Evaluation of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular identification

The sensitivity and specificity of the H2S test to detect fecal bacteria in water has been variable and uncertain in previous studies, partly due to its presence–absence results. Furthermore, in groundwater samples false-positive results have been reported, with H2S-positive samples containing no fecal coliforms or Escherichia coli. False-negative results also have been reported in other studies, with H2S-negative samples found to contain E. coli. Using biochemical and molecular methods and a novel quantitative test format, this research identified the types and numbers of microbial community members present in natural water samples, including fecal indicators and pathogens as well as other bacteria. Representative water sources tested in this study included cistern rainwater, a protected lake, and wells in agricultural and forest settings. Samples from quantitative H2S tests of water were further cultured for fecal bacteria by spread plating onto the selective media for detection and isolation of Aeromonas spp., E. coli, Clostridium spp., H2S-producers, and species of Salmonella and Shigella. Isolates were then tested for H2S production, and identified to the genus and species level using biochemical methods. Terminal Restriction Fragment Length Polymorphisms (TRFLP) was the molecular method employed to quantitatively characterize microbial community diversity. Overall, it was shown that water samples testing positive for H2S bacteria also had bacteria of likely fecal origin and waters containing fecal pathogens also were positive for H2S bacteria. Of the microorganisms isolated from natural water, greater than 70 percent were identified using TRFLP analysis to reveal a relatively stable group of organisms whose community composition differed with water source and over time. These results further document the validity of the H2S test for detecting and quantifying fecal contamination of water.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (122 K)Download as PowerPoint slideHighlights► First work to compare molecular and biochemical methods in validating the identity of microbes in H2S tests in natural water samples. ► Positive H2S tests consistently contained fecal bacteria and pathogens. ► Negative H2S tests contained few fecal bacteria or pathogens. ► There were strong relationships of agreement between the organisms identified by both molecular and biochemical methods.