Adv Biological Research, 6, 14-18.

10. Mariyappan, M., Bharathidasan, R., Mahalingam, R., Madhanraj, P., Panneerselvam, A. and Ambikapathy, V., 2011, “Antibacterial Activity of Cardiospermum halicacabum and Melothria Heterophylla”, Asian J Pharma Res, 1, 111-115. 11. Berghe, V., Cos, P., Hermans, N., Bruyne, S., Apers, J. B., Sindambiwe, D., Pieters, L. and Vlietinck, A. J. , 1991, “Further evaluation of Rwandan medicinal Plant extracts for their antimicrobial and antiviral activities”, J Ethnopharmacoly, 79, 155-163




Screening of Asymptomatic carriers for MRSA in the sports personnel and evaluation of Syzygium aromaticum (Clove oil) against MRSA. S.K. Jasmine Shahina 1, M. Durga Priyadarshini 1 and Padma Krishnan2* 1 2

Dept of Microbiology, JBAS college for women, Chennai- 600 018, India Dept of Microbiology, Dr. ALM PG IBMS, University of Madras, Tharamani, Chennai-600 113, India

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for several difficult-totreat infections in humans. Community acquired MRSA (CA-MRSA) causes infection in healthy individuals in communities with no traditional risk factors. CA-MRSA is rapidly spreading among healthy individuals and are more virulent. CA-MRSA is notably becoming more prevalent in athletic environments and can be easily transmitted via superficial abrasions and minor skin trauma. These infections are highly contagious and are associated with significant morbidity. Most of the invasive S. aureus infections are assumed to arise from nasal carriage. It is reported that approximately 25 to 30 percent of healthy population is colonized with S. aureus in their nose. A total of 54 samples were collected from the asymptomatic sports personnel. Out of the 54 samples, 21 were found to be positive for Staphylococci. A total of 11 were identified to be S. aureus and 10 were coagulase negative Staphylococci (CoNS). Among the eleven isolates of S. aureus three were MRSA (27%) and eight were MSSA (73%). Among the ten CoNS three were resistant to methicillin (30%) and seven were sensitive to methicillin (70%). MRSA were detected by multiplex PCR using mecA and femA along with the detection of pvl gene which is considered as marker for community acquired S. aureus infection. In this study pvl genes were detected in 6 isolates including 1 from MRSA and 5 from MSSA. Essential oils are among the most versatile and effective antimicrobial agents known. The present study shows that clove oil could inhibit the growth of MRSA and therefore these essential oils may be considered as a valuable weapon against MRSA. Key words: CA-MRSA, Sports, Clove oil, Athlete infections.


Screening of Asymptomatic carriers for MRSA and evaluation of Syzygium aromaticum 73

Introduction Staphylococcus aureus is commonly found on the skin and nares of healthy people. CA-MRSA strains have been responsible for outbreaks of skin and soft tissue infections amongst children, high school, collegiate, and professional athletes. Contact sports, particularly involving skin-to-skin contact, are the primary sources for CA-MRSA outbreaks, namely American football, wrestling, and rugby. The first documented sports related outbreak occured in 1993 in the United States in Vermont. Six high school wrestlers developed CA-MRSA abscesses [1]. Since then, several case reports have documented CA-MRSA among athletes. CA-MRSA, like all Staphylococci are transmitted to people from infected skin lesions or colonized nasal discharge. Transmission occurs from one person to another by direct physical contact or indirectly through contaminated objects, such as towels, bar soaps, wound dressings, clothes or sports equipment [2]. As most sports personnel are in frequent physical contact with others during both training and competition, they represent an “at risk” group for both typical Staphylococcal and CA-MRSA infections. High-contact sports inevitably bring about scrapes, cuts and bruises and lead to infections if not properly managed. Essential oils have been traditionally used for treatment of infections and diseases all over the world for centuries [3]. Thymol, carvacrol, linalool and eugenol are main constituents of some plant essential oils that have been shown to have a wide spectrum of antimicrobial activity against microbes [4,5]. The aim of this study was to screen the sports personnel for MRSA and to evaluate the antibacterial activity of clove oil against MRSA.

Materials and Methods Study subjects Community- Sports personnel belonging to the games such as cricket, throw-ball, basket-ball and kho kho were inducted in the study.

Demographic details The various details collected from the study subjects included name, age, sex, occupation, history of hospitalization and details of risk factors such as diabetes, COPD, if immunocompromised and if alcoholic or smokers.

Collection of Sample Sterile cotton swabs were used for nasal swabbing of the anterior nares of the healthy volunteer. The swabs were rubbed well by rotating five times over the inner wall of the nasal septum and were transported in salt nutrient broth to the laboratory and were inoculated on the mannitol salt agar as per the conventional technique. The culture plates were incubated at 37oC for 24-48 h in the incubator. Based on the colony morphology and gram staining and mannitol fermentation, the gram-positive cocci in clusters were further identified based on the biochemical methods as per standard protocols. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

74 Jasmine Shahina et al

Antibiotic sensitivity testing Antibiotic sensitivity testing was carried out by Kirby Bauer disc diffusion method for the following antibiotics- (in µg/disc): ampicillin (10), amikacin (30), chloramphenicol (30), ciprofloxacin (5), co-trimoxazole (25), erythromycin (15), gentamicin (10), linezolid (30), netilmicin (30), norfloxacin (10), ofloxacin (5), rifampicin (5), tetracycline (30), teicoplanin (30) and vancomycin (30). Screening for methicillin resistance was done by cefoxitin disc diffusion method and oxacillin agar screening method as per CLSI guidelines.

Polymerase chain reaction ( PCR) MRSA were detected using the genes mecA and femA by multiplex PCR as described by Kondo et al. [6] and Berger et al. [7] along with the detection of pvl gene by the method of Lina [8] which is considered as marker for community acquired S. aureus infection. The following were the primer sequences used in the study. femA (132bp): F: 5’ – AAA AAA GCA CAT AAC AAG CG – 3’ R: 5’ – GAT AAA GAA GAA ACC AGC AG – 3’ mecA (286bp): F: 5’ – TGC TAT CCA CCC TCA AAC AGG – 3’ R: 5’ – AAC GTT GTA ACC ACC CCA AGA – 3’ pvl (433bp): F: 5’ – ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A – 3’ R: 5’ – GCA TCA AST GTA TTG GAT AGC AAA AGC – 3’ PCR was performed in a 25µl reaction mixture with 10x standard PCR buffer [100 mM TrisHCl pH 8.3, 500 mM KCl; 1.5 mM MgCl2] (NEB), 200 mM dNTP mix (Sigma), 25pmol of each primer (Sigma), 2.5U of Taq DNA polymerase (NEB) and 1µl template DNA. Amplication was carried out with initial denaturation at 94°C for 5 min, followed by denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 5 mints. The PCR products were analyzed in a 2% agarose gel in 1xTBE buffer. Ethidium bromide stained DNA amplicons were visualized using a gel imaging system.

Anti-MRSA activity of clove oil Antibacterial activity of clove oil against MRSA isolates was determined by disc diffusion assay [9]. Standard suspension of the isolates was made and the turbidity was matched to Mc Farland standard 0.5. The inoculum was spread evenly on the Mueller Hinton agar surface using a sterile cotton swab and was allowed to dry for 5-10 min. 30µl of clove oil was placed on sterile 13 mm discs and were placed in the plate. The plates were incubated at 37°C for 24 h and the diameter of the zone of inhibition was measured. INDIAN JOURNAL OF APPLIED MICROBIOLOGY



Results Out of the 54 samples collected from the sports personnel, 21 were found harboring Staphylococci. Based on gram staining, mannitol fermentation, catalase test, tube coagulase test and DNase test the isolates were identified as S. aureus and coagulase negative Staphylococci (CoNS). A total of 11 were identified to be S. aureus and 10 were coagulase negative Staphylococci .

Phenotypic Method Screening for methicillin resistance was done according to the CLSI guidelines using cefoxitin disc diffusion method and oxacillin agar screening method. Three out of eleven of S.aureus (27 %) and three out of ten of CoNS (30%) were found to be methicillin resistant while 8/11 of S.aureus (73%) and 7/10 of CoNS (70%) were found to be methicillin sensitive.

Genotypic Method A total of 3/11 (27%) S.aureus were found to harbour mecA gene confirming them as MRSA. Three of the ten (30%) CoNS were found to harbour mecA gene confirming them as MRCoNS. Eleven out of twenty one (52%) Staphylococci were found to be positive for femA gene. The results were in accordance to the findings of the phenotypic methods. In this study pvl genes were detected in 6 isolates, 1 and 5 respectively were from MRSA and MSSA. (Fig.1)

Fig. 1. Gel Picture showing fem A, mec A and pvl gene


76 Jasmine Shahina et al

Fig. 2. Plate showing anti-MRSA activity of clove oil by disc diffusion method

Antibiotic sensitivity testing The Staphylococcal isolates showed high sensitivity to vancomycin, linezolid and teicoplanin (100%) followed by chloramphenicol (88%), genatmicin (85%), ciprofloxacin (82%), co-trimoxazole and rifampicin (80%) and erythromycin (78%). They were highly resistant to ampicillin (89%) followed by amikacin (86%), ofloxacin (80%), norfloxacin (75%), tetracycline (70%) and netilmicin (65%).

Anti-MRSA activity of Clove oil (Syzygium aromaticum) Clove oil showed high inhibitory activity against the 3 MRSA isolates giving a zone size of 20 mm diameter (Fig. 2).

Discussion MRSA infections have been reported from members of sports personnel in a variety of sports and related activities [10], at levels from high school to professional in a variety of sports. The frequency of these reports has suggested that sports personnel constitute a population at risk for MRSA infections and that sports facilities constitute a new environment for the transmission of MRSA outside the health care system. Risk factors identified among sports personnel have included sharing personal items, such as soap, towels (CDC, 2009), razors, sports training equipment, and clothing, in addition to poor hygiene [11,12]. In this study out of the 54 samples collected from sports personnel, 3 MRSA and 3 MRCoNS were detected. Of the 3 MRSA isolates 1 was found in basket ball player, 1 in cricket player and 1 in kho-kho player. Similarly the MRCoNS was isolated as one each from cricket player, throw ball player and volley ball player.




CA-MRSA isolates commonly possess genes for the Panton-Valentine leukocidin (PVL) toxin, which are rarely identified in HA-MRSA isolates [13]. Presence of pvl genes in S. aureus isolates has been associated with primary skin infections, severe necrotizing pneumonia [14,15] and increased complications of haematogenous osteomyelitis [16]. In the present study, a total of 6 isolates were positive for pvl genes, 1 and 5 respectively from MRSA and MSSA. Since infections caused by CA-MRSA and HA-MRSA are reportedly increasing, as there are therapeutic failures, new measures to treat and prevent these infectious diseases has become a need of the present scenario [17, 18]. There are also several clinical studies [19] and case reports advocating the successful use of essential oils in treating nasal carriage and infections of MRSA. AntiMRSA activity of clove oil was evaluated in this study and clove oil was found to be effective in inhibiting the growth of MRSA isolates. This report could be considered as a significant finding in view of public health as the clove oil can be suggested to treat MRSA infections.

Conclusion This study therefore concludes that three isolates each of MRSA and coagulase negative Staphylococci resistant to methicillin were obtained from a total of 54 sports personnel belonging to various sports. This indicates that MRSA is contracted and transmitted among sports personnel, which may be attributed to sharing of objects and poor hygiene. Therefore, maintaining good hygiene and avoiding contact with drainage from skin lesions of other players could be suggested as measures for preventing spread of Staphylococcal skin infections. Acknowledgement: The authors are thankful to all the sports personnels who voluntarily participated in this study. We acknowledge the help of Ms. Summera Rafiq, Head of the Department of Microbiology, JBAS College for Women, Chennai, India for the resources and facilities provided for this study.

References 1. Lindenmayer, J.M., Schoenfeld, S., O’Grady, R., and Carney, J.K.,1998, “Methicillin-resistant Staphylococcus aureus in a high school wrestling team and the surrounding community”, Arch Intern Med,158, pp.895–899 2. Stacey, A.R., Endersby, K.E., Chan, P.C., Marples, R.R., 1998, “ An outbreak of methicillin resistant Staphylococcus aureus infection in a rugby football team”, Br J Sports Med, 32, pp.153–4. 3. Rios, J.L., Recio, M.C., 2005, “Medicinal plants and antimicrobial activity”, J Ethnopharmacol, 100, pp.80–84. 4.

Kalemba, D., Kunicke, A., 2003, “Antibacterial and antifungal Properties of Essential Oils”, Curr Med Chem, 10, pp.813-829.


78 Jasmine Shahina et al 5. Dorman, H.J.D., Deans, S.G., 2000, “Antimicrobial agents from plants: antibacterial activity of plant volatile oils”, J Appl Microbiol., 88, pp.308-316. 6.

Kondo, Y., Ito, T., Ma, X. X., Watanabe, S., Kreiswirth, B. N., Etienne, J. and Hiramatsu, K.,2007, “Combination of multiplex PCRs for Staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions”, Antimicrob Agents Chemother, 51, pp.264–274.

7.

Berger-Bachi, B., Berberis-Maino, L., Strassle, A., & Kayser, F. H.,1989, “femA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization”, Mol Gen Genet, 219, pp.263-269.

8. Lina, G., Piemont, Y., Godail-Gamot, F., Bes, M., Peter, M., Gauduchon, V., Vandenesch, F. & Etienne, J., 1999, “Involvement of Panton-Valentine Leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia”, Clin Infect Dis, 29(5), pp.1128-1132. 9.

Sue Chao, Gary Young, Craig Oberg and Karen Nakaoka., 2008, “Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by essential oils”, Flavour Fragr J, 23, pp.444–449.

10. Borchardt, S. M., Yoder, J. S., and Dworkin, M. S, 2005, “ Is the recent emergence of communityassociated methicillin-resistant Staphylococcus aureus among participants in competitive sports limited to participants?”, Clin Infect Dis, 40, pp.906–907. 11. Hall, A. J., Bixler, D. and Haddy, L. E., 2008, “Multiclonal outbreak of methicillin-resistant Staphylococcus aureus on a collegiate football team”, Epidemiol Infect, 137, pp.85–93. 12. Nguyen, D.M., Mascola, L., Brancoft, E., 2005, “Recurring methicillin-resistant Staphylococcus aureus infections in a football team”, Emerg Infect Dis, 11, pp.526–32. 13. Naimi, T. S., LeDell, K. H., K. Como-Sabetti, S. M., Borchardt, D. J., Boxrud, J., Etienne, S. K., Johnson, F., Vandenesch, S., Fridkin, C., O’Boyle, Danila ,R. N. and Lyneld, R., 2003, “Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus infection”, JAMA, 290, pp.2976–2984. 14. Lina, G., Piemont, Y., Godail-Gamot, F., Bes, M., Peter, M.O., Gauduchon, V., Vandenesch, F. and Etienne, J., 1999, “Involvement of Panton-Valentine leukocidin–producing Staphylococcus aureus in primary skin infections and pneumonia”, Clin Infect Dis, 29, pp.1128–32. 15. Gillet, Y., Issartel, B., Vanhems, P., Fournet, J.C., Lina, G., Bes M, Vandenesch, F., Piemont, Y., Brousse, N., Floret, D. and Etienne, J., 2002, “Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients”, Lancet, 359, pp.753–9. 16. Caelli, M., J. Porteous, C. F. Carson, Heller, R. and T. V. Riley., 2000, “Tea tree oil as an alternative topical decolonization agent for methicillin-resistant Staphylococcus aureus”, J Hosp Infect,46, pp.236-237.



