Studies on Metal Resistant Bacteria Incochin Estuary and Its Responsetowards Antibiotics and Silvernanoparticles
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STUDIES ON METAL RESISTANT BACTERIA IN COCHIN ESTUARY AND ITS RESPONSE TOWARDS ANTIBIOTICS AND SILVER NANOPARTICLES Thesis Submitted to the Cochin University of Science and Technology in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy in Marine Microbiology Under the Faculty of Marine Sciences By JIYA JOSE Reg.No.3876 CSIR-NATIONAL INSTITUTE OF OCEANOGRAPHY REGIONAL CENTRE, KOCHI - 682018, INDIA February 2014 Dedicated to my dear parents and teachers This is to certify that the research work presented in this thesis entitled "STUDIES ON METAL RESISTANT BACTERIA IN COCHIN ESTUARY AND ITS RESPONSE TOWARDS ANTIBIOTICS AND SILVER NANOPARTICLES” is based on the original work done by Ms. Jiya Jose (Reg. No. 3876), under my supervision at CSIR- National Institute of Oceanography, Regional Centre, Kochi, 682018, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Faculty of Marine Sciences, Cochin University of Science and Technology, Kochi, 682018 and that no part of this work has previously formed the basis for the award of any degree, diploma, associateship, fellowship or any other similar title or recognition in any universities or institutes. Kochi -18 Dr. ShantaAchuthankutty February, 2014 (Supervising Guide) Chief Scientist CSIR-National Institute of Oceanography Regional Centre Kochi – 682018 The research work presented in this thesis entitled "STUDIES ON METAL RESISTANT BACTERIA IN COCHIN ESTUARY AND ITS RESPONSE TOWARDS ANTIBIOTICS AND SILVER NANOPARTICLES" submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, is a bonafide record of the research work done by me under the supervision of Dr. ShantaAchuthankutty, Chief Scientist, National Institute of Oceanography, Regional Centre, Kochi, 682018. No part of this work has previously formed the basis for the award of any degree, diploma, associateship, fellowship or any other similar title or recognition in any universities or institutes Kochi-18 Jiyajose February, 2014 First and foremost, praises and thanks to the Almighty GOD I will forever be thankful to my mentor and Research Guide Dr. Shanta Achuthankutty, Chief Scientist, Microbiology Department, CSIR-National Institute of Oceanography, Regional Centre, Kochi, for infallible guidance, encouragement and untiring efforts during the course of my study. I take this opportunity to convey my respect and indebtedness to her. I thank Dr. P. A. Lokabharathi, Emeritus Scientist, CSIR-NIO, Goa for her encouragement and constant support during the course of my work. I express my deep sense of gratitude to Dr. Anas Abdulaziz, Scientist, CSIR-NIO, RC, Kochi, for his constant guidance, support and encouragement. I am grateful to Dr. C.T. Achuthankutty, Retired Chief Scientist, CSIR-NIO, Goa and Visiting Scientist, National Centre for Antarctic and Ocean Research, Goa, for his support throughout the period of my study and especially for his gesture towards correcting my thesis drafts with valuable suggestions and critical comments which have helped in improving the quality and presentation of my thesis. I would like to thank Dr. Judith, Dr.J. Raveendran and Dr.Parvathi Scientists of CSIR-NIO for their kind support. I will fail in my duty if I do not acknowledge the support of Dr.Chandramohan,Retired Scientist CSIR-NIO and Mr. O. Raveendran, Retired Technical Officer, NIO RC, Kochi for initiating me into this field of research. My deep sense of gratitude to the Director, CSIR-NIO, Goa and the Scientist-in- Charge of this centre for providing me with the necessary infrastructure and excellent supporting facilities during the course of my Ph.D work. I express my gratefulness to Prof. Anantharaman , Department of Physics Cochin University of Science and Technology Kochi and Dr. Sujith, National Institute of Technology, Calicut for their support and encouragement. I am very much thankful to Dr.K.K.Balachandran and Mr.Kiran Krishna, Technical officers CSIR-NIO-Kochi for their valuable suggestions. I sincerely thank all my Seniors, friends and labmates Dr.Neetha, Dr.Divya, Dr.Feby, Shoji, Rajesh, Francis Sonali, Vijitha, Sheeba, Subina, Dr.Sheryl, Dr. Laiju, Dr.Sujith, Dr.Christy, Dr.Sini, Dr.Jasmin, Dr.Sara, Dr.Indhu, Dr.Kusum, Jagadeesh, Anusree, Sneha, Vipin and Neenu for their corporation and support. My special thanks to Dr.K.V.Jayalakshmi, Chief Scientist, CSIR-NIO, RC, Kochi for the help rendered in statistical analysis of data and interpretation. I wish to thank the students Suresh, Monson, Bina and Rameez who were associated with me during my tenure. I acknowledge the support rendered by Council of Scientific and Industrial Research (CSIR), New Delhi for providing me with Senior Research Fellowship. I