Evaluation of Methanobrevibacter smithii as a Human-Specific Marker of Fecal Pollution

Microbial source tracking has historically focused on the origin of traditional enteric indicators including coliforms, enterococci, or Escherichia coli. Recently, questions of genetic variability and environmental persistence have encouraged researchers to search for additional animal specific indicators of fecal pollution. To date only eubacteria have been utilized as markers of human and animal-specific pollution. In this study we developed a molecular primer pair specific for Methanobrevibacter smithii, a methanogen found only in the human intestine. PCR primers for the nifH gene of M. smithii were designed, tested, and used to detect the presence or absence of the organism in fecal and environmental samples. Product amplification was observed in 28.6% of all human fecal samples and 93% of sewer samples, and water contaminated with human sewage. No amplification was observed when primers were tested against 43 bacterial stock cultures and fecal samples from 204 animals. Sequencing of PCR products from sewers and M. smithii cells demonstrated that the 222bp product amplified was the nifH gene of M. smithii. Sensitivity assays demonstrated a detection limit of 10ng in human feces, 10ng in fecally contaminated water, and 5 ng in sewer samples. In addition, samples seeded with M. smithii established a lower detection limit of 13 cells/ml. The Mnif method for M. smithii detection appears to be a rapid, inexpensive, and reliable test for determining the presence or absence of human fecal pollution in recreational waters