ABSTRACT MAMMALIAN DERIVED RUMINOCOCCACEAE POPULATION AS MOLECULAR TARGET OF FECAL CONTAMINATION By Nilay J. Sheth Domesticated animal’s fecal material, as agricultural runoff is a contributor to decreased water quality and subsequent, human exposure to waterborne illness. Fecal contamination of water sources is currently monitored by bacteria indicators such as Escherichia coli (E. coli), enterococci, or fecal coliforms. However, these bacteria typically comprise less than 1% of the intestinal bacterial population, and may not precisely predict the human health risk associated with fecal contamination. The mammalian intestinal tract is dominated by the bacterial phylum Firmicutes and a major population is the family Ruminococcaceae. The main objective of this research was to determine if animal derived Ruminococcaceae can be used as an alternative molecular indicator target for the detection of fecal pollution in surface water. To address this objective a 16S rRNA molecular target for mammalian specific Ruminococcaceae was created using the characterized diversity of rumen gastrointestinal/fecal microbial community. The Ruminococcaceae target was amplified from the fecal DNA samples of dairy cow, pig, goat, camel, bison, and water buffalo. The target population was not amplified from horse, chicken, goose, and human sewage influent DNA samples. In natural water spiked with fresh cow fecal material; the culturable indicator E. coli was detected for four days as compared to one day post inoculation, illustrating rapid signal loss. Following rain events, this population was detected in surface water samples from Spring Brook creek (n=10) and only one sample from Fox River, indicating this creek receives animal fecal pollution. The bacterial community analysis of Spring Brook and Fox River water samples demonstrated an increased diversity in the Spring Brook samples. This is most likely attributed to the surrounding agricultural area, and may suggest a significant input of agricultural related fecal pollution. Overall, this molecular target may prove useful in detecting mammalian fecal contaminations, specially that of agricultural. ACKNOWLEDGMENTS First of all, I would like to thank my advisor, Dr. Sabrina Mueller-Spitz, for encouraging me and allowing me to work in her lab. Thank you for all your guidance, support, teaching, and encouragement for past three years. I would not be where I am today if it were not for you. I would also like to thank my committee member, Dr. Colleen McDermott, for her guidance and being there for me and answering my many questions. I also would like thank Dr. McDermott for helping me obtain dairy cow fecal samples which I was able to use in different aspects of my research. I would like to acknowledge my committee member, Dr. Toivo Kallas, for his guidance and support that played an important part in my development as a scientist. Dr. Kallas always welcomes questions and has played a vital role in completion of my thesis. I also want to thank numerous people for allowing me to come to their personal farm and letting me collect fecal samples from various different animals (who have requested to be kept anonymous). This has helped me fulfill my research and understandings to completion. There are not enough words to thank my family whose support and guidance has helped me to get where I am. They gave remarkable amount of time and personal sacrifice so that I could work towards my goals in life. ii TABLE OF CONTENTS Pages LIST OF TABLES.................................................................................... v LIST OF FIGURES.................................................................................. vii INTRODUCTION.................................................................................. 1 Global Perspective on Water Pollution………………………… 1 Sources of Water Pollution…………………………………….. 2 Fecal Indicator Bacteria……………………………………….. 3 Regulations for Water Quality………………………………… 5 Molecular Methods for Fecal Source Tracking……………….. 6 Bacterial Diversity in Mammalian GI tract…………………… 7 PROJECT FRAME WORK…………………………………………… 11 PROJECT OBJECTIVES……………………………………………… 12 MATERIALS AND METHODS............................................................ 13 Creation of Ruminococcaceae 16S rRNA Primers………… …. 13 Collection of Animals Fecal Samples………………………….. 17 Bacterial DNA Extraction and Concentration…………………. 18 Determination of Optimal Annealing Temperature……………. 18 Detection of Fecal Ruminococcaceae and Bacteroides Populations…………………………………………………….. 19 PCR Cloning of 16S rRNA Gene Sequences…………………. 20 Plasmid Extraction for Ruminococcaceae Animal Clones……. 20 DNA Concentrations and Confirmation of Extracted Plasmids… 21 DNA Sequencing and Analysis of Clones…………………… 21 Development of Real-time PCR Analysis for Ruminococcaceae……………………………………………. 