Siesta Key Beach, located on the Gulf Coast of Florida, is frequently mentioned among the top ten beaches in the US. In summer 2004, high levels of indicator bacteria caused health warnings to be posted, and a storm drainage system was implicated as a possible source of microbial contamination. A study was initiated to determine whether indicator bacteria that persisted in the stormwater system could contribute to high microbial loads in receiving waters. Two sampling events, one within 48 hours of a rain event and the other during dry conditions, were conducted. Water and sediment samples were taken at various sites from the storm drainage system to the beach. Fecal coliforms and Enterococcus spp. were enumerated, and genotypic fingerprints of E. coli and Enterococcus spp. were generated by BOX-PCR. Diversity of E. coli and Enterococcus populations was calculated with the Shannon-Weiner diversity index. Similarity of E. coli and Enterococcus populations was calculated with the population similarity coefficient. After the rain event, levels of fecal coliforms and Enterococcus spp. were high in sediments and exceeded the regulatory standard for all water samples. In dry conditions, levels were lower in water samples, but still high in sediment samples. Significantly greater population diversity was observed in the rain event compared to the dry event for both E. coli and Enterococcus populations, and greater population similarity was vi observed in dry conditions. Enterococcus population diversity was significantly higher in untreated sewage and the Siesta Key rain event when compared to dry conditions, and to a site on the Myakka River (no known human input or urban stormwater runoff). Siesta Key populations in dry conditions were most similar to Myakka, and sewage was the least similar to all other populations. Increased population similarity for E. coli and Enterococcus spp. during dry conditions suggests that a portion of the population is composed of “survivor” isolates. Persistence of survivor isolates in the storm drainage system, where urban runoff can sit for days, suggests a reservoir for indicator bacteria that can be flushed through the system to the Gulf, causing high levels of indicator bacteria in receiving waters.

A library of 18 coliform bacteria strains was obtained from different sampling points in the drinking water distribution system in Lexington, KY, over a three month period in 2006. To investigate the cause of the coliform occurrence we conducted a microbial source tracking study using phenotypic (API 20E, Biolog, and Vitek) and genotypic (pulsed field gel electrophoresis (PFGE) and ribotyping) analyses to determine the degree of genetic variation among isolates. Characterization of isolates by PFGE and ribotyping showed that coliform events in the distribution system were related and a regrowth problem may exist due to biofilm formation. The ability of a persistent Enterobacter cloacae strain to adhere and form biofilm was found to depend on environmental conditions such as temperature, pipe material, soiled surface, chlorine and nutrient levels with higher temperatures and nutrient levels promoting adherence. Considerable variation in adherence and biofilm formation was observed among representative Enterobacter isolates.

Waterborne pathogen presence caused by fecal pollution is a leading cause of morbidity and mortality worldwide. In developed countries, this problem can result in waterborne outbreaks. Research suggests that there is a need for better fecal indicators because current methods (total coliforms and E. coli) are insufficient. This study investigated Bacteroidales 16S rRNA markers as a microbial source tracking tool in an agricultural watershed. Correlations between pathogens and markers were also investigated. Water quality monitoring was conducted following assay validation of ruminant-, bovine-, human-specific, and universal Bacteroidales markers. Results revealed a positive relationship between E. coli and the universal marker. Ruminant- and bovine-specific marker detection was associated with increased runoff due to precipitation; however, the human associated marker was not detected. Furthermore, no correlations between Campylobacter, Salmonella, or E. coli O157:H7 could be made. Consequently, these techniques have potential to become a powerful tool; however, further research is needed

