Antimicrobial Activity of a Natural Product from Callistemon rigidus and its Mechanism of Action against Staphylococcus aureus A thesis submitted in fulfillment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY in BIOTECHNOLOGY by Charu Gomber (Regd. No.: 9051004) Department of Biotechnology & Environmental Sciences Thapar University Patiala- 147004, Punjab, INDIA (March, 2010) ACKNOWLEDGEMENTS There are many individuals and organizations that have contributed to my thesis and to my education, and it is now a great pleasure to take this opportunity to thank them. I wish to express my deepest gratitude to my revered supervisor, Dr. Sanjai Saxena, Assistant Professor, Department of Biotechnology & Environmental Sciences, Thapar University, Patiala for his encouragement to start this work and for the opportunity to be a part of his research group. It is in fact an honor to be his first Ph.D. student. I am indebted to him for his endless support, constructive criticism, and valuable guidance during my doctoral studies. I am thankful to him for his, inventive comments and suggestions especially during the writing phase. The professional experience he shared with me, his never failing support and his patience during all these years are gratefully acknowledged. Mrs Merry Saxena, his wife is a remarkable woman. I am thankful to her for her support, endurance and affection. I would like to thank Dr. Devendra Kumar who went through the entire thesis and helped to minimize errors. His support has been invaluable on both academic and personal level, for which I am awfully grateful. My special thanks to Mrs. Savita Saxena for giving motherly love and warmth. I express my sincere gratitude to Dr. Abhijeet Mukherjee, Director, Thapar University for providing academic and technical facilities at Thapar University. I wish to thank Dr. Susheel Mittal, Dean, Research & Sponsored Projects, Thapar University for his scientific guidance in getting the analysis work done. I also owe my gratitude to Dr. K.K. Raina, Deputy Director and Dean, Faculty Affairs, Thapar University for his precious guidance and constant motivation. I owe my heartfelt thanks to Dr. N. Das, Head, Department of Biotechnology & Environmental Sciences, Thapar University for providing excellent facilities for pursuing my work smoothly in the department and for his kind support and guidance throughout these years. My sincere thanks to Dr. D. Goyal, Executive Director, STEP (doctoral committee member), Thapar University for his valuable advice throughout my doctoral studies. I express my regards and gratitude to Dr. M.S. Reddy, Coordinator, Centre of Relevance in Agro and Industrial Biotechnology (TIFAC‐CORE), Thapar University for providing lab facility. It’s a pleasure to acknowledge Dr. O.P. Pandey, Head, Department of Physics, Thapar University for his guidance whenever required. I would like to acknowledge the technical support of Thapar University and its staff. The library facilities of the University have been indispensable. I also thank Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India for providing me fellowship under the sponsored project. My acknowledgements are due to Council of Scientific and Industrial Research (CSIR), Ministry of Science and Technology, Government of India for providing me Senior Research Fellowship and the necessary financial support for this research. My earnest thanks to Dr. Arti Kapil, Additional Professor, Department of Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi; Dr. Amarjit Kaur Gill, Professor & Head, Department of Microbiology, Government Medical College, Patiala; Dr. A. Staffan, Professor, Microbiology & Tumor Biology Centre, Karolinska Institute, Sweden; Dr. Geeta Mehta, Department of Microbiology, Lady Harding Medical College, New Delhi for providing cultures/microorganisms. My sincere gratitude to Dr. Rameshwar Singh, Professor and Registrar, National Dairy Research Institute, Karnal for his invaluable guidance in getting the analysis work done. I am also thankful to Dr. M. Sitaram Kumar, Vice President, Panacea Biotech, Mohali for gifting pure antibiotic powders. I am very grateful to Dr. Bernhard Krismer, Dr. Petry Genmedics, Germany for gifting lysostaphin. I wish to thank Mr. R.K. Sharma, Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh and Mrs Maninder Kaur, Centre with Potential for Excellence in Biomedical Sciences, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh for carrying MS analysis and elemental analysis respectively. Thanks are due to Mr. Ashok Aggarwal, Scientific Officer, Central Instrumentation Facility, National Institute of Pharmaceutical Education and Research (NIPER), Mohali for his kind support during the