Donald M. Stoeckel

Relative Decay of Bacteroidales Microbial Source Tracking Markers and Cultivated Escherichia coli in Freshwater Microcosms

ABSTRACT Fecal indicator bacteria (FIB), commonly used to regulate sanitary water quality, cannot discriminate among sources of contamination. The use of alternative quantitative PCR (qPCR) methods for monitoring fecal contamination or microbial source tracking requires an understanding of relationships with cultivated FIB, as contamination ages under various conditions in the environment. In this study, the decay rates of three Bacteroidales 16S rRNA gene markers (AllBac for general contamination and qHF183 and BacHum for human-associated contamination) were compared with the decay rate of cultivated Escherichia coli in river water microcosms spiked with human wastewater. The following five sets of microcosms were monitored over 11 days: control, artificial sunlight, sediment exposure, reduced temperature, and no autochthonous predation. Decay was characterized by estimation of the time needed to produce a 2-log reduction (t99). No treatment-associated differences in the decay of the 4 targets were evident except with reduced predation, where E. coli, qHF183, and BacHum markers had lower levels of decay by day 3. However, there were substantial target-associated differences. Decay curves for the AllBac marker indicated a larger persistent population than those of the other targets. Exposure to sunlight, sediment, and reduced predation resulted in more rapid decay of the human-associated markers relative to cultivable E. coli, but there were no differences in t99 values among the 4 targets under control conditions or at reduced temperatures. Further evaluation of epidemiological relationships will be needed in order to relate the markers directly to health risk. These findings suggest that the tested human-associated markers can complement E. coli as indicators of the human impact on sanitary water quality under the constrained conditions described in this paper.

Evaluation of two spike-and-recovery controls for assessment of extraction efficiency in microbial source tracking studies

Quantitative PCR (qPCR), applied to complex environmental samples such as water, wastewater, and feces, is susceptible to methodological and sample related biases. In this study, we evaluated two exogenous DNA spike-and-recovery controls as proxies for recovery efficiency of Bacteroidales 16S rDNA gene sequences (AllBac and qHF183) that are used for microbial source tracking (MST) in river water. Two controls--(1) the plant pathogen Pantoea stewartii, carrying the chromosomal target gene cpsD, and (2) Escherichia coli, carrying the plasmid-borne target gene DsRed2--were added to raw water samples immediately prior to concentration and DNA extraction for qPCR. When applied to samples processed in replicate, recovery of each control was positively correlated with the observed concentration of each MST marker. Adjustment of MST marker concentrations according to recovery efficiency reduced variability in replicate analyses when consistent processing and extraction methodologies were applied. Although the effects of this procedure on accuracy could not be tested due to uncertainties in control DNA concentrations, the observed reduction in variability should improve the strength of statistical comparisons. These findings suggest that either of the tested spike-and-recovery controls can be useful to measure efficiency of extraction and recovery in routine laboratory processing.

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence.

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections--augmented with local observations and land-use patterns--indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination.

Bacteriological water quality in and around Lake Pontchartrain following Hurricanes Katrina and Rita: Chapter 7H in Science and the storms-the USGS response to the hurricanes of 2005

The U.S. Geological Survey (USGS), in collaboration with the Louisiana Department of Environmental Quality (LDEQ), monitored sanitary water quality at 22 sites in and around Lake Pontchartrain, La., for 3 consecutive weeks beginning September 13, 2005, following Hurricanes Katrina and Rita. Fecal-indicator bacteria (Escherichia coli, enterococci, and fecal coliform) concentrations ranged from the detection limit to 36,000 colonyforming units per 100 mL on north shore streams (September 13–29). A survey of 30 sites in Lake Pontchartrain during the week of October 11–14, 2005, confirmed that there were little widespread, long-term effects on sanitary water quality of Lake Pontchartrain directly caused by Katrina and Rita. Concentrations of fecal-indicator bacteria in tributaries on the north shore of Lake Pontchartrain tended to be high relative to historical data.