University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Fall 2017 MICROBIAL SOURCE TRACKING IN COASTAL RECREATIONAL WATERS OF SOUTHERN MAINE: RELATIONSHIPS BETWEEN ENTEROCOCCI, ENVIRONMENTAL FACTORS, POTENTIAL PATHOGENS, AND FECAL SOURCES Derek Rothenheber University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Rothenheber, Derek, "MICROBIAL SOURCE TRACKING IN COASTAL RECREATIONAL WATERS OF SOUTHERN MAINE: RELATIONSHIPS BETWEEN ENTEROCOCCI, ENVIRONMENTAL FACTORS, POTENTIAL PATHOGENS, AND FECAL SOURCES" (2017). Master's Theses and Capstones. 1133. https://scholars.unh.edu/thesis/1133 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. MICROBIAL SOURCE TRACKING IN COASTAL RECREATIONAL WATERS OF SOUTHERN MAINE: RELATIONSHIPS BETWEEN ENTEROCOCCI, ENVIRONMENTAL FACTORS, POTENTIAL PATHOGENS, AND FECAL SOURCES BY DEREK ROTHENHEBER Microbiology BS, University of Maine, 2013 THESIS Submitted to the University of New Hampshire In Partial Fulfillment of the Requirements for the Degree of Master of Science in Microbiology September, 2017 This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Masters of Science in Microbiology by: Thesis Director, Dr. Stephen H. Jones Research Associate Professor Department of Molecular, Cellular, and Biomedical Sciences Dr. Jeffrey Foster Assistant Professor Department of Molecular, Cellular, and Biomedical Sciences Dr. W. Kelley Thomas Professor Department of Molecular, Cellular, and Biomedical Sciences Date Original approval signatures are on file with the University of New Hampshire Graduate School ii Table of Contents ABSTRACT ...................................................................................................................................v Chapter 1: Background and Literature Review ..............................................................................1 Introduction.................................................................................................................................1 1.1 Public Health Concern in Relation to Fecal Contamination..................................................4 1.1.1 Human/Sewage Fecal Source.........................................................................................5 1.1.2 Bird Fecal Source ...........................................................................................................6 1.1.3 Dog Fecal Source ...........................................................................................................6 1.1.4 Other Wildlife ................................................................................................................7 1.2 Enterococci in the Environment............................................................................................8 1.2.1 Enterococci in Soils........................................................................................................9 1.2.2 Enterococci in Sediments ...............................................................................................9 1.3 Current Microbial Source Tracking Methods. ....................................................................11 1.3.1 Mammal and Human Fecal Assays ..............................................................................11 1.3.2 Dog Fecal Assay...........................................................................................................14 1.3.3 Gull/Bird Fecal Assay ..................................................................................................16 1.3.4 Microbial Source Tracking with Next Generation Sequencing....................................18 1.3.5 Persistence of MST DNA Markers in the Environment...................................................20 1.4.1 Simple Models to Inform Decision Making. ................................................................22 1.4.2 Identification of Fecal Hotspots ...................................................................................23 1.4.3 Using Regional Microbial Source Tracking qPCR Data to Help Convey the Significance of Fecal Pollution to the Public. .......................................................................25 1.5 Study Site Background and Research Questions. ...............................................................27 1.5.1 York, ME......................................................................................................................28 1.5.2 Wells, ME ....................................................................................................................28 1.5.3 Old Orchard Beach, ME...............................................................................................29 1.5.3 Research Questions ......................................................................................................29 Chapter 2: Influences of Fecal Sources, Environmental Sources and Environmental Conditions on Enterococci Concentrations in Coastal Water .........................................................................32 Abstract:....................................................................................................................................32 1. Introduction:..........................................................................................................................34 2. Materials and Methods:.........................................................................................................37 iii 2.1 Site description................................................................................................................37 2.2 Water Sampling...............................................................................................................37 2.2 Soil, Sediment, and Marine Sediment Collection............................................................37 2.3 Enterococci and Total Suspended Solids Quantification.................................................38 2.4 DNA Extractions.............................................................................................................38 2.5 Microbial Source Tracking (MST) PCR and qPCR Assays. ...........................................39 2.6 16S Library Preparation. .................................................................................................40 2.6 Quality Filtering and OTU Picking. ................................................................................41 2.7 SourceTracker Analysis. .................................................................................................41 2.9 Partial Least Squares Regression Model. ........................................................................42 2.10 Routine Statistical Analysis and Data Visualizations....................................................43 3 Results: ..................................................................................................................................43 3.1 Total and Particle-Associated Enterococci Concentrations and Total Suspended Solids in Water.....................................................................................................................................43 3.2 Presence of Fecal Sources in Fresh, Estuarine, and Marine Waters................................45 3.3 Differences Between Water, Soil, and Sediment Bacterial Community Compositions. .47 3.4 Environmental Source Contribution to Water Samples...................................................50 3.5 Relationships Between Environmental Conditions, Fecal Source Concentrations, Environmental Sources and Enterococci Concentrations......................................................51 4 Discussion:.............................................................................................................................54 Supplementary Material 1: MST PCR Primer List, PCR Percent Detection, And qPCR Standard Curve Performance ....................................................................................................61 Supplementary Material 2: Diversity Measurements of OTU Picking Method ........................63 Supplementary 3: PLSR Validation and Diagnostic Plots ........................................................64 Supplementary Material 4: Taxa Key for all Class-level Assignments.....................................68 Chapter 3: Abundance of Fecal-Borne Potential Pathogenic Bacteria and Their Relationship to Fecal Sources in Three Maine Coastal Watersheds......................................................................69 Abstract:....................................................................................................................................69 1. Introduction:..........................................................................................................................70 2. Methods: ...............................................................................................................................72 2.1 Study Sites.......................................................................................................................72 2.2 Sample Collection. ..........................................................................................................73