analytical studies. A special thanks to Mr. Purushotam, lab superintendent, School of Physics & Material Science, Thapar University for his unending support for electron microscopic studies. My colleagues and friends in the Department of Biotechnology and Environmental Sciences deserve warm thanks for making my work easier and for providing a pleasant working atmosphere. My special thanks to Ms. Gunjan Chopra, Research Fellow for inspiring discussions while designing and planning experimental protocols. She would always be remembered as a great colleague to work with and as a special friend. My heartfelt thanks to Mr. Vinit Meshram, Ph.D. Scholar for providing a nice atmosphere in the lab. It was pleasure to work with people that have such a good sense of humour! Thanks to Mr. Santosh Karn for providing help and support whenever required. Thanks to Mr. Ashwani Aggarwal, lab superintendent, Mr. Babban, Mr. Phoolchand, Mr. Iqbal, Mr. Satpal , Mr. Bharat and Mr. Mohinder, lab attendants without whom’s help many experiments would not be have been possible. Last, but not least, I thank my family: my parents, Mr. G.L. Gomber & Mrs. V.P. Gomber for giving me life in the first place, for educating me, for unconditional support and encouragement to pursue my interests. I am thankful to them for being by my side during my tough times all these years, without which I would not have been able to accomplish this work in the present form. My sister, Divya Gomber deserves special thanks for her friendship, for listening to my complaints and frustrations, and for believing in me. I would be failing in my duty if I don’t appreciate the encouragement given to me by Mutreja Family, especially Sidharth Mutreja. Above all, I thank GOD for blessing me to complete this work successfully. Charu Gomber LIST OF PUBLICATIONS Publications Related to Thesis Work 1. Sanjai Saxena & Charu Gomber (2006): Antimicrobial potential of Callistemon rigidus R.Br. Pharmaceutical Biology, 44 (3), 194‐ 201. 2. Charu Gomber & Sanjai Saxena (2007): Antistaphylococcal potential of Callistemon rigidus. Central European Journal of Medicine, 2 (1), 79‐ 88. 3. Sanjai Saxena & Charu Gomber (2008): Comparative in vitro antimicrobial procedural efficacy for susceptibility of Staphylococcus aureus, Escherichia coli and Pseudomonas species to chloramphenicol, ciprofloxacin and cefaclor. British Journal of Biomedical Sciences, 65(4), 178‐ 183. 4. Sanjai Saxena & Charu Gomber (2010): Surmounting antimicrobial resistance in the millennium superbug: Staphylococcus aureus. Central European Journal of Medicine, 5 (1): 12‐ 29. 5. Sanjai Saxena & Charu Gomber (2010): Superoxide dismutase, protease and lipase expression of clinical isolates of Staphylococcus aureus: A tool for antimicrobial drug discovery. Molecular & Cellular Biochemistry. (Accepted for publication). Book Chapter 1. Sanjai Saxena and Charu Gomber (2009): Herbal pharmacogenomics: Understanding mechanism in development of rational herbal products to overcome antibacterial drug resistance in Staphylococcus aureus. Chapter 36, 811‐ 824. In Text Book on Molecular Biotechnology. Eds Ashok K. Chauhan & Ajit Varma, IK International, ISBN 978‐93‐80026‐37‐4. In Conference Proceedings 1. Charu Gomber and Sanjai Saxena (2006): Callistemon rigidus as a new plant providing potent phytochemicals in designing new antimicrobial drug and cosmeceutical formulation. In Proceedings of International Conference‐ Bot Expo 2006, 88‐ 91, NABARD‐ Mumbai and University of Rajasthan, Jaipur. Poster Presentations 1. Charu Gomber & Sanjai Saxena (2007): In vitro mechanisms to assess the mechanisms of staphylococcal inhibition by extract of Callistemon rigidus R.Br. Presented at the 48th Annual Conference of The Association of Microbiologists of India‐ Microbes: Biofactories of Future, 18‐ 21st Dec, 2007, Indian Institute of Technology Madras, Chennai. 2. Charu Gomber & Sanjai Saxena (2007) Bioassay guided fractionation of antibacterial compounds from Callistemon rigidus R.Br with potential Anti‐ MRSA activity. Presented at Joint Bilateral seminar on “Antimicrobial Drug Resistance & the Development of New Antibiotics” November 21‐ 25, 2007, Indian National Science Academy, New Delhi. 3. Charu Gomber and Sanjai Saxena (2006) Overcoming multidrug resistance Staphylococcus aureus by two bioactive fractions from Callistemon rigidus R. Br. Presented at the 47th Annual Conference of The Association of Microbiologists of India ‐ Microbiology: The Challenges Ahead, 6‐ 8th Dec, 2006, Barkatullah University, Bhopal. 4. Charu Gomber and Sanjai Saxena (2006) Callistemon rigidus as a new plant providing potent phytochemicals in designing new antimicrobial drug and cosmeceutical formulation.