title: Evaluation of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular identification.
abstract: The sensitivity and specificity of the H(2)S 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 H(2)S-positive samples containing no fecal coliforms or Escherichia coli. False-negative results also have been reported in other studies, with H(2)S-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 H(2)S 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., H(2)S-producers, and species of Salmonella and Shigella. Isolates were then tested for H(2)S 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 H(2)S bacteria also had bacteria of likely fecal origin and waters containing fecal pathogens also were positive for H(2)S 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 H(2)S test for detecting and quantifying fecal contamination of water.
authors.affiliation: University of North Carolina at Chapel Hill, Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, Chapel Hill, NC 27599, USA. kmcmahan@gmail.com
authors.forename: Lanakila
authors.initials: L
authors.lastname: McMahan
authors.forename: Amy M
authors.initials: AM
authors.lastname: Grunden
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Devine
authors.forename: Mark D
authors.initials: MD
authors.lastname: Sobsey
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D018929:Cell Culture Techniques; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D016680:Genome, Bacterial / Q000235:genetics; D006862:Hydrogen Sulfide / Q000494:pharmacology*; D060106:Lakes / Q000382:microbiology; D009657:North Carolina; D012150:Polymorphism, Restriction Fragment Length; D011891:Rain / Q000382:microbiology; D012984:Software; D012985:Software Design; D013045:Species Specificity; D014495:Universities; D014867:Water; D014871:Water Microbiology; D060986:Water Wells / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D014867:Water; D006862:Hydrogen Sulfide