Rana Abed El-Majeed Mahmoud Salameh

115 Pages·2015·2.79 MB·English

An-Najah National University
Faculty of Graduate Studies

Polymerase chain reaction for detection of
waterborne bacterial pathogens in potable
water in Tubas district-Palestine

By
Rana Abed El-Majeed Mahmoud Salameh

Supervisor
Dr. Raed Alkowni

Co-Supervisor
Dr. Shehde Jodeh

This Thesis is Submitted in Partial Fulfillment of the requirements for
the Degree of Master of Life Sciences (Biology), Faculty of Graduate
Studies, An-Najah National University, Nablus - Palestine.
2015

III
Dedication
I dedicate my thesis to science, enlightening us
I would like to dedicate my thesis to my parents whom I owe everything
since I was born. There is no doubt in my mind that without their continued
support and counsel I couldn’t have completed this process.
To my wonderful husband, thank you for your unconditional support with
my studies, who had given me the dreams to look forward. Thank you for
giving me a chance to prove and improve myself.
To my kids; Seedra and Kareem, you have made me stronger, better and
more fulfilled than I could have ever imagined who were supported me in
their childhood innocent and their patience has at times. I love you to the
moon and back.
IV
Acknowledgements
First of all praise is to Allah for blessings and guidance in fulfilling this
thesis. I would like to acknowledge and thank my supervisors Dr. Raed
Alkowni and Dr. Shehde Jodeh, who have assisted, guided and supported
me in my study.
I am extremely grateful to Palestinian Water Authority for their financial
support and for guiding me to search in this topic and Ministry of
Education and Research/ German BMBF for the fund from SMART
project.
Special thanks to all who made the lab environment friendly for working,
the technicians in Department of Biology and Biotechnology at An-Najah
National University and express my sincere gratitude to Hiba Bourini for
her help, patient and support. And for Health and Environment
Organization in Tubas district representative Mr. Mahmoud Bsharat who
helped me in data and water samples collection.
I humbly extend my thanks to Mother, Father, brothers and sisters who
have supported and assisted me throughout my studies.
Special thanks to my husband and my kids for pushing me farther than I
thought I could do.

