Technical Report

Sponsor: GWRI

Start Date: 2000-03-01; Completion Date: 2001-02-28;

Keywords: Coliform isolation, Pollution source assessment, Pollution transport assessment, Pollutant source control

Problem and research objective:

Fecal coliforms consist of several bacterial genera from the family Enterobacteriaceae that can grow on a selective medium at 44.5°C for 24 hours. Fecal coliforms normally inhabit the intestinal tract of warm-blooded animals and their presence in soil or water is a good indicator that the soil or water was contaminated by bacterial pathogens. For example, when numbers of fecal coliforms exceed 2,000 per 100 mL of water, the likelihood of bacterial pathogens in the water is 98.1% (Geldreich, 1970). Fecal coliform counts are typically used to monitor Georgia's recreational waters.

One of the most vexing problems in isolating fecal coliforms from water samples is not knowing the host origin of these bacteria. In the past, the only way to identify the host origin of a bacterium was to observe the bacterium's various phenotypic markers (i.e., characteristics expressed by the bacterium, like antibiotic resistance). The main problems with using phenotypic markers are their lack of reproducibility and lack of discriminatory power (ability to distinguish two closely related strains). However, in recent years, it has become possible to identify the host origin of a bacterium based on its DNA. This alternative method, called genotyping, not only has increased reproducibility, but also has increased discriminatory power. The most common of these genotypic methods include chromosomal DNA restriction analysis, plasmid typing, pulsed field gel electrophoresis, various polymerase chain reaction (PCR) methods, and ribotyping (Farber, 1996).

Each genotypic method has its advantages and disadvantages with respect to strains that can be typed, reproducibility, discriminatory power, ease of interpretation, and ease of performance. In this report, the genotypic method selected was ribotyping. Ribotyping is based on ribosomal RNA (rRNA). Ribosomal RNA is present in all bacteria, and is composed of three species, 5S, 16S, and 23S. The DNA in the bacterium that encodes for these three species of rRNA is usually present in 2 to 11 copies and is highly conserved (does not mutate; Grimont and Grimont, 1986). In ribotyping, the DNA is isolated from the bacterium and cut with a special enzyme that only recognizes certain DNA sequences (i.e., a restriction enzyme). The DNA is electrophoresed in a gel and the DNA transferred to a nylon membrane (this is called Southern blotting). The membrane is probed with a chemiluminescent copy of the 5S, 16S, and 23S portions of the DNA and, when properly treated, the membrane gives a pattern that can be scanned with an imager. As a method for distinguishing a subspecies of a bacterium, ribotyping is considered to have excellent reproducibility, good discriminatory power, excellent ease of interpretation, and good ease of performance (Farber, 1996).

Here, the fecal coliform selected for ribotyping was Escherichia coli. This bacterium was selected for five reasons. First, as a fecal coliform, E. coli is accepted by the American Public Health Association as a good indicator of pathogenic bacteria (Clesceri et al., 1998). Second, most environmental ribotyping has been done with this bacterium. As a result, the methodology for ribotyping this bacterium is established. Third, there is good scientific evidence that specific strains of E. coli are associated with different host species (e.g., Amor et al., 2000). Fourth, E. coli does not exist as a stable population in the environment unless the source of contamination is persistent. Fifth, E. coli is easy to isolate and easy to manipulate genetically.