A DNA microarray capable of detecting 445 virulence factors (VFs) and antibiotic resistance genes was used to assess human and animal fecal E. coli isolates for pathogenic potential and host specificity. The only enteric pathotype detected was atypical EPEC, which was found in 3.7% of all isolates. 17% of human isolates were extraintestinal pathotypes, with the majority of these being uropathogenic. Isolates from humans and chickens were the most likely to have resistance to at least one class of antibiotic. VFs that were found almost exclusively in human isolates, when compared to one other group, included sat (10% of human isolates and no animal isolates), iucD and iut (24% of human, <1% mammal) and iha (16% human, <1% wild avian). Decision trees utilizing multiple probes to identify the source of an E. coli isolate were able to correctly identify the source of 79% of validation isolates in a human vs. animal comparison. / Environment Canada

The field of molecular fecal source tracking in the water environment has developed rapidly since the first PCR assays for general and host-­‐specific Bacteroides 16s rRNA markers were published. Numerous host-­‐specific molecular markers and PCR assays have been developed, adding greater specificity, sensitivity and quantitative methods to the array of options. The public demand for readying methods for transfer to the commercial lab, so that they may be used to generate data for public utilities, citizen action groups and regulatory agencies, has fueled the development of an entire new research community. These methods, however plentiful, have not found community agreement and there is no consensus concerning the appropriate implementation of molecular fecal source tracking in the field. Some issues plaguing the implementation include imperfect marker specificity, environmental variability, DNA extraction variability, PCR inhibition and high cost of molecular analysis. This thesis presents an approach for locating hot spots of human fecal pollution in an urban watershed by using published methodologies for the collection of molecular fecal source tracking data along with a tiered watershed screening tool for cost reduction and two data normalization techniques which ameliorate several known sources of error and strengthen the efficacy of watershed application.

The City of Georgetown, Kentucky relies on a vast karst spring network as a drinking water source. This karst feature has several inputs from sinkholes and streams in the Cane Run Watershed: a watershed associated with a variety of land uses in the recharge area. The recharge area encompasses the area from North Lexington to Georgetown and is composed of urban, suburban, agricultural and industrial usage. A serious water quality issue exists with respect to the impact of fecal contamination within the spring recharge area. Identification of fecal contamination is quantified by microbial indicators adapted from surface water applications: fecal load (E. coli), fecal source (two human-host specific Bacteroides DNA markers) and fecal age (AC/TC ratio). These three criteria are used in a categorical Microbial Source Tracking (MST) model to assign a Sanitary Category Value (SCV) between 0 and 3 for each sample location. Low SCVs (1.5) are associated with high values of fecal load, low fecal age and detectable concentration of human-specific markers. SCV measured during dry weather conditions are indicative of potentially leaking human sewers. Due to retention and conservation of fecal load (E. coli) and age (AC/TC) microbial indicators in the karstic environment, ambiguous SCV model results cannot pinpoint, with statistical confidence, fecal sources in a karstic environment. Human-host specific genetic markers (HF183 and HuBac) were also detected at all sample sites above limits of detection, indicating steady inflow of fecal material during all sample events. By adding a flow multiplier and expressing HF183 and HuBac values as a load, it was strongly indicated that a human fecal source was entering the groundwater conduit and impacting Royal Spring independent from other upstream fecal sources. Interpretation of these trends, while strongly indicated, cannot be supported with statistical evidence.

Surface water is used for drinking by many people around the world. E. coli is the most frequently used bacterial indicator used for assessing water quality. The survival, sources, and concentrations of E. coli were examined through a series of experiments that investigated the survival of beef cattle E. coli on land and in water, and used bacterial source tracking (BST) to determine the sources of fecal contamination diurnally and annually in multiple watersheds in British Columbia. A fecal pat experiment was conducted to examine the survival of E. coli under 4 levels of solar exposure. E. coli survived longer with increasing shade. Age of fecal pats, as well as exposure to solar radiation negatively influenced the survival of E. coli. The survival of E. coli in stream water was examined in filtered and unfiltered stream water at 3 different temperatures (6, 20 and 26 ºC). There was no significant difference in the survival of E. coli in filtered versus non-filtered stream water. Lower water temperatures (6 ºC) increased the survival of E. coli. The addition of manure to the water substantially increased the nutrient concentrations and organics. BST is a rapidly growing area of research and technology development and many methods are being developed and tested. The choice of method used for BST depends on: question(s) to be answered, scale of identification needed, available expertise, cost of analysis, turnaround time, and access to facilities. The spatial, diurnal, and annual sources and concentrations of E. coli were investigated in several watersheds in British Columbia. Fecal coliforms and E. coli concentrations varied throughout the day, as well as by site, month and year. Ribotyping identified many different sources of E. coli within the watersheds. The majority of E. coli isolates classified were from wildlife sources in each watershed even though they had different land-use.