Screening of Asymptomatic carriers for MRSA and evaluation of Syzygium aromaticum 79 17. Saravolatz, L. D., Markowitz, N., Arking, L., Pohlod, D. and Fisher, E., 1982, “Methicillinresistant Staphylococcus aureus. Epidemiologic observations during a community-acquired outbreak”, Ann Intern Med, 96, pp.11-16. 18. Carson, C. F., Mee, B. J., and Riley, T. V., 2002, “ Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy”, Antimicrob Agents Chemother, 48, pp.1914-1920. 19. Sherry, E. and Warnke, P. H., 2004, “ Successful use of an inhalational phytochemical to treat pulmonary tuberculosis: A case report”, Phytomedicine., 11, pp.95-97.l




Antibacterial activity of Pyocyanin pigment produced by Clinical isolate, Pseudomonas aeruginosa WS1 T. Sudhakar1* , S. Karpagam2 and M. Lakshmipathy1 1 2

Department of Biomedical Engineering, Sathyabama University, Chennai 600 119, India. Department of Botany, Queen Mary’s College, Chennai 600 004, India.

Abstract: Drug resistant strains of Pseudomonas aeruginosa has become a major threat in medical care and has drawn the attention of the microbiologists to combat and contain the spread of infectious diseases. The characteristic feature of P. aeruginosa is the production of a soluble pigment pyocyanin, a secondary metabolite that exhibits antimicrobial activity. The present study was undertaken to evaluate and optimize conditions essential for the production of pyocyanin pigment from clinical isolates of Pseudomonas and to ascertain the antibacterial potency toward gram positive and gram negative pathogens. The study also involved the demonstration of pigment production on various solid and liquid media. Further, the pigment pyocyanin was extracted and confirmed by UV-visible spectrum. The pigment was then analyzed for antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis and Shigella sp., which concluded that pyocyanin pigment has the ability to inhibit the growth of pathogenic bacteria by arresting the electron transport in both in vitro and in vivo conditions. Key words: Pseudomonas aeruginosa, Pyocyanin, Antibacterial activity, Secondary metabolites.

Introduction The emergence of drug resistant strains due to the indiscriminate use of antibiotics and chemotherapeutic agents is posing a major threat in health care systems worldwide. Deleterious effects of resistance have been reported among the gram negative strains, notably those belonging to Pseudomonas aeruginosa and Acinetobacter species [1]. Although Pseudomonads are widely


Antibacterial activity of Pyocyanin produced by Pseudomonas aeruginosa WS1 81

distributed in nature, it has drawn much of attention in recent years with the increase in the incidence of hospital borne infections. The characteristic outer membrane barrier with low permeability and efficient multidrug efflux machinery contributes significantly to Pseudomonads’ resistance toward antimicrobials. Another peculiar feature of P. aeruginosa is the production of soluble pigments such as pyocyanin and fluorescein, of which pyocyanin is a water soluble blue green phenazine compound. The physiological significance of the pigment is not known but because of its inhibitory action it has been postulated that pigment production may give P. aeruginosa a selective advantage in growth situations. Ninety percent of Pseudomonas spp. produce pyocyanin pigment which is involved in iron acquisition and commonly used in diagnostics. Other pigments produced by Pseudomonas spp. are pyochelin, pyoverdin, pyorubrin and pyomelanin. Pinghui [2] isolated P. aeruginosa and its pigment pyocyanin demonstrating the antibacterial activity, the characteristic of which aids colonization, damages endothelial tissue and mediates formation of super oxide and peroxide. The present study was undertaken to evaluate and optimize conditions essential for the production of pyocyanin pigment from clinical isolates of Pseudomonas and to ascertain the antibacterial potency toward gram positive and gram negative pathogens.

Materials and Methods Isolation and Identification Forty two clinical isolates of P. aeruginosa used in this study were procured from Sharp Clinical Laboratory, Chennai, India. The bacteria was sub-cultured on Cetrimide Agar, Nutrient agar and Blood agar medium (Hi Media, India) and the cultural characteristics of these isolates was studied by standard gram staining and biochemical methods. The identified cultures were maintained in Nutrient agar slants at 4ºC and used for experimental.

Production and Extraction of Pigment Liquid Media: Pseudomonas spp. was screened for the production of soluble pigment pyocyanin in various liquid medium and it was extracted by adding 2 to 3ml of chloroform and further and confirmed by the addition of 0.2 N HCl. Solid Media: Pyocyanin production was demonstrated on various solid medium and incubated overnight at 37ºC. Extraction of the pigment was done using the solvent chloroform and few drops of 1N H2SO4, which parted red color to the water layer. It was filtered and then evaporated for dryness.

UV-visible spectrum of pigment The pigment produced was subjected to UV-visible spectrum in the absorbance range 200 – 800 nm and the maximum absorbance was recorded. The samples were analysed along with distilled water as blank. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

82 Sudhakar et al

Antibacterial activity – Well diffusion method The antibacterial activity of the pyocyanin pigment was determined by well diffusion method. The bacterial cultures used as test organisms included S. aureus, Enterococcus sp., Proteus mirabilis and Shigella spp. Three hours broth culture of each of the above these test pathogen was preferred for lawn culture on Muller Hinton Agar plates and wells of 3 mm diameter were made on the plates. 100 mg/ml of the pyocyanin pigment was prepared using distilled water and 10µl of the pigment was added to the wells using a sterile pipette. The plates were incubated at 37ºC for 24 h along with control.

Results Identification of Bacteria Out of fifty clinical samples processed 42 isolates of Pseudomonas spp. were confirmed in the laboratory based on Gram’s staining, motility, cultural characteristic and by various biochemical reactions. Among 42 isolates of Pseudomonas, only one strain P. aeruginosa WS1 was selected based on its growth characteristics in the whole set of medium.

Production and Extraction of pigment Pigment production was accomplished after overnight incubation of the bacterial culture. Production of soluble pigments namely, pyocyanin and fluorescein were indicated by color change in the solid media. In the case of liquid media, pyocyanin production was demonstrated in shades of green color and fluorescein production by visual observation under ultra violet trans-illuminator on both solid and liquid media. The percentage of pigment production by different strains of Pseudomonas sp. in solid and liquid media is shown in Fig. 1 and 2. Enhanced pigment production was observed in Potato glycerol broth (95.23%) followed by Cetrimide agar medium (90.4%) indicating the maximum diffusion and soluble nature of the pigment in broth culture.

UV-visible spectrum of pigment The change in color of the pigment to deep pink upon addition of chloroform and 0.2 N HCl confirmed the presence of pyocyanin. The absorbance of this solution was found be maximum at 278 nm (Fig. 3). This peak indicates the presence of pyocyanin compound.

Antibacterial activity of Pyocyanin pigment The pigment produced by P. aeruginosa showed maximum antibacterial activity toward Shigella sp. with 17 mm followed by P. mirabilis (16 mm), S. aureus and E. faecalis showing 14 mm zone of inhibition (Table 1).




PW- Peptone water; NB – Nutrient Broth; LB – Luria Bertani Broth; BEP – Beef extract peptone water; TS – Trypticase soya broth; FDM – Frank’s and Demoss broth; NB.5 - 2 – Nutrient broth + 0.5 – 2.0 glucose; NB.5G–2G – Nutrient Broth + 0.5 – 2.0 glycerol; PGB – Potato Glycerol Broth Fig. 1. Production of pyocyanin pigment in different liquid media

NA – Nutrient Agar; CA – Cetrimide agar; PF – Pseudomonas agar F; TSA – Trypticase soy agar; TGYA – Tryptone glucose yeast extract agar; FNA – Fluorescein nitrification medium; SDM – Seller’s Denitrification medium; SDA – Saboraud Dextrose agar; PCA – Plate count agar Fig. 2. Production of pyocyanin pigment in different solid media


84 Sudhakar et al

Fig. 3. UV Absorption spectra of P. aeruginosa WS1 showing Amax at 278nm Table 1. Antimicrobial activity of pyocyanin pigmentof P. aeruginosa WS1 Sl. No.

Test Pathogens

Diameter of Zone of Growth inhibition (in mm)

1.

S. aureus

14

2.

E. faecalis

14

3.

Proteus mirabilis

16

4.

Shigella spp.

17

Discussion The multidrug resistant bacteria are becoming more and more prevalent in recent years and the control these agents needs elaborate and comprehensive medication. The antibiotics available commercially are mainly focused for their action bacterial infection. In health care set ups meant for treating infectious diseases are complicated due to the emergence of antibiotic resistant strains concerned with hospiral acquired infections. In the present study biologically produced pyocyanin was purified, and the nature of its antibacterial action was determined for several isolates of bacteria. The pigment was shown to be bactericidal for all the test organisms tested. The bactericidal effect was in concordance with the pyocyanin concentration and resulted in decreases in viability. Both gram-positive and gram negative bacteria were susceptible to the pigment. The most widely used criteria for distinguishing P. aeruginosa from closely related organisms is by the production of pyocyanin pigment in the various solid and liquid media. In the present studypyocyanin pigments were produced using P. aeruginosa obtained from various clinical samples in different media. The results are in accordance with the work of Young [3] who reported that




P. aeruginosa isolated from various samples produced pigments in various media. Chang. et al. [4] reported that production of pyocyanin pigment on various media indicated a wide variation in yields depending on the composition of the media, but satisfactory yield of this pigment was obtained. This report was co-relating the present study. In the present study, pyocyanin pigment was produced by P. aeruginosa on various liquid and solid media. Pigment was further extracted by using chloroform and 0.2 N HCl. Pyocyanin production was maximum in liquid media namely Potato glycerol broth (95%), Beef extract peptone broth (90%) and Glycerol broth (90%). Pyocyanin production in solid media such as Cetrimide agar (90%), Trypticase soy agar (86%) and Nutrient agar (83%) was effective. Pyocyanin produced by P. aeruginosa exhibit satisfactory antibacterial activity. Ra’oof [5] reported that P. aeruginosa suppressed the growth of E. coli, S. typhi, S. aureus and K. pneumoniae in vitro. The Pseudomonas spp. produces a variety of metabolites that exhibit wide range of antimicrobial activity, among which pyrrolnitrin has been known to possess antifungal activity [6]. In the present study pigment produced by P. aeruginosa demonstrated antibacterial activity by inhibiting the growth of test bacteria including S. aureus, E. faecalis, P. mirabilis and Shigella spp. This finding is in correlation with the data of Ra’oof [5]. The present study suggests to use the pigment produced by P. aeruginosa along with antibiotics so as to inhibit the growth of pathogenic strains. This will help to achieve a positive step towards the management of infectious diseases.

References 1.

Nikaido, H., 2009, “Multidrug resistance in Bacteria”, Annu Rev Biochem, 78, pp.119–146.

2.

Pinghui. V. Kiu., 1962, “Non – motile varieties of P. aeruginosa melanin like pigment”, J Bacteriol, 16(2), pp. 369-378.

3. Young, G., 1947, “Pigment production and antibiotic activity in cultures of Pseudomonas aeruginosa”, J Bacteriol, 54, pp.109 – 117. 4.

Chang, P.C. and Black Wood, A.C., 1969, “Simultaneous production of three phenazine pigments by Pseudomonas aeruginosa Mac 436”, Can J Microbiol, 15, pp.439–444.

5.

Wa’ad Mahmood Ra’oof and Ibraheem Abudl Rahman Latif, 2010, “In vitro study of the swarming phenomena and antimicrobial acvitiy of pyocyanin produced by Pseudomonas aeruginosa isolated from different human infections”, Eur J Sci Res, 47 (3), pp.405-421.

6.

Pal, R.B. and Revathi, R., 1998, “Susceptibility of yeast to P. aeruginosa”, Ind J Med Microbiol, 16, pp. 72 – 74.



Copyright 2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, pp. 86-93

Assessment of Organic waste composting and designing of consortia to improve the compost efficiency as a Biofertilizer J. Jelin Ilayaraja1* and M.S. Dhanarajan2 1 2

Department of Biotechnology, Sathyabama University, Chennai, India Principal, Jaya College of Arts and Science, Chennai, India

Abstract: Waste management remains the major concern for many industries for years. The process of treating organic waste has several benefits such as improved sanitation and production of renewable products. Composting is associated with reclamation, recycling, treatment and disposal of wastes. The present study deals with the conversion of waste organic products into a useful by product, compost which is used to enhance growth of crops with combinations of microorganisms. In the present study the physical characteristics and microbiological analysis of the compost were established. The micro flora analysis explains the microbiota at various stages of the composting process, and its different characteristic degrading ability and efficacy at each stage. The compost reached a temperature of about 53ºC during the 30th day and reached room temperature during the 90th day. The ash content increased from 10.12% to 15.95% and a pH nearly neutral. The cellulose concentration reduced from 34.02% to 4.21% and a change in lignin content from 8.01% to 3.16%. the C:N ratio reduced from 161:33 to 28:10. A consortium of microorganisms were designed to cater different needs of growing plants so as to understand the interactions between consortium of microbial inoculants and plant systems. It is expected that this will pave way to harness for improving growth and yield of paddy plant. Combinations of microorganisms with compost act as a good biofertilizer, which could improve the fertility of soil and increases plant growth. Encouraging results were produced by mixed organisms like Azospirillum, Rhizobium and Pseudomonas (T4). The least growth was observed in the inoculation of Bacillus and Azotobacter with soil. Based on this study we conclude that the Rhizospheric organisms play a critical role as plant growth promoting agents and cause a better yield and growth of plants. Key words: PGPR, Compost Analysis, pot culture.


