influence of stresses on antibiotic resistance in lactobacilli from traditional dairy products master of

112 Pages·2013·3.85 MB·English

INFLUENCE OF STRESSES ON ANTIBIOTIC
RESISTANCE IN LACTOBACILLI FROM TRADITIONAL
DAIRY PRODUCTS

THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE DEGREE OF

MASTER OF SCIENCES
IN
DAIRY MICROBIOLOGY

BY
ARCHANA CHAUDHARY

B.Sc. (Ag. Sciences)

DIVISION OF DAIRY MICROBIOLOGY
NATIONAL DAIRY RESEARCH INSTITUTE
(I.C.A.R.)
KARNAL- 132001 (HARYANA), INDIA
2013

Regn. No. 2011109
DEDICATED
TO MY
BELOVED MUMMY
&
GOD

ACKNOWLEDGEMENTS

I pen down my feelings of indebtness and gratitude to all those who helped out during the course of
my study. I applaud their incredible support which accompanies me during my research work.

It gives me immense pleasure to express my heartfelt gratitude to my Guide and Major Advisor,
Dr. A. K. Puniya, Principal Scientist, Dairy Microbiology, National Dairy Research
Institute, Karnal, Haryana for his timely suggestions and continuous encouragement during the
entire period of this study.

I express my sincere thanks to the members of advisory committee Dr. S. K. Tomar, Principal
Scientist, D.M. Division, Dr. SurajitMandal, Scientist, D.M. Division, Dr. Rajan
Sharma, Senior Scientist, D.C. Division and Dr. P. N. Raju, Scientist, D.T. Division for
their keen interest, valuable suggestions and cooperation.

I am grateful to Dr. R. K. Mallik, Head of D.M. Division, Dr. ShilpaVij, Senior
Scientist, Dr. N. K. Goyal, Senior Scientist, Dr. Chand Ram, Senior Scientist and Dr.
Sunita Grover Principal scientist in D.M. Division at NDRI.

I am very much thankful to my caring and supportive lab seniors SwarnaDidi, SamleshDidi for
giving their valuable time and great support, JyotiDidi and ManpreetDidi for their moral support
throughout the research work, Prasanta Sir for teaching valuable lessons about life.

My acknowledgements will not be complete without thanking to my family members, Santosh,
Dheeraj, Khushi, Rajeeve, PankajBhaiya, Manisha and AnuFufaji for their moral support,
guidance and timely help in my need as well as being always with me during my tough time.

I feel the inadequacy to my dictionary a suitable word to pay regards to my friend Prashant, who was
always with me during my good and tough time.

No words will be sufficient to express my thanks to Narendra Sir and VandanaDidi to add
beautiful memories of NDRI in my life.

Thanks of more million tons to Chhaya and InduDidi for providing me their valuable time, guidance
and support in tough time at NDRI.
ACKNOWLEDGEMENTS

I am very much Thankful to NimishaDidi, Sabnam, Mahesh, Rohit, SumanDidi and
SonaliDidi for being friendly with me and helping me during my need. Very special thanks to
Rajani and siddhivinayaka for their help and moral support during my tough time in research work.

I am indeed highly indebted to Dr. A. K. Srivatsava, Director NDRI for providing me
requisite infrastructural facilities that enabled incessant compilation of this project.

I owe special gratitude and thankfulness to the valuable help rendered by Prem sir Technical Office,
Raminder sir is duly acknowledged. Thanks to all staffs and colleagues of Fungal Biotechnology
Laboratory for providing me a homely environment.

I sincerely appreciate the relentless by, Joginderji, Satveerji D.M. Division staff for their timely
help.

I record my deepest sense of respect and appreciation for my parents and well-wishers for their
blessings showered on me.

Financial assistance from ICAR in the form of JRF is greatly acknowledged.

A special note of thanks is for Dr. A. K. Mohanty for providing me need full help in research
work.

I feel the inadequacy to my dictionary a suitable word to pay regards to my papa, brothers (Santosh,
Dheeraj) and sister (khushi) who had sacrificed many things in their lives to nurture my ambition.
They were been my strength and support and moulded me the way I am, showed the noble path to tread
and trusted me.

I am thankful to all staff members of Dairy Microbiology Division and other Divisions of this
Institute, for their help in carrying out this research work.

Finally I thanks all those who rendered the help directly or indirectly during the course of my study.

