SR9071 - DNA Fingerprinting Analysis of Escherichia Coli Using Repetitive-Element and Toxin Specific Gene Amplification to Differentiate Pollution Sources in the Environment

Lead researchers

Michele Van Dyke and Jesse Crawford, GAP EnviroMicrobial Services, London

Objectives

To develop a method for tracing the source of fecal pollution in the environment by using DNA fingerprinting techniques called repetitive-element and toxin gene amplification to differentiate between E. coli strains.

Expected benefits

To provide a technique for accurate characterization and differentiation of fecal contamination sources so that preventative and remedial actions can be taken by the appropriate agencies.

Results

Rep-PCR analysis was performed on the database of E. coli and Ent. faecium isolates by amplifications using three different Rep-PCR primers sets, including BOX, REP and ERIC. The amplification conditions were shown to provide reproducible and reliable banding patterns for each strain.

Statistical analysis of banding patterns produced from E. coli strains showed that there was poor separation when grouped by individual host animals. When the data was then analyzed by selecting representative isolates from strains that gave the same pattern from individual animals, the category separation values were found to be decreased. This type of analysis was necessary to limit bias caused by the high degree of similarity between E. coli strains within individual animals.

When host animals were grouped into three main categories, including animals, birds and humans, the separation values were somewhat improved. The category separation values for humans, animals and birds were 54, 60 and 68%, respectively. Using the group violations analysis, it was found that there was a large amount of overlap between the groups. It was concluded that the group separation values were not sufficiently high to allow sensitive discrimination of an animal host.

Rep-PCR analysis was also performed on 50 Ent.faecium isolates from different sources. The ability of Rep-PCR to assign Ent.faecium isolates to individual animal sources was poor. However, the category separation of animals versus birds was high, resulting in values of 78 and 77%, respectively.

Three sites were selected to determine if E.coli strains isolated from defined sources could be tracked in real environmental situations. Results of the site studies showed that E.coli contamination could be successfully tracked from the point of origin through different watershed systems. This analysis showed that there is potential for using Rep-PCR fingerprinting techniques in defined environments where access to potential sources is available.

Related information

• Other projects funded through the New Directions Research Program in 2001