Assessment of Organic waste composting and designing of consortia 87

Introduction The use of microorganism with the aim of improving nutrients availability for plants is an important practice and essential for the success of agriculture. Some of the mechanisms, which function simultaneously or sequentially at different stages of plant growth are, increased mineral nutrient solubilization and nitrogen fixation and making nutrients available for the plants. One of the most important bacteria presenting one or more of these characteristics is Plant Growth Promoting Rhizobacteria – PGPR. According to Wu and Martinez [1], numerous species of soil bacteria which flourish in the rhizosphere of plants, which may grow in on or around plant tissues and stimulate plant growth by plethora of mechanisms are collective known as PGPR. Earlier researchers have recently shown the association of PGPR and categorized them into extracellular (PGPR), existing in the rhizosphere and on the rhizoplane, and intracellular (iPGPR) groups which exist inside the root cells generally in specialized nodular structures. PGPR inoculants currently commercialized seem to promote growth by improved nutrients acquisition (biofertilizers). During the past decades the use of plant growth promoting rhizobacteria (PGPR) for sustainable agriculture has increased tremendously in various parts of world. Significant increases in response to inoculation with PGPR have been frequently reported. Reclamation and recycling are the means of saving and reusing natural resources. Waste disposal has become a less desirable option because of environmental concerns. The degradation of organic waste is a natural process and begins almost as soon as the wastes are generated. Composting is a decomposition process influenced by various biochemical parameters such as temperature, pH, odor, biological survivors, types of organic matters, cellulose, lignin and enzyme action. The mineral nutrient status (including macro and micro) of the compost is considered as most essential for plant growth and yield. The mineral elements are known to be involved in metabolic functions of plants without which the plants cannot complete their life cycle. The nutrient status is known to vary depending upon the type, physico-chemical and biotic components of compost and soils. Intensive use of only chemical fertilizers to achieve high production has created various problems. To enhance the plant growth, application of microbial fertilizers in agriculture lands has been a regular practice by the farmers. Though the microbial fertilizers are known to enhance the NPK level of the soil to support the plant growth, continuous application of combined microbial fertilizer is reported to affect the soil fertility in the long run. Some of the biofertilizer viz., Azospirillum and Azotobacter are known to increase the growth, yield and quality of crops by fixing atmospheric nitrogen synthesis of plant growth hormone. Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that actively colonize plant roots and increase plant growth and yield. The mechanism by which PGPR promote plant growth are not fully understood, but are thought to induce the ability to produce phytohormones, asymbiotic nitrogen fixation, resistance against phytopathogenic microorganisms by the production of siderophores, synthesis of antibiotics, enzymes and fungicidal compounds. Significant increase in growth and yield by agronomical important crops in response to inoculation with PGPR have been reported. Another major benefit of PGPR is to produce antibacterial INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

88 Jelin Ilayaraja and Dhanarajan

compounds that are effective against certain plant pathogens and pest. Moreover, PGPR mediate biological control indirectly by enhancing systematic resistance against a number of plant diseases. Under salt stress, PGPR have shown positive effect in plants on such parameters as germination rate, tolerance of mineral uptake drought, yield and plant growth [2]. As such the scientific information available with respect to the effect of nitrogen fixing bacteria on growth and yield of crops are scanty [3]. Plant growth promoting rhizobacteria use one or more direct mechanisms of action to improve plant growth and health. This mechanism may be sequential or act simultaneously at different stages of plant growth [4]. The plant growth promoting rhizobacteria trigger an increase in root surface area which results in increased nutrient uptake [5,6]. The present study was designed to evaluate the activity of PGPR along with the compost by using pot culture technique.

Materials and Methods Compost Lay out The vegetable market waste was collected and laid in a pit of size 1m x 1m x 1m. Ten cm of vegetable waste was over laid with 10 cm of soil and the process was repeated to fill the pit. The pit contained 5 piles of vegetable waste and over laid with 5 piles of soil. It was moistened with water and 10 layers of pile were over laid with straw to maintain the moisture. The compost was turned once in15 days and the samples were collected for every 15 days and analysed from 0 day to 90th day.

Analysis of Characteristics of compost The physical characteristics such as temperature, pH, ash content, moisture content, carbon content and C:N ratio were determined. The microbiological characteristics such as standard plate count, biochemical and physiological tests were done to identity and characterize the strain according to Bergey’s manual of determinative Bacteriology [7]. The samples were taken from the central surface of composting material. The temperature of compost was measured by a thermometer at the sampling points. The compost sample (5 g) was suspended in ultra pure sterile water. The volume of suspension was adjusted to 50ml and shaken for 30 min at 1800 rev/min. After sub-sampling the suspension were centrifuged at 1500 x g, at 4°C for 10 min. The supernatant was filtered through 0.22 μm filter units. The filtrate was measured for pH and then used for analysis of dissolved components by the method of [8]. Water contents were determined by weight loss of triplicate 5 gm composting samples after drying at 105oC for longer than 24 h. The dried samples were ground and mixed well and large pieces (about > 0.1mm) of wood chips, pieces that were not finely ground were removed from samples for ash content and C:N ratio measurement,. Ash contents were determined by weight loss of 2 gm samples after burning at 550oC for 4 h [9]. Total dissolved organic carbon was measured by method suggested by Jackson [10]. Microbial load was enumerated by standard plate count method from 10-1 to 10-12 dilutions using Plate count agar, Nutrient agar, Sabraouds Dextrose Agar and PBYG medium. After incubation the plates were examined carefully for occurrence of morphologically similar and dominant bacterial INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Assessment of Organic waste composting and designing of consortia 89

colonies [11]. One gram of soil sample was weighed and dissolved in 99ml of distilled water and plated on Nutrient agar and YEMA medium. Colonies were confirmed by standard biochemical tests.

Analysis of PGPR The degrading efficacies of the isolated species were demonstrated in terms of their enzyme activity. The hydrolysis of starch, casein and phosphate, reduction of nitrate and ammonia, oxidative fermentation of glucose and production of hydrogen peroxide are substantial evidence for the production of various degrading enzymes. The isolated bacteria were tested for the production of ammonia in peptone water. Ten ml of fresh culture in peptone water were incubated for 48 h at 30oC. Nessler’s reagent, a filtrate of mercuric iodide (100 µg), potassium iodide (90 µg), distilled water (400µl) mixed with sodium hydroxide (100 µg) in 500µl of distilled water was prepared. Addition of Nessler’s reagent to the broth culture induced brown to yellow color indicating the ability of organism to produce ammonia. The isolated rhizospheric bacteria were inoculated in sterile nitrite broth and incubated at 37 oC form 24 h. Nitrate reagent A and B were added to observe a red colour change. The isolated organisms were tested for carbohydrate metabolism in a medium containing peptone (2g), NaCl (5g), K2HPO4 (0.3g), phenol red (3ml), agar (3g) and 10ml of distilled water. The oxidation and fermentation of carbohydrates were determined by change in colour [12].

Experimental Design of pot culture The seeds of the paddy plant were obtained from Agricultural office, Thiruvallur, Tamilnadu, India. A pot experiment was performed with different combinations of rooting media (10:1) viz., T 1 = Soil + Azotobacter (10:1); T 2 = Soil + Pseudomonas; T 3 = Soil + Bacillus; T 4 = Soil + Rhizobium; T 5 = Soil + Mixing of organisms. The treatments were replicated six times and laid out in a Randomized Block Design (RBD). Three replicates were harvested at 30 days of plant growth and checked for every 5 days interval. Plants were grown in a pot culture at day night temperature region of 15 to 25oC at natural tropical environment. After growth for 4 weeks, fresh weight and dry weight were measured [13]. Seed germination rate and vigor index were calculated and the results were compared to analyse the efficiency of the organisms and their combinations as PGPR [14].

Results and Discussion The physico-chemical and microbial characteristics of compost are presented in Table 1. The measurement of ranges of temperature indicated the highest occurring during 30th day. The pH did not show any significant change.


90 Jelin Ilayaraja and Dhanarajan Table 1 Physico-Chemical characteristics of Compost Days

Temp pH Moisture % Ash % °C

odour

Cellulose (%)

Lignin (%)

Carbon (%)

Nitrogen C:N ratio (%)

0

29

6.9 50 ± 0.05

10.12 ± 0.32

FO

34.02

8.01

48.4

0.3

161.33

15

49

6.7 56 ± 0.23

11.3 ± 0.65

FO

26.21

6.13

46.02

0.4

115.05

30

53

6.5 56 ±0.23

13.3 ± 0.65

FO

15.81

5.76

38.02

0.5.5.5

76.02

45

44

6.6 56 ±0.34

14.02 ±0.78 ST

09.54

3.54

33.12

0.9

34.57

60

35

6.9 58 ±0.56

14.5± 0.65

OL

07.21

4.75

32.56

0.9

36.17

75

31

7.1 57 ±0.56

15.2± 0.56

PES

5.21

03.42

31.13

1.1

30.11

90

56

7.4 59

15.95 ± 0.56 PES

4.21

03.16

30.92

1.1

28.10

All the values are averages of 5 observations (Mean ± SE); FO-fowl smell, ST-Slightly tolerable, OL-odorless,PES-Plesant earthy smell

The ash content increased with the age of the compost and indicated that mineralization of organic materials took place. The compost changed its odour with time during composting. The most effective compost managing plan is to maintain temperature at the highest level is possible without inhibiting the rate of microbial decomposition [1]. If the temperature is less than 20oC microorganism would be inhibited or killed and the reduce the diversity of organism, thus resulting in lower rate of decomposition [1]. In the present study the amount of moisture content was 50% in control and it was maintained till 90th day using sprinklers. Microorganisms require moisture to assimilate nutrients, metabolize new cells and reproduce [15]. They also produced water as a part of decomposition process. If water is accumulated faster than it is eliminated via evaporation, then oxygen flow is impeded and anaerobic condition result [16]. This usually occurs at moisture levels of above 65% in which the microbial activities decreases and the composting process slows down. The moisture level below 20% may lead to very little microbial activity in the media. There a was reduction in the odour from control, from the rotten smell to slightly tolerable on 45th day and it became odourless on 75th day with longer period of composting. The odour originate from three main sources such as odourless raw material, ammonium released from high nitrogen materials and anaerobic conditions within the windrow and piles. The ash content of the compost was analysed and showed significant increases with the time. The changes in ash content reflect trends in mineralization of organic matter. The similar result was reported by Wu. et al. [1]. The compost shows decrease in their organic carbon content with time (Table 1). C: N (%) ratio is a major parameter in composting process. The reduction in C: N ratio (Table 1) with increase in time is an indicative of compost maturation process. The compost showed increase in the bacterial load up to 15th day and there was reduction in bacterial load on 30th day which again raised on the 75th day. Compost showed increase in their fungal load up to 60th day and there was slight reduction on 90th day. Enumeration of different microorganisms in the compost is presented in Table 2. INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Assessment of Organic waste composting and designing of consortia 91 Table 2. Enumeration of microorganisms from the compost Days

Bacteria (CFU/ml)

Fungi (CFU/ml)

Actinomycete (CFU/ml)

0

4.7 × 10

5.13 × 10

4.96 × 105

15

9.3 × 10 6

1.13 × 1011

1.5 × 106

30

1.4 × 104

9.53 × 108

6.19 × 104

45

1.4 × 10 4

1.01 × 108

1.20 × 104

60

2.3 × 10 4

9.31 × 106

2.16 × 103

75

1.2 × 105

1.33 × 107

1.81 × 105

90

3.6 × 105

9.46 × 109

2.31 × 105

6

10

Table 3. Effect of PGPR on germination and seedling growth of Paddy Isolates

Days of Germination

Seed Germination (4th day)%

Germination Index

Root Length(cm)

Shoot Length (cm)

Vigor Index

2

4

6

C

-

3

5

66.7

2

0.8

0.9

113.39

T1

-

5

6

83.33

2.25

0.9

0.9

149.99

T2

-

5

6

83.33

2.25

0.9

0.8

141.66

T3

-

3

6

50

1.75

0.7

0.1

40

T4

-

4

6

66.7

2

0.5

0.6

73.37

T5

-

4

6

66.7

2

0.3

0.5

66.7

T6

2

4

6

100

4

0.9

0.9

149.99

The results suggested that mixing of organisms yield better results of growth of plants rather than other combinations or un-inoculated soil (C) (Table 3 and 4). The inoculation of Bacillus with soil combinations showed poor results in paddy plants. Based on this study we concluded that the rhizospheric organisms play crucial role as plant growth promoting agents and cause a better yield and growth of plants. It also increases the fertility of soil and does not cause any pollution to the environment. These organism when subjected to seed germination assay the seeds were observed to germinate more rapidly and the Azotobacter sp. yielded a vigor index of 149.99. Thus Azotobacter has been proved to initiate paddy germination. The height and weight of paddy root and shoot was measured and paddy plant growth efficiency was observed to increase by applying Azotobacter. Thus it is concluded from the present study that the Azotobacter, Bacillus sp., Azospirillum sp. and Pseudomonas sp. together as a consortium can be used as a biofertilizer for promoting the growth of paddy.


92 Jelin Ilayaraja and Dhanarajan Table 4. Effect of Azotobacter on Paddy seed germination 10 Days Inoculum

15 days

Length Fresh weight (gm) Dry weight (gm) (cm)

Length (cm)

Fresh weight (gm)

Dry weight (gm)

RL SL R

RL SL

R

R

S

TW R

S

TW

S

TW

S

TW

C

5.2 26 0.421 0.761 1.182 0.053 0.116 0.169 8.12 28

0.61 1.324 1.934 0.082 0.162 0.244

T1

5.2 25 0.621 0.841 1.732 0.047 0.18 0.237 8.84 25.4 0.75 1.621 2.371 0.131 0.393 0.524

T2

5.7 26 0.321 0.921 1.542 0.076 0.132 0.208 9.1 29.2 0.81 1.571 2.281 0.181 0.151 0.312

T3

5.5 23 0.461 0.916 1.377 0.091 0.141 0.232 8.6 28.9 0.69 1.321 2.011 0.152 0.126 0.278

T4

5.6 25 0.542 0.821 1.382 0.064 0.176 0.24 8.4 28.5 0.81 1.591 2.240 0.128 0.215 0.343

T5

5.1 26 0.616 0.811 1.427 0.031 0.189 0.22 8.9 28.1 0.85 1.541 2.291 0.176 0.212 0.388

T6

5.7 26 0.561 0.905 1.466 0.080 0.167 0.247 9.8 29.5 0.81 1.533 2.343 0.164 0.233 0.397

RL-Root length, SL-Shoot length, FRW-Fresh weight, R-Root, S-Shoot, TW-Total weight

References 1.

Wu. L., Ma, L.Q. and Martinez. G.A., 2000, “Comparison of methods for evaluating stability and maturity of biosolids compost”, J Environ Qua, 29, pp.424-429.

2.

Marmite Datta and Rakhi palit, 2011, “Plant growth promoting Rhizobacteria enhance growth and yield of chilli”, Austr J crop sci, 5(5), pp.531-536.

3. Ravikumar, M., Venkatesha, P., Gangadharppa G. and Munikrishnappa, P.M., 2010, “Effect of nitrogen fixing bacteria in combination with organic and chemical fertilizers on growth and yield of Coleus”, J Eco Biol, 2(4), pp. 349-358. 4.

