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Culture-independent techniques were used for the detection of pathogenic bacteria in drinking water at potentially critical control points along the production lines at a German dairy company and a Spanish dry cured ham company. Denaturing gradient gel electrophoresis (DGGE) was used to describe bacterial population shifts indicating biological instability in the drinking water samples. Autochthonous bacteria were identified by sequencing the excised DGGE DNA bands. More specifically, real-time PCR was applied to detect a number of pathogenic bacteria, i.e. Listeria monocytogenes, Mycobacterium avium subsp. paratuberculosis, Campylobacter jejuni, Enterococcus spp., Salmonella spp, Escherichia coli, and Pseudomonas aeruginosa. Due to the detection limits of the real-time PCR method, a specific protocol was established in order to meet the technical detection requirements and to avoid unwanted polymerase inhibitions. Autochthonous bacterial populations were found to be highly stable at most of the sampling points. Only one sampling point exhibited population shifts at the German dairy company. Enterococci and P.aeruginosa were detected in some water samples from these companies by molecular biology detection methods, but not by conventional culturing methods. Some opportunistic bacteria as Enterobacter sp., Acinetobacter, Sphingomonas sp. and non-pathogenic Bacillus, were also detected after DNA sequencing of DGGE bands., (Copyright 2010 Elsevier B.V. All rights reserved.)