Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge

In order to isolate antibiotic resistance plasmids from bacterial communiti es found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as a n identification marker, were used as recipients in filter crosses. Transco njugants were selected on agar plates containing 3-chlorobenzoate as the so le carbon source and the antibiotic tetracycline, streptomycin or spectinom ycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) pe r recipient. A total of 12 distinct plasmids, designated pB1-pB12, was iden tified. Their sizes ranged between 41 to 69 kb and they conferred various p atterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 pl asmids were identified as broad-host-range plasmids displaying extremely hi gh transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) pe r recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibili ty group, based on replicon typing using IncP group-specific PCR primers. D NA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncP beta subgroup, whereas two plasmids we re identified as IncP alpha plasmids. Analysis of the IncP-specific PCR pro ducts revealed considerable differences among the IncP beta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP p lasmids, restriction fragments were cloned and their DNA sequences establis hed. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic r esistance, two putative DNA invertases belonging to the Din family, a methy ltransferase of a type I restriction/modification system, a superoxide dism utase. parts of a putative efflux system belonging to the RND family, and p roteins of unknown function were identified.