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