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

A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the beta-D-glucuronidase gene (uid4) for specific detection and differentiation of Escherichia coli populations according to their uidA sequence variations. Detection of sequence variations by PCR-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 differences between alleles of the uidA gene fragments (screened fragment length, 126 hp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed E. coli sequence types was revealed, PCR-DGGE applied to fecally polluted river water samples simultaneously detected E. coli and generated a fingerprint of the mixed populations by separating the polymorphic uidA amplicons, Na significant differences between non-cultivation-based and cultivation-based profiles were observed suggesting that at least some members of all occurring sequence types could be cultivated. As E. coli is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters.