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Microbiology 155 (2009), 2306-2319; DOI  10.1099/mic.0.028233-0
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Microbiology 155 (2009), 2306-2319; DOI  10.1099/mic.0.028233-0
© 2009 Society for General Microbiology

Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics

Rafael Szczepanowski1, Burkhard Linke2, Irene Krahn1, Karl-Heinz Gartemann3, Tim Gützkow1, Wolfgang Eichler4, Alfred Pühler1 and Andreas Schlüter1

1 Institute for Genome Research and Systems Biology, Center for Biotechnology, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany
2 Bioinformatics Resource Facility, Center for Biotechnology, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany
3 Lehrstuhl für Gentechnologie und Mikrobiologie, Fakultät für Biologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany
4 Landesamt für Natur, Umwelt und Verbraucherschutz NRW, FB76.2, Auf dem Draap 25, 40221 Düsseldorf, Germany

To detect plasmid-borne antibiotic-resistance genes in wastewater treatment plant (WWTP) bacteria, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Subsequent PCR analyses on total plasmid DNA preparations obtained from bacteria of activated sludge or the WWTP's final effluents led to the identification of, respectively, 140 and 123 different resistance-gene-specific amplicons. The genes detected included aminoglycoside, β-lactam, chloramphenicol, fluoroquinolone, macrolide, rifampicin, tetracycline, trimethoprim and sulfonamide resistance genes as well as multidrug efflux and small multidrug resistance genes. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and WWTP bacteria. Sequencing of selected resistance-gene-specific amplicons confirmed their identity or revealed that the amplicon nucleotide sequence is very similar to a gene closely related to the reference gene used for primer design. These results demonstrate that WWTP bacteria are a reservoir for various resistance genes. Moreover, detection of about 64 % of the 192 reference resistance genes in bacteria obtained from the WWTP's final effluents indicates that these resistance determinants might be further disseminated in habitats downstream of the sewage plant.

Correspondence
Andreas Schlüter
Andreas.Schlueter{at}Genetik.Uni-Bielefeld.de

Abbreviations: WWTP, wastewater treatment plant

A supplementary table of primers is available with the online version of this paper.






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