Chaiharn, M., Chunhaleuchanon, S., Kozo, A. and Lumyong, S., 2008, “Screening of Rhizobacteria for their plant growth promoting activities”, KMITL Sci Technol, 81, pp.18-23

5. Sophie Mantalian and Bruno Touraine, 2000, “Plant growth promoting bacteria and nitrate availability impacts on root development and nitrate uptake”, J Experim Bot, 55, pp.27-34. 6.

Kenjiiyama, 1994, “Compositional changes in compost during composting and growth of Agaricus species”, J Environ Microbiol, 61, pp.194-199.

7.

John Holt, R., Noel King, H.A., Peter Sneath, T., James stanely, T. and Stanely Williams, T., 1994, Bergey’s Manual of determinative bacteriology Williams R. Hensyl, 9th ed. USA. pp. 562-575

8. Furke, G., A.Von Graevaitz, J.E.Claridge, and Bernard, K.A., 1997, “Clinical Microbiology of Corynebacteria”, Clin Microbiol Rev, 10, pp.125-159 9. Breed, R.S., Merry, E.G.D. and Parker Hitchines, A., 1957, Bergey’s manual of Determinative Bacteriology, 7thed, Ballimore, The Williams and Wilkins company.



Assessment of Organic waste composting and designing of consortia 93 10. Jackson,M.L., 1975, Soil chemical analysis, Prentice Hall of India, New Delhi, India. 11. Alsina,M., and Blanch, A.R., 1994, “A set of keys for biochemical identification of environmental Vibrio sp.” J Appl Bacteriol, 76, pp.79-85. 12. Ifran Afzal, Shahzad Basra, M.A. and Amir Iqbal, 2005, “The efficiency of wheat under salinity stress”, J Stress physiol Biochem, 1(1), pp.6-4. 13. Chanway, C.P., Nelson, L.M. and Holl, F.B., “Cultivar specific growth promotion of spring wheat by Co existent Bacillus species”, Can J Microbiol, 34, pp.925-929 14. Afzar, I., Basara, S.M.A., Ahmad Cheema, M.A., Warraich E.A. and Khalq, 2002, “Effect of Priming and growth regulator treatment on emergence and seedling growth of hybrid maize (ZEA maize)”, Int J Agric Biol, 13, pp.303-306. 15. Hakim.M.A., Abdul Shukor Juramini, Hanafi M.M., Selamat A., Mohd Razi Ismail, and Rezaul Karim, S.M., “Studies on seed geremination and growth in weed species of rice field under salinity stress”, J Environ Biol, 32, pp.529-536 . 16. Kiyohiko Nakasaki., 1985, “Change in number during thermophilic composting of sewage sludge with reference to carbon di oxide evaluation rate”, Appl Environ Microbiol, 49, pp.37-41.




Bioprospecting of Marine Micromonospora with special reference to Antibacterial metabolite production R. Balagurunathan1* and M. Radhakrishnan2 1 2

Department of Microbiology, Periyar University, Salem, Tamilnadu, India Department of Bacteriology, National Institute for Research in Tuberculosis, Chennai, India

Abstract The present study reports the bioprospecting potential of marine Micromonospora with special reference to antibacterial metabolite production. A total of 40 colonies with suspected Micromonospora morphology were isolated from Pichavaram mangroves and Andaman marine sediment samples by adopting selective isolation procedures. In the preliminary screening by cross streak method 19 isolates showed antagonistic activity. All the isolates showed good growth at NaCl concentrations ranging between 1.5% and 2.5%. During secondary screening by submerged fermentation, all the isolates showed better growth with pigmentation on soybean meal broth when compared to YEME and SGG medium. Totally 114 crude extracts were prepared from 19 isolates of Micromonospora using three different media and two different solvents. Among the 19 isolates, the ethyl acetate and methanol extract from the strain M104 showed maximum activity against all the bacterial pathogens tested. Further purification and characterization of active compound from the Micromonospora species isolated from Pichavaram mangrove rhizosphere will lead to the discovery of promising antibacterial candidates. Key words: Actinomycetes, marine, Micromonospora, antibacterial metabolites

Introduction Actinomycetes are the group of saprophytic bacteria that are widely distributed in different natural and man-made environments. Among the group actinomycetes, Streptomyces is the most dominant and well studied genus in terms of biodiversity and biological activity. Next to

*Author for correspondence; E-mail: [email protected]

Bioprospecting of Marine Micromonospora 95

Streptomyces, Micromonospora is the most significant source for secondary metabolites [1]. The genus Micromonospora is gram positive chemo-organotrophic, aerobic bacteria with high mol % G+C genomic content. Micromonospora species have long been recognized as important sources of antibiotics and also for their unusual spores. They are also playing important role in degradation complex substrates in the environments [2]. In addition, several studies demonstrate that Micromonospora species function in biocontrol, plant growth promotion, root ecology, and in the breakdown of plant cell wall material [3,4]. In India, there are very few reports on Micromonospora in general and from marine ecosystems in particular. Ellaiah and Reddy [5] isolated Micromonospora species from the sediment samples collected from Vishakapatnam coastal area, Andhra Pradesh. Raja and Prabakaran [6] isolated psychrophilic Micromonospora species from Manali icepoint, Himachala Pradesh and studied their antagonistic potential. Recently, Talukdar et al. [7] isolated about 100 Micromonospora strains from Kaziranga National Park of north-East India and reported their anti-infective activity. With this view, an attempt was made for bioprospecting of Micromonospora isolated from selected Indian marine ecosystems.

Materials and methods Isolation of marine Actinomycetes Sediment samples were collected from Pichavaram mangroves and Andaman marine ecosystems. The collected samples were dried at room temperature for 1 week and heat treated at 80oC for 1 h. About 0.1ml of serially diluted sediment samples were plated on Starch casein agar medium prepared in 50% sea water and supplemented with Nalidixic acid (100/ml) and Nystatin (20/ml). All the plates were incubated at 28oC for 1 month.

Selection of marine Micromonospora Colonies with the morphology of typical Micromonospora were selected from starch casein agar plates. Characteristics which are recorded include mucoid and leathery pin pointed colonies with brown, orange or yellow in color and presence of substrate mycelium alone with monospore at end of the mycelium. Cultural characteristics were studied by using Yeast extract malt extract (YEME) agar medium (ISP medium 2) prepared in 50% seawater [8]. Slide culture method was used for studying the micromorphology. Morphologically different isolates were preserved on YEME agar slants and also in 30% glycerol broth.

Effect of sodium chloride on growth Effect of sodium chloride on the growth of antagonistic marine Micromonospora isolates was studied by inoculating in to YEME broth supplemented with different concentration of sodium chloride (0% - 3.5%). About 10% of inoculum was transferred into all the flasks and incubated at 28oC in rotary shaker for 10 days. Biomass was collected by centrifugation at 5000 for 10 min. Wet weight of the biomass was calculated using preweighed tubes [9]. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

96 Balagurunathan and Radhakrishnan

Preliminary screening for antagonistic activity All the Micromonospora isolates were screened for antagonistic activity by cross streak method using modified nutrient glucose agar (MNGA) medium. Test organisms used in this study include Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi and Shigella flexneri. All the test organisms were obtained from Christian Medical College, Vellore, Tamil Nadu, India [10].

Production, extraction and bioassay of crude extracts Bioactive metabolites from antagonistic marine Micromonospora were produced by adopting submerged fermentation. Inoculum of all the isolates was prepared using YEME broth by incubating in rotary shaker for 72 h. About 10 % of inoculum was transferred in to each 100ml of soybean meal broth, YEME broth and SGG broth and incubated at 28°C in rotary shaker for 10-14 days. At the end of fermentation, the biomass was separated from culture supernatant by centrifugation at 10,000 rpm for 30 min. Intracellular metabolites present in the mycelial biomass were extracted overnight using methanol. Extracellular metabolites from culture supernatant were extracted overnight using equal volume of ethyl acetate. Both the methanol and ethyl acetate extracts were concentrated by evaporation. In the present study, totally 114 crude extracts were obtained from 19 marine Micromonospora isolates using three different media. Antimicrobial activity of crude extracts was tested by adopting disc diffusion method. 0.25 mg of crude extract was impregnated into 5 mm sterile filter paper disc and plated over nutrient agar plates inoculated with test pathogens. Zone of growth inhibition was measured after 24 h of incubation at 37oC and expressed in millimetre in diameter [10].

Results and Discussion Actinomycete colonies appeared on isolation media after one week of incubation. Totally 54 actinomycete colonies with the morphology of Micromonospora was selected. They belong to brown, yellow and orange, pin pointed mucoid or leathery colonies and the presence of monospore at the end of substrate mycelium. All the isolates showed good growth between 1.5% and 2.5% of NaCl supplemented ISP-2 medium (Table 1). This confirmed the marine nature of all the Micromonospora isolates. In the primary screening for antagonistic activity, 19 out of 40 Micromonospora isolates inhibited one or more of the bacterial pathogens tested. Broad spectrum activity was exhibited by 15 Micromonospora isolates (Table 2). In submerged fermentation for secondary screening, all the isolates showed good growth with pigmentation on soybean meal broth when compared to YEME and SGG medium.



Bioprospecting of Marine Micromonospora 97 Table 1. Effect of sodium chloride on the growth of Micromonospora isolates Strain No

Colony colour

SM

AM

MA1 MA2 M1 M3 M4 M5 M6 M11 M15 M16 M17 M19 M20 M22 M101 M102 M104 BR10 BR12

Brown Brown Orange Brown Orange Orange Yellow White Brown Brown Yellow Brown Yellow Yellow Yellow Orange Brown Brown White

+ + + + + + + + + + + + + + + + + + +

-

NaCl Tolerance [mycelial dry weight in mg/10ml] 0%

0.5%

1.0%

1.5%

2.0%

2.5%

3.0%

3.5%

15 17 21 16 19 20 23 15 17 20 18 22 20 21 20 24 20 20 15

40 38 41 39 42 45 43 35 41 45 39 46 49 47 45 48 50 43 40

70 69 73 68 75 75 74 70 72 80 69 76 81 80 75 79 83 73 70

105 96 110 103 108 106 105 110 112 120 113 110 123 120 115 124 125 107 103

145 130 150 137 142 140 139 146 150 154 148 145 160 158 147 148 160 146 140

124 115 120 112 121 118 115 120 123 121 120 118 125 98 120 118 140 105 107

98 80 86 79 85 95 90 93 96 102 97 88 95 85 100 90 104 79 81

60 53 56 50 55 65 64 67 60 73 66 54 55 60 73 55 73 49 60

Most of the crude extracts were brown in colour. In disc diffusion method, crude extracts from all the 19 Micromonospora isolates showed inhibitory activity against atleast one of the six bacterial pathogens tested. Among the three different media used, ethyl acetate extract of culture supernatant from soybean meal medium showed good antimicrobial activity. Soybean meal based medium is economically suitable medium for the production of bioactive metabolites from actinomycetes isolated from different rare ecosystems. Radhakrishnan et al. [9, 11] carried out the production of bioactive compounds from marine sediments and Himalayan mountain soils using soybean meal medium. Of the 19 Actinomycetes tested, ethyl acetate extract from 13 (68 %) Micromonospora isolates showed antimicrobial activity. This indicates the extracellular nature of the bioactive metabolites. Most of the secondary metabolites including antibiotics are extracellular in nature and extracellular products of actinomycetes exhibit potent antimicrobial activities [12, 13]. Among the 114 crude extract prepared from 19 Micromonospora using three different media and two different solvents, ethyl acetate and methanol extract from the strain M104 showed maximum activity against all the bacterial pathogens tested (Table 3). In India there are many reports on Actinomycetes from various marine ecosystems [14]. But reports on marine Micromonospora are very few. The antagonistic Micromonospora species reported in this study from Pichavaram mangroves ecosystem and Andaman marine ecosystem is a promising, newly added source for the isolation of novel anti-infective metabolites. INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

98 Balagurunathan and Radhakrishnan Table 2. Antibacterial activity of marine Micromonospora isolates Strain No MA1 MA2 M1 M3 M4 M5 M6 M11 M15 M16 M17 M19 M20 M22 M101 M102 M104 BR10 BR12

S. aureus + + + + + + + + + + + + +

Test organisms E. coli S. typhi + + + + + + + + + + + + + + + + + + + + + + + + + + -

B. subtilis + + + + + + + + + + + + + + + + +

S. flexneri + + + + -

Table 3. Antimicrobial activity of marine Micromonospora species M104 Medium

SM

YEME

SGG

Extracts

Zone of inhibition* against S. aureus

B. subtilis

E. coli

S. typhi

S. flexneri

EAE

14

13

13

14

13

ME

15

14

15

14

12

EAE

8

9

8

6

7

ME

9

7

7

10

7

EAE

7

7

8

9

10

ME

10

11

7

7

8

EAE - ethyl acetate extract; ME – methanol extract *expressed in millimetre in diameter

Acknowledgement: Authors thank the authorities of Sri Sankara Arts & Science College, Kancheepuram, Tamil Nadu, India for their encouragement and the research facilities provided.



Bioprospecting of Marine Micromonospora 99

References 1.

Berdy, J., 2005, “Bioactive microbial metabolites”, J Antibiot, (Tokyo) 58, pp.1-26.

2.

Hirsch, A.M. and Valdés, M., 2009, “Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels”, Soil Biol Biochem, 11, pp. 1-7.

3.

El-Tarabily, K.A., Sykes, M.L., Kurtböke, I.D., Hardy, G.E.St.J., Barbosa, A.M. and Dekker, R.F.H., 1996, “Synergistic effects of a cellulase-producing Micromonospora carbonaceae and an antibioticproducing Streptomyces violascens on the suppression of Phytophthora cinnamomi root rot of Banksia grandis”, Can J Botany, 74, pp. 618-624.

4.

Conn, V.M., Walker, A.R. and Franco, C.M.M., 2008, “Endophytic actinobacteria induce defense pathways in Arabidopsis thaliana”, Molecular Plant-Microbe Interactions, 21, pp. 208-218.

5. Ellaiah, P. and Reddy, A.P.C., 1987, “Isolation of actinomycetes from marine sediments of Visakhapatnam, east coast of India”, Ind J Marine Sci, 16, pp. 134-135. 6.

Raja, A. and Prabakaran, P., 2011, “Preliminary screening of antimycobacterial effect of pschrophilic actinomycetes isolated from manali ice point: Himachal Pradesh”, J Microbiol. Antimicrobials”, 3(2), pp. 41-46.

7.

Talukdar, M., Duarah, A., Talukdar, S., Gohain, M.B., Debnath, R., Yadav, A., Jha, D.K and Bora, T.C.,. 2012, “Bioprospecting Micromonospora from Kaziranga National Park of India and their antiinfective potential”, World J Microbiol Biotechnol, 28(8), pp.2703-2712.

8.

Shirling, E.B. and Gottileb, D., 1966, “Methods for characterization of Streptomyces species”, Int J Sys Bacteriol., 16, pp.313-340.

