Microbial Source Tracking: Tools for Refining Total Maximum Daily Load Assessments (2005)
This report summarizes the results of an investigation undertaken by Dr. Jody Harwood, University of South Florida, to test the usefulness of recently developed Microbial Source Tracking (MST) methods for determining the dominant sources of indicator bacteria in rural and urban land uses. The MST methods evaluated were (1) creation of a library of genotypic fingerprints of Enterococcus spp., (2) detection of the human-associated esp gene of Ent. faecium by PCR, (3) detection of a human-associated Bacteroides strain by PCR for the 16S rRNA gene, and (4) detection of a ruminant-associated Bacteroides strain by PCR for the 16S rRNA gene.
For over 100 years, indicator bacteria of fecal origin have been used to assess water quality and alert managers to increased risk of the presence of human pathogens. While these indicator organisms, including total coliforms and fecal coliforms, have helped protect public health for decades, they are now being utilized as surrogates for pathogens in total maximum daily load (TMDL) programs that are mandated by the US Environmental Protection Agency. The goal of TMDL assessment, which is carried out by the Florida Department of Environmental Protection in Florida, is ultimately to identify the sources of contaminant loading to Florida waters that are listed as “impaired”, and to determine how the loading can be reduced in order to return each water body to its designated use.
Many of Florida’s impaired waters are listed due to high concentrations of fecal indicator bacteria (total coliforms and/or fecal coliforms). Simply measuring the concentration of these organisms provides no information about their source, as they may originate from a variety of warm-blooded, or even cold-blooded animal feces. Mounting evidence indicates that some of these indicator bacteria are capable of long-term survival and/or slow growth outside of their host’s gastrointestinal tract. Although there are major questions about the continued use of indicator bacteria for water quality monitoring in the long term, State and Federal regulations and programs mandate their use for the foreseeable future.
Microbial source tracking (MST) is a term that includes a “toolbox” of methodologies that are designed to determine the dominant source(s) of fecal pollution to environmental waters. Knowledge of fecal source is essential to TMDL assessment, and is also crucial for refining best management practices (BMPs) for various land uses. Furthermore, because certain types of fecal material, particularly that originating from humans, poses a relatively great health risk, knowledge of the source of fecal loading from a given watershed can help refine risk assessment for better prediction of human health risk posed by water used for recreational and shellfishharvesting purposes.
This project was designed to test the usefulness of recently developed MST methods for determining the dominant sources of indicator bacteria in two Florida counties which represent contrasting land use: Nassau County is largely rural, and the most probable sources of impacts to 4 water quality include poorly functioning septic systems, livestock (mainly cattle), stormwater runoff and bacterial resuspension from sediments. The Duval County sites were located in urban Jacksonville on the Cedar River, and the most probable sources of fecal contamination were poorly functioning septic systems and aging central sewer infrastructure, wild birds, dogs and bacterial resuspension from sediments. One of the sample sites was positioned at a creek that was the site of a major sewage spill several months before the study started. The MST methods utilized were (1) creation of a library of genotypic fingerprints of Enterococcus spp., (2) detection of the human-associated esp gene of Ent. faecium by PCR, (3) detection of a humanassociated Bacteroides strain by PCR for the 16S rRNA gene, and (4) detection of a ruminantassociated Bacteroides strain by PCR for the 16S rRNA gene. Because of the expense of building the library, the library-dependent method (BOX-PCR) was utilized only in Duval County. An inter-laboratory study was conducted by the University of South Florida (USF) and Biological Consulting Services of North Florida (BCS). The USF and BCS laboratories carried out the MST methodologies separately to assess the inter-laboratory variability of the methods.
Indicator bacteria concentrations (fecal coliforms, Escherichia coli and enterococci) were measured in the water column and sediments at all sites. Bacteriological water quality levels at the Nassau County sites differed significantly (few exceedances of Florida State standards) from those in Duval County (majority of samples exceeded standards) (Executive Summary Fig. 1; see Fig. 1 in Methods section for GPS coordinates for all sites). MST analyses suggested the presence of human fecal contamination at all sites except for N3 (Nassau County), at the headwaters of the St. Mary’s River. The human-associated esp gene was detected most frequently at Duval site D1 (five times), followed by D2 and N1 (three times each), and was most frequently detected in urban (Duval) waters. The human-associated Bacteroides marker was detected most frequently at D1 and D2 (four times each). The ruminant-specific Bacteroides marker was detected infrequently in both watersheds.