Despite current efforts to reduce fecal loads into aquatic environments, the problem persists, partially due to the inability to reliably identify the origin of fecal pollution. The swine industry has come under increasing environmental scrutiny due to augmented production and concentration of farming operations, resulting in large amounts of more concentrated waste products. Swine waste often harbors several human pathogens and thus presents a great risk to human health. Recently, methods targeting host-specific microbial populations have been proposed to identify specific sources and loads of fecal pollution entering environmental waters. Currently, there are a limited number of swine-specific markers available for source tracking studies. This is in part explained by our limited understanding of phylogenetic and functional diversity present within the swine gut microbiome. Several selective pressures imposed at the host and microbe level are predicted to result in unique microbial populations within the swine gastrointestinal system, which could serve as useful targets for swine fecal source tracking purposes.
Two popular targets utilized for fecal source tracking are Bifidobacterium and Bacteroidales 16S rRNA genes. In this study, the 16S rRNA gene sequence diversity of these two bacterial groups was examined to determine identity and distribution of swine-specific populations. The occurrence and abundance of these fecal bacterial populations were studied using samples from several geographically diverse host fecal types and impacted environmental samples. This molecular ecology approach revealed the diversity patterns of these popular fecal source tracking targets and unveiled previously unknown swine fecal source-specific populations relevant to environmental swine fecal pollution.
Since 16S rRNA gene approaches underestimate functional diversity, the swine fecal microbiome was analyzed using metagenomics-based (i.e., collective fecal microbial genomes) approaches to reveal the identity and functionality of the uncultivable majority within the swine distal gut. Function-specific genes represent another potential pool of swine-specific genetic targets. The use of this in silico comparative metagenomic approach facilitated the discovery of several genetic targets harbored exclusively in the swine gut. Coupling the detection of these function-specific targets to the 16S rRNA-based assays will help further validate if a swine fecal source is present within a given environmental sample. Using the approaches herein described has lead to the design of more comprehensive swine fecal source-specific assays, which will ultimately facilitate the accurate identification and relative contribution of swine fecal pollution in environmental waters.
Chapter one of this dissertation will discuss the detrimental impacts of swine fecal pollution, microbial source tracking methods, and how comparative metagenomics can be utilized for uncovering source-specific targets. Chapter two focuses on analyzing bifidobacterial diversity within different mammalian and avian fecal sources with the purpose of testing this bacterial group as a potential fecal source tracking target. Chapter three evaluates the currently available swine-specific fecal source tracking PCR-based assays within surface and groundwater surrounding swine farms. Chapter four examines Bacteroidales host distribution and presence within fecally contaminated environmental samples with the purpose of revealing populations both specific to swine fecal sources that can also be detected in environmental monitoring scenarios. Chapter five discusses the application of comparative metagenomics as an approach to expose bacterial populations and functional attributes unique to the swine distal gut. Chapter six discusses future directions of the field of microbial source tracking.