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Applied and Environmental Microbiology, April 2000, p. 1340-1346, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Simultaneous Detection and Differentiation of Escherichia coli Populations from Environmental Freshwaters by Means of Sequence Variations in a Fragment of the beta -D-Glucuronidase Gene

Andreas H. Farnleitner,1,* Norbert Kreuzinger,2 Gerhard G. Kavka,3 Sonja Grillenberger,4 Johannes Rath,5 and Robert L. Mach1

Institute of Biochemical Technology and Microbiology 172/5, Technical University of Vienna, 1060 Vienna,1 Institute of Water Quality and Waste Management, Department of Chemistry and Microbiology, Technical University of Vienna, 1040 Vienna,2 Institute of Water Quality, Federal Agency for Water Management, 1220 Vienna,3 Yppenplatz 5/18, 1160 Vienna,4 and Institute of Zoology, University of Vienna, 1090 Vienna,5 Austria

Received 31 August 1999/Accepted 29 December 1999

A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the beta -D-glucuronidase gene (uidA) 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 polymorphic sites and an average number of pairwise differences between alleles of the uidA gene fragments (screened fragment length, 126 bp) 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. No 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.

* Corresponding author. Mailing address: Institute of Biochemical Technology and Microbiology 172/5, Technical University of Vienna, Getreidemarkt 9, 1060 Vienna, Austria. Phone: 43 2630 30650. Fax: 43 2630 363439. E-mail: A.FARNLEITNER{at}aon.at.

Applied and Environmental Microbiology, April 2000, p. 1340-1346, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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