In this approach, individual colonies, usually of E. coli or enterococci, are isolated from water samples, and also from potential sources of contamination such as human sewage or animal faeces. These isolates are then fingerprinted using either DNA-based or phenotypic/biochemical based methods.
DNA based methods, such as ribotyping, pulsed field gel electrophoresis (PFGE), and Rep-PCR generate bar code-like DNA fingerprints that differ according to the DNA of individual isolates. Biochemical or phenotypic methods generate a profile of each isolate based on characteristics such as resistance to a range of antibiotics, or utilisation of different carbon sources.
Once a library of known sources has been constructed – usually from hundreds, if not thousands of isolates from known sources — it can be used as a reference database with which to compare isolates from unknown sources.
The key attraction of library methods is that the results relate directly to the E. coli or enterococci which have prompted the investigation. However these indicator organisms do not appear to be particularly host specific, and the same fingerprint types are often found in multiple sources. This can be partially overcome by the application of statistical techniques, such as discriminate analysis, to whole populations, allowing the relative contributions of different sources to be estimated.
The approach also relies on the library containing all possible sources of the selected indicator, and the usefulness of libraries tends to be limited to particular geographical areas and time periods. In addition, creating and maintaining libraries large enough to provide source discrimination is expensive (e.g., the largest ribotyping database in the USA has over 250,000 isolates).
At ESR we can offer antibiotic resistance analysis (ARA), PFGE and Rep-PCR as additional methods for distinguishing faecal sources. Furthermore, these particular library-based methods maybe useful where there are questions arising from high levels of faecal indicator bacteria but further testing does not implicate faecal contamination as a source. Controversy surrounds the ability of E. coli to replicate in temperate environments. Library-based methods that identify a clonal population of an E. coli subtype may support the premise of E. coli replication, in contrast to a faecal source polluting a particular waterway.
Prices associated with these additional library-based tests are available on application to ESR.
Additional literature references on library-based methods can be accessed here.