9. Radhakrishnan, M., Deeparani, K. and Balagurunathan, R., 2006, “Production of Bioactive Compounds by Solid State Fermentation from Actinomycetes of Andaman sediments”, Ind J Appl Microbiol, 6(1), pp. 92-98. 10. Balagurunathan, R. and Subramanian, A., 2001, “Antagonistic streptomycetes from marine sediments”, Advances in Biosci, 20(II), pp. 71–76. 11. Radhakrishnan, M., Balaji, S. and Balagurunathan, R., 2007, “Thermotolerant actinomycetes from Himalayan mountain – Antagonistic potential, characterization and identification of selected strains”, Malaysian Appl Biol, 36, pp. 59-65. 12. Radhakrishnan, M., Balagurunathan, R., Selvakumar, N., Mukesh Doble. and Vanaja Kumar., 2011 “Bioprospecting of marine derived actinomycetes with special reference to antimycobacterial activity”, Ind J Geo-Mar Sci, 40(3), pp. 407-410. 13. Mohanraj, D., Bharathi, S., Radhakrishnan, M. and Balagurunathan, R., 2011, “Bioprospecting of Actinobacteria from Yelagiri Hills with special reference to antibacterial activity”, J Chem Pharma Res, 3(3), pp.439-446. 14. Sivakumar, K., Sahu, M.K., Thangaradjou, T. and Kannan, L.K., 2007, “Research on marine actinobacteria in India”, Ind J Microbiol, 47, pp. 186-196.




Bacteriological examination of Backwater from Fishing Harbour of Ennore Creek in Chennai coast C. Ganga Baheerathi and K. Revathi* 1 2

Department of Biotechnology, Sathyabama University, Chenna - 600119, India Dept. of Zoology, Ethiraj College for Women, Chennai – 600 008, India

Abstract: Ennore creek is a major fishing area in the northern coast of Chennai, India. This area is being impacted by pollution from both industries and human wastes affecting the aquatic animals and the human population. These wastes carry enormous number of microbial pathogens and other heavy metals which result in greater economic loss. The current study is aimed at analyzing the total viable count of bacteria and pathogenic bacterial species in the water samples taken from different places of Ennore fishing area, from where many kind of fishes, oysters, mussels and clams are regularly caught for human consumption. The samples were collected in different seasons like pre monsoon, monsoon and post monsoon. MPN values of the water samples were analysed by Multiple tube test. The bacteria were isolated using Zobell’s agar medium, selective and non selective media and identified using biochemical tests. The bacteria were identified upto genus level. Results showed a higher distribution of pathogenic and non pathogenic bacteria in the water sample in all the seasons. The present study showed the prevalence of human pathogens and fecal indicator organisms like Escherichia coli, Salmonella sp., Vibrio sp., Shigella sp. in the water sample in all the seasons in varying degree. Keywords: Back water, Human pathogens, indicator organisms, Multiple tube test.

Introduction Coastal water bodies are contaminated by the sewage and industrial effluents which ultimately affect the plant, animals and human population. These wastes carry enormous level of microbial pathogens to the coastal environment causing an adverse impact on the marine resources. Some microbial

* Author for Correspondence; E-mail: [email protected]

Bacteriological examination of Backwater from Fishing Harbour 101

pathogens in the coastal environment are indigenous to the oceans, including Vibrios, whereas others like Escherichia coli, Salmonella sp. and Shigella sp. are allochthonous which are introduced through agriculture, urban surface runoff, waste water discharges and industrial effluents. Most of the Vibrios and Salmonella sp. are pathogenic to humans and some are incriminated with fatal infections [1-4]. Bacteriological analysis of water is a comprehensive and prolonged process. The microbiological analysis of water primarily involve the detection of fecal indicator bacteria which might indicate the presence of other human pathogenic bacteria of intestinal origin. Marine bivalves accumulate large number of bacteria from the immediate environment due to their filter feeding nature [5]. Filter feeding shellfishes such as scallops and oysters concentrate bacteria from the overlying water, land drainages, domestic sewage outfalls and other discharges. The present study was carried out to examine the bacteriological quality of backwater of Ennore creek located in the northern coastal region of Chennai coast along the Coromandal coast of the Bay of Bengal. It is located 20 km north of the city and 2.6 km south of the Ennore port. The creek area stretches over 3 km in to the sea and 5 km along the coast. Studies reveal that Ennore Creek is a metropolitan water body that receives treated and untreated waste from surrounding industries and domestic set up. The objective of the present study was to enumerate the total viable count of bacteria and to identify predominant pathogenic bacteria occurring in the backwater samples taken from different places of Ennore fishing area, from where many kind of fishes and molluscans are caught for human consumption.

Materials and Methods Sampling of backwater was done during different seasons such as premonsoon, monsoon and post monsoon during the year 2010 - 2011. The surface water sample was collected in sterile screw capped bottles following aseptic procedures. All the samples were transported to the laboratory in portable icebox with in 2 h and further processed for bacteriological assessment.

Multiple tube test for Most Probable Number of bacteria Presumptive test Most probable number test was carried out with three different volumes of the sample. The different dilutions used were 10ml, 1ml and 0.1ml. Each dilution was inoculated in five tubes of MacConkey broth media so as to achieve the inoculation of the sample in five replicas of each dilution. The samples were inoculated respectively in 10ml each of MacConkey broth including one set of double strength and two sets of single strength. After 24-48 h of incubation at 37oC the results were noted for acid production and/or gas production in the tubes. Acid production during the fermentation was noted by the change in the dye color indicating the the change of pH. Small tubes, called Durham’s tubes, were placed inverted in each tube of MacConkey to collect the gas bubbles formed during the fermentation. Based on the observation of INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

102 Ganga Baheerathi and Revathi

acid/gas production the values were recorded and compared with MPN standard chart. The presence of fecal indicator organism E. coli was also checked by inoculating the sample into two tubes of brilliant green lactose bile broth and incubating them separately at 44.5ºC and 37ºC for 24 h.

Confirmed test The presence of fecal indicator coliform bacteria, E.coli was further confirmed by inoculating the broth culture of positive presumptive test in Eosin Methylene Blue agar and it was incubated at 37º C for 24 h.

Completed test The isolated bacteria from EMB agar plates were further inoculated onto nutrient agar slants and in Lactose broth with Durham’s tube for detecting acid and gas production. The colonies isolated on nutrient agar slants were stained using Gram’s staining technique.

Isolation and identification of bacteria The water samples were also serially diluted and plated on Zobells agar media. Nutrient agar plates prepared with different composition of NaCl and some selective media were used to isolate and identify specific pathogens. Subsequent to inoculation the plates were incubated in an inverted position in a bacteriological incubator at a temperature of 28±2oC for 24 to 48 h. Individual colonies from Zobell’s agar, Nutrient agar media and Macconkey agar plates were randomly selected and sub cultured. After purification, the identification of bacteria isolated from different samples was carried out using classical bacteriological techniques including Gram’s reaction, motility and IMVIC tests [6]. Oxidative or fermentative metabolisms of the bacterial isolates were determined following the methods of Hugh and Leifson [7]. The ability of bacteria to ferment sucrose, lactose and mannitol was determined by a standard method [8]. Based on these results the organisms were identified upto generic or group level according to Bergey’s manual [9]. Selective and non selective media were also used for identification.

Results and Discussion The results of enumeration of bacteria - quantitative test is presented in Table 1, which indicates that the occurrence of bacteria in monsoon is higher than that of other seasons. It could be attributed to the reason that during monsoon the flow of rain water and the chances of mixing up of the domestic sewage containing coliforms with the creek is more. In general the total heterotrophic bacterial population varies from seasons to season. The high bacterial population during monsoon in the backwater creek may be due to rain water flow which brings in huge quantities of nutrients along with mixing up of the sediment [10]. The bacterial load during pre monsoon season as observed from the results was low. Natarajan et al. [11] had reported similar observation of occurrence of low levels of bacterial population during premonsoon season. Several factors have been proposed that




considerably reduce the survival of fecal bacteria during certain seasons in aquatic environment. Sunlight, which increases the overall temperature, is thought to be the critical factor contributing to the death of these bacteria in seawater [12]. Other factors of similar kind include high salinity [13], presence of toxic agents [14], predation and parasitism [15] and low nutrient availability [16] in estuarine and marine waters during summer season. Table 1. Enumeration of bacteria isolated in different seasons Sample*

Premonsoon (CFU/ml)

Monsoon (CFU/ml)

Postmonsoon (CFU/ml)

Water sample

5.9 × 105

6.8 × 106

7.2 × 105

*All figures represent an average of 10 samples.

Table 2 shows the results of the test for determination of Most probable numbers of total coliforms. The present study reports higher amount of occurrence of coliforms than the permissible numbers. In all the samples analysed in the present study most of the pathogenic coliforms and in particular E. coli were detected. Flavobacterium sp. and Enterobacter sp. are considered to have little sanitary significance and are reportedly common in surface run off. Klebsiella sp. is ubiquitous and may be found in waters receiving carbohydrate rich effluents.The presumptive test carried out with the samples of Ennore creek shows more number of total coliforms. Presence of bottle green colour colonies with metallic sheen on EMB agar in the confirmed test and detection of gram negative bacteria in the completed test revealed the presence of fecal coliforms mainly E. coli. This clearly indicates fecal contamination of human origin during all the seasons. Table 2. Results of MPN test of water samples Period of sampling and results Sl.No

Premonsoon

Monsoon

Postmonsoon

1 2 3 4

10ml + + + +

1ml + +

0.1ml +

10ml + + + +

1ml + -

0.1ml +

10ml + + + +

1ml + + +

0.1ml + -

5

+

+

+

+

+

+

+

+

-

MPN value

532= 150*

542=225*

541 = 150*

*Most propable number of coliforms in 100ml of water; (+) Acid and Gas produced; (-) No Acid and Gas produced

Edible shell fishes like mussels, oysters, crabs and other fishes that are sold in the Chennai market are caught mostly from Ennore backwaters, where considerable dilution occurs with fresh water, thus bringing in high nutrient load. Filter feeding shellfish such as mussels and oysters generally concentrate bacteria from the over lying water [17]. The heterotrophic bacterial species INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

104 Ganga Baheerathi and Revathi

in estuaries are characterized by the mixture of marine, fresh water and soil which are adapted to organic rich environment [18]. Mussels, oysters and other shell fishes that are harvested from these areas are reportedly often eaten raw or smoked with minimal cooking, which emphasizes the fact that less sterilization of the animal is done. Microbiological analysis is therefore necessary on a routine basis for understanding the impact of sewage and effluent discharge which affect the edible quality of these animals. Microbiologists rely on the principle that the higher the incidence of sewage indicator bacteria in any environment, greater would be the chances for occurrence of human pathogenic bacteria [19,20]. It is generally acknowledged that higher sewage contamination would lead to enhance the number of coliform in natural water body and also in the parts of animal living in these water bodies. Table 3. Occurrence of bacteria in Backwater in different seasons Sl. No.

Bacterial species

Premonsoon

Monsoon

Postmonsoon

1.

Shigella sp.

+

+

-

2.

Escherichia coli

+

+

+

3.

Vibrio sp.

_

+

+

4.

Bacillus sp.

+

+

+

5.

Aeromonas sp.

-

+

+

6.

Salmonella sp.

+

-

+

7.

Flavobacterium sp.

+

+

+

8.

Alcaligens sp.

+

+

+

9.

Pseudomonas sp.

+

+

-

10.

Streptococci.sp.

+

+

+

11.

Micrococcus sp.

+

+

+

12.

Proteus sp.

+

+

+

13.

Klebsiella sp.

-

+

+

The present study showed the occurrence of differnt bacteria in backwater as listed in Table 3. If the fishes and shell fishes caught from these areas are not properly cooked or sterilized, the presence of certain bacteria such as Shigella sp. may lead to food poisoning in the consumer. Presence of Streptococcus sp. may lead to skin infections and in severe cases meningitis. Aeromonas sp. are commonly encountered with septicemic conditions. Consumption of food contaminated with pathogenic E.coli may lead to gastric disorder. Consumption of raw oyster infected with Vibrio sp. may lead to severe gastroenteritis with abdominal cramps, vomiting, fever and diarrhea. The Vibrio contaminated oyster, crabs and mussels, if eaten undercooked, may lead to primary septicemia, cellulites, cholera etc. and in susceptible cases it may even be fatal. Many earlier researcher had reported the predominance of Vibrio sp. and Aeromonas sp. has been observed in the digestive tract of oysters and fishes [21]. INDIAN JOURNAL OF APPLIED MICROBIOLOGY



From the bacteriological analysis carried out in the present study it is concluded that Ennore backwater is mixed heavily with domestic sewage, fecal contaminants and industrial waste frequently. Thus the present study has brought about certain useful data, which could be applied for framing strategies in managing the water pollution occurring in this area. The present study also suggests that effort on priority basis has to be taken to reduce these kinds of pollutions so as to protect and conserve the welfare of aquatic animals and human beings.

References 1.

Blacke, P.A., Weaner, R.E. and Hollis, A.G., 1981, “Diseases of Humans (other than cholera) caused by Vibrio”, Annu Rev Microbiol, 34, pp. 341-367.

2. Grimes, D.J., 1975, “Release of sediment found coli forms by dredging”, Appl Microbiol, 29, pp.109-111. 3.

Carlson, G.F., Woodard, F.E. , Wentworth, J. and Sproul, S., 1968, “A name 572160 Virus inactivation on clay articles in natural waters”, J Water Pollut Control Fed, 40, pp.R89-R106

4. Gerba, C.P. and Sehalberger, F.E., 1975, “Effect of particulates on virus survival in sea water” J Water Pollut Control Fed, 47, pp.93-103. 5. Jan A. Olafsen, Helene, V., Mikkelsen, Hanne, M., Glever and Gei Hovik Hansen, 1993, “Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures”, Appl EnvironMicrobiol, 18, pp.1848-1854 6. Leifson, E., Cosenza, B.J., Murchelano, R. and Cleverdon, R.C, 1964, “Motile marine bacteria I Techniques ecology and general characteristics”, J Bacteriol, 89, pp.652 – 666 7. Hugh, R. and Leifson, E., 1953, “The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria”, J Bacteriol, 66, pp.24-26 8.

Martin, W.J., Washington, J.A II., 1980, “Entero Bacteriaceae”, In: Manual of Clinical Microbiology 3rd ed, Lennette, E.H., Balows, A., Hausler, WlJ. Jr. and Truant. J.P, pp. 195 – 219. Washington D.C., American Society of Microbiology.

9.

Buchanan, R.E. and Gibbons, N.E. (ed.), 1974, “Bergey’s Manual of Dertminative Bacteriology” 8th edn., Genus Pseudomons Duodoroff, Palleroni, N.J., Genus Vibrio shewan, Veron, M., pp.217 – 221, 340 – 345 Baltimore, William and Wikins.

