4. Bacteria Identification Methods

4.1 Identification using permissive growth media and labeling approaches

Contemporary, commonly-used methods for measuring concentrations of the fecal indicator bacteria E. coli and Enterococcus sp. rely on culturing bacteria obtained in grab samples to allow their detection. These approaches are valid for measuring the concentrations of live, culturable E. coli or Enterococcus, but don’t permit strain identification or measuring the concentrations of live, but non-culturable bacteria.

Bonadonna et al 2006

4.2 Identification using genetic approaches

There are several DNA-based approaches to detecting the presence of fecal organisms, as well as methods using antibodies to detect bacteria-specific proteins.

The DNA approaches can be lumped into the following categories: polymerase chain reaction (PCR) amplification of DNA using specific DNA primer sequences (e.g., Al-Ajmi et al., 2006); ribosomal RNA sequence detection (“Ribotyping”, e.g., Anderson et al 2005); DNA microarray (Hamelin et al., 2006); fluorescence in situ hybridization (“FISH”) for environmental bacterial cell counting (Tank et al., 2005); and antibody labeling and microscopic detection (e.g., Li and Su, 2006).

These approaches are used to identify particular strains of fecal bacterial species in order to determine the host organism sources of fecal material (Yan et al., 2007). Any of E. coli, Enterococcus sp., or Bacteroides (a common fecal bacterium) can be identified using one or more of these approaches. In a recent study of fecal contamination in tributaries to the San Joaquin River, Johnson et al., (2007) used polymerase chain reaction with DNA primers specific to human, bovine, and chicken fecal Bacteroides to track potential sources of fecal contamination. They used Bacteroides because it is an indicator of recent fecal contamination, due to the fact that this genus is an anaerobic obligate and dies quickly in surface waters. They were able to use this approach to identify human fecal contamination of surface waters during the summer-months study. Bovine contamination from lots and pastures was less prevalent, though the authors noted that there was little overland flow to mobilize dispersed cow manure into waterways. There was not good agreement between Bacteroides (detected by PCR) and E. coli (detected using conventional methods) results, probably due to the longer survival times of E. coli in the environment.

DNA primers exist to identify Bacteroides, E. coli, and Enterococcus strains and thus isolate host organisms for the original feces inputs to waterways. DNA microarrays can be used to test for many strains simultaneously in a water sample. These DNA-based tests are a relatively inexpensive way to forensically point to potential fecal matter inputs, though they may be too expensive for a regular monitoring program across a large area.