Simultaneous detection and differentiation of Escherichia coli populationsfrom environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene

A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was app lied to a partial sequence of the beta-D-glucuronidase gene (uid4) for spec ific detection and differentiation of Escherichia coli populations accordin g to their uidA sequence variations. Detection of sequence variations by PC R-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50 E. coli freshwater isolates and reference strains revealed 11 sequence types, showing nine poly-morphic sites and an average number of pairwise di fferences between alleles of the uidA gene fragments (screened fragment len gth, 126 hp) of 2.3%. Among the analyzed strains a range of dominating to m ore rarely and/or uniquely observed E. coli sequence types was revealed, PC R-DGGE applied to fecally polluted river water samples simultaneously detec ted E. coli and generated a fingerprint of the mixed populations by separat ing the polymorphic uidA amplicons, Na significant differences between non- cultivation-based and cultivation-based profiles were observed suggesting t hat at least some members of all occurring sequence types could be cultivat ed. As E. coli is frequently used as a fecal indicator, this work is consid ered an important step towards a new, practical tool for the differentiatio n and tracing of fecal pollution in all kinds of waters.