The Evaluation of PCR-Based Techniques for the Detection of Fecal Indicator Organisms in Environmental Samples
Escherichia coli is the indicator organism of choice for the detection of fecal contamination. Standard methods for detecting fecal contamination often rely on detecting fecal coliforms. While this practice if highly efficient in most applications, when used with the sediment-laden waters of Louisiana, free-living soil bacteria, such as Klebsiella pneumoniae were shown to inflate the fecal coliform counts. An accurate assessment of the contamination problem requires methods that detect E. coli. To detect E. coli, several of species-specific genes were tested before the gadAB genes were selected. The polymerase chain reaction (PCR) was chosen to detect these genes using a nested primer array. Using the nested PCR assay, E. coli was routinely detected in laboratory samples down to the single-cell level. This level of detection was deemed essential for a quantitative or semiquantitative assay to determine E. coli numbers. The most beneficial use of PCR-based techniques may be the use of specific fecal indicator organisms to differentiate the sources of fecal contamination. E. coli is the indicator of fecal contamination from essentially all sources posing a risk to human health. The Bacteroides fragilis group (BFG) demonstrates strong potential as a fecal indicator organism of human waste. While the levels of E. coli in humans and cattle are comparable, the levels of BFG are approximately 105 times greater in humans than cattle. By determining the ratio of E. coli to BFG in contaminated water, one should be able to distinguish fecal contamination from humans and common farm animals, such as dairy cattle. This requires the determination of the relative levels of E. coli and BFG and not the absolute levels in the sample. Using the nested PCR method, a region of the 16S rRNA genes specific to members of the BFG was targeted. In the laboratory, B. fragilis cells were detected down to the single-cell level. When environmental water samples were tested, impurities in the template preparations limited the amount of DNA template that could be added to the reactions. Within those limitations the level of detection was comparable to laboratory studies using cultured cell suspensions.
Advisor:Caye M. Drapcho; Alan J. Biel; Ronald J. Siebeling
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Keywords:microbiology biological sciences
Date of Publication:11/15/2001