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Abstract
In this study real-time PCR assays, based on the LUX™-technique, were developed for quantification of genes mediating resistance to aminoglycosides [aac(6´′)-Ie + aph(2´′´′)], β-lactams (mecA), and tetracyclines (tetA and tetB), for use in wastewater environments. The developed assays were applied on DNA extracted from three wastewater-associated environments: soil from an overland flow area treating landfill leachates, biofilm from a municipal wastewater treatment plant, and sludge from a hospital wastewater pipeline. The highest concentration of all genes was observed in the hospital pipeline and the lowest in the overland flow system. TetA and aac(6´′)-Ie + aph(2´′´′) could be detected in all environments. The tetB gene was detected in the overland flow area and the hospital wastewater pipeline and mecA was detected in the wastewater treatment plant and the hospital pipeline. The developed LUX™ real-time PCR assays were shown to be fast and reproducible tools for detection and quantification of the four genes encoding antibiotic resistance in wastewater.
Acknowledgements
Sture Löfgren and Michael Toepfer, Ryhov County Hospital, Jönköping, and Elisabet Hollén, Linköping University, are acknowledged for comments and linguistic revision of the manuscript. Reference strains were kindly provided by Lennart Nilsson, Linköping University, Lars-Olof Hedén and Aftab Jasir, Lund University. Financial support was received from The Swedish Federations of County Councils, The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas; contract no 245-2005-860), The Swedish Strategic Programme for the Rational Use of Antimicrobial Agents and Surveillance of Resistance (STRAMA) and the Health Research Council of South-East Sweden (FORSS).