Plasmid-encoded quinolone resistance was previously reported for different bacteria isolated from patients not only in the United States and Asia but also in Europe. Here we describe the isolation, by applying a new selection strategy, of the quinolone resistance plasmid pGNB2 from an activated sludge bacterial community of a wastewater treatment plant in Germany. The hypersensitive Escherichia coli strain KAM3 carrying a mutation in the multidrug efflux system genes acrAB was transformed with total plasmid DNA preparations isolated from activated sludge bacteria and subsequently selected on medium containing the fluoroquinolone norfloxacin. This approach resulted in the isolation of plasmid pGNB2 conferring decreased susceptibility to nalidixic acid and to different fluoroquinolones. Analysis of the pGNB2 nucleotide sequence revealed that it is 8,469 bp in size and has a G؉C content of 58.2%. The plasmid backbone is composed of a replication initiation module (repA-repC) belonging to the IncQ-family and a two-component mobilization module that confers horizontal mobility to the plasmid. In addition, plasmid pGNB2 carries an accessory module consisting of a transposon Tn1721 remnant and the quinolone resistance gene, qnrS2, that is 92% identical to the qnrS gene located on plasmid pAH0376 from Shigella flexneri 2b. QnrS2 belongs to the pentapeptide repeat protein family and is predicted to protect DNA-gyrase activity against quinolones. This is not only the first report on a completely sequenced plasmid mediating quinolone resistance isolated from an environmental sample but also on the first qnrS-like gene detected in Europe.Quinolones and especially fluoroquinolones are among the most often prescribed antimicrobial drugs worldwide (1). As a consequence, high resistance rates have developed due to the persisting selection pressure. Resistances are mainly attributed to chromosomal mutations in the genes gyrA and parC, which encode, respectively, subunit A of DNA-gyrase (GyrA) and topoisomerase IV (ParC), representing the target enzymes for quinolones (25). Nevertheless, plasmid-encoded quinolone resistance is of great concern since these resistance determinants potentially can be disseminated among bacteria due to plasmid mobility. The isolation of the multiresistance plasmid pMG252 from a clinical strain of Klebsiella pneumoniae in Birmingham, Alabama, in 1994 was the first documented discovery of a plasmid-encoded resistance to quinolones (19). The quinolone resistance gene qnrA, located on pMG252, encodes a 218-amino-acid protein of the pentapeptide repeat family. Members of this protein family are characterized by the repetition of the consensus sequence (C/A)-(D/N)-(L/F)-X-X (3). It was shown that Qnr protects DNA-gyrase and topoisomerase IV activity against inhibition by quinolones (38-40). Recently, other qnr genes, namely, qnrS and qnrB, were identified on plasmids originating from clinical isolates (12, 16).Several multiresistance plasmids were previously isolated from the activated sludge bacterial com...

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1␤ plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the ␣-proteobacterium Sinorhizobium meliloti and the ␥-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1␤ plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.