Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, naliclixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (replpar) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-Iike plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB1 01 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfrl for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and b/a(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-su11-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase 1), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB1 01 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101 -like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB1 01 -like plasmids are released with the final effluents into the environment.