10. Sathiyamurthy, K., Purushothaman, A. and Ramaiyan, V. , 1992, “Heavy metal and drug resistant bacteria in the vellar estuary, south east coast of India” Mahasagar, 25, pp.119-122. 11. Natarajan, R.M., Abraham and Nair, G.B. ,1980, “Distribution of Vibrio parahaemolyticus in PortoNovo environment, India”, J Med Res, 71, pp.679-687. 12. Chamberlin, C. E. and Mitchell, R., 1978, “A decay model for enteric bacteria in natural waters”, pp. 325–348. In: Water pollution Microbiology, Mitchell, R. (ed.), vol. 2, Wiley, New York. 13. Pike, E. B., Gameson, A. H. L. and Gould, D. J., 1970, “Mortality of coliform bacteria in seawater samples in the dark”, Rev Int Oceanogr Med, 18/19, pp.97–107


106 Ganga Baheerathi and Revathi 14. Jones, G. E., 1964, “Effect of chelating agents on the growth of Escherichia coli in seawater”, J Bacteriol, 87, pp.484–499 15. Enzinger, E. M. and Cooper, R. C. , 1976, “Role of bacteria and protozoa in the removal of Escherichia coli from estuarine waters”, Appl Environ Microbiol, 31, pp.758–763 16. Gauthier, M. J., Munro, P. M. and Breittmayer, V. A., 1989, “Influence of prior growth conditions on low nutrient response of Escherichia coli in seawater”, Can J Microbiol, 35, pp.379–383. 17. Slanets, L.W., Bertley, C.H. and Stanley, K.W., 1968, “Coliform, fecal Streptococci and Salmonella in Sea water and shellfish”, Health Lab Sci, 5, pp.66-78. 18. Keuh, C.S.W. and Chan,K.Y., 1975, “The distribution of heterotrophic bacteria related to some indicators of marine pollution in Tolo harbour, Hong Kong, In: The proceeding of the special Symposium on marine Sciences, December 1973 pp.95 – 99. Hong kong: The Pacific Science Association. 19. Brock,T., Madigan, M.T., Maetinko,J.M. and Parker,J., 1994, “Biology of Microorganism” (7th edn.), Prentice Hall ,New Jersey 20. Fujioka,R., 2002, “Microbial indicators of marine recreational water quality”, In: Manual of Environmental Microbiology, (2nd edn.), American society for Microbiology Press, Washington DC, pp.234 – 243. 21. Okuzumi, M. and Horie, S., 1968, “Studies on the bacterial flora in the intestine of various marine fish”, Bullet Japan Soc Scient Fisheries, 35, pp.93-100.





Antibacterial studies of Smilax zeilanica against Rifampicin and Methicillin resistant Staphylococcus aureus K. Kavitha1* and K. Murugan2 1 2

Dept of Microbiology, Madras Christian College, Tambaram, Chennai – 600 059, India. Dept of Microbiology, KSR College of Arts and Science, Tiruchengode – 637215, Tamil Nadu, India.

Abstract: The worldwide emergence of hospital and community acquired pathogen Methicillin-resistant Staphylococcus aureus (MRSA) warrants new agents for treatment of its infections. The present study investigated the methanol, ethyl acetate and aqueous extract of Smilax zeilanica for its ability to inhibit seven strains of multi-drug and common beta-lactam antibiotic resistant clinical MRSA. The ethyl acetate extract of S. zeilanica exhibited higher antibacterial inhibitory potency with an inhibition zone size of 13 mm to 19 mm. The potential of this plant extract was compared with antibiotic Ciprofloxacin. TLC phytochemical evaluation of these extracts deciphered the presence of alkaloids, carbohydrates, flavonoids, quinones, cardiac glycosides, terpenoids, thytosteeroids, coumarins and proteins. The demonstration of promising anti-MRSA potential of S. zeilanica is a primary attempt which will facilitate further validation this drug for treatment of skin infection, activity-guided fractionation, isolation and characterization of active principle to establish its mode of action. Keywords: Antimicrobial activity, well cut method, clinical pathogens, phytoconstituents.

Introduction The infectious diseases and oncology therapeutic areas have gained rich scaffold diversity in natural products (NP). The NP discovery leads are better and often more biologically friendly owing to their co-evolution with the target sites in biological systems. The uses of NP templates are still a viable source of new drug candidates and have been the source of inspiration for the


108 Kavitha and Murugan

majority of FDA approved drugs [1]. Herbs are widely used to treat the microbial infections for their less side effects and relatively low cost [2]. Therefore investigation of some active principles from medicinal plants has become more important [3]. The WHO (1980) recommends the evaluation of the effectiveness of plants in treating microbial diseases where we lack even in managing safe modern drugs [4]. Traditional medicine, which provides health service to 75–80% of world’s population, is given lot of significance in recent days. Plant-based traditional knowledge serves as recognized tool for new sources of drugs and neutraceuticals search. India is rich in vegetation with a wide variety of plants due to the prevailing variations in geographical and climatic conditions. The plants have been used since ancient times for the treatment of various ailments [5]. Nevertheless, the folk and ethno medicinal uses in rural India have been playing a great role in treatment of diseases, which is now becoming less important and endemic due to various reasons. The ethanomedicinal use of these plants does not necessarily imply efficacy, but it does give a list of species that can be studied pharmacologically for its active principles and bioactive potential [6]. Smilax zeylanica Linn., a member of Liliaceae family commonly known as Chopachinee (Sanskrit), chobchini (Hindi), Kaaduhambuthaavare (Kannada), Kummeritheega (Telugu), Cherunchakayagavalli (Malayalam), Gholbel (Marathi), Kumari (Bengali), Ayadi, Tirunamappalai, Periyakanni and Karuvilanchikudam (Tamil) [7] is a widely used ethanomedicinal herb throughout India. It is a climber with slender 4-angular branches found throughout the tropical hilly areas from the Himalayas, Southward to Kerala. Leaves are used as vegetable. Roots are eaten for the treatment of venereal diseases, skin troubles and a decoction of the bulbous root is given for sores, swellings and abscesses [8]. Found on hills, at higher elevation in South India, they were used to treat rheumatism, urinary complaints and dysentery [9]. In Bangladesh, it is used for the treatment of fever, headache, and wounds [10]. Apart from skin disorders, swellings and abscess, it is also applied for rheumatism and pain in the lower extremities [11]. It possess the property of rejuvenator and blood purifier and in the treatment of Parkinson, polyuria, psychosis [12]. In Tamilnadu, tribal people in Theni District (Western Ghats) use shade dried stem bark powder to cure skin diseases and arthritis[13]. Whereas, to the best of our knowledge no report is available in literature on antibacterial activities of S. zeylanica on multidrug resistant MRSA and hence in this study an attempt was made to study the antibacterial activity of this plant against community acquired clinical MRSA strains.

Materials and Methods Collection of plants The plant was collected from Tirunelveli district, Tamilnadu. The identification of the plant was done following the descriptions of Flora available in Madras Christian College (Aut.), Chennai. Drying was done under shade by spreading over the paper or on a wooden table for a week.



Antibacterial studies of Smilax zeilanica against Rifampicin and Methicillin resistant S.aureus 109

Preparation of plant extract All the assays were performed using the root of the plant. The powdered root was extracted with non-polar to polar solvents like hexane, ethylacetate, chloroform and methanol. A fine dried powder sample (250 gm) was near extracted using 1:3 of hexane for 72 h and filtered through Whatman No.1 filter paper. The hexane was allowed to evaporate in reduced pressure and to dry. Using the dry powder different extracts were prepared using different solvents (ethyl acetate, chloroform and Methanol) by repeating same procedure. The extracts were used for determining phytochemical constituents and antibacterial activity.

Collection of MRSA cultures Twenty one clinical isolates were collected and screened for methicillin resistant S.aureus, out of which seven cultures were identified as MRSA. These cultures were incoulated by inoculating into NA , MA plates were incubated at 37oC for 24 h. After the overnight incubation the colony morphology was noted and identified for Biochemical test.

Antibiotics used Antibiotics such as Methicillin and Rifampicin were used against all the strains of bacteria. The inoculums of necessary size was performing antibiotic sensitivity test preparation of inoculum was done.

Assay of Antibacterial Activity using Agar Well Diffusion Method (cup plate method) All the seven selected strains of MRSA isolated from clinical specimens were inoculated into 5ml of sterile nutrient broth and incubated at 37oC for 16 to 18 h. The broth culture was swabbed on Mueller Hinton Agar (MHA). The wells were made by using sterile well culter (size 8mm) using micropipette, 4 mg/100ml conc. of plant extract diluted in dimethyl sulfoxide were added to different wells in the plate. They were incubated at 37oC for 24 h. The diameter of inhibition zone was measured in millimeter and the results were recorded. The growth inhibition zones with diameter less than 7 mm were not considered for antibacterial activity.

Evaluation of MIC by well diffusion method Using sterile swabs the nutrient broth culture was swabbed on the surface of the MHA plates. The wells were made using sterile culture each strain of bacteria. Using Micro pipette varying concentration of the extract such as 3.5 mg/100ml, 4.0 mg/ml, 4.5 mg/ml and 5.0 mg/ml were added to respective wells and incubated 37oC for 24 h.

Results and Discussion As the primary objective of this study is to determine the antibiotic resistance of MRSA against methicillin and Refiampicin, the antibiotic sensitivity test was carried out using standard antibiotics INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012


(Table 1). The results of the antibacterial activity of the extracts are shown in Tables 2 and 3. The extract of the plant was found to be effective against all the seven clinical isolates of MRSA. Methanol and chloroform extracts showed the highest activity against strain 1 and strain 7; however chloroform extract showed only moderate activity against the remaining strains. Ethyl acetate and hexane extracts showed comparatively better activity against almost all strains. In particular, ethyl acetate extract showed the highest activity against Strain 2. Table 1. Antibiotic sensitivity test Culture Antibiotic Discs

MRSA 1 MRSA 2

MRSA 3

MRSA 4

MRSA 5

MRSA 6

MRSA 7

Z

Re

Z

R

Z

R

Z

R

Z

R

Z

R

Z

R

Methicillin

_

R

_

R

_

R

_

R

_

R

_

R

_

R

Rifampacin

16

R

14

R

18

M

17

M

19

M

18

M

19

M

Z - Zone size (mm); Re – Result; R-resistant; _ no zone formation Standard zone measurement: methicillin (5 mcg): R, 9mm; M, 10-13mm; S,14mm Rifampacin (5mcg): R; 16mm, M; 17-19mm, S; 20mm

Table 2. Antibacterial activity of plant extracts (4mg/100μl) Culture MRSA 1

Plant extract

MRSA 2

MRSA 3

MRSA 4

MRSA 5

MRSA 6

MRSA 7

Z

Re

Z

R

Z

R

Z

R

Z

R

Z

R

Z

R

16

+

15

+

14

+

14

+

13

+

15

+

16

+

Ethyl acetate 18

+

19

+

13

+

14

+

16

+

13

+

15

+

Hexane

+

_

R

_

R

_

R

_

R

_

R

13

+

Methanol

12

Z - Zone size (mm); Re – Result; R-resistant; _ no zone formation +; susceptibility (inhibition zone ≥7mm)

Phytochemical screening of plant extract (Table 4) showed the presence of carbohydrates [14], flavonoids, alkaloids [15,16], terpenoids [15], cardiac glycosides [16], quinines [17], phytosteriods [18] and protein [19]. These compounds may be suggested to contribute to the antibacterial activity as has been shown in studies. Further research is needed to isolate the bioactive compounds from plant for the preparation of new drugs.



Antibacterial studies of Smilax zeilanica against Rifampicin and Methicillin resistant S.aureus 111 Table 3. Minimum Inhibitory Concentration of plant extracts Plant extract Methanol extract Zone diameter in mm

Culture

Ethyl acetate Zone diameter in mm

5.0 mg

4.5 mg

4.0 mg

3.5 mg

5.0 mg 4.5 mg 4.0 mg

MRSA 1

20

19

16

13

20

19

MRSA 2

15

14

14

12

19

MRSA 3

15

14

13

10

MRSA 4

16

14

13

MRSA 5

16

15

MRSA 6

18

MRSA 7

19

Positive control

3.5 mg

Cp

18

16

31

19

18

17

31

15

14

13

12

29

11

16

15

14

13

29

13

13

18

17

16

14

30

17

16

12

15

15

14

12

29

17

15

13

17

16

15

13

38

Cp – Ciprofloxacin; MIC of plant extract is 4.5 mg/100µl (methanol); 4mg/100µl (ethyl acetate)

Table 4. Phytochemical profile of plant extracts Test Extract Ca

Ta

Sa

Fl

Al

Qu

Gl CaGl

Tr

Trt

Ph

Co

Pr

Ph

Phl

An

Ethyl acetate

+

+

_

_

_

+

_

_

+

_

_

_

_

+

+

_

Methanol

+

_

_

_

_

+

_

+

+

_

_

_

_

_

_

_

Hexane

+

_

_

_

+

+

_

+

+

_

_

+

_

_

_

_

Chloroform

+

_

_

+

_

+

_

+

_

_

_

+

+

+

_

_

Ca – Carbohydrate; Ta – Tannins; Sa – Sapponins; Fl – Flavanoids; Al – Alkaloids; Qu – Quinones; Gl – Glycosides; CaGl - Cardiac glycosides; Tr – Terpenoids; Trt – Triterpenoids; Ph – Phhenols; Co – Coumarins; Pr – Protiens; Ph – Phytosteroids; Phl – Phlobatanins; An Anthraquinones



References 1.

Mishra, B.B. and Tiwari,V.K, 2011, “Natural products: An evolving role in future drug discovery”, Europ J Med Chem, 46, pp.4769-4807.

2. Venkatesh, S., Reddy, G.O., Reddy, B.M., Ramesh, M. and AppaRao, A.V.N., 2003, “Antihyperglycemic activity of Caralluma attenuates”, Fitoterapia, 74, pp.274-279 3. Suba, V., Murugesan, T., Arunachalam, G., Mandal, S.G. and Saha, B.P., 2004, “Hypoglycemic potential of Barlerialupulina extract in rats”, Fitoterapia, 75, pp.18-26. 4.

Upathaya,V., and Pandey, K., 1984, “Ayurvedic approach to Diabetes Mellitus and its management by Indigenous Resources. In: Diabetes Mellitus in Developing countries”, Bajaj,J.S. (ed.), Interprint, New Delhi, pp.375-377.

5. Ayyanar, M., and Ignacimuthu, S., 2005, “Traditional knowledge of Kanitribals in Kouthalai of Tirunelveli hills, Tamil Nadu, India”, J Ethnopharmacol, 102, pp.246–255. 6.

