Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system

Abstract

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, nalidixic 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 (rep/par) module and the pXAC33-replicon from the phytopathogenXanthomonas axonopodispv.citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1αplasmid RP4 providing transfer functionsin trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genesdhfr1for a dihydrofolate reductase,aadA2for a spectinomycin/streptomycin adenylyltransferase andblaTLA-2for a so far unknown Ambler class A extended spectrumβ-lactamase. The integron-specific 3′-segment (qacEΔ1-sul1-orf5Δ) is connected to a macrolide resistance operon consisting of the genesmph(A) (macrolide 2′-phosphotransferase I),mrx(hydrophobic protein of unknown function) andmphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genestetA(tetracycline efflux pump) andtetRwas identified upstream of the Tn402-specific transposase genetniA. The second ‘genetic load’ region on pRSB101 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 genesmph(A),mrxandmphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-likerepAamplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.