A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking

Abstract

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.

Basin-Wide Analysis of the Dynamics of Fecal Contamination and Fecal Source Identification in Tillamook Bay, Oregon

“…We previously showed that seawater did not affect the PCR detection limit for HF134, HF183, CF128, and CF193 (6,12,17), as well as two other Bacteroidales host-specific markers, PF163 and DF475 (6,12,17), but we did not examine the effect of seawater on survival of the marker cells. However, PCR assays could in theory detect dead cells, in contrast to indicator bacteria assays that depend on live cells.…”

“…1). Previous studies demonstrated that 100-ml water samples gave detection levels similar to those for indicator bacterium assays (5, 6,13,17). Therefore, for each sample, 100 ml of water was filtered through 0.2-l-pore-size Supor-200 filters (Whatman).…”

“…One of the most widely used FST approaches is a PCR-based method that targets host-specific Bacteroidales 16S rRNA genes (4, 5). This technique has been used successfully to discriminate between ruminant and human fecal sources in fresh and marine waters (5, 8,17,19), and it can also identify fecal sources such as dogs, pigs, and horses (12,14).…”

Impacts of Migratory Sandhill Cranes (Grus canadensis) on Microbial Water Quality in the Central Platte River, Nebraska, USA

“…Despite early attempts to utilize the higher ratio of fecal coliforms (E. coli) to enterococci to discriminate human from animal (bird) fecal sources (Geldreich and Kenner 1969;Geldreich 1976), due to varying differential die-off (and even growth) of either E. coli or enterococci in different aquatic environments (Davies et al 1995;Genthner et al 2005), interpretation of such ratios is generally confounded. Hence, alternative approaches have been explored, and despite continued efforts, no robust E. coli nor enterococci markers have been reported (Field et al 2003;Jiang et al 2007;Whitman et al 2007;Mohapatra et al 2008;Lanthier et al 2010). There are promising bird markers, however, which come from Catellicoccus 16S rRNA gene markers (Lu et al 2011;Green et al 2012;Ryu et al 2012a, b), and one demonstrated to identify Sandhill Crane excreta (Ryu et al 2012a, b) was used and supported in the current study (Table 1).…”

Detection of Genetic Markers of Fecal Indicator Bacteria in Lake Michigan and Determination of Their Relationship to Escherichia coli Densities Using Standard Microbiological Methods

“…These methods include detection of human enteroviruses or adenoviruses (16,22), host-specific species of Bacteroides (1), or virulence genes in E. coli (6). Several of these approaches were shown to be useful in detecting human sources of fecal contamination but did not necessarily identify all of the contributing hosts when multiple sources were present (11).…”