VI
List of Contents
No Subject Page
Dedication III
Acknowledgement IV
Declaration V
List of contents VI
List of Tables IX
List of Figures X
List of Abbreviations XI
Abstract XIII
Chapter One: Introduction 1
1.1 General Background 2
1.2 S tudy Area 4
1.2.1 G e ographical Location 4
1.2.2 T emperature and Annual Rainfall 6
1.2.3 P opulation 7
1.2.4 W ater Resources 7
Chapter Two: Literature Review 8
2.1 W ater Quality 9
2.2 Physicochemical Characteristics of Potable Water 9
2.3 Microbiological Characteristics of Potable Water 11
2.4 Water-Related Illnesses 13
2.5 Water Borne Diseases 14
2.5.1 B acteria 15
2.5.1.1 E s cherichia coli 16
2.5.1.2 S a l monella spp. 17
2.5.1.3 K l e bsiella spp. 18
2.5.2 V iruses 19
2.5.3 P rotozoa 19
2.6 M icrobial Indicator of Water Quality 20
2.6.1 I n dicator Organisms (Coliform) 20
2.6.2 T o tal Coliform TC 22
2.6.3 F a ecal Coliform FC 23
2.6.4 O t h er Less Commonly Used Bacterial Indicators 26
2.6.4.1 I n t e stinal Enterococci 26
2.6.4.2 S u l f ite-Reducing Clostridia 27
2.7 Water Disinfection by Chlorination 28
2.8 B iochemical Tests for Identification Water Borne 30
Bacteria
2.9 Molecular Analysis of Water Borne Pathogens 32
2.10 Justification 37
VII
2.11 Aims of study 38
Chapter Three: Materials and Methods 40
3.1 C ollection of Samples 41
3.2 M edia Preparation 42
3.2.1 M acConkey Agar 42
3.2.2 S almonella- Shigella Agar 42
3.2.3 S elenite Broth 42
3.2.4 M -Endo Agar 43
3.2.5 M-Fc Agar 43
3.2.6 L auryl Sulfate Tryptose Broth 43
3.2.7 Eosin Methylene Blue Agar 44
3.2.8 Citrate Agar 44
3.2.9 SIM Medium 44
3.2.10 M ethyl Red Voges Proskauer Medium 45
3.3 Detection of Bacteria by Culture Methods and 45
Biochemical Tests
3.3.1 Detection of Total Coliform (TC) 45
3.3.2 Detection of Fecal Coliform (FC) 46
3.3.3 I s o lation of E. coli and Klebsiella 47
3.3.4 I s o lation of Salmonella 47
3.4 DNA Extraction 47
3.5 Detection of water borne pathogens by Polymerase 48
Chain Reaction
3.5.1 Detection of E. coli Mdh gene 48
3.5.2 Detection of Salmonella IpaB gene 49
3.5.3 Detection of Klebsiella Gap A gene 51
Chapter Four: Results 53
4.1 Bacterial Enumeration and Culture Characterization 54
4.1.1 TC and FC Enumeration 54
4.1.2 Culture Characterization and Colony Morphology of TC 59
and FC
4.2 Biochemical Tests Results 61
4.3 Results of DNA extraction purity by using 64
Spectrophotometer
4.4 Detection of E. coli by PCR 65
4.5 Detection of Klebsiella by PCR 66
4.6 Detection of Salmonella by PCR 67
4.7 Comparison between waterborne bacterial pathogens 68
Culture and PCR Assay
4.8 Prevalence of E. coli, Klebsiella and Salmonella 69
distribution in different water resources
VIII
Chapter Five: Discussion, Conclusion and 71
Recommendation
5.1 Discussion 72
5.2 C onclusion 79
5.3 Recommendations 80
References 82
صلخلولا ب
IX
List of Tables
No. Table Title Page
E. coli target genes for PCR amplification, amplicon
Table 1 size, primer sequences and annealing temperature 94
(AT).
Salmonella target genes for PCR amplification,
Table 2 51
amplicon size, primer sequences and (AT).
Klebsiella target genes for PCR amplification,
Table 3 52
amplicon size, primer sequences and (AT).
Comparison of TC (3 CFU/100ml) range of water
samples from tap water (houses, restaurants and
Table 4 57
school), ground water wells, rain fed cisterns, network
distribution systems and tankers
Comparison of FC (0 CFU/100ml) range of water
samples from tap water (houses, restaurants and
Table 5 58
school), ground water wells, rain fed cisterns, network
distribution systems and tankers
Biochemical Test Results of E.coli, Klebsiella and
Table 6 64
Salmonella Spp
Table 7 TSI results of E.coli, Klebsiella and Salmonella 64
Results of absorbance at 260 nm and A260nm/A280nm
Table 8 65
by spectrophotometer
Comparison between culture and PCR assay in
Table 9 detecting waterborne bacterial pathogens (E. coli, 69
Klebsiella and Salmonella)
X
List of Figures
No Figure Title Page
Figure 1 Geographic location of Tubas governorate 5
Distribution of samples collection localities in Tubas
Figure 2 41
Governorate
Percentage of TC and FC counts in water samples
Figure 3 54
from different water resources.
Results of (TC) and (FC) distribution in drinking
water samples from rain fed cisterns, tap water
Figure 4 55
(houses, restaurants and schools), ground water wells,
network distribution systems and tankers
TC with bright green metalic sheen color colonies in
Figure 5 59
M-Endo culture medium
Figure 6 FC with blue color colonies in M-Fc culture medium 60
Colony morphology on different selective and
Figure 7 differential media; M-Fc media, MacConkey agar, SS 60
Agar and EMB
Figure 8 Biochemical Test Results of E. coli 62
Figure 9 Biochemical Test Results of Klebsiella 63
Figure 10 Biochemical Test Results of Salmonella 63
Figure 11 PCR Results of E. coli 66
Figure 12 PCR Results of Klebsiella 67
Figure 13 PCR Results of Salmonella 68
Prevalence of E. coli, Klebsiella and Salmonella
distribution in water samples from rain fed cisterns,
Figure 14 70
tap water and tankers