abstract: Water quality in surface water is frequently degraded by fecal contamination from human and animal sources, imposing negative implications for recreational water use and public safety. For this reason it is critical to identify the source of fecal contamination in bodies of water in order to take proper corrective actions for controlling fecal pollution. Bacteroides genetic markers have been widely used to differentiate human from other sources of fecal bacteria in water. The results of this study indicate that many assays currently used to detect human-specific Bacteroides produce false positive results in the presence of freshwater fish. To further characterize Bacteroides from fish and human, the fecal samples were cultured, speciated, and identified. As a result, forty six new Bacteroides 16S rRNA gene sequences have been deposited to the NCBI database. These sequences, along with selected animal fecal sample Bacteroides, were aligned against human B. volgatus, B. fragilis, and B. dorei to identify multi-segmented variable regions within the 16S rRNA gene sequence. The collected sequences were truncated and used to construct a cladogram, showing a clear separation between human B. dorei and Bacteroides from other sources. A proposed strategy for source tracking was field tested by collecting water samples from central AZ source water and three different recreational ponds. PCR using HF134 and HF183 primer sets were performed and sequences for positive reactions were then aligned against human Bacteroides to identify the source of contamination. For the samples testing positive using the HF183 primer set (8/13), fecal contamination was determined to be from human sources. To confirm the results, PCR products were sequenced and aligned against the four variable regions and incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped within the human clade. As an outcome of this study, a tool box strategy for Bacteroides source identification relying on PCR amplification, variable region analysis, human-specific Bacteroides PCR assays, and subsequent truncated cladogram grouping analysis has been developed. The proposed strategy offers a new method for microbial source tracking and provides step-wise methodology essential for identifying sources of fecal pollution. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2012

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22762号 / 工博第4761号 / 新制||工||1745(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 米田 稔, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Microbial Source Tracking (MST) is a field of study that attempts to identify the source of fecal contamination in waterways in order to assist with development of remediation strategies. Biologists at Cal Poly Center for Applications in Biotechnology (CAB) are developing a new MST method using microbes from the genus Bacteroides. Bacteroides species are host-specific microorganisms that can theoretically be used to trace back to a single host species. After fecal samples are collected, biologists use Next-Generation Sequencing (NGS) techniques to obtain only the genetic sequences of microorganisms belonging to the phylum Bacteroidetes. Investigators hypothesize that similar sequences belong to the same phlyogenetic group (i.e., the same genus) and can therefore be computationally clustered. Each cluster of related sequences, typically 97% similar, is called an Operational Taxonomic Unit (OTU). Theoretically, an OTU acts as a molecular signature that can be traced back to a specific host genus. This thesis presents LOTUS, the Library of OTUs, a web-based computational tool for the preliminary investigation of the use of the Bacteroides OTU library as an MST method. This work discusses the four contributions of LOTUS: a database design which accurately models OTUs and the underlying relationships necessary for source tracking, a pipeline to create OTUs from raw sequencing reads, a method of assigning taxonomy to OTUs, and a web-based user interface. In preliminary testing for a reference library of twelve samples, LOTUS produced 1,431 OTUs, of which 891 were single-source (OTUs derived from sequences from a single host species). Using these OTUs, LOTUS was able to accurately taxonomically match four of five unknown test samples, showing promise for using OTUs as an MST method.