Open AccessSimultaneous Detection and Differentiation of Escherichia coli Populations from Environmental Freshwaters by Means of Sequence Variations in a Fragment of the β- d -Glucuronidase Gene
Author(s) -
Andreas H. Farnleitner,
Norbert Kreuzinger,
Gerhard G. Kavka,
Sonja Grillenberger,
Johannes Rath,
Robert L. Mach
Publication year - 2000
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.66.4.1340-1346.2000
Subject(s) - biology , escherichia coli , amplicon , gene , genetics , sequence analysis , polymerase chain reaction , feces , sequence (biology) , temperature gradient gel electrophoresis , microbiology and biotechnology , 16s ribosomal rna
A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the β-d -glucuronidase gene (uidA ) for specific detection and differentiation ofEscherichia coli populations according to theiruidA sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50E. coli freshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of theuidA gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observedE. coli sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detectedE. coli and generated a fingerprint of the mixed populations by separating the polymorphicuidA 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. AsE. 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.