also acknowledge the support extended by the COMAPS MMRF Project and Supra Institutional Project (SIP 1302) during my Ph.Dwork . I am very much thankful to all the members of Doctoral and Research Committee for their valuable suggestions. I sincerely thank my parents, brothers, and in-laws for their unconditional support and hope that they would feel proud of this effort. Last but not the least, I find no appropriate words to express my feelings and gratitude to my beloved husband, Jinish John and son Jokuttan. Their unconditional love was my inspiration and the driving force. Their selfless sacrifice and deep affection gave me the strength to complete my Ph.D work. Jiya Jose Page No Chapter 1 – INTRODUCTION .................................................................................................. 1 Chapter 2 – REVIEW OF LITERATURE ................................................................................... 6 2.1 Metal –Microbe interaction ................................................................. 6 2.1.1 Introduction ...................................................................................... 6 2.1.2 Role of Heavy Metals in bacteria ........................................................ 6 2.1.3 Toxic effect of heavy metals on bacteria ............................................ 8 2.1.4 Mode of resistance in bacteria ........................................................... 9 2.2 International scenario ........................................................................ 11 2.2.1 Heavy metal pollution ....................................................................... 11 2.2.2 Ecological Studies on Metal–Microbe interaction ............................. 12 2.2.3 Metal Tolerance in Bacteria ............................................................. 13 2.2.4 Effect of metal on bacterial abundance ............................................ 14 2.2.5 Effect of metals on bacterial diversity .............................................. 15 2.2.6 Effect of metal on bacterial enzymes ................................................ 16 2.2.7 Transfer of metal resistance among bacteria .................................... 17 2.2.8 Co-occurrence of metal and antibiotic resistance in bacteria............. 17 2.2.9 Effect of Silver Nanoparticles (AgNPs) on bacteria .......................... 19 2.2.10 Mode of inhibition of AgNPs ........................................................... 20 2.3 National Scenario .............................................................................. 21 2.3.1 Heavy Metal Pollution ..................................................................... 21 2.3.2 Metal –Microbe interaction ............................................................. 22 2.3.2.1 Ecological studies ......................................................................... 22 2.3.2 Cochin Estuary ................................................................................. 25 Chapter 3 – MATERIALS AND METHODS ........................................................................... 27 3.1 Study Area ....................................................................................... 27 3.1.1 Sampling locations ........................................................................... 27 3.1.2 Sample collection ............................................................................ 29 3.2 Analysis of Environmental Parameters .............................................. 29 3.2.1 Water ............................................................................................... 29 3.2.1.1. Salinity and Temperature ............................................................ 29 3.2.1.2. Dissolved oxygen (DO) .................................................................. 29 3.2.1.3. Nitrite ......................................................................................... 30 3.2.1.4. Nitrate ........................................................................................ 30 3.2.1.5. Phosphate ................................................................................... 30 3.2.2 Metal Analysis ................................................................................. 30 3.3 Bacterial Variables ............................................................................ 31 3.3.1 Abundance ...................................................................................... 31 3.3.1.1 Total Counts (TC) .......................................................................... 31 3.3.1.2 Total Viable Count (TVC) ............................................................... 32 3.3.1.3 Culturable Aerobic Bacteria ........................................................... 32 3.3.1.4 Metal Resistant Bacteria (MRB) .................................................