ArchanaChaudhary
Chapter Title Page No.
1 Introduction 1-2
2 Review OF Literature 3-22
2.1 Stress 3
2.1.1 Heat Stress 4
2.1.2 Acid Stress 5
2.1.3 Bile Salt Stress 6
2.2 Antibiotics 7
2.2.1 Development of Antibiotic Resistant in Bacteria 8
2.2.2 Antibiotic Resistance of Lactobacilli 9
2.2.3 Antibiotics resistance in the food chain 12
2.2.4 Ecological Roles in Antibiotics Resistance 14
2.2.4.1 Effect of stress on antibiotics resistance 14
2.2.5 Antibiotic Targets
2.2.5.1 Mechanisms of antibiotics resistance 15
2.2.5.2 Horizontal gene transfer 15
2.2.5.3 Intrinsic resistance 16
2.2.5.4 Acquired resistance 17
2.3 Microbiological Breakpoints 18
2.4 Phenotypic Methods 19
2.5 Lactic Acid Bacteria 19
2.5.1 The Genus Lactobacilli 20
3.0 Materials and Methods 23-34
3.1 Requirements 23
3.1.1 Equipments Used 23
3.1.2 Reagents Used 23
3.2 Procedures 23
3.2.1 Isolation Of Lactobacilli 23
3.2.1.1 Sample Collection 23
3.2.1.1.1 Collection of Fermented Milk and Milk 23-24
Based Sweets Samples
3.2.1.2 Processing of Samples 24
3.2.1.2.1 Processing of Raw Milk 24
Samples 24
3.2.1.2.2 Processing of Fermented Milk
Samples
3.2.1.2.3 Processing of Milk Based Burfi 24
and Rasogolla
3.2.1.2.4 Processing of Kulfi 24
3.2.1.3 Isolation Procedure 25
3.2.2 Characterization of Lactobacilli 25
3.2.2.1 Morphological Characterization 25
3.2.2.2 Microscopic Characterization 25
3.2.2.3 Catalase Test 25
3.2.2.4 Maintenance and Preservation of Cultures 25
3.2.2.5 Molecular Characterization 26
3.2.2.5.1 Genomic DNA extraction 26
3.2.2.5.2 Determination of DNA 26
Concentration and Purity
27
3.2.2.5.3 PCR reaction

3.2.2.5.4 Analysis of PCR Product by
28
Agarose Gel Electrophoresis
3.2.3 SPECIES LEVEL CHARACTERIZATION 28
3.2.3.1 Biochemical Characterization 28-29
3.2.3.2 Confirmation Using Species Specific PCR 29
3.2.3.2.1 PCR reactions 29
3.3 Antibiotics Susceptibility Test 29
3.3.1 Preparation of McFarland 0.5Standards 30
3.3.2 Preparation of Bacterial Inoculum 30
3.3.3 Etest Method 30
3.3.4 Preparation of Antibiotics Dilution Range 31
3.3.4.1 Preparation of dilution range 31
3.3.4.2 Preparation of microdilution plate 31
3.3.4.3 Broth Microdilution Method 32
3.4 Assessment of Stresses 32-33
3.4.1 Assessment of Heat Stress 32
32
3.4.2 Assessment of Low pH Stress
33
3.4.3 Assessment of Bile Salt Stress
33
3.5 Assessment of Antibiotics MICs In Stressed Cultures 33-34
3.5.1 Assessment of MIC After Heat Stress 33
3.5.2 Assessment of Antibiotics MIC After Low pH Stress 33
3.5.3 Assessment of Antibiotics MIC after Bile Salt Stress 33-34
4.0 RESULTS AND DISCUSSION 35-61
4.1 35
Isolation of Lactobacilli Isolates
35
4.1.1
35
Sample Collection
35
4.2
Characterization of Lactobacilli Isolates
35
4.2.1

Morphological Characterization
35
4.2.2
Microscopic Characterization 35
35
4.2.3 Catalase Test
35
4.2.4
40
Molecular Characterization Using Genus Specific

Primers
42
Species Level Characterization
4.2.4.1
42
4.2.4.1.1 Biochemical Characterization

44-45
4.2.4.1.2 Confirmation Using Species

Specific PCR
46-50
Determination of Susceptibility Pattern and Breakpoint
Concentration to Different Antibiotics
4.4
Assessment of Different Stress Conditions on Antibiotic
50-51
Resistance Susceptibility Pattern

4.4.1
51-52
Effect of Acid Stress

4.4.2 Effect of Bile Salt
52-53
4.6 4.4.3 Effect of Heat Stress 53-55
4.5 Assessment of MIC For Stressed Culture to Different 55-61
Antibiotics
5.0 SUMMARY AND CONCLUSION 62-65

6.0 BIBLOGRAPHY i-xvii

7.0 APPENDIX I i-ii
APPENDIX II iii-V
APPENDIX III VI