23 Microcosm Experiment to Detect Signal Loss of E. coli And Ruminococcaceae………………………………………….. 25 Characterization of Fecal Pollution in Upper Fox Watershed….. 26 Bacterial Community Analysis of Surface Waters……………… 27 RESULTS………………………………………………………………. 29 Specificity of Ruminococcaceae Primer Set……………………. 29 Sensitivity of Ruminococcaceae Primer Set……………………. 34 iii TABLE OF CONTENTS (Continued) Pages Gene Sequence Analysis of PCR Ruminococcaceae 16S rRNA Animal Clones…………………………………………………... 34 Microcosm Experiment to Detect Signal Loss of E. coli and Ruminococcaceae........................................................................... 39 Field Test of Ruminococcaceae as Fecal Pollution Target……… 41 Bacterial Community Analysis………………………………….. 46 Bacterial Populations Associated with Fecal Signature of Mammals……………………………………………………… 52 DISCUSSION………………………………………………………….. 56 APPENDIX…………………………………………………………….. 62 REFERENCES………………………………………………………… 79 iv LIST OF TABLES Page Table 1. Twelve out groups within the known Clostridiales ……….. 15 Table 2. Sequences and the annealing temperatures of the primers used in this study…………………………………………… 16 Table 3. Mammalian specific Ruminococcaceae 16S rRNA primer sequences ………………………………………………….... 31 Table 4. The predicated overall genera for the family of Ruminococcaceae sequences for 120RF, CF and 430R primer Sequences…………………………………………………... 32 Table 5. Detection of Ruminococcaceae in various agricultural animals and human fecal material……………………………………. 33 Table 6. Diversity of Ruminococcaceae population targeted with 120RF/430R primer set……………………………………....… 36 Table 7. The predicated genera for Ruminococcaceae sequences at 95% similarity level for cow, goat and pig clone library origin using RDP classifier …………………………………… 36 Table 8. Ruminant animal sources matching cow, goat and pig fecal clones ……………………………………………….. 37 Table 9. Non-ruminant animal sources matching cow, goat and pig fecal clones …………………………………………... 38 Table 10. Detecting signal loss of fecal indicators in seven day microcosm … 40 Table 11. Spring Brook samples were analyzed for average E. coli count and real-time SYBR green PCR of Ruminococcaceae target………………………………………… 42 v LIST OF TABLES (Continued) Page Table 12. Fox River samples were analyzed for average E. coli count and SYBR green real-time PCR of Ruminococcaceae target………………………………………………………… 44 Table 13. Average E. coli for the five different water samples used for pyrosequence analysis ………………………………………… 51 Table 14. Number of sequences of Gram positive species commonly identified within the GI tract of mammals from five different surface water samples. …………………………………………. 53 Table 15. Number of sequences of Gram negative species commonly identified within the GI tract of mammals from five different surface water samples. ………………………………………… 54 vi LIST OF FIGURES Page Figure 1. The major steps in the CAP3 assembly algorithm…………….. 14 Figure 2. Standard curve for SYBR Green real-time qPCR analyses of cowB1 plasmid clone……………………………… 24 Figure 3. Map of the lower Fox basin (Oshkosh, WI)…………………… 26 Figure 4. The correlation between rainfall totals and culturable E. coli levels for Spring Brook surface water samples………… 43 Figure 5. Relationship between Ruminococcaceae abundance and culturable E. coli levels in Spring Brook surface water samples……..………………………………………………….. 43 Figure 6. The correlation between rainfall totals and culturable E. coli levels for Fox River surface water samples……………. 44 Figure 7. The correlation between Spring Brook and the Fox River E. coli levels for surface water samples ………………………. 45 Figure 8. Relative abundance of dominant bacteria phylum from five different water sample sequences…………………………… 48 Figure 9. Rarefaction analysis to determine species richness and diversity………………………………………………………… 49 Figure 10. Principal coordinate plot generated utilizing MG-RAST analysis comparing five different sample dates………………… 50 Figure 11. Principal component analysis using UniFrac metric to analyze fecal population cluster sequence diversity ………………………. 55 vii 1 INTRODUCTION Global Perspective on Water Pollution Currently, 1.1 billion people lack access to safe water and over 2.6 billion people do not have proper sanitation. These conditions exist primarily in developing countries, leading to unsafe drinking water (90). Globally, the restricted access to safe drinking water results in 1.6 million deaths per year, with more than 99% of those deaths occurring in developing countries (109). These easily preventable diarrheal diseases contribute to 6.1% of all health-related deaths (3). The World Health Organization (WHO) estimates that there are four billion cases of waterborne diarrhea each year