Behera, S.K. and Misra, M.K., 2005, “Indigenous phytotherapy for genito-urinary diseases used by the Kandha tribe of Orissa, India”, J Ethnopharmacol, 102, pp.319–325.

7.

Gurudeva, M. R., 2001, “Botanical and Vernacular names of South Indian Plants”, Divya Chandra Prakashana, 21, pp367.

8.

Koyamma in advance front, 1963, Pl. Sci., IV, 50,Rama Rao, Santapau, H., Henry, A.N., A Dictionary of the flowering plants in India (reprint), New Delhi. (SIR 1976 : 58).

9.

Siddique,N.A., Bari,M.A., Naderuzzaman, A.T.M., Khatun.N, Rahman, M.H., 2004, “Collection of indigenous knowledge and identification of endangered medicinal plant by questionnaire survey in Barind tract of Banglaesh”, J Biol Sci, 4, pp.72-80.

10. Madhavan, V., Hemalatha, H.T., Anita Murali Yoganarasiman, S.N, 2008, “Antiepileptic activity of alchohol and aqueous extract of roots an rhizomes and Smilax zeylanicalin”, Pharmacology online, 3, pp.263-272. 11. Gurudeva, M. R, 2001, “Botanical and Vernacular names of South Indian Plants”, Divya Chandra prakashana, pp.367. 12. Sharma, P. V. and Dravyaguna Vijnana, 2005, Vol II (in Hindi), Chaukambhabharatiya Academy, Varanasi, Reprint, p. 802. 13. Jeyaprakash, K., Ayyanar, M., Geetha, K.N. and Sekar, T, 2011, “Traditional uses of medicinal plants among the tribal people in Theni District (Western Ghats), Southern India”, Asian Pacific J Trop Biomed, pp.S20-S25 14. Sofowora, A., 1993, Medicinal plants and traditional medicinal in Africa. 2nd Ed. Sunshine house, Ibadan, Nigeria: Spectrum Books Ltd; Screening plants for bioactive agents; pp.134-156 15. Sonali Jana and Shekhawat, G.S., 2010, “Phytochemical analysis and antibacterial screening of invivo and in-vitro extracts of Indian medicinal herbs: Anethumgraveolens”, Research J Med plants, 4(4):206-212, ISSN 1819-3455.



Antibacterial studies of Smilax zeilanica against Rifampicin and Methicillin resistant S.aureus 113 16. Ayoolal, G.A., Cocker, H.A.B., Adesegun, S.A., Adepoju-Bello1, AA., Obaweya1, K., Ezennial, E.C. and Atangbayilal, T.O., 2008, “Phytochemical Screening and Antioxidant Activities of Some Selected Medicinal Plants Used for Malaria Therapy in South-western Nigeria”, Trop J Pharma Res, 7 (3), pp.1019-1024. 17. Suresh Kumar, C.A., Vardharajan, R., Muthumani, P., Meera, R., Devi, P. and Kameshawari, B., 2009, “Pharmacognist and Preliminary Phytochemical Investigations on the stem of Saccharumspontaneum”, J Pharm Sci Res, 1 (3), pp.129-136. 18. Kolawole, O.M., Oguntoye, S.O, Agbede, O. and Olayemi, A.B., 2006, “Studies on the efficacy of Brideliaferruginea Benth - Bark extract in reducing the coliform load and BOD of domestic waste water”, Ethnobotan Leaflets, 10, pp.28-238. 19. Manasboxi, Y., Rajesh, V., Raja Kumar, B., Praveen and K Mangamma, 2010, “Extraction phytochemiacl screening and in-vitro evaluation of anti-oxidant properties of chommicarpuschinesis (aqueous leaf extract)”, Int J Pharma Bio Sci , 1(4), pp. 75-89.




Isolation of Vibrio spp. and other Predominant bacteria from Green Mussels in Muttukadu, Tamilnadu V. Gayathri1 and K. Revathi2* 1 2

Department of Biotechnology, Sathyabama University, Chennai – 600 119, India Dept. of Zoology, Ethiraj College for Women, Chennai -600 008, India

Abstract: The microflora in the estuarine and marine environments include various members of the family Vibrionaceae, some of which are pathogenic to humans and constitute a potential health threat for consumers of raw or partially cooked mussels. In this present investigation, Green Mussels were collected from Chennai Coast – Muttukadu. Bacteria were isolated using selective and non-selective agar medium and further confirmed by biochemical tests. The organisms were identified upto generic or group level according to Bergey’s manual of systematic Bacteriology. Vibrio spp. have been isolated from processed samples. Escherichia coli, Vibrio alginolyticus, V. parahaemolyticus and V. harveyi were the dominant species. Vibrio parahaemolyticus, a facultative pathogen widely implicated in outbreaks of gastroenteritis related to the consumption of improperly processed seafood was present in the Green Mussels. Keywords: Vibrio spp., Green Mussels, gastroenteritis, outbreak

Introduction Marine bivalves accumulate large number of bacteria from the immediate environment due to their filter feeding nature [1]. The Vibrios constitute a considerable part of marine halophilic bacterial populations, which require high concentrations of salt for growth in the sea. Vibrios are found less frequently when the temperature drops and salinity increases. In addition to V. cholerae, the most widely known pathogenic Vibrio, a number of Vibrio species can cause diseases. The Vibrio parahaemolyticus, V. minicus, and V. vulnificus are food-poisoning bacteria frequently isolated from


Isolation of Vibrio spp. and other Predominant bacteria from Green Mussels 115

seawater and shellfish. Among halophilic vibrios V. alginolyticus, V. fluvialis, and V. metschnikovii are also pathogenic to humans, while V. anguillarum is a pathogen that affects fish and other aquatic animals. V. parahaemolyticus has been reported responsible for one-fourth of all gastrointestinal pathologies caused by food. The Green mussel Pernaviridisis, one of the commercially important marine bivalves found all along the East and the West Coasts [2] of India. These bivalve molluscs typically inhabit the estuaries and coastal areas that are increasingly contaminated with anthropogenic chemicals. Marine bivalves accumulate large number of microorganisms including Gram-negative Achromobacter spp., Aeromonas spp., Alcaligenes spp., Flavobacteium spp., Pseudomonas spp. and Vibrio spp. and Gram-positive Bacillus spp., Corynebacterium spp. and Micrococcus spp. [1]. The members of the family Vibrionaceae contribute 60% of the total bacterial population [3]. Since Vibrio species are isolated from water, sediment, invertebrates and fishes they are considered as autochthonous marine and estuarine microflora [4]. The bacteria responsible for the early stage of spoilage in bivalve shellfish are thought to be derived mainly from their natural flora [5]. The genus Vibrio includes more than 35 species, mostly of marine origin. A number of Vibrio species other than V. cholerae, many cause disease in man mainly by ingestion of raw sea food. V. parahaemolyticus, V.mimicus and V.vulnificus are food poisoning bacteria which are normal habitants in estuarine and marine environments and are frequently isolated from seawater and seafood. Among halophillic Vibrios V. alginolyticus, V. fluvialis and V. metschnikovii are also pathogenic for humans, while V. anguillarum represents a pathogen for fishes and other marine animals [6]. The presence of specific human pathogenic species of Vibrio can serve as an indicator of public health safety of water and food destined for human consumption [7]. The present study was carried out with Muttukadu back waters (12°47’N, 80°15’E) located 36 km from Chennai city, runs parallel to the east coast of India and opens into the Bay of Bengal, from where lot of fishes, shellfishes are harvested and the area is surrounded by many aquaculture farms. Sewage and industrial effluents from the surrounding area have a greater impact on the Muttukadu backwater affecting the aquatic animals and in turn the human population. These wastes carry enormous number of microbial pathogens and other heavy metals resulting in greater economic loss. Therefore a study was conducted to assess and determine the presence of Vibrio spp. and other predominant bacteria in green mussels in Muttukadu back water.

Materials and Methods Sampling was done during premonsoon, monsoon and post monsoon seasons of the year 2010. With proper precautions 10-15 mussel samples were collected, taken in an ice box and transported to Microbiological Laboratory within 4 h for further examination. A total of 12 mussel samples were taken for observation. Cleaning, shucking and preparation of the mussel for bacteriological examination were done aseptically. The shell liquor of the mussel was discarded and the flesh was INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 15 No. 2 July-Dec. 2012

116 Gayathri and Revathi

removed. The flesh was aseptically transferred to a sterile bottle. The flesh was then transferred to a sterile test tube containing 10ml of sterile sea water having Tween 80. The tube was kept on a rotary shaker at 150 rpm. Then the mixture was homogenized, serially diluted and plated in Zobell Marine Agar media and Nutrient agar with different concentrations of NaCl. Predominantly 4-5 organisms were isolated and identified.

Isolation and Identifiction Individual colonies isolated on Zobell Marine Agar media were randomly selected and sub cultured. After purification, the organisms were tested for Gram reaction, motility, Biochemical tests (IMViC), H2S production, sucrose, lactose and mannitol fermentations. Vibrio species was selected using Thiosulphate citrate bile sucrose (TCBS) agar. The organisms were identified up to generic or group level according to Bergey’s manual of systematic Bacteriology [8].

Results and Discussion The results of microbiological analysis of the Green mussel Pernaviridis is summarized in Tables 1 and 2. Bacteria such as Vibrio cholerae, V. parahaemolyticus, V. damsela, V. harveyi, Klebsiella pneumoniae, Aeromonas hydrophila, Yersinia rohdei, Escherichia coli, Citrobacter freundii, Pseudomonas spp. and Proteus mirabilis were isolated from mussel. The present study reports the occurence of different Vibrio sp. in Mussel samples. Among the different Vibrio spp., V. cholerae is considered as the part of natural bacterial flora in aquatic environments. It causes diarrhea, which results in loss of body fluids and minerals. This bacteria does not multiply in water but can survive for upto two weeks. It is salt tolerant, heat sensitive and destroyed by cooking. The possible reason for the predominance of other bacteria over V. cholera could be attributed to its fresh water nature. Similar results were reported by Wilson and Moore et al. [9]. In another research, 26 out of 200 fresh seafood samples contained Vibrio spp., and the highest percentage of contamination was found in mussels [10] . V. parahaemolyticus is found in warm coastal waters of countries throughout the world. These organisms cause severe abdominal pain, nausea, diarrhea and vomiting. The diseases caused by this bacteria are usually associated with the ingestion of raw or insufficiently cooked seafood, improper post-harvest storage conditions or poor handling of seafood during preparation. The isolation of V. parahaemolyticus in the present study from mussel samples is in accordance to the findings of Baffone et al. [10]. The presence of V. parahaemolyticus in marine environment is due to its nature of acclimatization with a broad range of salinity especially in backwater. Recent studies have shown that V. cholera naturally occurs in temperate estuaries and that cases of cholera in the Gulf coast region of the United states have resulted from the ingestion of contaminated shellfish, including inadequately cooked crab meat [11]. The most common halophillic Vibrio species isolated from both clinical and environmental samples were V. parahaemolyticus. Environmental strains of V. parahaemolyticus are typically INDIAN JOURNAL OF APPLIED MICROBIOLOGY


Isolation of Vibrio spp. and other Predominant bacteria from Green Mussels 117

not human pathogens. However these strains cause diseases in shrimps, oysters, mussels and other marine invertebrates. These results suggest consumption of Mussels without proper cooking might lead to the infection by V. parahaemolyticus. This bacteria is commonly found on shellfishes and all varieties of fin fishes that are traditionally caught from marine and shore areas [12]. These strains cause diseases in Shrimps, oysters, mussels and other marine invertebrates [13]. Other Vibrio spp. isolated from the green mussel were V. damsela, V. harveyi and V. alginolyticus. Table 1. Vibrio spp. isolated from green mussel of Muttukadu Period

Vibrio spp. isolated

Pre monsoon

Vibrio harveyi Vibrio parahaemolyticus

Monsoon

Vibrio harveyi Vibrio parahaemolyticus

Post monsoon

Vibrio harveyi Vibrio parahaemolyticus Vibrio damsela Vibrio cholera

Table 2. Occurrence of different bacteria in green mussel of Muttukadu Period

Bacteria

Pre monsoon

Klebsiella pneumoniae Aeromonas hydrophila Yersinia rohdei Escherichia coli Citrobacter freundii Pseudomonas spp.

Monsoon

Klebsiella pneumoniae Aeromonas hydrophila Yersinia rohdei Escherichia coli Proteus mirabilis

Post monsoon

Klebsiella pneumoniae Aeromonas hydrophila Yersinia rohdei Escherichia coli Proteus mirabilis


118 Gayathri and Revathi

Population density of Vibrio sp. in the marine environment is usually higher than the other organisms as they can occur in a wide range of aquatic environments including estuaries, marine and coastal waters and sediments [14]. Common soil bacteria like Aeromonas spp. also grow on green mussels. Similar observations were made on bivalves from the west coast of the United States and Japan [15,16]. Aeromonas is a facultatively anaerobic, Gram negative organism that is commonly found in fresh water and sewage. It can be pathogenic to Frogs, fish and mammals, including man [17]. In general, abundance of all groups of bacteria were more during post monsoon period which shows that inflow of surface waters together with sewage influences diversity of microbes. Among the bacteria E.coli was detected throughout the sampling period. This shows that it is reasonable to assume that faecal pollution is more influencing the number of coliforms in the water which indirectly affect the green mussels and other shell fishes which are filter feeders consuming the bacterial pathogens along with the water. It is generally acknowledged that Microbiologists rely on the principle that higher the incidence of sewage indicator bacteria in any environment, the greater would be the chances for human pathogenic bacteria to be present [18,19]. The present study suggests that necessary stringent proceducres to be adopted to prevent effluent outfall in to coastal ecosystem and control the microbes of human health concern.

References 1.

Jan A.Olafsen, Helene, V., Mikkelsen, Hanne M. Glever and GeiHovik Hansen, 1993, “Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures”, Applied Environ Microbiol, 21, pp.1848-1854.

2.

Rao, K.S., 1974, “Edible bivalves: Mussels and oysters: Commercial Molluscs of India”, Bull Cent Mar Fish Res Inst, 15, pp.34-39.

3. Simidu, U. and Tsukamoto, K., 1985, “Habitat segregation and biochemical activities of marine members of the family Vibrionaceae”, Appl Environ Microbiol, 50, pp.781-790. 4. Grimens, D.J., Brayton, P., Colwell, R.R. and Gruber, S.H., 1986, “Vibrios as autochthonous flora of neretic sharks”, Syst Appl Microiol, 6, pp.221-226. 5. Jay, J.M., 1978, “Modern food Microbiology”, 2nd edn., Newyork. D van Noshand, “Potentially pathogenic Vibrios in brackish waters and Mussels”, Maugeri, T.L. , Coccamo, D., Cugliandolo, C., J Appl Microbiol, 89(2), pp.261-6. 6. Farmer. J.J, 1992, III & HichmanBrenner,F.N., “The genera Vibrio and Photobacterium. In the Prokaryotes” vol II 2ndedn. Eds. Balows, Truper, H.G., Dworkin, M., Harder,W. and Schleifer, K.H., Newyork, NY: Springer verlang, pp. 295. 7. Colwell, R.R. and Kaper, J. , 1977, “Vibrio spp. as bacterial indicators of potential health hazards associated with water”, Am Soc Test Mat, pp. 115-123.



