studies on metal resistant bacteria incochin estuary and its responsetowards antibiotics and ...

201 Pages·2014·18.36 MB·English

by  Jiya Jose

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) .................................................. 32
3.4. Studies on MRB ................................................................................ 32
3.4.1 Identification ................................................................................... 33
3.4.2 Multiple Metal Resistances ................................................................. 33
3.4.3 Antibiotic Resistances ...................................................................... 34
3.4.4 Enzyme Expression .......................................................................... 35
3.4.4.1Amylase ........................................................................................ 35
3.4.4.2 Gelatinase ..................................................................................... 35
3.4.4.3 Lipase ........................................................................................... 36
3.4.4.4 DNase ........................................................................................... 36
3.4.4.5 Phosphatase ................................................................................. 36
3.4.4.6 Urease .......................................................................................... 36
3.4.4.7 Protease ........................................................................................ 36
3.5. Effect of Silver Nanoparticles (AgNPs) On Multiple Drug Resistant (MDR)
Pathogenic MRB ............................................................................................... 37
3.5.1 Selection of MDR pathogenic MRB ........................................................... 37
3.5.2 Preparation of AgNPs ....................................................................... 37
3.5.3 Effect of AgNPs on MDR pathogenic MRB ...................................... 37
3.5.3.1 Cell wall integrity ......................................................................... 37
3.5.3.2. Viability ....................................................................................... 38
3.5.3.3 Metabolic active cells .................................................................... 38
3.5.3.4 Whole ell fatty acid composition ................................................... 39
3.5.3.5 Genetic stability ........................................................................... 39
3.5.4 Mechanism of resistance by MDR Staphylococcus aureus ................ 40
3.6 Statistical Analysis ............................................................................ 40
Chapter 4 – RESULTS ........................................................................................................... 41
4.1 Water quality .................................................................................... 42
4.2 Bacterial Abundance ......................................................................... 43
4.2.1 TC, TVC and TCC ............................................................................ 43
4.3 Studies on MRB................................................................................. 44
4.3.1 Abundance ..................................................................................... 44
4.3.2 Multiple Resistance ......................................................................... 45
4.3.3. Antibiotic Resistance ...................................................................... 48
4.3.4 Enzyme expression profile .............................................................. 51
4.3.5 Identification of MRB ...................................................................... 52
4.4. Studies on “MDR” pathogenic MRB ................................................. 59
4.4.1 Effect of AgNPs ............................................................................... 60
4.4.2 Cell wall Integrity ............................................................................ 60
4.4.3 Metabolic activity of cells ............................................................... 62
4.4.4 Cell wall composition ....................................................................... 63
4.4.5 DNA damage ................................................................................... 64
4.5 Mechanisms of resistance of S. aureus to AgNPs .............................. 66
Chapter 5 – DISCUSSION .................................................................................................... 68
5.1 Metal Pollution ................................................................................. 68
5.2 Effect of heavy metal pollution on bacterial abundance ...................... 70
5.3 Studies on Metal resistant Bacteria (MRB) ........................................ 72
5.3.1 Abundance ....................................................................................... 72
5.3.2 Multiple resistances ........................................................................ 74
5.3.3 Co-occurrence of antibiotic resistance ............................................. 76