Isolation of Vibrio spp. and other Predominant bacteria from Green Mussels 119 8.

Buchanan, R.E., Gibbons, N.E. (ed), 1974, Bergey’s Manual of Dertminative Bacteriology 8thedn., ‘Genus Pseudomons Duodoroff, M. and Palleroni, N.J., “Genus Vibrio shewan”, J Veron, .M M., Baltimore: William &Wikins.

9. Wilson, I.G. and Moore,J.E., 1996, “Presence of Salmonella spp. and Campylobacter spp. in shellfish”, Epidemiol infect, 116, pp.147-153 10. Baffone,W., Pianetti,A., Bruscolini,F., Barbieri,F. and Citterio, B., 2000, “Occurrence and expression of virulence related properties of Vibrio species isolated from widely consumed seafood products”, Int J Food Microbiol, 54, pp.9-18. 11. Blake, P.A., Weaner, R.E. and Hollis, A.G., 1980, “Diseases of Humans (other than cholera) caused by Vibrio”, Ann Rev Microbiol, 34, pp.341-367. 12. Syndam, D.R. and Gorbach, S.L., 1991, “Bacterial food poisoning in bacterial infections of Humans, Evans, A. S. and Brahman, P.S., (ed.), Plenum Press, New York, p 87-113. 13. Puente, M.E., Vegavillasante, F., Holguin, G. and Bashan, Y., 1992, “Susceptibilty of the brine shrimp Artemia and its pathogen Vibrio parahaemolyticus to chlorine dioxide in contaminated seawter”, J Appl Bacteriol, 73(6), pp.465-471. 14. Urakawa ,H., Yoshida, T., Nishimura, M. and Ohwada, K., 2000, “Characterzation of depth related population variation in Microbial communities of coastal marine sediment using 16srDNA based approaches and quiniae profiling”, Environ Microbiol, 5, pp.542-554 15. Sugita, H., Tanaami, H., Kobashi, T. and Deguchi, Y., 1981, “Bacterial flora of coastal bivalves”, Bull Japan Soc Sci Fish, 47, pp.655-661. 16. Vasconcelos, G.J. and Lee, J.S., 1972, “Microbial flora of pacific oysters (Crassostreagigas) subjected to ultraviolet irradiated sea water”, Appl Microbiol, 23, pp.11-16 17. Krieg, N.R. and Holt, J.G., 1984, “Bergeys Manual of Systematic Bacteriology” Vol.1 Williams and Wilkings, p.964 18. Brock,T., Madigan,M.T., Maetinko,J. M. and Parker,J., 1994, Biology of Microorganism (7th edn.), Prentice Hall, New Jersey. 19. Fujioka, R., 2002, “Microbial indicators of marine recreational water quality”, In : Manual of Environmental Microbiology, (2nd edn.), Am Soc Microbiology Press, Washington DC, pp.234 – 243.


INDIAN JOURNAL OF APPLIED MICROBIOLOGY Copyright  2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-Dcember 2012, pp.120-121.

Biography of a Renowned Microbiologist: Heinrich Hermann Robert Koch (1843 – 1910) Robert Koch was a German physician. He became famous for isolating Bacillus anthracis (1877), the Tuberculosis bacillus (1882) and Vibrio cholerae (1883) and for his development of Koch’s postulates. Working with very limited resources, he became one of the founders of bacteriology, the other major figure being Louis Pasteur, Paul Ehrlich and Gerhard Domagk. Koch was born in Clausthal-Zellerfeld in the Harz Mountains, then part of Kingdom of Hanover, as the son of a mining official. He studied medicine at the University of Gottingen and graduated in 1866. He then served in the Franco-Prussian War and later became district medical officer in Prussian Poland. After Casimir Davaine demonstrated the direct transmission of the anthrax bacillus between cows, Koch studied anthrax more closely. He invented methods to purify the bacillus from blood samples and grow pure cultures. He found that anthrax bacilli forming endospores that could last a long time in soil, were the cause of unexplained “spontaneous” outbreaks of anthrax. Koch published his findings in 1876 and was rewarded with a job at the Imperial Health Office in Berlin in 1880. In 1881, he urged the sterilization of surgical instruments using heat. In Berlin, he improved the methods he used in Wollstein, including staining and purification techniques and bacterial growth media, including agar plates and the Petri dish (named after its inventor, his assistant Julius Richard Petri). These devices are still used today. With these techniques, in 1882 he was able to discover the bacterium causing tuberculosis (Mycobacterium tuberculosis) which was the cause of one in seven deaths in the mid-19th century.


Vol. 15 No. 2 July.-Dec. 2012

Biography of a Renowned Microbiologist 121

In 1883, Koch worked with a French research team in Alexandria, Egypt, studying cholera. Koch identified the vibrio bacterium that caused cholera, though he never managed to prove it in experiments. In 1885, he became Professor of hygiene at the University of Berlin, then in 1891 he was made Honorary Professor of the medical faculty and Director of the new Prussian Institute for Infectious Diseases (eventually renamed as the Robert Koch Institute). He started travelling around the world, studying diseases in South Africa, India, and Java. He visited what is now called the Indian Veterinary Research Institute (IVRI), Mukteshwar on request from the Government of India to investigate cattle plague. The microscope used by him during that period was kept in the museum maintained by IVRI. Probably as important as his work on tuberculosis, are Koch’s postulates, which say that to establish that an organism is the cause of a disease. Koch’s pupils found the organisms responsible for diphtheria, typhoid, pneumonia, gonorrhoea, cerebrospinal meningitis, leprosy, bubonic plague, tetanus, andsyphilis, among others, by using his methods. Koch was invited to Veliki Brijun Island which was jeopardized by malaria outbreaks and spent two years, from 1900 to 1902 studying different forms of malaria and quinine-based treatments. People of this island erected a monument to Koch, which still stands in the vicinity of the 15th-century Church of St. German on Veliki Brijun. He was awarded the Nobel Prize in Physiology / Medicine in 1905 for his findings of tuberculosis. Koch on the Moon is named after him. The Robert Koch Prize and Medal were created to honour Microbiologists who make groundbreaking discoveries or who contribute to global health in a unique way. As for Koch’s personal life, he had no interest in politics and religion did not play a role in his life. He married Emmy Fraaze after graduation from medical school in 1866. On his 28th birthday, his wife gave him a microscope which he used frequently in his experiments and other discoveries. Koch remarried to Hedwig Freiberg in 1893. Robert Koch died on 27 May 1910 from a heart-attack in Baden-Baden, aged 66.



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References Citations in the text are given using numerals [1, 2, 3…]. If necessary, use surname(s) to identify publication(s) by single or two authors. Publications by three or more authors are cited by the first author’s surname followed by “et al.” (e.g. Neumann et al.). In taxonomic papers complete species citations including authors and year of description are required. Please list publications in the References section in the same order in which they appear in the text. The authors are advised to adhere to the following examples: 1. Article within a Journal Koonin, E.V., Altschul, S.F. and Bork, P., 1996, “BRCA1 Protein Products: Functional Motifs”, Nat. Genet., 13, pp. 226-267. 2. Article within a Journal Suppliement Orengo, C.A., Bray, J.E., Hubbard, T., LoConte, L. and Sillitoe, I., 1999, “Analysis and Assessment of ab initio Three-dimensional Prediction, Secondary Structure, and Contacts Prediction”, Proteins, 43 (Suppl 3), pp. 149170. 3. In Press Article Kharitonov, S.A. and Barnes, P.J., “Clinical Aspects of Exhaled Nitric Oxide”, Eur. Respir. J., in press. 4. Published Abstract Zvaifler, N.J., Burger, J.A., Marinova-Mutafchieva, L., Taylor, P. and Maini, R.N., 1999, “Mesenchymal Cells, Stromal Derived Factor-1 and Rheumatoid Arthritis”, Abstract, Arthritis Rheum., 42, s250. 5. Article within Conference proceedings Jones, X., 1996, “Zeolites and Synthetic Mechanisms”, In Proceedings of the First National Conference on Porous Sieves, Smith Y. Stoneham (Ed.), 27-30 June 1996, Baltimore, Butterworth-Heinemann, pp. 16-27. 6. Book Chapter, or Article within a Book Schnepf, E., 1993, “From Prey via Endosymbiont to Plastids: Comparative Studies in Dinoflagellates”, In Origins of Plastids, Vol. 2, 2nd Edn., R.A. Lewin (Ed.), Chapman and Hall, New York, pp. 53-76. 7. Whole Issue of Journal Ponder, B., Johnston, S. and Chodosh, L. (Eds.), 1998, “Innovative Oncology”, In Breast Cancer Res., 10, pp. 1-72. 8. Whole Conference Proceedings Smith, Y. (Ed.), 1996, Proceedings of the First National Conference on Porous Sieves, 27-30 June 1996, Butterworth-Heinemann, Baltimore, Stoneham. 9. Complete Book Margulis, L., 1970, Origin of Eukaryotic Cells, Yale University Press, New Haven. 10. Monograph or Book in a Series Hunninghake, G.W. and Gadek, J.E., 1995, “The Alveolar Macrophage”, In Cultured Human Cells and Tissues, T.J.R. Harris (Ed.), Academic Press, New York, pp. 54-56; Stoner, G. (Series Editor), Methods and Perspectives in Cell Biology, Vol. 1. 11. Book with Institutional Author Advisory Committee on Genetic Modification, 1999, Annual Report, London.



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The “Author’s Declaration Form” must be printed on an official letterhead of either the Corresponding Author or any one of the co-authors.

•

Mention the name and correct address of the Corresponding Author in the paper. All the author(s) are required to sign independently in this form in the sequence given.

•

No addition or deletion or change in the sequence of the authorship will be permissible at a later stage without valid reasons.


Copyright  2012 Indian Association of Applied Microbiologists, Chennai, India Volume 15 Number 2 July-December 2012, pp. 127-128.

Forthcoming Events Programme and Dates

Organizer / Venue / Website

International Conference on Public Health and Social Rajagiri College of Social Sciences, Kochi, Work Kerala, India [3-5, January 2013] Website: http://www.dyuti2013.blogspot.in 5-Days Hands-on Workshop on Molecular Biotechnology and Bioinformatics [7-11, January 2013]

International Center for Stem Cells, Cancer and Biotechnology (ICSCCB), Pune, India Website: http://www.icsccb.org/workshops

3rd International Conference on Advances in Biotechnology and Pharmaceutical Sciences (ICABPS’2013) [7-11, January 2013]

PSRC, Kuala Lumpur, Malaysia Website: http://psrcentre.org/listing.php? subcid=189&mode=detail

First International Conference on Bio-resource and Stress Management [6-9, February 2013]

Ratikanta Maiti Foundation and Bose Institute Kolkata, West Bengal, India Website: http://bsmconf.in

International conference on Advances in Biotechnology and patenting [18-21, February 2013]

Department of Biotecnology and Genetic engineering, Bharathidasan University, Tiruchirappalli-620024, India Website: http://icabp2013.webnode.com

International Conference on Global Scenario in Environment and Energy [14-16, March 2013]

Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India Website: http://www.manit.ac.in/manitbhopal/ index.php? option=com_content&view=article& id=494&Itemid=228



128 Forthcoming

2nd International Conference on Medical Information IACSIT, Bali Island , Indonesia and Bioengineering (ICMIB 2013) Website: http://www.icmib.org [16-17, March 2013] International Conference on Nanoscience and Nanotechnology-ICONN2013 [18-20, March 2013]

SRM University, Chennai, Tamilnadu, India Website: http://www.srmuniv.ac.in/SRM/ downloads/ICON2013.pdf

4th International Conference on Biotechnology and Food Science (ICBFS 2013) [21-22, April 2013]

CBEES, Beijing, China Website: http://www.icbfs.org

3rd International Conference on Biomedical Engineering and Technology – ICBET 2013

CBEES, Copenhagen, Denmark Website: http://www.icbet.org

[19-20, May 2013] 21st International Biodetection Technologies 2013 [18-19, June 2013]


Knowledge Foundation, Washington DC, DC, United States of America Website: http://knowledgefoundation.com/ viewevents.php?event_id=290&act=evt


Indian Association of Applied Microbiologists (IAAM) (Registered under Tamil Nadu Societies Registration Act 27 of 1975)

APPLICATION FOR MEMBERSHIP

To,

Dr. S. Anbalagan Treasurer, IAAM Head, Department of Microbiology Muthayammal College of Arts and Science Rasipuram – 637 408, Namakkal District Tamil Nadu, India

Passport size Photo

Sir, I wish to enroll as a Life/Annual Member# of the Indian Association of Applied Microbiologists (IAAM). I will abide by all the rules and regulations of IAAM as are in force from time to time. I furnish hereunder particulars about myself and enclose my Bio-data and Demand Draft* for membership. Full Name ____________________________________________________________________ Sex (F/M) ________ Age (Yrs) ________ Date of Birth (dd/mm/yyyy) ______________________ Academic Qualification(s) _________________________________________________________ Field of Specialisation ___________________________________________________________ Designation ___________________________________________________________________ Address (office) ________________________________________________________________ _____________________________________________________________________________________ ____ Pincode ________ State _____________ Country _____________________________________ Address (residence) ____________________________________________________________ ____________________________________________________________________________ Pincode _________ State ____________ Country _____________________________________ Phone (Office) _____________________ (Residence) _________________________________ Mobile ___________________________ Email ______________________________________ Preferred Address for Communication: Office/Residence (choose one) _______________________ Place _________________

Proposed by __________________________________________

Date (dd/mm/yyyy)_______

Life Membership No. ___________________________________

Seconded by _________________________________________

Life Membership No. ___________________________________

(signature)

Membership Fee Life Membership: 1000.00 (US$ 50.00 for individuals from outside India) Annual membership: 100.00 (US$ 50.00 for individuals from outside India) * Demand Draft to be made in favour of “India Association of Applied Microbiologists” payable at Tiruchengode, Tamil Nadu, India. #

Subscription Information Indian Journal of Applied Microbiology (IAAM) is a peer-reviewed journal published two times each year (as of 2011) by Wisdom Academic Publishers (No. 10/8, Dr. Nammalvar Street, Triplicane, Chennai – 600 005, India) on behalf of Indian Association of Applied Microbiologists (IAAM), Chennai, India. Annual Subscription for Volume 15 (No. 1 January-June 2012 and No. 2 July-December 2012) (Print ISSN 2249-8400) (Print Only) SAARC countries (Afghanistan, Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka) Institutional

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